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At least 19 recordsLinked to original sources

PubMed2004

Incorporation of pol into human immunodeficiency virus type 1 Gag virus-like particles occurs independently of the upstream Gag domain in Gag-pol.

By using particle-associated reverse transcriptase (RT) activity as an assay for Pol incorporation into human immunodeficiency virus type 1 (HIV-1) Gag virus-like particles (VLPs), it has been found that truncated, protease-negative, Gag-Pol missing cis Gag sequences is still incorporated into Gag VLPs, albeit at significantly reduced levels (10 to 20% of the level of wild-type Gag-Pol). In this work, we have directly measured the incorporation of truncated Gag-Pol species into Gag VLPs and have found that truncated Gag-Pol that is missing all sequences upstream of RT is still incorporated into Gag VLPs at levels approximating 70% of that achieved by wild-type Gag-Pol. Neither protease nor integrase regions in Pol are required for its incorporation, implying an interaction between Gag and RT sequences in the Pol protein. While the incorporation of Gag-Pol into Gag VLPs is reduced 12-fold by the replacement of the nucleocapsid within Gag with a leucine zipper motif, this mutation does not affect Pol incorporation. However, the deletion of p6 in Gag reduces Pol incorporation into Gag VLPs four- to fivefold. Pol shows the same ability as Gag-Pol to selectively package tRNA(Lys) into Gag VLPs, and primer tRNA(3)(Lys) is found annealed to the viral genomic RNA. These data suggest that after the initial separation of Gag from Pol during cleavage of Gag-Pol by viral protease, the Pol species still retains the capacity to bind to both Gag and tRNA(3)(Lys), which may be required for Pol and tRNA(3)(Lys) to be retained in the assembling virion until budding is completed.

Cell Line↗

PubMed2001

Interaction of human immunodeficiency virus type 1 Vif with Gag and Gag-Pol precursors: co-encapsidation and interference with viral protease-mediated Gag processing.

Interactions of human immunodeficiency virus type 1 (HIV-1) Vif protein with various forms of Gag and Gag-Pol precursors expressed in insect cells were investigated in vivo and in vitro by co-encapsidation, co-precipitation and viral protease (PR)-mediated Gag processing assays. Addressing of Gag to the plasma membrane, its budding as extracellular virus-like particles (VLP) and the presence of the p6 domain were apparently not required for Vif encapsidation, as non-N-myristoylated Deltap6-Gag and Vif proteins were co-encapsidated into intracellular VLP. Encapsidation of Vif occurred at significantly higher copy numbers in extracellular VLP formed from N-myristoylated, budding-competent Gag-Pol precursors harbouring an inactive PR domain or in chimaeric VLP composed of Gag and Gag-Pol precursors compared with the Vif content of Pr55Gag VLP. Vif encapsidation efficiency did not seem to correlate directly with VLP morphology, since these chimaeric VLP were comparable in size and shape to Pr55Gag VLP. Vif apparently inhibited PR-mediated Pr55Gag processing in vitro, with preferential protection of cleavage sites at the MA-CA and CA-NC junctions. Vif was resistant to PR action in vitro under conditions that allowed full Gag processing, and no direct interaction between Vif and PR was detected in vivo or in vitro. This suggested that inhibition by Vif of PR-mediated Gag processing resulted from interaction of Vif with the Gag substrate and not with the enzyme. Likewise, the higher efficiency of Vif encapsidation by Gag-Pol precursor compared with Pr55Gag was probably not mediated by direct binding of Vif to the Gag-Pol-embedded PR domain, but more likely resulted from a particular conformation of the Gag structural domains of the Gag-Pol precursor.

Animals↗

PubMed2004

Basic residues of the retroviral nucleocapsid play different roles in gag-gag and Gag-Psi RNA interactions.

