WO2002044204A2 - Cell-free assembly of lentiviral capsids - Google Patents

Abstract

Methods and compositions for assembling full-sized, stable lentiviral capsids from purified lentiviral Gag polypeptides in vitro are disclosed. The inclusion of phosphorylated compounds in the assembly reaction yields spherical particles indistinguishable from the viral capsids produced by infected cells. Such particles are useful to deliver nucleic acids or other therapeutic molecules to cells. Also provided are methods to identify other compounds that promote capsid assembly, as well as methods to identify therapeutic compounds that interfere with viral assembly. Pharmaceutical compositions containing the in vitro assembled lentiviral capsids are also provided.

Description

Cell-Free Assembly of Lentiviral Capsids

CROSS-REFERENCES TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S.S.N. 60/250,786, filed December 1, 2000, which is incorporated herein by reference and for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT Not Applicable

FIELD OF THE INVENTION

The invention relates to methods for producing lentiviral capsids from purified proteins in a cell-free system. Also provided are screening assays to identify compounds that inhibit or promote capsid assembly and pharmaceutical compositions containing the in vitro assembled lentiviral capsids.

BACKGROUND OF THE INVENTION The assembly of virus particles occurs through the organized multimerization of numerous protein subunits, although in some cases nucleic acid is also required (for a review, see Harrison, in Fields et al. (eds.), Virology p.37-61 (1990)). In retrovirases of the lentivirus genus, such as human immunodeficiency virus type 1 (HIV-1), a virus-like particle can be assembled in eukaryotic cells from the viral Gag polyprotein. The first step in assembly which is visible by electron microscopy (EM) is the accumulation of Gag proteins into electron-dense patches beneath the plasma membrane of the cell. These patches enlarge and project outward from the cell to form spherical, budding particles, which pinch off and are released into the environment. Freshly budded particles have an immature morphology; the Gag proteins are located around the periphery of the particle, under the plasma membrane-derived lipid envelope, giving a doughnut-shaped appearance. Soon after budding, the viral protease (PR) is activated and some of the peripheral material condenses into a cone-shaped core at the center of the HIV-1 particle. This is a mature virus particle.

The HIV-1 Gag polyprotein is composed of separate domains, which are (from the N to the C terminus) the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 domains. Short spacer peptides are also present between the CA and NC domains (p2) and between the NC and p6 domains (pi). The viral protease cleaves Gag at the junctions of these domains to produce the mature structural proteins MA, CA, NC, and p6 as well as p2 and pi.

The functions of these domains during assembly are different from the functions of the mature proteins. The MA domain is important for the transport of Gag from within the cell to the plasma membrane. This domain is cotranslationally modified by the addition of myristic acid to the N terminus, and mutations or drug treatments which prevent myristylation also prevent the association of Gag with the plasma membrane. In these cases, particles assemble in the cytoplasm rather than on the plasma membrane (Morikawa et al, J. Biol. Chem. 271:2868-2873 (1996), Royer et al, Virology 184:417-422 (1991)). The CA domain appears to guide the arrangement of the Gag molecules during assembly. Even small mutations within the CA domain can prevent particle assembly (Chazal et al, J. Virol. 68:111-122_.(1994), Dorfman et al, J. Virol. 68:8180-8187 (1994), Zhao et al, Virology 199:403-408 (1994)) or alter the size of the particle (Chazal et al, J. Virol. 68:111-122 (1994), Dorfman et al, J. Virol. 68:8180-8187 (1994)). As a mature protein, CA forms the shell around the viral core. The NC domain packages the viral RNA genome and promotes Gag-Gag interactions, presumably mediated by RNA binding (Bennett et al, J. Virol. 67:6487-6498 (1993)). As a mature protein within the virus particle, NC protects the RNA genome at the center of the core. The function of the p6 domain, during or after assembly, remains unclear. When Gag alone is expressed, the p6 domain can be deleted without causing any significant defect in particle assembly (Hockley et al, J. Gen. Virol. 75:2985- 2997 (1994), Royer et al, Virology 184:417-422 (1991)). However, in the context of the complete viral genome, deletion of p6 results in a late-assembly defect. In this case, particle assembly appears to proceed normally except that the particles remain tethered to the plasma membrane (Gottlinger et al, Proc. Natl. Acad. Sci., 88:3195-3199 (1991) ). This defect is dependent on the expression of an active PR (Huang et al, J. Virol, 69:6810-6818 (1995)). The functions of these domains within HTV-1 Gag have primarily been identified through genetic analysis and examination of the properties of the mutant particles in vivo. The cellular environment is difficult to manipulate, and thus it is not clear whether the observed Gag mutant phenotypes are purely the result of defective Gag-Gag interactions or are due to interactions with cellular factors. Numerous cellular proteins have been confirmed (e.g., cyclophilin A) or are suspected to interact with Gag during assembly (for a review, see D.E. Ott, Rev. Med. Virol. 7:167-180 (1997)). Cell-free systems, involving the use of wheat germ or reticulocyte lysates, have recently been developed to study retroviral assembly (Lingappa et al, J. Cell. Biol. 136:567-581 (1997), Sakalian et al, J. Virol. 70:3706-3715, (1996), Spearman et al, J. Virol 70:8187-8194 (1996), Weldon et al, J. Virol. 72:3098-3106 (1998)). These systems are more amenable to manipulation, and the results from these studies suggest that at least one cellular protein, which requires ATP for activity, is critical for HIV-1 and Mason-Pfizer monkey virus Gag assembly (Lingappa et al, J. Cell. Biol. 136:567-581 (1997), Weldon et al, J. Virol. 72:3098-3106 (1998)). However, these systems also contain numerous cellular factors, making it difficult to identify purely Gag-Gag interactions.

A fully defined, in vitro assembly system which uses Gag protein, or fragments of Gag, expressed in Escherichia coli has been previously developed. The viral proteins can be purified in a soluble form, without denaturation, and used for in vitro assembly studies. Using this system for RSV Gag proteins, it was observed that purified proteins by themselves did not efficiently assemble into organized, virus-like particles. Efficient assembly required the addition of nucleic acid (only RNA, not DNA, was used in these studies). The CA-NC fragment of RSV Gag formed cylindrical particles with RNA (Campbell et al, J. Virol. 69:6481-6491 (1995)), but when the protein was extended N terminally to also include the MA-p2-plO domains of RSV, spherical particles were formed (Campbell et al., J. Virol. 71:4425-4435 (1997)). The spherical particles that assembled in vitro were similar in appearance to authentic, immature particles which had been stripped of their lipid envelopes (Stewart et al, J. Virol. 64:5076-5092 (1990)). Further analysis showed that the pi 0 domain was responsible for the spherical, rather than cylindrical, shape of these particles (Campbell et al, J. Virol. 71:4425-4435 (1997)). The CA-NC and CA-NC-p6 fragments of HIV-1 Gag also formed cylindrical particles with RNA (Campbell et al, J. Virol. 69:6487-6497 (1995), Gross et al, Eur. J. Biochem., 249:592-600 (1997)).

In vitro assembly of purified, recombinant full-length HIV-1 Gag proteins, or Gag proteins lacking the p6 domain, yielded 25-30 nm spherical particles (Campbell and Rein, J. Virology, 73(3):2270-2279 (1999)). Assembly into spherical particles required nucleic acid. However, the 25-30 nm spherical particles were smaller than HIV-1 spherical particles assembled in vivo (100-120 nm), and were easily disrupted by high salt or nuclease treatment. When capsids were assembled from HIV-1 Gag proteins with a deletion in the MA domain, spherical particles ~ 90 nm in diameter were produced (Gross et al. , EMBO J. , 19(1): 103-113 (2000)). However, assembly was relatively inefficient, and the stability of the assembled capsids was not reported. Thus, stable, full-sized HIV capsids have not been successfully assembled from full-length Gag proteins in vitro under defined conditions. There remains a need to have a fully defined in vitro assembly system for lentiviral capsids in which compounds which modulate capsid assembly can be identified; in which the capsid proteins can be produced recombinantly in E. coli and recombinantly modified capsid proteins can be incorporated in vitro into immature capsids; in which in vitro assembled capsids can be used as delivery vehicles for nucleic acids or other molecules; and in which in vitro assembled capsids can be used to generate lentiviral vaccines which this invention solves.

SUMMARY OF THE INVENTION In one aspect of the invention, a lentiviral capsid is assembled by expressing a lentiviral Gag protein in a recombinant host, and assembling the lentiviral Gag protein into a capsid in a reaction mixture containing an exogenously added phosphate compound in an amount able to facilitate assembly compared to the absence of the phosphate compound.

In another aspect of the invention, a test compound is assayed for its ability to modulate assembly of a lentiviral capsid, by including the test compound in an assembly reaction containing an exogenously added phosphate compound in an amount able to facilitate assembly compared to the absence of the phosphate compound, and detecting the effect of the test compound on capsid assembly.

In yet another aspect of the invention, a test compound is assayed for its ability to affect lentiviral capsid assembly, by expressing a lentiviral Gag protein comprising a functional MA domain, contacting the test compound with the lentiviral Gag protein, and detecting the effect of the test compound on the assembly of the lentiviral Gag protein into lentiviral capsids.

In still another aspect of the invention, a test compound is assayed for its ability to modulate the effect of phosphate compounds on lentiviral capsid assembly, by contacting the test compound with a cell-free lentiviral assembly system, and detecting the effect of the test compound on phosphate compound-mediated capsid assembly.

In another aspect of the invention, a nucleic acid or other therapeutic molecule is delivered to a cell, by assembling a lentiviral capsid in the presence of the nucleic acid or therapeutic molecule, and contacting a cell with the assembled capsid. An additional aspect of the invention provides a lentiviral capsid of diameter at least about 90 nm, comprising a lentiviral Gag protein which is substantially free of fatty acid chains and comprises a functional MA domain.

Another aspect of the invention provides lentiviral capsids, assembled by expressing a lentiviral Gag protein in a recombinant host, and assembling the lentiviral Gag protein into a capsid in the presence of exogenously added phosphate compound in an amount able to facilitate assembly compared to the absence of the phosphate compound.

Still another aspect of the invention provides a cell-free system for assembling lentiviral capsids, comprising a recombinantly produced lentiviral Gag protein at a concentration greater than about 20 μg/ml, and a phosphate compound in an amount sufficient to promote capsid assembly.

In still another aspect of the invention, the cell-free assembled lentiviral capsids are included in pharmaceutical compositions for either prophylactic or therapeutic treatment of lentiviral infections. In one embodiment of the invention, the lentiviral Gag protein is the Gag protein of HIV-1.

In an embodiment of the invention, the effect of a compound on capsid assembly is detected by an increase in the size of the assembled capsids, while in another embodiment, the effect on assembly is detected by a decrease in the size of the assembled capsids.

In one embodiment of the invention, the effect of a compound on capsid assembly is detected by an increase in the salt resistance of the assembled capsids, while in another embodiment the effect on assembly is detected by a decrease in the salt resistance of the assembled capsids. In an embodiment of the invention, the effect of a compound on viral assembly is detected by a change in the sensitivity of the lentiviral Gag protein to a protease. In another embodiment, the effect on assembly is detected by change in the sensitivity of the assembled capsids to a nuclease.

