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PubMed1990

Expression of the avian gag-myc oncogene in Saccharomyces cerevisiae.

The gag-myc oncogenic sequence of the avian retrovirus MC29 was first inserted in a multicopy expression vector allowing its expression in Saccharomyces cerevisiae. The oncogene transcripts were detected in yeast by Northern blot hybridization and gag-myc proteins were revealed by immunoprecipitation. On solid medium, the average size of gag-myc transformant colonies was smaller than control. In liquid cultures, the gag-myc strains had a doubling time of 4.7 h compared with 3.1 h in the controls. In one of the recipient strains, and after an initial transient period of 5 days, the gag-myc transformants became physiologically indistinguishable from control. In another recipient strain, the slow-growth phenotype is permanent. Plasmid instability is increased in gag-myc transformants. When a single copy of the gag-myc gene was inserted in a yeast chromosome, no phenotype was observed, showing that slow growth is the consequence of plasmid loss.

Animals↗

PubMed2006

Mutations in the alpha-helix directly C-terminal to the major homology region of human immunodeficiency virus type 1 capsid protein disrupt Gag multimerization and markedly impair virus particle production.

The X-ray crystallographic structure of HIV-1 capsid protein suggests that the dimer interface of the dimerization domain is mainly formed from a putative alpha-helix structure of 14 amino acids (Gag residues 311-324) and lies directly C-terminal to the capsid major homology region. We found that a deletion mutation in the alpha-helix drastically reduces virus particle production. Alanine-scanning mutagenetic analysis indicated that substitution mutations at residues Q311, V313, K314, W316, and M317 all impair virus particle production markedly. Membrane flotation assays suggested that some mutations in the dimer interface have slight effects on the efficient binding of Gag to membranes. Indirect immunofluorescence studies revealed that mutants defective in virus production exhibit a subcellular distribution pattern similar to that of wild-type. However, velocity sedimentation analysis showed that mutations significantly impairing virus particle production were also detrimental to Gag multimerization, suggesting that the impaired virus production may be due to a defect in Gag multimerization. These results support the proposal that residues in the capsid dimer interface play a crucial role in promoting Gag multimerization, possibly by facilitating stable Gag-Gag interactions.

Capsid Proteins↗

PubMed1990

Synthetic HIV-2 protease cleaves the GAG precursor of HIV-1 with the same specificity as HIV-1 protease.

A 99-amino acid protein having the deduced sequence of the protease from human immunodeficiency virus type 2 (HIV-2) was synthesized by the solid phase method and tested for specificity. The folded peptide catalyzes specific processing of a recombinant 43-kDa GAG precursor protein (F-16) of HIV-1. Although the protease of HIV-2 shares only 48% amino acid identity with that of HIV-1, the HIV-2 enzyme exhibits the same specificity toward the HIV-1 GAG precursor. Fragments of 34, 32, 24, 10, and 9 kDa were generated from F-16 GAG incubated with the protease. N-terminal amino acid sequence analysis of proteolytic fragments indicate that cleavage sites recognized by HIV-2 protease are identical to those of HIV-1 protease. The verified cleavage sites in F-16 GAG appear to be processed independently, as indicated by the formation of the intermediate fragments P32 and P34 in nearly equal ratios. The site nearest the amino terminus is quite conserved between the two viral GAG proteins (...VSQNY-PIVQN...in HIV-1,...KGGNY-PVQHV...in HIV-2). In contrast, the putative second site (...IPFAA-AQQKG...) of HIV-2 GAG shares minimal sequence identity with site 2 of HIV-1 GAG (...SATIM-MQRGN...). These sequence variations in the substrates suggest higher order structural features that may influence recognition by the proteases. Pepstatin A inhibits HIV-2 protease, whereas 1,10-phenanthroline and phenylmethylsulfonylfluoride do not; these results are in agreement with the finding that proteases of HIV and other retroviruses are aspartyl proteases.

Amino Acid Sequence↗

PubMed2005

AP-3 directs the intracellular trafficking of HIV-1 Gag and plays a key role in particle assembly.