The Orthoretrovirus Gag interaction (I) domain maps to the nucleocapsid (NC) domain in the Gag polyprotein. We used the yeast two-hybrid system to analyze the role of Alpharetrovirus NC in Gag-Gag interactions and also examined the efficiency of viral assembly and release in vivo. We could delete either or both of the two Cys-His (CH) boxes without abrogating Gag-Gag interactions. We found that as few as eight clustered basic residues, attached to the C terminus of the spacer peptide separating the capsid (CA) and NC domains in the absence of NC, was sufficient for Gag-Gag interactions. Our results support the idea that a sufficient number of basic residues, rather than the CH boxes, play the important role in Gag multimerization. We also examined the requirement for basic residues in Gag for packaging of specific packaging signal (Psi)-containing RNA. Using a yeast three-hybrid RNA-protein interaction assay, second-site suppressors of a packaging-defective Gag mutant were isolated, which restored Psi RNA binding. These suppressors mapped to the p10 or CA domains in Gag and resulted in either introduction of a positively charged residue or elimination of a negatively charged one. These results imply that the structural interactions of NC with other domains of Gag are necessary for Psi RNA binding. Taken together, our results show that while Gag assembly only requires a certain number of positively charged amino acids, Gag binding to genomic RNA for packaging requires more complex interactions inherent in the protein tertiary structure.

Amino Acid Sequence↗

PubMed2006

Mutation of dileucine-like motifs in the human immunodeficiency virus type 1 capsid disrupts virus assembly, gag-gag interactions, gag-membrane binding, and virion maturation.

The human immunodeficiency virus type 1 (HIV-1) Gag precursor protein Pr55(Gag) drives the assembly and release of virus-like particles in the infected cell. The capsid (CA) domain of Gag plays an important role in these processes by promoting Gag-Gag interactions during assembly. The C-terminal domain (CTD) of CA contains two dileucine-like motifs (L189/L190 and I201/L202) implicated in regulating the localization of Gag to multivesicular bodies (MVBs). These dileucine-like motifs are located in the vicinity of the CTD dimer interface, a region of CA critical for Gag-Gag interactions during virus assembly and CA-CA interactions during core formation. To study the importance of the CA dileucine-like motifs in various aspects of HIV-1 replication, we introduced a series of mutations into these motifs in the context of a full-length, infectious HIV-1 molecular clone. CA mutants LL189,190AA and IL201,202AA were both severely impaired in virus particle production because of a variety of defects in the binding of Gag to membrane, Gag multimerization, and CA folding. In contrast to the model suggesting that the CA dileucine-like motifs regulate MVB targeting, the IL201,202AA mutation did not alter Gag localization to the MVB in either HeLa cells or macrophages. Revertants of single-amino-acid substitution mutants were obtained that no longer contained dileucine-like motifs but were nevertheless fully replication competent. The varied phenotypes of the mutants reported here provide novel insights into the interplay among Gag multimerization, membrane binding, virus assembly, CA dimerization, particle maturation, and virion infectivity.

Amino Acid Motifs↗

PubMed1988

The gag gene products of human immunodeficiency virus type 1: alignment within the gag open reading frame, identification of posttranslational modifications, and evidence for alternative gag precursors.

Seven human immunodeficiency virus gag polypeptides were identified in the purified virus and in infected CD4+ lymphocytes by peptide mapping and limited amino acid sequencing of immune-purified proteins. Two gag polyproteins of 55,000 (p55) and 41,000 (p41) daltons were rapidly labeled and readily processed into the major internal gag proteins that were aligned within the gag open reading frame (ORF) as NH2-p16 (MA)-p24 (CA)-p9 (NC)-p7-COOH. The myristoylated p16 (matrix, MA) protein was processed from the myristoylated p55 gag precursor protein. The immunoreactivity of the p16 (MA) protein with region-specific gag antisera and the conservation of the N-terminal myristyl group of the p55 precursor protein in p16 (MA) confirmed its position as the N-terminal-most protein. The p9 (nucleocapsid, NC) protein was localized to residue 378 of the gag ORF, next to the C terminus of the p24/p25 (core antigen, CA) protein. The p9 protein had a repeating Cys residue containing motif which is found in the nucleic acid-binding Cys residue-containing proteins of retroviruses. The p24 (CA) protein, which was localized to residue 133 of the gag ORF, was apparently derived by C-terminal processing of an intermediate polypeptide, p25. Both the mature p24 (CA) and p16 (MA) proteins were phosphorylated at Ser residue(s). We also identified two forms of gag p41 species, one resulting from the C-terminal processing of p55 and the other originating either from N-terminal processing of p55 or from de novo synthesis.

