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PubMed2006

Effects of mutations in the human immunodeficiency virus type 1 Gag gene on RNA packaging and recombination.

Human immunodeficiency virus type 1 (HIV-1) recombination occurs during reverse transcription when parts of the two co-packaged RNAs are used as templates for DNA synthesis. It was previously hypothesized that HIV-1 Gag polyproteins preferentially encapsidate the RNA from which they were translated (cis-packaging hypothesis). This hypothesis implies that mutants encoding Gag that cannot efficiently package viral RNA are selected against at two levels: these mutants do not generate infectious virus, and these mutants are not efficiently rescued by the wild-type virus because the mutant RNAs are packaged at much lower levels than are those of the wild-type genome. Therefore, genetic information encoded by gag mutants can be rapidly lost in the viral population. To test this prediction of the cis-packaging hypothesis, we examined several gag mutants by measuring the efficiencies of the mutant RNAs in being packaged in trans in the presence of wild-type virus and determining the rates of recombination between gag mutants and wild-type viruses. We observed that the viral RNAs from the nucleocapsid zinc finger or the capsid truncation mutant were packaged efficiently in trans, and these mutant viruses also frequently recombined with the wild-type viruses. In contrast, viral RNAs from mutants containing a 6-nucleotide substitution encompassing the gag AUG were not efficiently encapsidated, resulting in a low rate of recombination between the mutants and wild-type viruses. Further analyses revealed that other, more subtle mutations changing the gag AUG and abolishing Gag translation did not interfere with efficient encapsidation of the mutant RNA. Our results indicated that neither the gag AUG sequence nor Gag translation is essential for viral RNA encapsidation, and Gag can package both wild-type and gag mutant RNAs with similar efficiencies. Therefore, we propose that HIV-1 RNA encapsidation occurs mainly in trans, and most gag mutants can be rescued by wild-type virus; therefore, they are unlikely to face the aforementioned double-negative selection.

Cell Line↗
PubMed1994

Localization of the Vpx packaging signal within the C terminus of the human immunodeficiency virus type 2 Gag precursor protein.

Viral protein X (Vpx) is a human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus accessory protein that is packaged into virions in molar amounts equivalent to Gag proteins. To delineate the processes of virus assembly that mediate Vpx packaging, we used a recombinant vaccinia virus-T7 RNA polymerase system to facilitate Gag protein expression, particle assembly, and extracellular release. HIV genes were placed under control of the bacteriophage T7 promoter and transfected into HeLa cells expressing T7 RNA polymerase. Western immunoblot analysis detected p55gag and its cleavage products p39 and p27 in purified particles derived by expression of gag and gag-pol, respectively. In trans expression of vpx with either HIV-2 gag or gag-pol gave rise to virus-like particles that contained Vpx in amounts similar to that detected in HIV-2 virus produced from productively infected T cells. Using C-terminal deletion and truncation mutants of HIV-2 Gag, we mapped the p15 coding sequence for determinants of Vpx packaging. This analysis revealed a region (residues 439 to 497) downstream of the nucleocapsid protein (NC) required for incorporation of Vpx into virions. HIV-1/HIV-2 gag chimeras were constructed to further characterize the requirements for incorporation of Vpx into virions. Chimeric HIV-1/HIV-2 Gag particles consisting of HIV-1 p17 and p24 fused in frame at the C terminus with HIV-2 p15 effectively incorporate Vpx, while chimeric HIV-2/HIV-1 Gag particles consisting of HIV-2 p17 and p27 fused in frame at the C terminus with HIV-1 p15 do not. Expression of a 68-amino-acid sequence of HIV-2 containing residues 439 to 497 fused to the coding regions of HIV-1 p17 and p24 also produced virus-like particles capable of packaging Vpx in amounts similar to that of full-length HIV-2 Gag. Sucrose gradient analysis confirmed particle association of Vpx and Gag proteins. These results demonstrate that the HIV-2 Gag precursor (p55) regulates incorporation of Vpx into virions and indicates that the packaging signal is located within residues 439 to 497.

Amino Acid Sequence↗
PubMed1999

Intracellular interaction of simian immunodeficiency virus Gag and Env proteins.