DEFINITIONS

A "capsid" refers to an ordered proteinaceous shell primarily composed of a lentiviral Gag protein. Immature capsids contain unprocessed Gag protein, and are produced during virion budding from cells infected by lentiviruses or transfected to express lentiviral Gag proteins, or by in vitro assembly of Gag proteins in the present invention. Immature lentiviral capsids are typically spherical assemblies, with a diameter of approximately 90 nm. When produced from infected or transfected cells, immature capsids are enclosed within a membrane envelope derived from the host cell plasmid membrane and themselves enclose the viral RNA. Upon virion maturation, the viral protease cleaves the Gag protein forming the immature capsid into matrix (MA), capsid (CA), and nucleocapsid (NC) domains. While the processed MA and NC polypeptides are closely associated with the viral envelope and nucleic acid, respectively, the processed CA polypeptide forms the shell of the mature or core viral capsid. Maturation is associated with a condensation of the immature viral capsid shell into a smaller, denser protein shell. While capsids of immature viral particles are roughly spherical, the mature capsid assumes a virus-specific morphology, which may be conical or cylindrical.

"Lentiviral Gag protein" refers to a polyprotein of at least between about 450 and 550 amino acids and is composed of distinct domains, which are ( from the N to C terminus) the matrix (MA), capsid (CA), nucleocapsid (NC), and p6. In addition, CA and NC are separated by a short spacer peptide p2 and NC and p6 are separated by a short spacer peptide pi . During viral maturation, the Gag polyprotein is cleaved by the viral protease enzyme into distinct peptides, MA, CA, NC, p6, p2 and pi. Lentiviral Gag protein is not stable when expressed in bacterial expression systems such as the system described in Example 1. Lentiviral Gag protein can assemble into capsids with a diameter at least about 80 nm in an in vitro system such as the system described in Example 3.

A "truncated lentiviral Gag protein" refers to a lentiviral Gag protein which has a functional MA domain and in which the p6 domain has been modified such that the truncated lentiviral Gag is stably expressed in E. coli expression systems such as the system described in Example 1 (see Campbell and Rein supra) and can assemble into immature capsids of at least about 80 nm in an in vitro system such as the system described in Example 3. A preferred modification is a deletion of the p6 domain.

"Stable expression" of lentiviral Gag in a bacterial system refers to expressed protein which is greater than 50% homogeneous when purified and assayed as described by Campbell and Rein supra. A lentiviral Gag protein lacks a "functional MA domain" if, on account of mutations or deletions within the segment ordinarily forming the MA polypeptide after cleavage by the viral protease, it will assemble into capsids with diameter at least about 80 nm in the in vitro assembly system described by Gross et al, EMBO J. 19(1): 103-113 (2000). "Assembly" and "capsid assembly" refer to the process by which Gag proteins polymerize into an ordered macromolecular complex. Typically, approximately 1500 monomeric Gag proteins assemble into a spherical immature capsid particle. In infected or transfected cells, assembly of lentiviral Gag proteins takes place at the cytoplasmic face of the plasma membrane, concurrent with viral budding. Assembly may be distinguished from aggregation by the organization of the Gag proteins into a regular macromolecular structure such as a spherical capsid, with a relatively well-defined number of subunits.

A "lentivirus" refers to a member of the genus of retrovirases including HIV-1 and HIV-2, the simian immunodeficiency viruses (SIVs), feline immunodeficiency virus (FIN), bovine immunodeficiency virus (BIN), Maedi/Nisna virus (MW), caprine arthritis encephalitis virus (CAEN) and equine infectious anemia virus (El AN).

"Lentiviral" refers to a gene, gene product, component, structure or other characteristic of a lentivirus.

A "phosphate compound" refers to a compound, other than a nucleic acid, which bears one or more phosphate groups and facilitates the assembly of lentiviral capsids, as determined by the methods described herein, when present in an effective amount in a capsid assembly reaction. Phosphate compounds include polyphosphates, inositol phosphates, and phosphatidyl inositol derivatives. Some phosphate compounds, and their efficacy in promoting capsid assembly, are listed in Table 1. "Inositol phosphates" are phosphorylated derivatives of inositol

(hexahydroxycyclohexane), bearing one to six phosphate groups attached to the hydroxy groups of the hexane ring. Naturally occurring inositol and inositol derivatives are found as myo-inositol (1,2,3,5/4,6-hexahydroxycyclohexane), rather than epi-inositol (1,2,3,4,5/6- hexahydroxycyclohexane) or scyllo-inositol (1,3,5/2,4,6-hexahydroxycyclohexane). Inositol phosphates also include molecules with fatty acid chains, e.g., phosphatidyl inositol phosphates such as PI(3,4,5)P3.

A compound "facilitates assembly" of a capsid if Gag polypeptides assemble into capsids to a greater extent in the presence of the compound than in its absence. Compounds may also facilitate capsid assembly by increasing the rate at which Gag polypeptides are assembled into capsids, by increasing the diameter of the assembled capsids, or by increasing the stability of the assembled capsids.

The "stability" of an assembled capsid refers to the ability of the macromolecular complex to resist dissociation by an agent such as heat, ionic strength, detergents, chaotropic agents, or dilution. The stability of an assembled complex against a dissociative agent correlates with the ability of the monomeric Gag proteins to assemble in the presence of the dissociative agent.

"Salt resistance" refers to the ability of an assembled capsid to resist dissociation by high ionic strength, or the ability of Gag polypeptides to assemble into capsids in solutions of high ionic strength. A "test compound" is a compound added to an assembly reaction in order to assay its ability to facilitate or inhibit capsid assembly. Test compounds may be added to an assembly reaction either in the presence or the absence of a phosphate compound, in order to determine whether a test compound affects capsid assembly on its own, or modulates the effect of phosphate compounds on capsid assembly.

The terms "isolated," "purified," or "biologically pure" refer to material that is substantially or essentially free from components that normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified. In particular, an isolated Gag nucleic acid is separated from open reading frames that flank the Gag gene and encode proteins other than Gag. The term "purified" denotes that a nucleic acid or protein gives rise to essentially one band in an elecfrophoretic gel. Particularly, it means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.

"Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2- O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al, Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al, J. Biol Chem. 260:2605-2608 (1985); Rossolini et al, Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

A particular nucleic acid sequence also implicitly encompasses "splice variants." Similarly, a particular protein encoded by a nucleic acid implicitly encompasses any protein encoded by a splice variant of that nucleic acid. "Splice variants," as the name suggests, are products of alternative splicing of a gene. After transcription, an initial nucleic acid transcript may be spliced such that different (alternate) nucleic acid splice products encode different polypeptides. Mechanisms for the production of splice variants vary, but include alternate splicing of exons. Alternate polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any products of a splicing reaction, including recombinant forms of the splice products, are included in this definition.

The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ- carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention. The following eight groups each contain amino acids that are conservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Naline (N);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).

Macromolecular structures such as polypeptide structures can be described in terms of various levels of organization. For a general discussion of this organization, see, e.g., Alberts et al, Molecular Biology of the Cell (3^rd ed., 1994) and Cantor and Schimmel, Biophysical Chemistry Part I: The Conformation of Biological Macromolecules (1980). "Primary structure" refers to the amino acid sequence of a particular peptide. "Secondary structure" refers to locally ordered, three dimensional structures within a polypeptide. These structures are commonly known as domains. Domains are portions of a polypeptide that form a compact unit of the polypeptide and are typically 15 to 350 amino acids long. Typical domains are made up of sections of lesser organization such as stretches of β-sheet and α- helices. "Tertiary structure" refers to the complete three dimensional structure of a polypeptide monomer. "Quaternary structure" refers to the three dimensional structure formed by the noncovalent association of independent tertiary units.

A "label" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins for which antisera or monoclonal antibodies are available (e.g., the polypeptide of SEQ ID NO:l can be made detectable, e.g., by incorporating a radiolabel into the peptide, and used to detect antibodies specifically reactive with the peptide).

As used herein a "nucleic acid probe or oligonucleotide" is defined as a nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation. As used herein, a probe may include natural (i.e., A, G, C, or T) or modified bases (7-deazaguanosine, inosine, etc.). In addition, the bases in a probe may be joined by a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization. Thus, for example, probes may be peptide nucleic acids in which the constituent bases are joined by peptide bonds rather than phosphodiester linkages. It will be understood by one of skill in the art that probes may bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions. The probes are preferably directly labeled as with isotopes, chromophores, lumiphores, chromogens, or indirectly labeled such as with biotin to which a streptavidin complex may later bind. By assaying for the presence or absence of the probe, one can detect the presence or absence of the select sequence or subsequence. A "labeled nucleic acid probe or oligonucleotide" is one that is bound, either covalently, through a linker or a chemical bond, or noncovalently, through ionic, van der Waals, electrostatic, or hydrogen bonds to a label such that the presence of the probe may be detected by detecting the presence of the label bound to the probe. The term "recombinant" when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non- recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.

A "promoter" is defined as an array of nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation. The term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence. The term "heterologous" when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

An "expression vector" is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the expression vector includes a nucleic acid to be transcribed operably linked to a promoter.

The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, 65%, 70%, 75%, 80%, preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to an amino acid sequence such as SEQ ID NO:l), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be "substantially identical." This definition also refers to the compliment of a test sequence. Preferably, the identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. For sequence comparison of nucleic acids and proteins to Gag nucleic acids and proteins, the BLAST and BLAST 2.0 algorithms and the default parameters discussed below are used. A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol Bio 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat 'I Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel etal, eds. 1995 supplement)). A preferred example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al, Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al, J. Mol. Biol. 215:403-410 (1990), respectively. BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat 'I. Acad. Sci. USA 90:5873- 5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)). which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

The phrase "selectively (or specifically) hybridizes to" refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular or library DNA or RNA).

The phrase "stringent hybridization conditions" refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry arid Molecular Biology— Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). Generally, stringent conditions are selected to be about 5-10°C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength pH. The T_m is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T_m, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For high stringency hybridization, a positive signal is at least two times background, preferably 10 times background hybridization. Exemplary high stringency or stringent hybridization conditions include: 50%> formamide, 5x SSC and 1% SDS incubated at 42° C or 5x SSC and 1% SDS incubated at 65°C, with a wash in 0.2x SSC and 0.1% SDS at 65°C. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides that they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Exemplary "moderately stringent hybridization conditions" include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in IX SSC at 45°C. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency.

"Pharmaceutically acceptable" refers to a generally non-toxic, inert, and/or physiologically compatible composition.

A "pharmaceutical excipient" comprises a material such as an adjuvant, a carrier, pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservative, and the like.