Amino Acid Sequence↗

PubMed1996

Gag-Gag interactions in the C-terminal domain of human immunodeficiency virus type 1 p24 capsid antigen are essential for Gag particle assembly.

Seven internal deletions within the p24 domain of the human immunodeficiency virus type 1 Gag precursor have been assessed for their effect on Gag particle formation following their expression using recombinant baculoviruses. In addition, each deleted molecule was assessed for its ability to bind soluble p24 antigen in vitro. The mutants fell into three different phenotypic groups: (i) three mutants that had no effect on either p24 binding or Gag particle assembly, (ii) three mutants that abolished both features and (iii) one mutant that bound p24 in vitro but failed to assemble particles. Mutations that abolished both in vitro p24 binding and particle assembly mapped to the C terminus of p24 confirming this region as critical for virion assembly. We suggest the division of virion assembly into at least two distinct phases and suggest a model in which the critical sequences mapped to date are combined with available structural information.

Animals↗

PubMed2005

Association of human immunodeficiency virus type 1 gag with membrane does not require highly basic sequences in the nucleocapsid: use of a novel Gag multimerization assay.

Human immunodeficiency virus type 1 (HIV-1) particle production, a process driven by the Gag polyprotein precursor, occurs on the plasma membrane in most cell types. The plasma membrane contains cholesterol-enriched microdomains termed lipid rafts, which can be isolated as detergent-resistant membrane (DRM). Previously, we and others demonstrated that HIV-1 Gag is associated with DRM and that disruption of Gag-raft interactions impairs HIV-1 particle production. However, the determinants of Gag-raft association remain undefined. In this study, we developed a novel epitope-based Gag multimerization assay to examine whether Gag assembly is essential for its association with lipid rafts. We observed that membrane-associated, full-length Gag is poorly detected by immunoprecipitation relative to non-membrane-bound Gag. This poor detection is due to assembly-driven masking of Gag epitopes, as denaturation greatly improves immunoprecipitation. Gag mutants lacking the Gag-Gag interaction domain located in the N terminus of the nucleocapsid (NC) were efficiently immunoprecipitated without denaturation, indicating that the epitope masking is caused by higher-order Gag multimerization. We used this assay to examine the relationship between Gag assembly and Gag binding to total cellular membrane and DRM. Importantly, a multimerization-defective NC mutant displayed wild-type levels of membrane binding and DRM association, indicating that NC-mediated Gag multimerization is dispensable for association of Gag with membrane or DRM. We also demonstrate that different properties of sucrose and iodixanol membrane flotation gradients may explain some discrepancies regarding Gag-raft interactions. This report offers new insights into the association of HIV-1 Gag with membrane and with lipid rafts.

Base Sequence↗

PubMed2002

Coding sequences upstream of the human immunodeficiency virus type 1 reverse transcriptase domain in Gag-Pol are not essential for incorporation of the Pr160(gag-pol) into virus particles.