In polarized epithelial cells, the assembly and release of human immunodeficiency virus type 1 (HIV-1) occur at the basolateral side of the plasma membrane, and the site of assembly is determined by the site of expression of the Env protein. In order to investigate whether the expression of the Env proteins exclusively in the endoplasmic reticulum (ER) can alter the site of virus assembly, we coexpressed the simian immunodeficiency virus (SIV) Gag protein and mutant SIV Env proteins having an ER retrieval signal (KKXX motif). In cells expressing the wild-type (wt) Env protein or coexpressing Env and Gag proteins, the Env protein was processed into the surface (SU) and transmembrane (TM) proteins. In contrast, in cells expressing the mutant Env proteins alone or in combination with Gag, the Env proteins were retrieved to the ER and were not proteolytically processed. Coexpression of the Gag and ER-retained mutant Env proteins resulted in a transient decrease in the release of the Gag protein into the medium, suggesting an interaction between the Gag and ER-retrieved Env proteins. Using saponin-permeabilized cells coexpressing Gag and Env proteins, we obtained further evidence for Env-Gag interaction. A monoclonal antibody specific to the SIV Gag protein was found to coimmunoprecipitate both the Gag and Env proteins. The interaction was specific, as coexpressed SIV Env proteins without the cytoplasmic tail or a chimeric HIV-1 Env proteins with the CD4 cytoplasmic tail were not coimmunoprecipitated by the Gag-specific antibody. Electron microscopic analyses indicated that assembly of virus particles occurred only at the surfaces of cells in which the Gag protein was coexpressed with either the wt or ER-retrieved mutant Env protein. These data indicate that although the Env and Gag proteins interact intracellularly, the site of assembly of SIV is not redirected to an intracellular organelle by the retrieval of the Env protein to the ER.

Gene Products, gag↗
PubMed2002

The Mason-Pfizer monkey virus internal scaffold domain enables in vitro assembly of human immunodeficiency virus type 1 Gag.

The Mason-Pfizer monkey virus (M-PMV) Gag protein possesses the ability to assemble into an immature capsid when synthesized in a reticulocyte lysate translation system. In contrast, the human immunodeficiency virus (HIV) Gag protein is incapable of assembly in parallel assays. To enable the assembly of HIV Gag, we have combined or inserted regions of M-PMV Gag into HIV Gag. By both biochemical and morphological criteria, several of these chimeric Gag molecules are capable of assembly into immature capsid-like structures in this in vitro system. Chimeric species containing large regions of M-PMV Gag fused to HIV Gag sequences failed to assemble, while species consisting of only the M-PMV p12 region, and its internal scaffold domain (ISD), fused to HIV Gag were capable of assembly, albeit at reduced kinetics compared to M-PMV Gag. The ability of the ISD to induce assembly of HIV Gag, which normally assembles at the plasma membrane, suggests a common requirement for a concentrating factor in retrovirus assembly. Despite the dramatic effect of the ISD on chimera assembly, the function of HIV Gag domains in that process was found to remain essential, since an assembly-defective mutant of HIV CA, M185A, abolished assembly when introduced into the chimera. This continued requirement for HIV Gag domain function in the assembly of chimeric molecules will allow this in vitro system to be used for the analysis of potential inhibitors of HIV immature particle assembly.

Animals↗
PubMed2003

Human immunodeficiency virus type 1 assembly and lipid rafts: Pr55(gag) associates with membrane domains that are largely resistant to Brij98 but sensitive to Triton X-100.

The assembly and budding of human immunodeficiency virus type 1 (HIV-1) at the plasma membrane are directed by the viral core protein Pr55(gag). We have analyzed whether Pr55(gag) has intrinsic affinity for sphingolipid- and cholesterol-enriched raft microdomains at the plasma membrane. Pr55(gag) has previously been reported to associate with Triton X-100-resistant rafts, since both intracellular membranes and virus-like Pr55(gag) particles (VLPs) yield buoyant Pr55(gag) complexes upon Triton X-100 extraction at cold temperatures, a phenotype that is usually considered to indicate association of a protein with rafts. However, we show here that the buoyant density of Triton X-100-treated Pr55(gag) complexes cannot be taken as a proof for raft association of Pr55(gag), since lipid analyses of Triton X-100-treated VLPs demonstrated that the detergent readily solubilizes the bulk of membrane lipids from Pr55(gag). However, Pr55(gag) might nevertheless be a raft-associated protein, since confocal fluorescence microscopy indicated that coalescence of GM1-positive rafts at the cell surface led to copatching of membrane-bound Pr55(gag). Furthermore, extraction of intracellular membranes or VLPs with Brij98 yielded buoyant Pr55(gag) complexes of low density. Lipid analyses of Brij98-treated VLPs suggested that a large fraction of the envelope cholesterol and phospholipids was resistant to Brij98. Collectively, these results suggest that Pr55(gag) localizes to membrane microdomains that are largely resistant to Brij98 but sensitive to Triton X-100, and these membrane domains provide the platform for assembly and budding of Pr55(gag) VLPs.