A "prophylactic immune response" or "therapeutic immune response" refers to an immune response to an antigen on a lentiviral capsid which prevents or a least partially arrests disease symptoms or progression. The immune response may include a T cell response and or a B cell response and may include an antibody response which has been facilitated by the stimulation of helper T cells.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Electron micrographs of particles assembled in vitro. Panel A shows particles assembled in buffer alone. Panel B shows particle assembled in buffer with reticulocyte lysate. Scale bar = 100 nm

Figure 2. Stabilization of HIV-1 capsids by reticulocyte lysate. HIV-1 or MoMuLV particles produced by infected cells, HIV-1 capsids assembled in vitro, or HIV-1 capsids assembled in vitro with reticulocyte lysate, were treated with RNase or high salt. Assembled capsids were pelleted by centrifugation. The total (T), pellet (P), or supernatant (S) fractions were analyzed by SDS-PAGE followed by Coomassie Blue staining (particles assembled in vitro) or immunoblotting with polyclonal sera recognizing the viral capsid (particles isolated from infected cells). Particles isolated from infected cells were treated with 1% NP-40 to remove the viral membrane; digestion of the HIV-1 particles by HIV-1 protease confirms that the viral membrane has been removed.

Figure 3. Stabilization of HIV-1 capsids by active assembly fraction or inositol phosphate. Capsids assembled in vitro in the presence of fractionated reticulocyte lysate or inositol pentakisphosphate were treated with RNase or high salt. Assembled capsids were pelleted by centrifugation and analyzed by SDS-PAGE followed by Coomassie Blue staining.

Figure 4. Proteolytic digest of HIV-1 particles assembled in vitro and in vivo. Anti-capsid immunoblot of in vitro assembled particles and immature HIV-1 particles produced from cells, treated with trypsin, HIV protease, or trypsin and HIV protease together.

DETAILED DESCRIPTION

INTRODUCTION

The present invention provides methods and compositions for assembling lentiviral capsids from purified components in vitro. The addition of inositol phosphates, and other compounds bearing phosphate groups, to the assembly reaction yields full-sized capsid particles with high efficiency. The resulting particles are indistinguishable from capsids assembled in vivo, as measured by their resistance to salt, nuclease, and protease treatment. Since assembly takes place from recombinant proteins under defined conditions, large amounts of pure capsids may be produced. These capsids are useful not only in the field of viral morphogenesis, but also may be used as delivery vehicles to transfect cells with therapeutic nucleic acids and in pharmaceutical compositions. Methods for identifying compounds that affect capsid assembly are also disclosed. The assembly reactions of the present invention are rapid and large amounts of starting material are easily synthesized. Such reactions provide convenient assay systems for screening libraries of compounds that inhibit or promote capsid assembly. Moreover, the discovery that inositol phosphates participate in viral assembly suggests new metabolic pathways as targets for antiviral drags. Compounds identified as inhibitors of viral capsid assembly, or that affect cellular inositol phosphate metabolism, represent potential therapeutics useful against viral infection. LENTIVIRUSES

The present invention provides methods and compositions for assembling capsids from lentiviral Gag proteins. Lentivirases are a genus of retrovirases characterized by distinct morphological features (e.g., a cylindrical or cone-shaped nucleoid in the mature virion), by several regulatory genes (e.g., tat and rev) not present in simpler retrovirases, and by a biphasic course of viral gene expression. As is the case with other retrovirases, lentiviral genomes are encoded by a single-stranded RNA molecule present in two copies in each virion. Unlike other retrovirases, lentivirases are not themselves oncogenic, but lentiviral infections are known to cause disease in primate, bovine, feline, equine, caprine, and ovine species. Common features of diseases caused by lentivirases include long and variable incubation periods, persistent viral replication, neurologic manifestations, and destruction of specific hematologic or immunologic cells. Lentivirases include the human pathogens HIV-1 and HrV-2, the simian immunodeficiency viruses (SIVs), feline immunodeficiency virus (FIN), bovine immunodeficiency virus (BIN), Maedi/Nisna virus (MNN), caprine arthritis encephalitis virus (CAEN) and equine infectious anemia virus (EIAV).

Lentivirases share a characteristic pathway of virion assembly, known as C- type assembly, with retrovirases in the HTLV/BLV group. In all retrovirases, the viral Gag protein is synthesized by cellular ribosomes. In B-type and D-type retrovirases, Gag spherical shells of Gag form deep in the cytoplasm, and are subsequently transported to the plasma membrane where they become enveloped and are released by budding. In contrast, in C-type retrovirases, Gag assembly takes place only on the inner face of the plasma membrane, and formation of the spherical Gag shell takes place concomitant with the budding process. Assembly begins with association of Gag monomers and the plasma membrane. Gag proteins of many C-type retrovirases bear both a myristic acid group and a cluster of basic amino acids near the Ν-terminus to facilitate interaction with the plasma membrane. Notably, Gag proteins of avian C-type retrovirases and non-primate lentivirases lack the myristic acid modification.

T .ENTIVIRAL GAG PROTEINS

The capsids of the present invention are primarily composed of lentiviral Gag polypeptides. Like the Gag proteins of all retrovirases, the lentiviral Gag proteins are the precursors to the internal structural proteins of mature virions. While Gag proteins of retrovirases display considerable sequence divergence, the Gag protein is easily recognizable in all retrovirases by its domain structure and position in the viral genome. See Coffin et al, Retroviruses, at 27-69. In all known retrovirases, the Gag protein is encoded by the 5'-most open reading frame of the viral genomic RNA. For example, in HIV-l, the Gag ORF occupies bases 336 to 1836 of the viral genome. Accession numbers for the sequences of lentiviral Gag proteins may be found in Coffin, supra, at p. 804.

Despite a lack of strong sequence conservation, all retroviral Gag proteins contain the MA, CA, and NC domains in the order (NH2)-MA-CA-NC-(COOH). Cleavage of Gag by the viral protease (PR) yields the MA, CA, and NC polypeptides found in mature viral particles. In unprocessed lentiviral Gag polypeptides, short spacer peptides also flank the NC polypeptide sequence. Translational readthroughs and frameshifts produce Gag-Pro and Gag-Pro-Pol precursor proteins, which yield the viral protease and reverse transcription polypeptides upon cleavage.

Lentiviral Gag proteins contain an additional polypeptide (p6) which resides at the carboxy terminus of Gag and is released by PR cleavage. Gag polypeptides containing the p6 domain suffer degradation when expressed in bacteria (see Campbell and Rein, supra; Gross et al, supra). For efficient expression of Gag proteins in prokaryotic systems, derivatives of Gag lacking the p6 domain may be expressed. The p6 domain of Gag does not affect in vitro assembly of immature capsids, and the methods and compositions of the invention may be practiced with either full-length Gag proteins or Gag proteins lacking the p6 sequence.

The matrix (MA) domain at the amino terminus of Gag gives rise to the MA polypeptide of the mature virion, which underlies the viral membrane and may be associated with the viral env protein. HIV Gag proteins with a deletion in the MA domain have been used to assemble full-sized capsids (~ 90 nm) in the apparent absence of assembly modulators (Gross et al, supra). In contrast to the capsids assembled by the methods of the present invention, capsids assembled from Gag with this mutated MA domain incorporate a relatively low fraction of the total Gag protein in the assembly reaction, and the stability of the assembled capsids is unknown. Without wishing to be bound by a particular theory, determinants in the Gag MA domain may mediate the effect of the phosphate compounds of the present invention on capsid assembly. Such determinants may be identified by assembling capsids from Gag polypeptides bearing MA mutations or deletions (e.g., the deletion of residues 16-99 reported by Gross et al). The ability to assemble a full-sized capsid in the absence of phosphate compounds, or to assemble a stable capsid in the absence of phosphate compounds, indicates that functional determinants of the Gag MA domain have been mutated.

Minor mutations or deletions in the Gag protein may be made without compromising its ability to assemble into full-sized capsids. Foreign or synthetic amino acid sequences may be fused to the amino or carboxy termini of Gag, inserted within the Gag open reading frame, or substituted for portions of the Gag sequence. Amino acids fused to the amino terminus of the MA domain will be displayed on the surface of the assembled capsid, while amino acids fused to the carboxy-terminal NC domain will be positioned on the inner surface of the assembled capsid. The regions between the MA, CA, and NC domains of Gag are preferred sites for internal insertion of foreign sequences, as insertion between the protein domains is less likely to disturb the structure of the Gag protein. However, the ability of any modified Gag protein to assemble into stable capsids may readily be tested by expressing the modified Gag protein, assembling it into capsids in the presence of a phosphate compound, and assaying the size and stability of the assembled particles as described herein.

When expressed in eukaryotic cells, primate lentiviral Gag proteins are modified cotranslationally by the addition of myristate, a 14-carbon fatty acid, at their amino termini. In contrast, prokaryotic cells lack equivalent myristylation enzymes and Gag expressed in prokaryotic cells is free of fatty acid chains. In eukaryotes, myristylation is directed by a consensus sequence (Met-Gly-X-X..Ser/Thr) at the amino terminus of Gag.

Myristate is attached the glycine residue of the consensus sequence following removal of the initiating methionine residue. Addition of a myristate group helps direct Gag to the plasma membrane, the ordinary site of capsid assembly. Upon virion maturation, the myristate group anchors the MA polypeptide to the viral envelope, although a cluster of positive charges at the amino terminus of MA is also important for association with the viral membrane.

When capsids are assembled in association with membranes, myristylation of Gag is required for efficient assembly if the Gag protein is normally myristylated (see supra). See Lingappa et al, supra. The present invention provides methods for assembling capsids in the absence of cells or membranes. Hence, the invention may be practiced with either myristylated or unmyristylated Gag polypeptides. Gag proteins without attached myristate groups may be prepared from eukaryotic cells by mutating the glycine residue encoded by the second codon of Gag. EXPRESSION OF GAG PROTEINS

This invention requires lentiviral Gag proteins as the starting material for an assembly reaction. Gag proteins are most easily obtained by recombinant DNA methodology. To obtain high level expression of a cloned gene, such as those cDNAs encoding lentiviral Gag proteins, one typically subclones Gag into an expression vector that contains a strong promoter to direct transcription, a transcription/translation terminator, and a ribosome binding site for translational initiation. Suitable bacterial promoters are well known in the art and described, e.g., in Sambrook et al. and Ausubel et al. Bacterial expression systems for expressing lentiviral Gag protein are available in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al, Gene 22:229-235 (1983); Mosbach et al, Nature 302:543-545 (1983). Kits for such expression systems are commercially available. Especially preferred are bacterial expression systems offering high level, tightly regulated expression, such as the pET series of vectors and compatible expression hosts available from Novagen (pET System Manual, Novagen, Madison, WI). Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available.

Selection of the promoter used to direct expression of a heterologous nucleic acid depends on the particular application. The promoter is preferably positioned about the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.

In addition to the promoter, the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the Gag encoding nucleic acid in host cells. A typical expression cassette thus contains a promoter operably linked to the nucleic acid sequence encoding Gag, ribosome binding sites, transcription or translation termination signals, and, in cassettes adapted to eukaryotic hosts, signals required for efficient polyadenylation of the transcript. Additional elements of a eukaryotic expression cassette may include enhancers and, if genomic DNA is used as the structural gene, introns with functional splice donor and acceptor sites.

In addition to a promoter sequence, the expression cassette should also contain a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes. The particular expression vector used to transport the genetic information into the cell is not particularly critical. Any of the conventional vectors used for expression in eukaryotic or prokaryotic cells may be used. Standard bacterial expression vectors include plasmids such as pBR322 based plasmids, pSKF, pET23D, and fusion expression systems such as GST and LacZ. Epitope tags can also be added to recombinant proteins to provide convenient methods of isolation, e.g., c-myc.