Incorporation of the human immunodeficiency virus type 1 (HIV-1) Gag-Pol into virions is thought to be mediated by the N-terminal Gag domain via interaction with the Gag precursor. However, one recent study has demonstrated that the murine leukemia virus Pol can be incorporated into virions independently of Gag-Pol expression, implying a possible interaction between the Pol and Gag precursor. To test whether the HIV-1 Pol can be incorporated into virions on removal of the N-terminal Gag domain and to define sequences required for the incorporation of Gag-Pol into virions in more detail, a series of HIV Gag-Pol expression plasmids with various extensive deletions in the region upstream of the reverse transcriptase (RT) domain was constructed, and viral incorporation of the Gag-Pol deletion mutants was examined by cotransfecting 293T cells with a plasmid expressing Pr55(gag). Analysis indicated that deletion of the N-terminal two-thirds of the gag coding region did not significantly affect the incorporation of Gag-Pol into virions. In contrast, Gag-Pol proteins with deletions covering the capsid (CA) major homology regions and the adjacent C-terminal CA regions were impaired with respect to assembly into virions. However, Gag-Pol with sequences deleted upstream of the protease, or of the RT domain but retaining 15 N-terminal gag codons, could still be rescued into virions at a level about 20% of the wild-type level. When assayed in a nonmyristylated Gag-Pol context, all of the Gag-Pol deletion mutants were incorporated into virions at a level comparable to their myristylated counterparts, suggesting that the incorporation of the Gag-Pol deletion mutants into virions is independent of the N-terminal myristylation signal.

Capsid↗

PubMed2003

Murine leukemia virus particle assembly quantitated by fluorescence microscopy: role of Gag-Gag interactions and membrane association.

In order to track the assembly of murine leukemia virus (MLV), we used fluorescence microscopy to visualize particles containing Gag molecules fused to fluorescent proteins (FPs). Gag-FP chimeras budded from cells to produce fluorescent spots, which passed through the same pore-size filters and sedimented at the same velocity as authentic MLV. N-terminal myristylation of Gag-FPs was necessary for particle formation unless wild-type Gag was coexpressed. By labeling nonmyristylated Gag with yellow FP and wild-type Gag with cyan FP, we could quantitate the coincorporation of two proteins into single particles. This experiment showed that nonmyristylated Gag was incorporated into mixed particles at approximately 50% the efficiency of wild-type Gag. Mutations that inhibit Gag-Gag interactions (K. Alin and S. P. Goff, Virology 216:418-424, 1996; K. Alin and S. P. Goff, Virology 222:339-351, 1996) were then introduced into the capsid (CA) region of Gag-FPs. The mutations P150L and R119C/P133L inhibited fluorescent particle formation by these Gag-FPs, but Gag-FPs containing these mutations could be efficiently incorporated into particles when coexpressed with wild-type Gag. When these mutations were introduced into nonmyristylated Gag-FPs, no incorporation into particles in the presence of wild-type Gag was detected. These data suggest that two independent mechanisms, CA interactions and membrane association following myristylation, cooperate in MLV Gag assembly and budding.

Amino Acid Sequence↗

PubMed2004

A novel fluorescence resonance energy transfer assay demonstrates that the human immunodeficiency virus type 1 Pr55Gag I domain mediates Gag-Gag interactions.

Human immunodeficiency virus type 1 (HIV-1) assembly takes place at the plasma membrane of cells and is directed by the Pr55(Gag) polyprotein (Gag). One of the essential steps in the assembly process is the multimerization of Gag. We have developed a novel fluorescence resonance energy transfer (FRET) assay for the detection of protein-protein interactions between Gag molecules. We demonstrate that Gag multimerization takes place primarily on cellular membranes, with the majority of these interactions occurring on the plasma membrane. However, distinct sites of Gag-Gag interaction are also present at punctate intracellular locations. The I domain is a functional assembly domain within the nucleocapsid region of Gag that affects particle density, the subcellular localization of Gag, and the formation of detergent-resistant Gag protein complexes. Results from this study provide evidence that the I domain mediates Gag-Gag interactions. Using Gag-fluorescent protein fusion constructs that were previously shown to define the minimal I domain within HIV-1 Pr55(Gag), we show by FRET techniques that protein-protein interactions are greatly diminished when Gag proteins lacking the I domain are expressed. Gag-Tsg101 interactions are also seen in living cells and result in a shift of Tsg101 to the plasma membrane. The results within this study provide direct evidence that the I domain mediates protein-protein interactions between Gag molecules. Furthermore, this study establishes FRET as a powerful tool for the detection of protein-protein interactions involved in retrovirus assembly.