Cell Membrane↗
PubMed2005

The retroviral capsid domain dictates virion size, morphology, and coassembly of gag into virus-like particles.

The retroviral structural protein, Gag, is capable of independently assembling into virus-like particles (VLPs) in living cells and in vitro. Immature VLPs of human immunodeficiency virus type 1 (HIV-1) and of Rous sarcoma virus (RSV) are morphologically distinct when viewed by transmission electron microscopy (TEM). To better understand the nature of the Gag-Gag interactions leading to these distinctions, we constructed vectors encoding several RSV/HIV-1 chimeric Gag proteins for expression in either insect cells or vertebrate cells. We used TEM, confocal fluorescence microscopy, and a novel correlative scanning EM (SEM)-confocal microscopy technique to study the assembly properties of these proteins. Most chimeric proteins assembled into regular VLPs, with the capsid (CA) domain being the primary determinant of overall particle diameter and morphology. The presence of domains between matrix and CA also influenced particle morphology by increasing the spacing between the inner electron-dense ring and the VLP membrane. Fluorescently tagged versions of wild-type RSV, HIV-1, or murine leukemia virus Gag did not colocalize in cells. However, wild-type Gag proteins colocalized extensively with chimeric Gag proteins bearing the same CA domain, implying that Gag interactions are mediated by CA. A dramatic example of this phenomenon was provided by a nuclear export-deficient chimera of RSV Gag carrying the HIV-1 CA domain, which by itself localized to the nucleus but relocalized to the cytoplasm in the presence of wild type HIV-1 Gag. Wild-type and chimeric Gag proteins were capable of coassembly into a single VLP as viewed by correlative fluorescence SEM if, and only if, the CA domain was derived from the same virus. These results imply that the primary selectivity of Gag-Gag interactions is determined by the CA domain.

Animals↗
PubMed1992

Morphogenesis of recombinant HIV-2 gag core particles.

The gag-pol coding region of the HIV-2BEN genome was expressed in CV-1 cells infected with four recombinant vaccinia viruses (VV). These recombinant VV encoded either the whole gag-pol region or the gag gene including the protease-coding region of the pol gene or the gag gene truncated at its 3'-end or only the pol gene. The HIV-2BEN gag precursor p55, its mature cleavage products p24 and p17 as well as the pol reverse transcriptase (RT) p66 were detected in VV-infected CV-1 cells. The p55 and two intermediate cleavage products p40 and p35 were myristilated. Comparison to lysates of permanently HIV-2BEN-infected Molt 4 clone 8 cells revealed that several additional gag and pol proteins were present in the VV-infected CV-1 cells. Deletion of the gag and pol overlapping region coding for the viral protease prevented cleavage of the recombinant gag precursor. Electron microscopy of VV-infected CV-1 cells revealed budding structures and immature as well as mature retroviral particles formed by the recombinant gag proteins. Striking differences in the ability to form complete particles were observed between the different recombinant VV. Expression of the truncated gag gene led to the formation of budding structures, but completely budded circular particles were not detectable. Such particles were produced by expression of the whole gag gene and the protease. Mature virions with an internal core structure were only detected in VVgagpol-infected cells. From these findings we conclude that the 3'-end of the gag gene coding for the p16 protein is essential for the formation of complete HIV-2 particles and that the pol proteins support the assembly of the viral core.

Base Sequence↗
PubMed2006

A domain directly C-terminal to the major homology region of human immunodeficiency type 1 capsid protein plays a crucial role in directing both virus assembly and incorporation of Gag-Pol.