Expression vectors containing regulatory elements from eukaryotic viruses are typically used in eukaryotic expression vectors, e.g., SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Barr virus. Other exemplary eukaryotic vectors include pMSG, ρAV009/A⁺, pMTO10/A⁺, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the CMV promoter, SV40 early promoter, S V40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells. Expression of proteins from eukaryotic vectors can also be regulated using inducible promoters. With inducible promoters, expression levels are tied to the concentration of inducing agents, such as tetracycline or ecdysone, by the incorporation of response elements for these agents into the promoter. Generally, high level expression is obtained from inducible promoters only in the presence of the inducing agent; basal expression levels are minimal. Inducible expression vectors are often chosen if expression of the protein of interest is detrimental to eukaryotic cells.

Some expression systems have markers that provide gene amplification such as thymidine kinase and dihydrofolate reductase. Alternatively, high yield expression systems not involving gene amplification are also suitable, such as using a baculovirus vector in insect cells, with a Gag encoding sequence under the direction of the polyhedrin promoter or other strong baculovirus promoters.

The elements that are typically included in expression vectors also include a replicon that functions inE. coli, a gene encoding antibiotic resistance to permit selection of bacteria that harbor recombinant plasmids, and unique restriction sites in nonessential regions of the plasmid to allow insertion of eukaryotic sequences. The particular antibiotic resistance gene chosen is not critical. Any of the many resistance genes known in the art are suitable. The prokaryotic sequences are preferably chosen such that they do not interfere with the replication of the DNA in eukaryotic cells, if necessary. Standard transfection methods are used to produce bacterial, mammalian,^' yeast or insect cell lines that express large quantities of Gag protein, which are then purified using standard techniques (see, e.g., Colley et al, J. Biol. Chem. 264:17619-17622 (1989); Guide to Protein Purification, in Methods in Enzymology, vol. 182 (Deutscher, ed., 1990)). Transformation of eukaryotic and prokaryotic cells are performed according to standard techniques (see, e.g., Morrison, J Bad. 132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology 101:347-362 (Wu et al, eds, 1983)).

Any of the well-known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, liposomes, microinjection, plasma vectors, viral vectors and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al, supra). It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell capable of expressing Gag.

After the expression vector is introduced into the cells, the transfected cells are cultured under conditions favoring expression of Gag, which is recovered from the culture using standard techniques identified below.

PURIFICATION OF GAG POLYPEPTIDES

Either naturally occurring or recombinant Gag can be purified for use in the methods and compositions of the invention. Gag proteins may be purified to substantial purity by standard techniques, including selective precipitation with such substances as ammonium sulfate; column chromatography, immunopurification methods, and others (see, e.g., Scopes, Protein Purification: Principles and Practice (1982); U.S. Patent No. 4,673,641; Ausubel et al, supra; and Sambrook et al, supra).

A number of procedures can be employed when recombinant Gag proteins are being purified. For example, proteins having established molecular adhesion properties can be reversibly fused to the Gag proteins. With the appropriate ligand, the Gag proteins can be selectively adsorbed to a purification column and then freed from the column in a relatively pure form. The fused protein is then removed by enzymatic activity. Finally the Gag proteins may be purified using immunoaffinity columns. A. Purification of Gag proteins from recombinant bacteria

Recombinant proteins are expressed by transformed bacteria in large amounts, typically after promoter induction; but expression can be constitutive. Promoter induction with IPTG is a one example of an inducible promoter system. Bacteria are grown according to standard procedures in the art. Fresh or frozen bacteria cells are used for isolation of protein.

Proteins expressed in bacteria may form insoluble aggregates ("inclusion bodies"). Several protocols are suitable for purification of the Gag proteins from inclusion bodies. For example, purification of inclusion bodies typically involves the extraction, separation and/or purification of inclusion bodies by disraption of bacterial cells, e.g., by incubation in a buffer of 50 mM TRIS/HCL pH 7.5, 50 mM NaCl, 5 mM MgCl₂, 1 mM DTT, 0.1 mM ATP, and 1 mM PMSF. The cell suspension can be lysed using 2-3 passages through a French Press, homogenized using a Polytron (Brinkman Instruments) or sonicated on ice. Alternate methods of lysing bacteria are apparent to those of skill in the art (see, e.g., Sambrook et al. , supra; Ausubel et al. , supra).

If necessary, the inclusion bodies are solubilized, and the lysed cell suspension is typically centrifuged to remove unwanted insoluble matter. Proteins that formed the inclusion bodies may be renatured by dilution or dialysis with a compatible buffer. Suitable solvents include, but are not limited to urea (from about 4 M to about 8 M), formamide (at least about 80%, volume/volume basis), and guanidine hydrochloride (from about 4 M to about 8 M). Some solvents which are capable of solubilizing aggregate-forming proteins, for example SDS (sodium dodecyl sulfate), 70% formic acid, are inappropriate for use in this procedure due to the possibility of irreversible denaturation of the proteins, accompanied by a lack of immunogenicity and/or activity. Although guanidine hydrochloride and similar agents are denaturants, this denaturation is not irreversible and renaturation may occur upon removal (by dialysis, for example) or dilution of the denaturant, allowing re-formation of immunologically and/or biologically active protein. Other suitable buffers are known to those skilled in the art. Gag proteins are separated from other bacterial proteins by standard separation techniques, e.g., with Ni-NTA agarose resin in the case of Gag proteins comprising a metal-chelating moiety.

Alternatively, it is possible to purify the Gag proteins from bacterial periplasm. After lysis of the bacteria, when the Gag proteins are exported into the periplasm of the bacteria, the periplasmic fraction of the bacteria can be isolated by cold osmotic shock in addition to other methods known to skill in the art. To isolate recombinant proteins from the periplasm, the bacterial cells are centrifuged to form a pellet. The pellet is resuspended in a buffer containing 20% sucrose. To lyse the cells, the bacteria are centrifuged and the pellet is resuspended in ice-cold 5 mM MgSO₄ and kept in an ice bath for approximately 10 minutes. The cell suspension is centrifuged and the supernatant decanted and saved. The recombinant proteins present in the supernatant can be separated from the host proteins by standard separation techniques well known to those of skill in the art.

B. Standard protein separation techniques for purifying the Gag proteins Solubility fractionation

Often as an initial step, particularly if the protein mixture is complex, an initial salt fractionation can separate many of the unwanted host cell proteins (or proteins derived from the cell culture media) from the recombinant protein of interest. The preferred salt is ammonium sulfate. Ammonium sulfate precipitates proteins by effectively reducing the amount of water in the protein mixture. Proteins then precipitate on the basis of their solubility. The more hydrophobic a protein is, the more likely it is to precipitate at lower ammonium sulfate concentrations. A typical protocol includes adding saturated ammonium sulfate to a protein solution so that the resultant ammonium sulfate concentration is between 20-30% of saturation. This concentration will precipitate the most hydrophobic of proteins. The precipitate is then discarded (unless the protein of interest is hydrophobic) and ammonium sulfate is added to the supernatant to a concentration known to precipitate the protein of interest. The precipitate is then solubilized in buffer and the excess salt removed if necessary, either through dialysis or diafiltration. Other methods that rely on solubility of proteins, such as cold ethanol precipitation, are well known to those of skill in the art and can be used to fractionate complex protein mixtures.

Size differential filtration

The molecular weight of the Gag proteins can be used to isolate it from proteins of greater and lesser size using ultrafiltration through membranes of different pore size (for example, Amicon or Millipore membranes). As a first step, the protein mixture is ultrafiltered through a membrane with a pore size that has a lower molecular weight cut-off than the molecular weight of the protein of interest. The retentate of the ultrafiltration is then ultrafiltered against a membrane with a molecular cut off greater than the molecular weight of the protein of interest. The recombinant protein will pass through the membrane into the filtrate. The filtrate can then be chromatographed as described below.

Column chromatography The Gag proteins can also be separated from other proteins on the basis of its size, net surface charge, hydrophobicity, and affinity for ligands. In addition, antibodies raised against proteins can be conjugated to column matrices and the proteins immunopurified. All of these methods are well known in the art. It will be apparent to one of skill that chromatographic techniques can be performed at any scale and using equipment from many different manufacturers (e.g. , Pharmacia Biotech).

ASSEMBLY REACTIONS

Assembly of Gag proteins into viral capsids typically takes place in solution. Assembly may be initiated by dialyzing or diluting the Gag protein from storage buffers not permissive of capsid assembly (e.g., containing 0.5 mM NaCl) into assembly buffers permissive of capsid assembly (e.g., containing 0.1 mM NaCl).

Assembly reactions include, in addition to Gag protein, nucleic acid, and buffer components, either a phosphate compound, phosphate compound plus test compound, or a test compound alone. Assembly preferably takes place at a temperature from 0°C to 37°C, more preferably at 25°C. Temperature and other reaction conditions or components may be optimized for a particular application by varying the parameter in question over a specified range and monitoring for the desired effect on capsid assembly or capsid stability. Gag protein is typically present in the assembly reaction at a concentration greater than 0.02 mg/ml, preferably from 0.1 mg/ml to 2 mg/ml, and most preferably at 1 mg/ml. Protein concentration may be ascertained by methods known to those skilled in the art.

Nucleic acids are generally included in the assembly reaction, as the presence of nucleic acid facilitates the assembly of capsids and the nucleic acid becomes packaged within the capsid particle. However, the identity and sequence of the nucleic acid are generally unimportant, so long as the nucleic acid is single-stranded; see Campbell and Rein, supra; Gross et al, supra. Single-stranded nucleic acid molecules between approximately 15 nt and 20,000 nt in length, preferably at least 4,000 nt or 10,000 nt, may be included in the assembly reaction. Thus, viral RNA, ribosomal RNA, prokaryotic and eukaryotic tRNA, synthetic DNA or RNA oligonucleotides, and DNA or RNA molecules produced by recombinant methods are all suitable for inclusion in assembly reactions, either as mixtures or as pure preparations. Yeast tRNA, an inexpensive and abundant nucleic acid, is suitable for practice of the invention. Nucleic acids are typically present at a nucleic acid/Gag protein ratio of 1 - 16% (wt/wt), preferably 4%.

Standard physiological buffers are suitable for practice of the invention. Typically, an assembly buffer will include salts at a concentration of 50 - 150 mM, preferably NaCl at 100 mM. The pH of the assembly reaction is maintained within the range of 7 - 8, preferably 7.5, with buffer systems known in the art (e.g., 20 mM Tris-HCl). The assembly reaction may also include protein stabilizing reagents (e.g., 2 -10 % glycerol), reducing agents (e.g., 1 - 10 mM DTT), or detergents to reduce protein aggregation or adsorption. A preferred detergent is Nonidet P-40 (NP-40), preferably present at a concentration of 0.5 - 2% (vol/vol), most preferably at 1%.