Cell Line↗

PubMed2006

Elicitation of immunity to HIV type 1 Gag is determined by Gag structure.

The gag gene of the human immunodeficiency virus type 1 (HIV-1) encodes for viral proteins that self-assemble into viral particles. The primary Gag gene products (capsid, matrix, and nucleocapsid) elicit humoral and cellular immune responses during natural infection, and these proteins are included in many preclinical and clinical HIV/AIDS vaccines. However, the structure (particulate or soluble) of these proteins may influence the immunity elicited during vaccination. In this study, mice were inoculated with four different HIV-1 Gag vaccines to compare the elicitation of immune responses by the same Gag immunogen presented to the immune system in different forms. The immunity elicited by particles produced in vivo by DNA plasmid (pGag) was compared to these same proteins retained intracellularly (pGag(DMyr)). In addition, the elicitation of anti- Gag immunity by Gag(p55) virus-like particles (VLPs) or soluble, nonparticulate Gag(p55) proteins was compared. Enhanced cellular responses, but almost no anti-Gag antibodies, were elicited with intracellularly retained Gag proteins. In contrast, DNA vaccines expressing VLPs elicited both anti-Gag antibodies and cellular responses. Mice vaccinated with purified Gag(p55) VLPs elicited robust humoral and cellular immune responses, which were significantly higher than the immunity elicited by soluble, nonparticulate Gag(p55) protein. Overall, purified particles of Gag effectively elicited the broadest and highest titers of anti-Gag immunity. The structural form of Gag influences the elicited immune responses and should be considered in the design of HIV/AIDS vaccines.

AIDS Vaccines↗

PubMed2002

Naturally occurring amino acid polymorphisms in human immunodeficiency virus type 1 (HIV-1) Gag p7(NC) and the C-cleavage site impact Gag-Pol processing by HIV-1 protease.

Human immunodeficiency virus type 1 (HIV-1) protease activity is targeted at nine cleavage sites comprising different amino acid sequences in the viral Gag-Pol polyprotein. Amino acid polymorphisms in protease and in regions of Gag, particularly p7(NC) and the C-cleavage site between p2 and p7(NC), occur in natural variants of HIV-1 within infected patients. Studies were designed to examine the role of natural polymorphisms in protease and to identify determinants in Gag that modulate protease processing activity. Closely related Gag-Pol regions from an HIV-1-infected mother and two children were evaluated for processing in an inducible expression system, for protease activity on cleavage-site analogues, and for impact on replication by recombinant viruses. Gag-Pol regions displayed one of three processing phenotypes based on the appearance of Gag intermediates and accumulation of mature p24(CA). Gag-Pol regions that were processed rapidly to produce p24(CA) resulted in high-level replication by recombinant viruses, while slow-processing Gag-Pol variants resulted in recombinant viruses that replicated with reduced kinetics in both T cell lines and peripheral blood mononuclear cells. Direct impact by Gag sequences on processing by protease was assessed by construction of chimeric Gag-Pol regions and by site-directed mutagenesis. Optimal protease activity occurred when Gag and Pol regions were derived from the same gag-pol allele. Heterologous Gag regions generally diminished rates and extent of protease processing. Natural polymorphisms in novel positions in p7(NC) and the C-cleavage site have a dominant effect on protease processing activity. Accumulation of Gag products after processing at the C site appears to delay subsequent cleavage and production of mature p24(CA).

Alleles↗

PubMed1998

Proteolytic processing and assembly of gag and gag-pol proteins of TED, a baculovirus-associated retrotransposon of the gypsy family.