We demonstrate here that a deletion of 14 amino acid residues directly C-terminal to the major homology region (MHR) of the HIV-1 capsid (CA) in Gag-Pol markedly affects the incorporation of Gag-Pol into virions. The 14-amino acid deletion also significantly impaired virus assembly. In agreement with previous reports, mutations at the very C-terminus of CA resulted in a remarkable reduction in virus production. However, HIV-1 Gag-Pol precursors containing the C-terminal CA mutation were still capable of being incorporated into virions at a level of about 50% that of the wild-type. These results suggest that the domain immediately C-terminal to the MHR is functionally involved in Gag-Gag and Gag/Gag-Pol interaction, and this supports the notion that Gag or Gag-Pol mutants blocked in assembly into virus particles can be rescued into virions provided they retain the domains that are able to interact with the Gag precursor. An HIV-1 Gag-Pol deletion mutant retaining a minimal sequence consisting of the MHR and the adjacent CA-SP1 was efficiently incorporated into virions. Analysis by immunofluorescence staining indicated that the subcellular localization patterns shown by the Gag-Pol mutants were not fully compatible with their efficiency in being incorporated into virions, suggesting that the ability of Gag-Pol mutants to be incorporated into virions largely depends on their interactions with the Gag precursor.

Blotting, Western↗
PubMed2004

Human immunodeficiency virus type 1 Gag assembly through assembly intermediates.

Human immunodeficiency virus Gag protein self-assembles into spherical particles, and recent reports suggest the formation of assembly intermediates during the process. To understand the nature of such assembly intermediates along with the mechanism of Gag assembly, we employed expression in Escherichia coli and an in vitro assembly reaction. When E. coli expression was performed at 37 degrees C, Gag predominantly assembled to a high order of multimer, apparently equivalent to the virus-like particles obtained following Gag expression in eukaryotic cells, through the formation of low orders of multimer characterized with a discreet sedimentation value of 60 S. Electron microscopy confirmed the presence of spherical particles in the E. coli cells. In contrast, expression at 30 degrees C resulted in the production of only the 60 S form of Gag multimer, and crescent-shaped structures or small patches with double electron-dense layers were accumulated, but no complete particles. In vitro assembly reactions using purified Gag protein, when performed at 37 degrees C, also produced the high order of Gag multimers with some 60 S multimers, whereas the 30 degrees C reaction produced only the 60 S multimers. However, when the 60 S multimers were cross-linked so as not to allow conformational changes, in vitro assembly reactions at 37 degrees C did not produce any higher order of multimers. ATP depletion did not halt Gag assembly in the E. coli cells, and the addition of GroEL-GroES to in vitro reactions did not facilitate Gag assembly, indicating that conformational changes rather than protein refolding by chaperonins, induced at 37 degrees C, were solely responsible for the Gag assembly observed here. We suggest that Gag assembles to a capsid through the formation of the 60 S multimer, possibly a key intermediate of the assembly process, accompanied with conformational changes in Gag.

Capsid↗
PubMed1994

Proteolytic cleavage at the Gag-Pol junction in avian leukosis virus: differences in vitro and in vivo.

In avian leukosis virus, processing by the viral protease (PR) appears to activate reverse transcriptase (RT), since PR-defective virions have extremely feeble reverse transcriptase activity. We showed previously that when such detergent-treated virions are digested in vitro with PR, the Gag precursor is completely and properly matured, but the Gag-Pol precursor is not. In particular, the junction between Gag and Pol, i.e., between the PR and RT domains in Gag-Pol, remains refractory to cleavage, and reverse transcriptase is hardly activated. We have now investigated processing between Gag and Pol in greater detail, both in vitro and in vivo. In vivo, three mutations designed to destroy or alter the cleavage site at the N-terminus of RT failed to abrogate processing, suggesting that nearby cryptic cleavage sites can be used by PR, and thus that in virions this portion of Gag-Pol is in an extended conformation. By contrast, resistance to cleavage was observed in vitro in a series of N- and C-terminally truncated Gag-Pol substrates, produced by in vitro translation or in the baculovirus-insect cell system. This resistance was maintained even in short polypeptides, implying that the inability to be processed in vitro is a consequence of local conformation. In the previously described Gag mutant cs22, which is unable to undergo full activation of PR, we found that in vivo in quail cells the only cleavages made in the Gag-Pol polypeptide are at the NC-PR and the PR-RT junctions, suggesting that in wild-type avian leukosis virus, processing of Gag-Pol begins by cleavage immediately upstream and downstream of the PR domain. Taken together, these results suggest a model in which in immature virions the segment of polypeptide between PR and RT is held in an extended but inherently unstable conformation, and that in vivo the first cleavage in Gag-Pol must occur in this region. In the absence of virion structure this segment of polypeptide collapses into its most stable conformation, preventing cleavage. Based on amino acid sequence, we predict that this portion of Gag-Pol adopts a coiled coil conformation reminiscent of a leucine zipper.