Phosphate compounds or other compounds that modulate capsid assembly are included in the reaction at the concentration required to achieve the desired effect. Table 1 shows the concentration of several phosphate compounds required to stabilize 100% of assembled capsids against salt dissociation when Gag is present in the assembly reaction at 1 mg/ml. Lesser concentrations of phosphate compounds stabilize a lesser proportion of capsids in a linear fashion; thus decreasing the concentration of phosphate compound in a typical assembly reaction by half reduces the amount of Gag protein assembled into salt- resistant capsids by 50%.

The kinetics of capsid assembly may be monitored by sampling the assembly reaction at various time points and assaying for the extent of capsid assembly, or the quality of the assembled capsids, as described below. Under the conditions described in Example 3, the assembly solution becomes turbid within minutes of adding the protein to the reaction, indicating that particulate matter is being formed in the reaction. Typically, assembly reactions performed at room temperature are allowed to proceed for at least 30 minutes. Following principles of macromolecular assembly known to the art, or simply by empirical adjustment, assembly time may be shortened or lengthened depending on the temperature, protein concentration, or other parameters of the assembly reaction. Assembly generally incorporates at least 30% of the Gag present in the reaction into spherical capsids, preferably at least 60%, more preferably at least 90%, and most preferably at least 95% of the Gag protein. ASSAYING CAPSID ASSEMBLY AND STABILITY

The phosphate compounds of the invention and other modulators of capsid assembly identified by the methods of the invention may affect both the extent of capsid assembly and the nature of the resulting particles. Hence, assays that monitor the amount of capsids generated in an assembly reaction, and assays that monitor the size, stability, or other properties of the assembled capsids are useful in the practice of the invention.

A. Detecting assembly of Gag polypeptides into capsids Capsid assembly may be detected by monitoring the incorporation of monomeric Gag polypeptides into macromolecular complexes. Such assays typically involve separation of monomeric Gag polypeptides from assembled capsids on the basis of size, followed by detection and quantitation of the Gag polypeptides. Thus, in Example 3, assembled capsids are pelleted by centrifugation at 21,000 x g for 60 minutes, and the amount of Gag polypeptide in the pellet and supernatant fractions is determined by SDS-PAGE followed by Coomassie Blue staining. Alternatively, assembled capsids may be separated from monomeric Gag polypeptides and incompletely assembled capsids by sedimentation through sucrose gradients or other media (see, e.g., Lingappa et al, PCT publication WO 98/35062). Where the Gag protein is present at a low concentration or in an impure preparation, the presence of Gag protein in a particular fraction may be detected by means known in the art, e.g., immunoblotting, radiolabeled Gag protein, or detecting the nucleic acid incorporated into the assembled particle.

Capsid assembly may also be detected and analyzed by changes in the physiochemical properties of the assembly solution. Particularly preferred for high- throughput screening procedures are optical methods to monitor capsid assembly such as light scattering (see, e.g., Zlotnick et al, Virology 277(2):450-456 (2000)). Assembled capsids may also be visualized directly by electron microscopy of the assembly reaction.

B. Stability and size of the assembled capsids Capsids assembled in vitro from lentiviral Gag proteins that comprise a functional MA domain are smaller and less stable than authentic lentiviral capsids in the absence of phosphate compounds. See Campbell and Rein, 1999, supra; Gross et al, supra; Example 3. When added to in vitro assembly reactions, the phosphate compounds of the present invention yield capsid particles similar in size and stability to authentic lentiviral capsids (Examples 3 and 5). Other modulators of capsid assembly identified by the methods of the present invention may increase or decrease the size or stability of the assembled capsids. The present invention therefore provides methods to assay the size and stability of assembled capsids. The effect of an assembly modulator on particle size may be determined directly by electron microscopy using methods known in the art; see Campbell and Rein, supra. The size of the assembled particle is determined by measurements on the electron micrographs. The diameter of the image of the particle is divided by the magnification of the image to obtain the actual diameter of the particle itself. For example, as shown in Figure 1A, an assembly reaction lacking assembly modulators shows spherical particles 25-30 nm in diameter when examined by thin-section electron microscopy. When cellular extracts containing inositol phosphates are added to the assembly reaction, electron microscopy shows 100-120 nm particles similar to those produced from HIV-infected cells.

Other techniques that yield information about particle size are also suitable to detect the effect of an assembly modulator on capsid size. Examples include light scattering, and sedimentation techniques such as density gradient analysis and rate-zonal gradient analysis (see, e.g., Gamier et al, J. Virology, 73(3):2309-2320 (1999)).

It is a discovery of the present invention that authentic lentiviral capsids, or capsids assembled in the presence of phosphate compounds, are more stable than capsids assembled in vitro in the absence of phosphate compounds. Particle stability may be determined by subjecting the assembled particles to treatments that dissociate protein-protein or protein-nucleic acid interactions and assaying the amount of Gag polypeptide remaining in a macromolecular complex. Assembly in the presence of the dissociative agent may also be used to detect effects on capsid assembly. Salts, chaotropic agents, detergents, temperature, and other dissociative agents may be employed to test the stability of the assembled capsids. For example, treatment of in vitro assembled capsids with 0.5 M NaCl drastically reduces the amount of Gag protein precipitable by centrifugation, indicating that the capsids have been disrupted (Figure 2). Salt treatment also eliminates the turbidity associated with particle assembly. However, 0.5 M NaCl does not affect the distribution of Gag when assembly takes place in the presence of inositol phosphates, indicating that the assembled capsids have been stabilized (Figure 3).

Nuclease resistance may also be employed to determine the stability of the assembled complex. Unstable capsids are dissociated to Gag monomers when the nucleic acid complexed with the capsid is degraded. In contrast, the Gag monomers of stable capsids remain associated in a macromolecular complex when the nucleic acid is degraded, although the spherical morphology of the capsids may be lost. The nuclease chosen depends on the identity of the nucleic acid included in the assembly reaction. Thus, when tRNA or other RNA molecules are included in the assembly reaction, RNase A or other RNases may be used to assess capsid stability. If DNA is included in the assembly reaction, DNase resistance provides a measure of capsid stability. Thus, as shown in Figure 2, RNase A treatment of capsids assembled in buffer alone eliminates Gag from the pelleted fraction, indicating that Gag monomers are released when the RNA complexed with the capsid is degraded. When stable capsids assembled with inositol phosphates are treated with RNase, Gag remains in the pelleted fraction, indicating that at least some protein-protein interactions of the stabilized capsids remain even when the RNA is degraded.

Resistance to proteolysis provides another measure of the stability and authenticity of capsids assembled according to the invention. Capsid stability may be determined by the extent to which the Gag polypeptide is accessible to a protease added after assembly is complete. Digestion with any protease that differentially degrades the Gag polypeptide in unstable capsids is suitable to practice the invention. For example, as shown in Figure 4, treatment with HIV protease or HIV protease plus trypsin yields a similar Gag digestion pattern with authentic capsids, capsids assembled in buffer alone, or capsids assembled with inositol phosphates. Digestion with trypsin alone completely degrades Gag assembled in buffer alone, while authentic capsids or capsids assembled with inositol phosphates yield a resistant fragment upon trypsin digestion. Thus, the resistance of Gag to trypsin provides a measure of whether the Gag protein exists in a conformation or packing arrangement characteristic if authentic viral particles.

IDENTIFYING MODULATORS OF CAPSID ASSEMBLY

A. Inhibitors of capsid assembly

The phosphate compounds of the present invention, or compounds that promote assembly as identified by the methods of the present inventions, may be used to identify inhibitors of lentiviral capsid assembly. Inhibitors may be identified by their ability to block phosphate compound-mediated assembly of full-size, stable capsid particles. For example, a test compound may be added to a reaction containing recombinant Gag protein, nucleic acid, buffer components, and one or more phosphate compounds. The stability and size of the assembled capsids, the extent of capsid assembly at equilibrium, and the kinetics of capsid assembly are all measured in the presence and absence of the test compound. An inhibitor of assembly will affect at least one measure of capsid assembly when added to the assembly reaction in an effective amount.

An inhibitor that affects capsid stability will reduce the average size of the assembled particles as measured by electron microscopy or sedimentation, or render the assembled capsid susceptible to disraption by high salt or nuclease treatment as described herein. Inhibitors affecting the extent of capsid assembly at equilibrium will reduce the amount of Gag polypeptide that is incorporated into capsids and precipitable by centrifugation. Inhibitors that affect assembly kinetics will reduce either the extent or stability of capsid assembly when the assembly reaction is sampled at a particular time point. Assembly inhibitors may specifically antagonize the effect of phosphate compounds on capsid assembly, or they may interfere with capsid assembly at a site or pathway unrelated to the phosphate compound effect. These possibilities may be distinguished by standard biochemical measurements. For example, an inhibitor that competes with phosphate compounds for a binding site on Gag may be overcome by increasing the concentration of phosphate compound. An inhibitor that blocks assembly by binding to an entirely different site on the Gag protein will be insensitive to increased phosphate compound concentration.

B. Promoters of capsid assembly

The methods of the present invention can also be used to identify additional compounds that facilitate lentiviral capsid assembly. Like inhibitors, such compounds may affect the stability of assembled capsids, the extent of capsid assembly, or the kinetics of capsid assembly. To identify additional compounds that facilitate capsid assembly, a test compound is added to an assembly reaction containing Gag protein, nucleic acids, and buffer components. As shown in Examples 3-6, capsids assembled in the absence of an assembly modulator are smaller than authentic lentiviral capsids and are sensitive to disraption or digestion by high salt, nucleases, or proteases. In contrast, when a compound that facilitates assembly is present in the assembly reaction, the capsids are the same size as authentic particles and are resistant to salt, nuclease, and protease treatment.

Additional modulators of capsid assembly may be identified by adding test compounds in assembly reactions that already contain a known assembly modulator, such as inositol phosphates. These assays identify compounds that do not themselves promote assembly by the same pathway as inositol phosphates, but facilitate assembly by an independent pathway. The effects of such compounds might not be detectable without the stabilization provided by phosphate compounds. Alternatively, some assembly modulators may amplify the effect of phosphate compounds on capsid assembly, and may only be detected in assembly reactions containing a phosphate compound.

PHOSPHATE COMPOUNDS

It is a discovery of the present invention that phosphorylated compounds facilitate the assembly of lentiviral capsids in vitro. Some phosphate compounds identified by the methods of the invention are listed in Table 1. Phosphate compounds effective in the practice of the invention generally possess at least three phosphate groups, and may either be polyphosphate molecules or multiply phosphorylated ring compounds. Polyphosphate compounds suitable for practicing the invention have the general formula Na_n+2P_nO_3n+ι, and typically contain between 15 and 75 phosphorous atoms preferably 18 or more phosphorous atoms.