TED (transposable element D) is an env-containing member of the gypsy family of retrotransposons that represents a possible retrovirus of invertebrates. This lepidopteran (moth) retroelement contains gag and pol genes that encode proteins capable of forming viruslike particles (VLP) with reverse transcriptase. Since VLP are likely intermediates in TED transposition, we investigated the roles of gag and pol in TED capsid assembly and maturation. By using constructed baculovirus vectors and TED Gag-specific antiserum, we show that the principal translation product of gag (Pr55(gag)) is cleaved to produce a single VLP structural protein, p37(gag). Replacement of Asp436 within the retrovirus-like active site of the pol-encoded protease (PR) abolished Pr55(gag) cleavage and demonstrated the requirement for PR in capsid processing. As shown by expression of an in-frame fusion of TED gag and pol, PR is derived from the Gag-Pol polyprotein Pr195(gag-pol). The PR cleavage site within Pr55(gag) was mapped to a position near the junction of a basic, nucleocapsid-like domain and a C-terminal acidic domain. Once released by cleavage, the C-terminal fragment was not detected. This acidic fragment was dispensable for VLP assembly, as demonstrated by the formation of VLP by C-terminal Pr55(gag) truncation proteins and replacement of the acidic domain with a heterologous protein. In contrast, C-terminal deletions that extended into the adjacent nucleocapsid-like domain of Pr55(gag) abolished VLP recovery and demonstrated that this central region contributes to VLP assembly or stability, or both. Collectively, these data suggest that the single TED protein p37(gag) provides both capsid and nucleocapsid functions. TED may therefore use a simple processing strategy for VLP assembly and genome packaging.

Amino Acid Sequence↗

PubMed2000

Differential budding efficiencies of human T-cell leukemia virus type I (HTLV-I) Gag and Gag-Pro polyproteins from insect and mammalian cells.

In this study, we examined the ability of human T-cell leukemia virus type I (HTLV-I) Gag and Gag-Pro to assemble immature virus-like particles (VLPs) and bud from insect and mammalian cells. Transmission electron microscopy of insect cells infected with a recombinant baculovirus carrying the entire gag gene revealed that Pr53(Gag) is targeted to the plasma membrane, where it extensively accumulates and forms electron-dense evaginations. However, no particles could be detected either inside the cells or in the culture supernatants. With the Gag-Pro-expressing construct, we observed HTLV-I-specific cytoplasmic proteolysis of the Gag precursor, but again no particle released in the culture supernatants. Transmission electron microscopic analysis of insect cells expressing Gag-Pro polyprotein revealed large vacuoles in the cytoplasm and no budding particles at the plasma membrane. In contrast, human immunodeficiency virus type 1 Gag polyprotein expressed in insect cells is able to release VLPs. These data showed that unlike other retroviruses, Pr53(Gag) is unable to be released as immature VLPs from insect cells. To determine whether the block in particle budding and release is due to an intrinsic property of Pr53(Gag) or the absence of essential cellular factors in insect cells, we expressed Gag and Gag-Pro polyproteins in human 293 cells. The results indicate that Pr53(Gag) and p24 capsid are released within particles into the culture supernatants of human 293 cells. We found that the myristylation of the N-terminal glycine residue is essential for Gag release. Altogether, these results strongly suggest that the proper assembly of HTLV-I particles is dependent on mammalian host cell factors.

Animals↗

PubMed1998

The Gag domain of the Gag-Pol fusion protein directs incorporation into the L-A double-stranded RNA viral particles in Saccharomyces cerevisiae.

The L-A double-stranded RNA virus of yeast encodes its major coat protein, Gag, and a Gag-Pol fusion protein made by a -1 ribosomal frameshift, a coding strategy used by many retroviruses. We find that cells expressing only Gag from one plasmid and only Gag-Pol (in frame) from a separate plasmid can support the propagation of M1 double-stranded RNA, encoding the killer toxin. We use this system to separately investigate the functions of Gag and the Gag part of Gag-Pol. L-A contains two fusion protein molecules per particle, and although N-terminal acetylation of Gag is essential for viral assembly, it is completely dispensable for function of Gag-Pol. In general, the requirements on Gag for viral assembly and propagation are more stringent than on the Gag part of Gag-Pol. Finally, we directly show that it is Gag that instructs the incorporation of Gag-Pol into the viral particles.