Amino Acid Sequence↗
PubMed2006

Interactions between HIV-1 Gag molecules in solution: an inositol phosphate-mediated switch.

Retrovirus particle assembly is mediated by the Gag protein. Gag is a multi-domain protein containing discrete domains connected by flexible linkers. When recombinant HIV-1 Gag protein (lacking myristate at its N terminus and the p6 domain at its C terminus) is mixed with nucleic acid, it assembles into virus-like particles (VLPs) in a fully defined system in vitro. However, this assembly is defective in that the radius of curvature of the VLPs is far smaller than that of authentic immature virions. This defect can be corrected to varying degrees by addition of inositol phosphates to the assembly reaction. We have now explored the binding of inositol hexakisphosphate (IP6) to Gag and its effects upon the interactions between Gag protein molecules in solution. Our data indicate that basic regions at both ends of the protein contribute to IP6 binding. Gag is in monomer-dimer equilibrium in solution, and mutation of the previously described dimer interface within its capsid domain drastically reduces Gag dimerization. In contrast, when IP6 is added, Gag is in monomer-trimer rather than monomer-dimer equilibrium. The Gag protein with a mutation at the dimer interface also remains almost exclusively monomeric in IP6; thus the "dimer interface" is essential for the trimeric interaction in IP6. We discuss possible explanations for these results, including a change in conformation within the capsid domain induced by the binding of IP6 to other domains within the protein. The participation of both ends of Gag in IP6 interaction suggests that Gag is folded over in solution, with its ends near each other in three-dimensional space; direct support for this conclusion is provided in a companion manuscript. As Gag is an extended rod in immature virions, this apparent proximity of the ends in solution implies that it undergoes a major conformational change during particle assembly.

5-Hydroxytryptophan↗
PubMed2003

Enhanced cellular immune responses to SIV Gag by immunization with influenza and vaccinia virus recombinants.

An effective vaccination strategy against human immunodeficiency virus type 1 (HIV-1) should include the induction of potent cellular immune responses against conserved HIV-1 antigens. We have generated five replication competent recombinant influenza viruses (rFlu/SIV Gag nos. 1-5) expressing different portions of Gag of simian immunodeficiency virus (SIV). Single intranasal immunizations in mice with each rFlu/SIV Gag viruses resulted in different degrees of protection against a challenge with recombinant vaccinia virus expressing SIV Gag. Immunized BALB/c mice had detectable CD8+ T cell responses specific for Gag peptide 185-199 when mice were vaccinated with rFlu/SIV Gag no. 3 virus, and for Gag peptides 281-295 and 285-299 when vaccinated with rFlu/SIV Gag no. 4 virus. Cellular immune responses against SIV Gag were further enhanced by a booster with a recombinant vaccinia virus expressing SIV Gag in both the spleen and local lymph node tissues, resulting in the induction of robust Gag-specific CD8+ T cell responses at both systemic and mucosal levels. We suggest that a prime-boost immunization regimen using recombinant influenza and vaccinia viruses expressing HIV Gag might represent an effective means to induce potent HIV-specific, protective CD8+ T cell responses.

Amino Acid Sequence↗
PubMed1999

Incorporation of Vpr into human immunodeficiency virus type 1 requires a direct interaction with the p6 domain of the p55 gag precursor.

The 96-amino acid Vpr protein is the major virion-associated accessory protein of the human immunodeficiency virus type 1 (HIV-1). As Vpr is not part of the p55 Gag polyprotein precursor (Pr55(gag)), its incorporation requires an anchor to associate with the assembling viral particles. Although the molecular mechanism is presently unclear, the C-terminal region of the Pr55(gag) corresponding to the p6 domain appears to constitute such an anchor essential for the incorporation of the Vpr protein. In order to clarify the mechanism by which the Vpr accessory protein is trans-incorporated into progeny virion particles, we tested whether HIV-1 Vpr interacted with the Pr55(gag) using the yeast two-hybrid system and the maltose-binding protein pull-down assay. The present study provides genetic and biochemical evidence indicating that the Pr55(gag) can physically interact with the Vpr protein. Furthermore, point mutations affecting the integrity of the conserved L-X-S-L-F-G motif of p6(gag) completely abolish the interaction between Vpr and the Pr55(gag) and, as a consequence, prevent Vpr virion incorporation. In contrast to other studies, mutations affecting the integrity of the NCp7 zinc fingers impaired neither Vpr virion incorporation nor the binding between Vpr and the Pr55(gag). Conversely, amino acid substitutions in Vpr demonstrate that an intact N-terminal alpha-helical structure is essential for the Vpr-Pr55(gag) interaction. Vpr and the Pr55(gag) demonstrate a strong interaction in vitro as salt concentrations as high as 900 mM could not disrupt the interaction. Finally, the interaction is efficiently competed using anti-Vpr sera. Together, these results strongly suggest that Vpr trans-incorporation into HIV-1 particles requires a direct interaction between its N-terminal region and the C-terminal region of p6(gag). The development of Pr55(gag)-Vpr interaction assays may allow the screening of molecules that can prevent the incorporation of the Vpr accessory protein into HIV-1 virions, and thus inhibit its early functions.