Inositol phosphates are particularly preferred phosphate compounds for practice of the invention. Inositol phosphates are generally derivatives of myo-inositol (1,2,3,5/4,6-hexahydroxycyclohexane). Inositol phosphates such as IP3 (d-myo-inositol 1, 4,5-tris-phosphate), IP4 (d-myo-inositol 1,3,4,5-tetrakis phosphate and d-myo-inositol 3,4,5,6-tetrakis-phosphate) play roles in cellular signaling pathways (for review see Shears, Biochim. Biophys. Acta 1436:49-67 (1998)). IP5 (myo-inositol 1,3,4,5,6-pentakis phosphate) and IP6 (inositol hexaphosphate; phytic acid) are particularly effective at facilitating capsid assembly. Without wishing to be bound by a particular theory, the ability of IP6 to stimulate capsid assembly may relate to the fact that lentiviral capsids normally assemble at the inner surface of the plasma membrane, as much of the cellular IP6 pool may be tightly associated with cell membranes (Poyner et al, J. Biol. Chem. 268:1032-1038 (1993)). Likewise, PIP3 (phosphatidylinositol 3,4,5-triphosphate) is naturally found in the plasma membrane and facilitates capsid assembly. Thus, phosphate compounds ordinarily found in association with cellular membranes, and analogues of such compounds, are particularly suited for practice of the invention. DELIVERY VEHICLES

The capsids of the invention are also suitable as vehicles for delivering nucleic acids or other molecules to cells, e.g., to deliver therapeutically useful genes. Self- assembling spheres of viral capsid proteins have been exploited to deliver nucleic acids to cells both in vitro and in vivo. See, e.g., Krauzewicz et al, Gene Tlierapy 7:1094-1102 (2000). A wide variety of natural and synthetic RNA and DNA molecules may be packaged into capsids by including the appropriate nucleic acid in the assembly reaction. Nucleic acids ranging from 24 nt to approximately 20,000 nt may be packaged into or associated with particles formed from HIV Gag proteins. Nucleic acids suitable for packaging into the capsids of the invention include therapeutic genes, oligonucleotides, antisense molecules, and catalytically active RNA molecules. Other substances, such as drags or toxins, may be encapsulated by the Gag particles if included in the assembly reaction. Alternatively, drags or toxins may be chemically or biosynthetically attached to the Gag polypeptides or nucleic acids present in the assembly reaction. As the capsids of the present invention are relatively permeable to small molecules, small molecule drugs or toxins may be covalently or non- covalently attached to the Gag polypeptide or nucleic acids to ensure their retention within the capsid.

The efficiency and specificity with which cells take up the capsid vehicles may be increased by replacing or fusing the viral MA domain with a foreign peptide. As the MA domain resides at the outer surface of the capsid particle, polypeptides fused to MA will be displayed on the surface of the capsid particle. For example, capsids may be assembled with chimeric Gag proteins, where Gag is fused to a protein that binds to a cellular receptor or other specific cell surface protein. Targeting molecules may also be chemically conjugated to Gag polypeptides. Examples of molecules that may be attached to Gag include antibodies, cell adhesion molecules, hormones, and receptors. Attaching these molecules to Gag can improve the efficiency or specificity of cellular uptake. Uptake is improved by employing a targeting molecule that interacts with a chosen cellular molecule, either located on the surface of the targeted cell population, or located intracellularly, in a targeted cellular vesicle or compartment.

Particles containing the nucleic acid or therapeutic to be delivered are prepared by mixing purified Gag protein, the chosen nucleic acid or therapeutic, a phosphate compound, and the appropriate buffer components in an assembly reaction. Assembled particles may be separated from unincorporated polypeptides and nucleic acids by centrifugation. For transfer to a mammal, the purified particles may be mixed with common pharmaceutical carriers and administered intravenously, intradermally, intramuscularly, or orally. Following administration, the particles are taken up by cells through non-receptor- mediated mechanisms, such as phagocytosis by macrophages, or receptor-mediated uptake. Within the cell, delivery of the therapeutic substance occurs as the capsid disassembles in a cellular vesicle or compartment.

CAPSID VACCINES

Once the lentiviral Gag proteins are assembled into capsids in vitro, the capsids of the invention are useful as prophylactics, or vaccines for administration to mammals, particularly humans, to treat and/or prevent lentiviral infection. Vaccine compositions containing the capsids of the invention are administered to a patient infected with a lentivirus or to an individual susceptible to, or otherwise at risk for, lentiviral infection to elicit an immune response against lentiviral capsid antigens and thus enhance the patient's own immune response capabilities. In therapeutic applications, capsid compositions are administered to a patient in an amount sufficient to elicit an effective immune response to the capsid antigen and to cure or at least partially arrest or slow symptoms and/or complications. An amount adequate to accomplish this is defined as "therapeutically effective dose." Amounts effective for this use will depend on, e.g., the particular composition administered, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician.

The vaccine compositions of the invention may also be used purely as prophylactic agents. Generally the dosage for an initial prophylactic immunization generally occurs in a unit dosage range where the lower value is about 1, 5, 50, 500, or 1000 μg of capsid and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a human typically range from about 500 μg to about 50,000 μg per 70 kilogram patient. This is followed by boosting dosages of between about 1.0 μg to about 50,000 μg of capsid administered at defined intervals from about four weeks to six months after the initial administration of vaccine. The immunogenicity of the vaccine may be assessed by measuring for example, the specific activity of cytotoxic T lymphocytes (CTL) and/or helper T lymphocytes (HTL) specific for capsid proteins obtained from a sample of the patient's blood. Alternatively, the immungenicity of the vaccine may be assessed by measuring the levels of antibody obtained from a sample of the patient's blood specific for lentiviral capsid proteins. Additionally, the ability of the vaccine to induce an immunoprotective immune response may be tested in an animal model specific for the lentivirus capsid proteins present in the vaccine.

As noted above, capsids comprising CTL and/or HTL epitopes induce immune responses when presented by HLA molecules and contacted with a CTL or HTL specific for the capsid epitope. The manner in which the capsid epitope is contacted with the CTL or HTL is not critical to the invention. For instance, the capsid can be contacted with the CTL or HTL either in vivo or in vitro. If the contacting occurs in vivo, the capsid itself can be administered to the patient, or other vehicles, e.g., DNA vectors encoding one or more capsids, viral vectors encoding the capsids, liposomes and the like, can be used, as described herein. When the capsid is contacted in vitro, the vaccinating agent can comprise a population of cells, e.g., peptide-pulsed dendritic cells, or capsid-specifϊc CTLs, which have been induced by pulsing antigen-presenting cells in vitro with the capsid. Such a cell population is subsequently administered to a patient in a therapeutically effective dose. For pharmaceutical compositions, the immunogenic capsids of the invention, or DNA encoding them, are generally administered to an individual already infected with a lentivirus. Those in the incubation phase or the acute phase of infection can be treated with the immunogenic capsids separately or in conjunction with other treatments, as appropriate. For therapeutic use, administration should generally begin at the first diagnosis of lentivirus infection. This is followed by boosting doses until at least symptoms are substantially abated and for a period thereafter. In chronic infection, loading doses followed by boosting doses may be required.

The capsid or other compositions used for the treatment or prophylaxis of lentivirus infection can be used, e.g., in persons who have not manifested symptoms of disease but who act as a disease vector. In this context, it is generally important to provide an amount of the capsid epitope delivered by a mode of administration sufficient to effectively stimulate a cytotoxic T cell response; compositions which stimulate helper T cell responses can also be given in accordance with this embodiment of the invention.

The dosage for an initial therapeutic immunization generally occurs in a unit dosage range where the lower value is about 1, 5, 50, 500, or 1000 μg of capsid and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a human typically range from about 500 μg to about 50,000 μg per 70 kilogram patient. Boosting dosages of between about 1.0 μg to about 50000 μg of capsid pursuant to a boosting regimen over weeks to months may be administered depending upon the patient's response and condition as determined by measuring the specific activity of CTL and HTL obtained from the patient's blood. The capsids and compositions of the present invention may be employed in serious disease states, that is, life-threatening or potentially life threatening situations. In such cases, as a result of the minimal amounts of extraneous substances and the relative nontoxic nature of the capsids in preferred compositions of the invention, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these peptide compositions relative to these stated dosage amounts.

The pharmaceutical compositions for therapeutic treatment are intended for parenteral, topical, oral, intrathecal, or local administration. Preferably, the pharmaceutical compositions are administered parentally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly. Thus, the invention provides compositions for parenteral administration which comprise a solution of the immunogenic capsids dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers may be used, e.g., water, buffered water, 0.8% saline, 0.3%> glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

The concentration of capsids of the invention in the pharmaceutical formulations can vary widely, i. e., from less than about 0.1 %, usually at or at least about 2% to as much as 20% to 50%) or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.

A human unit dose form of the capsid composition is typically included in a pharmaceutical composition that comprises a human unit dose of an acceptable carrier, preferably an aqueous carrier, and is administered in a volume of fluid that is known by those of skill in the art to be used for administration of such compositions to humans (see, e.g., Remington's Pharmaceutical Sciences. 17^th Edition, A. Gennaro, Editor, Mack Publising Co., Easton, Pennsylvania, 1985). The capsids of the invention may also be administered via liposomes, which serve to target the capsids to a particular tissue, such as lymphoid tissue, or to target selectively to infected cells, as well as to increase the half-life of the peptide composition. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations, the capsid to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to a receptor prevalent among lymphoid cells, such as monoclonal antibodies which bind to the CD45 antigen, or with other therapeutic or immunogenic compositions. Thus, liposomes either filled or decorated with a desired capsid of the invention can be directed to the site of lymphoid cells, where the liposomes then deliver the peptide compositions. Liposomes for use in accordance with the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka, et al, Ann. Rev. Biophys.

Bioeng. 9:467 (1980), and U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

For targeting cells of the immune system, a ligand to be incorporated into the liposome can include, e.g., antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells. A liposome suspension containing a peptide may be administered intravenously, locally, topically, etc. in a dose which varies according to, z^'nter alia, the manner of administration, the capsid being delivered, and the stage of the disease being treated.

For solid compositions, conventional nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more capsids of the invention, and more preferably at a concentration of 25%-75%. For aerosol administration, the immunogenic capsids are preferably supplied in finely divided form along with a surfactant and propellant. Typical percentages of capsids are 0.01%-20% by weight, preferably 1%-10%. The surfactant must, of course, be nontoxic, and preferably soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute 0.1%-20% by weight of the composition, preferably 0.25-5%. The balance of the composition is ordinarily propellant. A carrier can also be included, as desired, as with, e.g., lecithin for intranasal delivery.

EXAMPLES The following examples are offered to illustrate, but not to limit the claimed invention.

Example 1: Expression and Purification of HIV Gag Δp6 protein

To provide Gag protein for capsid assembly, the Gag Δp6 expression vector pET 3xc HIV Gag Δp6 (Campbell and Rein, supra), based on pET-3xc (Novagen), was transformed into E. coli BL21(DE3)ρLysS (Novagen). The sequence of the expressed HIV Gag Δp6 protein is set forth in SEQ ID NO: 1. Cells bearing the pET 3xc HIV Gag Δp6 plasmid were grown in LB-Amp overnight and inoculated into a fresh culture at a dilution of 1 :100. After growth for 2 hr at 37°C with shaking, protein expression was induced by the addition of isopropyl-β-D-thiogalactopyranoside to 0.4 mM as well as an extra 20 μg/ml of ampicillin. After four hours of induction, the cells were harvested by centrifugation and frozen at -20°C.