Fusion Proteins, gag-pol↗

PubMed2004

The virus-associated human immunodeficiency virus type 1 Gag-Pol carrying an active protease domain in the matrix region is severely defective both in autoprocessing and in trans processing of gag particles.

We have previously demonstrated that a human immunodeficiency virus (HIV) chimeric Gag protein containing a partial replacement of the matrix domain by the viral protease domain (PR) could undergo autoprocessing with no virus particle production [J. Virol. 74 (2000) 3418]. To further analyze the effects of repositioned PR on virus particle production and Gag-Pol incorporation, we introduced the chimeric PR construct into a PR-negative Gag-Pol expression plasmid and coexpressed the resultant construct with a Pr55(gag) expression plasmid (pGAG) in 293T cells. Analysis indicated that the chimeric PR was similar to native PR in that both could prevent virus particle production in cotransfections with an equivalent amount of pGAG plasmid DNA, suggesting an efficient trans processing of Pr55(gag) by the chimeric PR. In cotransfections with the pGAG at a DNA ratio of 1:10 to 1:20, which resembles the normal intracellular expression ratio of Gag-Pol to Gag, Gag-Pol carrying the PR in the Gag coding region could undergo autoprocessing in cells and was incorporated into virus particles at a level about 20-40% of that of wild-type Gag-Pol. However, the incorporated chimeric Gag-Pol was unable to autocleave and unable to process the Gag particles properly, as mature particle-associated reverse transcriptase (RT) and p24(gag) proteins were barely detected. Our data strongly suggest that positioning an active HIV PR in the matrix region significantly affects the PR-mediated virus particle maturation.

Catalytic Domain↗

PubMed2000

Enhancement of primary and secondary cellular immune responses against human immunodeficiency virus type 1 gag by using DNA expression vectors that target Gag antigen to the secretory pathway.

In this study, we have investigated the influence of antigen targeting after DNA vaccination upon the induction of cellular immune responses against human immunodeficiency virus type 1 (HIV-1) Gag. In addition to the standard version of HIV-1 Gag, we constructed Gag expression vectors that encode a secreted (Sc-Gag) and a cytoplasmic (Cy-Gag) Gag molecule. Although all three HIV-1 Gag expression vectors induced detectable humoral and cellular immune responses, after intramuscular injection the DNA vector encoding the Sc-Gag generated the highest primary cytotoxic T-lymphocyte (CTL) and T-helper responses. Mice immunized with one of the HIV-1 Gag DNA vectors (but not with the control vector pcDNA3. 1) developed a protective immune response against infection with recombinant vaccinia virus expressing HIV-1 Gag, and this response persisted for 125 days. The magnitude of the protection correlated with the levels of Gag-specific ex vivo CTL activity and the number of CD8(+) T cells producing gamma interferon. The DNA vector encoding the Sc-Gag induced higher levels of protection and greater secondary CTL responses than did the DNA vector encoding Cy-Gag.

AIDS Vaccines↗

PubMed2003

Enhanced cellular immune response against SIV Gag induced by immunization with DNA vaccines expressing assembly and release-defective SIV Gag proteins.

Codon-optimized genes were synthesized for the SIVmac239 Gag, a mutant Gag with mutations in the major homology region, and a chimeric Gag containing a protein destruction signal at the N-terminus of Gag. The mutant and chimeric Gag were expressed at levels comparable to that observed for the wild-type Gag protein but their stability and release into the medium were found to be significantly reduced. Immunization of mice with DNA vectors encoding the mutant or chimeric Gag induced fourfold higher levels of anti-SIV Gag CD4 T cell responses than the DNA vector encoding the wild-type SIV Gag. Moreover, anti-SIV Gag CD8 T cell responses induced by DNA vectors encoding the mutant or chimeric Gag were found to be 5- to 10-fold higher than those induced by the DNA construct for the wild-type Gag. These results indicate that mutations disrupting assembly and/or stability of the SIV Gag protein effectively enhance its immunogenicity when expressed from DNA vaccines.

3C Viral Proteases↗