Amino Acid Sequence↗
PubMed1991

Immune response of mice infected with recombinant vaccinia viruses carrying the HIV gag gene.

We examined mouse immune response to 4 kinds of recombinant vaccinia viruses carrying the HIV gag gene, including vac-gag/pol, which produces HIV-like particles with processed gag proteins; vac-gag, which also produces HIV-like particles but with unprocessed gag protein; and vac-gag-pol-fuse and vac-es-gag/pol, neither of which produces such particles but releases reverse transcriptase and gag protein, respectively, from infected cells. Although infection of mice with recombinant vaccinia viruses induced production of the anti-p24 antibody in all mice, vac-gag/pol and vac-es-pol induced higher production than the other two recombinants. Increase in [3H]thymidine uptake by splenic lymphocytes following p24 antigen stimulation was most evident in mice infected with vac-gag/pol. Thus, the highest immune reaction, both humoral and cellular, was elicited by vac-gag/pol, indicating that among those tested, this recombinant vaccinia virus is the best candidate for a vaccine that induces anti-HIV gag immunity.

Animals↗
PubMed2006

Vaginal protection and immunity after oral immunization of mice with a novel vaccine strain of Listeria monocytogenes expressing human immunodeficiency virus type 1 gag.

Natural transmission of human immunodeficiency virus (HIV) occurs at mucosal surfaces. During acute infection, intestinal and other mucosae are preferential sites of virus replication and rapidly become depleted of CD4(+) T cells. Therefore, mucosal immunity may be critical to control both initial infection and the massive early spread of virus. An attenuated D-alanine-requiring strain of the oral intracellular microorganism Listeria monocytogenes expressing HIV type 1 gag was shown to induce protective cell-mediated immunity in mice against viruses that express HIV gag when immunization occurs in the presence of a transient supply of D-alanine. In this study, we examined the efficacy of new attenuated strains that are able to synthesize d-alanine from a heterologous dal gene tightly regulated by an actA-promoted resolvase recombination system. In the absence of d-alanine, Gag-specific cytotoxic T lymphocytes (CTLs) were induced systemically after intravenous immunization, and one strain, Lmdd-gag/pARS, induced strong dose-dependent Gag-specific CTLs after oral immunization. A significant level of Gag-specific CD8(+) T cells was induced in the mucosal-associated lymphoid tissues (MALTs). Upon intravaginal challenge of these orally immunized mice with recombinant vaccinia virus (rVV) expressing HIV gag, gamma interferon- and tumor necrosis factor alpha-secreting Gag-specific CD8(+) T cells were dramatically increased in the spleen and MALTs. Oral immunization with Lmdd-gag/pARS led to complete protection against vaginal challenge by a homologous clade B gag-expressing rVV. In addition, strong cross-clade protection was seen against clades A and C and partial protection against clade G gag-expressing rVV. These results suggest that Lmdd-gag/pARS may be considered as a novel vaccine candidate for use against HIV/AIDS.

Administration, Oral↗
PubMed2006

The transdominant endogenous retrovirus enJS56A1 associates with and blocks intracellular trafficking of Jaagsiekte sheep retrovirus Gag.