The frozen bacterial pellet was resuspended on ice in buffer A (20 mM Tris- HC1, pH 7.5), 10% glycerol, 1 mM EDTA, 0.1% Nonidet P-40, 1 mM phenylmethysulfonyl fluoride, 10 mM dithiothreitol) plus 0.5 M NaCl at 25 ml/liter of culture. The cells were lysed by sonication. Insoluble debris was removed by centrifugation, and the soluble protein was precipitated with 33% saturated ammonium sulfate. The precipitate was resuspended in buffer B (buffer A without glycerol, EDTA, or NP-40) plus 0.5 M NaCl at 2 ml/liter of cell culture and incubated on ice for 30 min. Buffer B was slowly added until the final NaCl concentration was 0.1 M. Insoluble protein was removed by centrifugation, and the supernatant was mixed with phosphocellulose (Whatman PI 1) at a volume ratio of 10: 1. The resin with bound protein was washed with buffer B plus 0.1 M NaCl and then with Buffer B plus 0.3 M NaCl. The volumes of the washes were approximately 3 ml per ml of packed resin. Protein was eluted with buffer B plus 0.5 M NaCl. The eluted protein was precipitated with 50%. ammonium sulfate, resuspended in buffer B plus 0.5 M NaCl, and dialyzed at 4°C overnight against the same buffer. Gag Δp6 was soluble to about 10 mg/ml and was determined to be about 85 to 90%) homogenous by SDS-PAGE. A slightly smaller degradation product, presumably the result of cleavage by a bacterial protease, was also present. The purified protein was stored at -80°C at a concentration of 5 mg/ ml in a buffer comprising 20 mM Tris, pH 7.5, 0.5 M NaCl, 10 mM dithiothreitol (DTT), 10% glycerol and 1 mM phenylmethylsulfonylflouride (PMSF).

Example 2: Preparation of reticulocyte lysate Reticulocyte lysate provides the starting material to isolate factors that facilitate capsid assembly. To prepare reticulocyte lysate, rabbits were made anemic and bled according to the protocol of Jackson and Hunt (Methods Enzymology, 96:50-74 (1983)). The whole blood was centrifuged in a JA 14 rotor at 6000 rpm for 10 minutes. The resulting pellet of cells was washed three times with phosphate buffered saline (PBS) solution. The volume of the washed pellet was determined by resuspending the pellet in a known volume of PBS and measuring the total volume of the suspension. Subtracting the known volume of PBS from the total volume of the suspension yielded the volume of the pellet. The cells were then repelleted and lysed by the addition of 1.5 volumes of ice cold distilled water. This lysate was then centrifuged at 10,000 rpm for 20 minutes in a JA 14 rotor. The supernatant was filtered through gauze pads. 7,500 units of S7 (micrococcal) nuclease and CaCl₂, to a final concentration of 2 mM, were then added prior to an overnight centrifugation at 20,000 rpm in an SW 28 rotor at 4 degrees. The final supernatant was then dialyzed overnight at 4°C against 5 volumes of buffer consisting of 20 mM Tris, pH 7.5, 50 mM NaCl, 10 mM DTT.

Example 3: In vitro assembly of immature viral capsids

To assemble capsids in vitro, HIV Gag Δp6 protein in storage buffer was slowly diluted fivefold (to 1 mg/ml protein and 0.1 M NaCl) into 80 μl of assembly buffer at room temperature. Final reaction conditions were 1 mg/ml Gag protein, 0.04 mg/ml yeast tRNA, 20 mM Tris pH 7.5, 0.1 M NaCl, 10 mM DTT, and 2% glycerol. The assembly reaction became turbid within minutes of protein addition, indicating that particulate matter was being formed in the reaction. Capsid assembly was allowed to proceed for at least 30 minutes at room temperature, at which point assembled Gag proteins were pelleted by centrifugation (60 min at 21,000 x g). The pellet Gag complexes were either directly visualized by transmission electron microscopy, or assayed by denaturing gel elecfrophoresis (SDS-PAGE) and Coomassie Blue staining. Typically, 95% of the Gag protein in the reaction assembled into a pelletable complex, and the extent of complex formation was not affected by addition of assembly factors.

Purified Gag Δp6 assembled in buffer alone formed uniform, spherical particles 25-30 nm in diameter (Figure 1 A). These particles are clearly much smaller than the 100-120 nm particles produced in vivo from HIV-1 infected cells or cells expressing recombinant Gag proteins (Campbell and Rein, supra). However, when rabbit reticulocyte lysate was included in the assembly reaction, Gag assembled into particles equivalent in size to HIV-1 particles assembled in vivo (Figure IB), indicating that a component of the reticulocyte lysate promoted the assembly of full-sized viral capsids in vitro.

Stability of the assembled capsids was assessed by their resistance to high salt conditions or nuclease digestion. Unstable particles will be dissociated into soluble, nonprecipitable proteins, while resistant particles will remain intact as precipitable complexes. Salt resistance of assembled capsids was assayed by the addition of NaCl to 0.5 M, while nuclease resistance was assayed by the addition of 50 μg of RNAse A and a 30-60 minute incubation at room temperature. In both cases, the amount of resistant particles was determined by centrifugation and SDS-PAGE of the pelleted complexes.

As shown in Figure 2, assembly in the presence of tRNA yields a pelletable complex containing the Gag Δρ6 protein. The addition of either RNAse A or 0.5 M NaCl to assembled capsids eliminates Gag from the pellet, indicating that the in vitro assembled capsids are easily disrupted by salt or nuclease treatment. In contrast, when authentic HIN capsids from infected cells are treated with salt or nuclease, Gag remains in the pellet, indicating that authentic capsids are more stable than capsids assembled in vitro. However, when reticulocyte lysate was included in the in vitro assembly reaction, Gag protein remained precipitable, indicating that the assembled Gag complexes were now stable in the presence of salt and nuclease. Reticulocyte lysate without added tRΝA yielded some precipitable Gag protein, although this material lacked the spherical morphology of particles assembled with tRΝA. Therefore, assembly factors in reticulocyte lysate not only increased the size of the in vitro assembled particles to that of authentic capsids, it also conferred the stability of authentic capsids on the in vitro assembled particles Example 4: Purification of an HIN-1 assembly factor

To purify the assembly-facilitating factor in reticulocyte lysate, the lysate was fractionated by cation exchange chromatography (Q Sepharose, Amersham Pharmacia) and eluted with a 0.05-lM gradient of ammonium bicarbonate. Fractions from the column were assayed by their ability to stabilize assembled capsids in the presence of 0.5 M ΝaCl (see Example 3), and active fractions were pooled and concentrated by lyophilization. The lyophilized powder was dissolved in one tenth the original volume of distilled water and heated for 20 minutes at 80°C to denature proteins and drive off residual ammonium bicarbonate. Denatured proteins were removed by centrifugation for 10 minutes at 10,000 rpm in a JA 25.5 rotor.

Ethanol was then added to the supernatant to a final concentration of 80%o ethanol v/v, and centrifuged for 10 minutes at 10,000 rpm in a JA 25.5 rotor. The pellet from the ethanol precipitation was dissolved in 10 ml of 0.1 M ammonium bicarbonate and fractionated by gel filtration on a Superdex 30 (Amersham Pharmacia) column. Active fractions were again identified by the ΝaCl resistance assay, pooled, lyophilized and dissolved in distilled water prior to precipitation by 80% ethanol.

This last ethanol precipitation resulted in a phase separation, with a dense liquid droplet at the bottom of the tube. This liquid had assembly activity. Analysis of the liquid droplet by gas chromatography-mass spectrometry (GCMS) indicated that it consisted primarily of the compound 2, 3-diphosphoglycerate (2, 3 DPG). The majority of the 2, 3 DPG was removed by digestion with 2, 3 DPG phosphatase and fractionation by cation exchange chromatography. Active fractions were identified and concentrated as described above. GCMS analysis of these fractions revealed that the 2, 3 DPG component had been significantly reduced, but no other compounds were detected. However, GCMS only detects volatile compounds. Polyphosphorylated compounds are not sufficiently volatile to be detected by GMCS. Therefore, the fractions were treated with alkaline phosphatase to dephosphorylate any such compounds that might be present. GCMS analysis of the alkaline phosphatase treated samples revealed the presence of inositol. Alkaline phosphatase treatment also destroyed the assembly-promoting activity.

The inositol that was detected must have been derived from dephosphorylation of an inositol phosphate, since inositol was not detected in untreated samples. As shown in Figure 3, an exogenously added inositol phosphate compound (inositol pentakisphospate) could substitute for the active fraction purified from reticulocyte lysate, and stabilize assembled HIV Gag Δp6 complexes against dissociation by high salt or nuclease. As was the case with reticulocyte lysate, assembly reactions with active fraction or inositol phosphate yielded some precipitable Gag protein even in the absence of nucleic acid, suggesting that inositol phosphates stabilize Gag-Gag interactions. Since the exogenously added inositol phosphate could substitute for the active reticulocyte fraction, and since alkaline phosphatase treatment destroyed the assembly activity of the active fraction, at least part of the assembly- promoting activity of reticulocyte lysates likely derived from phosphorylated compounds in the reticulocyte lysates.

Example 5: Proteolytic resistance of assembled Gag

To determine whether the increased stability of Gag Δp6 complexes assembled in the presence of phosphate compounds was equivalent to the increased stability of authentic HIV particles produced in vivo, the proteolytic resistance of particles assembled in vivo and in vitro was compared. Immature HIV-1 particles were collected from cultured cells (Fu et al, J. Virology 68:5013-5018 (1994)) and resuspended in assembly buffer plus 1% NP-40. Viral particles were incubated with high salt or protease as described in Example 3. Trypsin or HIN-1 protease, each at a molar ration of 1 : 100 enzyme: Gag, was added to authentic particles from cells, particles assembled in buffer alone, particles assembled in the presence of reticulocyte lysate, or particles assembled in the presence of IP5. All reactions included 1%> ΝP-40. Following digestion, the Gag proteins were analyzed by SDS-PAGE and immunoblotting with a polyclonal serum that recognizes the Gag protein.

The results (Figure 4) showed that HIV-1 protease was able to process the Gag in all HIV-1 particles into a smaller fragment, and that these fragments were completely digested by trypsin. Digestion with trypsin alone completely degraded the Gag in particles assembled in buffer alone. However, digestion of particles assembled in vivo, or particles assembled in the presence of reticulocyte lysate or IP5, yielded a Gag fragment that was resistant to trypsin digestion. Therefore, when capsids were assembled with lysate or inositol phosphate, the Gag protein assumed a protease-resistant conformation or packaging arrangement that is characteristic of authentic HIV-1 virions. Example 6: Activity of phosphorylated compounds in capsid assembly

To investigate whether compounds other than inositol phosphates could promote the assembly of Gag proteins into stable immature capsids, a variety of mono- and polyphosphorylated compounds were tested for their ability to stabilize assembled Gag proteins against dissociation by 0.5 M NaCl. The concentration of each compound required to stabilize 100% of the assembled HIV-1 Gag against salt dissociation is shown in Table 1. The data indicate that a single IP 5 or IP 6 molecule can bind to approximately 10 HIV Gag Δp6 molecules in order to confer stability on the capsid structure.