The sheep genome harbors approximately 20 endogenous retroviruses (enJSRVs) highly related to the exogenous Jaagsiekte sheep retrovirus (JSRV). One of the enJSRV loci, enJS56A1, acts as a unique restriction factor by blocking JSRV in a transdominant fashion at a late stage of the retroviral cycle. To better understand the molecular basis of this restriction (termed JLR, for JSRV late restriction), we functionally characterized JSRV and enJS56A1 Gag proteins. We identified the putative JSRV Gag membrane binding and late domains and determined their lack of involvement in JLR. In addition, by using enJS56A1 truncation mutants, we established that the entire Gag protein is necessary to restrict JSRV exit. By using differentially tagged viruses, we observed, by confocal microscopy, colocalization between JSRV and enJS56A1 Gag proteins. By coimmunoprecipitation and molecular complementation analyses, we also revealed intracellular association and likely coassembly between JSRV and enJS56A1 Gag proteins. Interestingly, JSRV and enJS56A1 Gag proteins showed distinct intracellular targeting: JSRV exhibited pericentrosomal accumulation of Gag staining, while enJS56A1 Gag did not accumulate in this region. Furthermore, the number of cells displaying pericentrosomal JSRV Gag was drastically reduced in the presence of enJS56A1. We identified amino acid residue R21 in JSRV Gag as the primary determinant of centrosome targeting. We concluded that JLR is dependent on a Gag-Gag interaction between enJS56A1 and JSRV leading to altered cellular localization of the latter.

Amino Acid Sequence↗
PubMed1996

A critical proteolytic cleavage site near the C terminus of the yeast retrotransposon Ty1 Gag protein.

Cleavage of the Gag and Gag-Pol polyprotein precursors is a critical step in proliferation of retroviruses and retroelements. The Ty1 retroelement of Saccharomyces cerevisiae forms virus-like particles (VLPs) made of the Gag protein. Ty1 Gag is not obviously homologous to the Gag proteins of retroviruses. The apparent molecular mass of Gag is reduced from 58 to 54 kDa during particle maturation. Antibodies raised against the C-terminal peptide of Gag react with the 58-kDa polypeptide but not with the 54-kDa one, indicating that Gag is proteolytically processed at the C terminus. A protease cleavage site between positions 401 and 402 of the Gag precursor was defined by carboxy-terminal sequencing of the processed form of Gag. Certain deletion and substitution mutations in the C terminus of the Gag precursor result in particles that are two-thirds the diameter of the wild-type VLPs. While the Ty1 protease is active in these mutants, their transposition rates are decreased 20-fold compared with that of wild-type Ty1. Thus, the Gag C-terminal portion, released in the course of particle maturation, probably plays a significant role in VLP morphogenesis and Ty1 transposition.

Antibodies↗
PubMed1997

Equine infectious anemia virus utilizes a YXXL motif within the late assembly domain of the Gag p9 protein.

We have previously demonstrated that the Gag p9 protein of equine infectious anemia virus (EIAV) is functionally homologous with Rous sarcoma virus (RSV) p2b and human immunodeficiency virus type 1 (HIV-1) p6 in providing a critical late assembly function in RSV Gag-mediated budding from transfected COS-1 cells (L. J. Parent et al., J. Virol. 69:5455-5460, 1995). In light of the absence of amino acid sequence homology between EIAV p9 and the functional homologs of RSV and HIV-1, we have now designed an EIAV Gag-mediated budding assay to define the late assembly (L) domain peptide sequences contained in the EIAV p9 protein. The results of these particle budding assays revealed that expression of EIAV Gag polyprotein in COS-1 cells yielded extracellular Gag particles with a characteristic density of 1.18 g/ml, while expression of EIAV Gag polyprotein lacking p9 resulted in a severe reduction in the release of extracellular Gag particles. The defect in EIAV Gag polyprotein particle assembly could be corrected by substituting either the RSV p2b or HIV-1 p6 protein for EIAV p9. These observations demonstrated that the L domains of EIAV, HIV-1, and RSV were interchangeable in mediating assembly of EIAV Gag particles in the COS-1 cell budding assay. To localize the L domain of EIAV p9, we next assayed the effects of deletions and site-specific mutations in the p9 protein on its ability to mediate budding of EIAV Gag particles. Analyses of EIAV Gag constructs with progressive N-terminal or C-terminal deletions of the p9 protein identified a minimum sequence of 11 amino acids (Q20N21L22Y23P24D25L26S27E28I29K30) capable of providing the late assembly function. Alanine scanning studies of this L-domain sequence demonstrated that mutations of residues Y23, P24, and L26 abrogated the p9 late budding function; mutations of other residues in the p9 L domain did not substantially affect the level of EIAV Gag particle assembly. These data indicate that the L domain in EIAV p9 utilizes a YXXL motif which we hypothesize may interact with cellular proteins to facilitate virus particle budding from infected cells.

Amino Acid Sequence↗