Table 1. Relative Activity of phosphorylated compounds in NaCl res iistance assay

Compound Concentration for Ratio compound/ 100%o resistance (mM) HIV Gag

Phosphoglycerates

2-phosphoglycerate >2 >100

2,3-diphosphoglycerate 1 50

Polyphosphates

Tripolyphosphate >1 >50 Tetrapolyphosphate 0.1 5 Polyphosphate glass 18-mer 0.035 2

Inositol Phosphates

IP3 0.54 27

IP4 0.08 4

IP5 0.002 0.1

IP6 0.002 0.1

Inositol Sulfates

IS6 >1 >50

Phosphatidyl Inositol Phosphates diC4PtdIns(3,4,5)P3 0.057 3

Example 7: HIV Gag Δ16-99/Δp6 assemble stable viras like particles in the absence of IP's

A recombinant vector capable of expressing HIV Gag protein lacking residues 16-99 within its MA domain and also lacking p6 (HIV Gag Δ16-99/Δp6) was constructed and expressed as described in Example 1 and by Campbell, et al, Proc. Nat. Acad. Sci. USA 98: 10875-10879 (2001). Gag protein was purified as described above and used to produce viral capsids as described in Example 3. The assembled particles were visualized as described. HIV Gag Δ16-99/Δp6 assembles into nearly full-size (~90 nm diameter) particles in vitro without the addition of inositol phosphates (IP's). The stability of the HIN Gag Δ16-99/Δp6 particles were analyzed as described in Example 3. These HIN Gag Δ16-99/Δp6 particles are not solubilized by the addition of RΝase or 0.5M ΝaCl following assembly. In both size and stability, these particles resemble authentic particles released from mammalian cells. HIN Gag Δ16-99/Δp6 particles differ from those assembled from Gag Δp6 protein with an intact MA domain in that the HIN Gag Δ16-99/Δp6 particles do not require reticulocyte lysate fractions or phosphorylated compounds to assemble stable capsids. These findings suggest that IP's are only needed for proper assembly of particles if the region between residues 16 and 99 is present.

Example 8: Gag ΔP6 and HIN Gag Δ16-99/Δp6 bind IP6

Gag Δp6 and HIN Gag Δ16-99/Δp6 bind inositol hexakisphosphate (IP6). These binding studies were performed using ³H-IP6 in 0.2M ΝaCl, following the protocol of Gaidarov et al, J. Biol. Chem. 271: 20922-20929 (1996). Briefly, recombinant proteins were incubated at 4°C for 15-20 min. in lOOμl of 25 mM Tris HCl pH 7.4, 200 mM ΝaCl, 1 mM EDTA, 1 mM dithiothreitol, 1 mg/ml bovine γ-globulin and 0.01 μCi of [³H]IP6. The samples were then mixed with sufficient ice cold 30% (w/v) PEG-8000 to yield a final 20% solution concentration and incubated on ice for 10 minutes. After centrifugation for 10 minutes at 10,000 x g at 4°C, the supernatants were carefully aspirated and the tubes were centrifuged again for 1 minute. The residual supernatant was aspirated and the pellet was dissolved in 200 μl of 1% SDS, transferred to scintillation vials with 10 ml of BCS scintillation fluid (Amersham Corp.) and counted in a scintillation counter. Non-specific binding of [³H]IP6 was measured in the presence of 10 μM unlabeled IP6. Under these conditions, Gag Δp6 binds 5 IP6 molecules, while HIN Gag Δ16-99/Δp6 binds 4 indicating that the region of MA encompassed by residues 16-99 contain an IP6 binding site.

Example 9: Gag Δp6 contains a high affinity IP6 binding site absent in HIN Gag Δ16-99/Δp6

Prior analysis of the effect of IP's on capsid assembly discussed in Examples 3-8 indicated that one IP5 or IP6 molecule was sufficient to induce correct assembly of -10 Gag Δp6 molecules. This strongly suggests that, although several IP molecules can apparently bind to the capsid protein, it is the highest-affinity binding site, that will be occupied when the IP concentration is very low, that is significant for assembly. In order to identify the highest-affinity IP6 binding site in Gag Δp6 a binding analysis as described in Example 8 was performed using varying concentrations of NaCl, assuming for this analysis, that binding of IP6, which has a very high charge-density, to the protein will have a very high electrostatic component, and, therefore, it will be relatively salt-resistant. Furthermore, the highest-affinity site will be the most resistant to the addition of salt. The binding of ³H-IP6 to Gag Δp6 and HIN Gag Δ16-99/Δp6 protein was measured at a series of increasing ΝaCl concentrations. Addition of ΝaCl gradually impairs the binding of ³H-IP6 to Gag Δp6. In reactions containing 0.5M ΝaCl, only one IP6 molecule is bound to Gag Δp6. The data suggest that these conditions eliminate all but the highest-affinity site.

As discussed in Example 7, the behavior of the HIN Gag Δ16-99/Δp6 protein indicates that IP's are not needed to modulate the assembly of stable capsids from this deleted protein. It is possible that this protein lacks the high-affinity site by which IP's modulate the assembly of Gag Δp6 protein. To test this possibility, the binding of IP6 to HIN Gag Δ16- 99/Δp6 was measured in a series of increasing ΝaCl concentrations. Results indicate that IP6 exhibits no detectable binding to HIN Gag Δ16-99/Δp6 in 0.5M ΝaCl. These results are fully consistent with the hypothesis that the site in Gag Δp6 to which IP6 binds with the highest affinity is between residues 16 and 99 and is the site responsible for the effect of IP's upon stable capsid assembly.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

WHAT IS CLAIMED IS: 1. A method of assembling a lentiviral capsid, comprising the steps of: (a) expressing a truncated lentiviral Gag protein in a recombinant host, and (b) assembling the truncated lentiviral Gag protein into a capsid in the presence of exogenously added phosphate compound in an amount able to facilitate assembly compared to the absence of the phosphate compound.

2. The method of claim 1 , wherein the truncated lentiviral Gag protein is the Gag protein of HIN.

3. A method of assaying a test compound for its ability to modulate the assembly of lentiviral capsids, comprising the steps of: (a) expressing a truncated lentiviral Gag protein in a recombinant host (b) assembling the truncated lentiviral Gag protein into a capsid in the presence of said test compound and exogenously added phosphate compound in an amount able to facilitate assembly compared to the absence of phosphate compound, and (c) detecting the effect of said test compound on the assembly of lentiviral capsids.

4. The method of claim 3, wherein the truncated lentiviral Gag protein is the Gag protein of HTV.

5. The method of claim 3, wherein the effect on capsid assembly is an increase in the size of the assembled capsids.

6. The method of claim 3, wherein the effect on capsid assembly is an decrease in the size of the assembled capsids.

7. The method of claim 3, wherein the effect on capsid assembly is an increase in the salt resistance of the assembled capsids.

8. The method of claim 3, wherein the effect on capsid assembly is a decrease in the salt resistance of the assembled capsids.

9. The method of claim 3 , wherein the effect on capsid assembly is detected by a change in the sensitivity of the truncated lentiviral Gag protein to a protease.

10. The method of claim 3 , wherein the effect on capsid assembly is detected by a change in the sensitivity of the capsid to a nuclease.

11. The method of claim 3, wherein the effect on capsid assembly is detected by the light scattering of a solution of the assembled capsids.

12. A method of assaying a test compound for its ability to affect lentiviral capsid assembly, comprising the steps of (a) expressing a truncated lentiviral Gag protein comprising a functional MA domain in a recombinant host (b) contacting the test compound with the truncated lentiviral Gag protein, and (c) detecting the effect of the test compound on the assembly of the truncated lentiviral Gag protein into lentiviral capsids.

13. The method of claim 12, wherein the truncated lentiviral Gag protein is the Gag protein of HIV.

14. The method of claim 12, wherein the effect on capsid assembly is an increase in the size of the assembled capsids.

15. The method of claim 12, wherein the effect on capsid assembly is an decrease in the size of the assembled capsids.

16. The method of claim 12, wherein the effect on capsid assembly is an increase in the salt resistance of the assembled capsids.

17. The method of claim 12, wherein the effect on capsid assembly is a decrease in the salt resistance of the assembled capsids.

18. The method of claim 12, wherein the effect on capsid assembly is detected by a change in the sensitivity of the truncated lentiviral Gag protein to a protease.

19. The method of claim 12, wherein the effect on capsid assembly is detected by a change in the sensitivity of the capsid to a nuclease.

20. The method of claim 12, wherein the effect on capsid assembly is detected by the light scattering of a solution of the assembled capsids.

21. A method of assaying a test compound for its ability to modulate the effect of phosphate compounds on lentiviral capsid assembly, comprising the steps of: (a) contacting the test compound with a cell-free lentiviral capsid assembly system, and (b) detecting the effect of the test compound on phosphate compound- mediated lentiviral capsid assembly.

22. The method of claim 21 , wherein the lentiviral capsid is the capsid of HIV.

23. The method of claim 21 , wherein the effect on capsid assembly is an increase in the size of the assembled capsids.

24. The method of claim 21, wherein the effect on capsid assembly is an decrease in the size of the assembled capsids.

25. The method of claim 21 , wherein the effect on capsid assembly is an increase in the salt resistance of the assembled capsids.

26. The method of claim 21, wherein the effect on capsid assembly is a decrease in the salt resistance of the assembled capsids.

27. The method of claim 21, wherein the effect on capsid assembly is detected by a change in the sensitivity of the truncated lentiviral Gag protein to a protease.

28. The method of claim 21, wherein the effect on capsid assembly is detected by a change in the sensitivity of the capsid to a nuclease.

29. The method of claim 21 , wherein the effect on capsid assembly is detected by the light scattering of a solution of the assembled capsids.

30. A method of delivering a nucleic acid to a cell, comprising the steps of (a) assembling a lentiviral capsid in a reaction containing the nucleic acid and an exogenously added phosphate compound, and (b) contacting the cell with the assembled capsid.

31. A method of delivering a therapeutic molecule to a cell, comprising the steps of (a) assembling a lentiviral capsid in a reaction containing the therapeutic molecule and an exogenously added phosphate compound, and (b) contacting the cell with the assembled capsid.

32. A lentivirus capsid of diameter at least about 90 nm, comprising a truncated lentiviral Gag protein, wherein the Gag protein is substantially free of fatty acid chains and comprises a functional Gag MA domain.

33. The lentiviral capsid of claim 32, wherein the lentiviral capsid is that ofHTV.

34. A lentiviral capsid produced by the method of claim 1.

35. A cell-free system for assembling lentiviral capsids, comprising (a) a recombinantly produced truncated lentiviral Gag protein at a concentration greater than about 20 μg/ml, and (b) a phosphate compound in an amount sufficient to promote capsid assembly.

36. The cell-free system of claim 35, wherein the truncated lentiviral Gag protein is the Gag protein of HIV.

37. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an in vitro assembled lentiviral capsid in an amount sufficient to induce an immune response in a susceptable organism.

38. A method of inducing an immune response against a lentiviral capsid, the method comprising administering to a susceptible organism a pharmaceutical composition comprising an in vitro assembled lentiviral capsid in an amount sufficient to induce an. immune response.