Gag proteins direct the process of retroviral particle assembly and form the major protein constituents of the viral core. The matrix region of the HIV-1 Gag polyprotein plays a critical role in the transport of Gag to the plasma membrane assembly site. Recent evidence indicates that Gag trafficking to late endosomal compartments, including multivesicular bodies, occurs prior to viral particle budding from the plasma membrane. Here we demonstrate that the matrix region of HIV-1 Gag interacts directly with the delta subunit of the AP-3 complex, and that this interaction plays an important functional role in particle assembly. Disruption of this interaction eliminated Gag trafficking to multivesicular bodies and diminished HIV particle formation. These studies illuminate an early step in retroviral particle assembly and provide evidence that the trafficking of Gag to late endosomes is part of a productive particle assembly pathway.

Adaptor Protein Complex 3↗

PubMed2006

Association of Gag cleavage sites to protease mutations and to virological response in HIV-1 treated patients.

OBJECTIVES: The sequence variability in the protease and in the 5 Gag cleavage sites (CS) were explored to look for eventual associations between the mutations. Moreover, we have evaluated associations between the Gag region sequence and the virological response to Protease Inhibitors (PI). METHODS: The protease and the 5 Gag CS sequences from 98 PI-experienced patients were sequenced and compared to the HXB2 reference sequence. Sixty patients, treated by Saquinavir plus Ritonavir, were studied to evaluate the clinical impact of the Gag region variability. RESULTS: The relationship between 63 protease mutations and 21 Gag CS mutations were explored. Two patterns of mutations in the protease were identified: (M46I/L, I54V, V82A/T/F) was associated to the A431V and (K20I/R/M, L89M/I) to the S373Q and L449P. None of the Gag CS mutations resulting from PI treatment was associated to the virological response to SQV/r. On the other hand, the S373P mutation had a negative impact on the virological response that remained statistically significant in a multivariate analysis after adjustment on the number of PI resistance mutations. CONCLUSIONS: These results evoke the pertinence to introduce some mutations found in the Gag CS in the algorithms used for the interpretation of resistance testing.

Amino Acid Sequence↗

PubMed2004

SIVmac Gag p27 capsid protein gene expression in potato.

A cDNA encoding the Simian immunodeficiency virus type (SIV(mac)) Gag capsid protein was introduced into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation methods. The gag gene was detected in the genomic DNA of transformed leaf tissues by PCR DNA amplification. Immunoblot analysis of transformed potato plant extracts with anti-Gag monoclonal antibody showed that biologically active Gag protein was synthesized in transformed tuber tissues. Based on ELISA results, recombinant Gag protein made up 0.006-0.014% of total soluble tuber protein. The synthesis of SIV Gag in transformed potato tubers opens the way for development of Gag-based edible plant vaccines for protection against SIV and potentially HIV-1 infection.

AIDS Vaccines↗

PubMed2005

Potent immunogenicity of an HIV-1 gag-pol fusion DNA vaccine delivered by in vivo electroporation.

A plasmid DNA vaccine containing a fusion gene consisting of an HIV-1 subtype C gag and a modified subtype C pol was compared to a mixture of gag plus pol or gag plus HIV env plasmids. Plasmid DNA was delivered by intramuscular injection followed by electroporation in vivo. Two vaccinations were sufficient to induce high levels of Gag- and Pol-specific CD4 and CD8 T cells in peripheral blood. The gag-pol fusion plasmid was as immunogenic as the plasmid mixtures. Thus, DNA vaccination by intramuscular electroporation was an effective means for inducing high levels of Gag- and Pol-specific T cells, and a single gag-pol fusion DNA vaccine was sufficient for eliciting immune responses against both antigens.

AIDS Vaccines↗

PubMed2005

Early detection of dominant Env-specific and subdominant Gag-specific CD8+ lymphocytes in equine infectious anemia virus-infected horses using major histocompatibility complex class I/peptide tetrameric complexes.

Cytotoxic T lymphocytes (CTL) are critical for control of lentiviruses, including equine infectious anemia virus (EIAV). Measurement of equine CTL responses has relied on chromium-release assays, which do not allow accurate quantitation. Recently, the equine MHC class I molecule 7-6, associated with the ELA-A1 haplotype, was shown to present both the Gag-GW12 and Env-RW12 EIAV CTL epitopes. In this study, 7-6/Gag-GW12 and 7-6/Env-RW12 MHC class I/peptide tetrameric complexes were constructed and used to analyze Gag-GW12- and Env-RW12-specific CTL responses in two EIAV-infected horses (A2164 and A2171). Gag-GW12 and Env-RW12 tetramer-positive CD8+ cells were identified in nonstimulated peripheral blood mononuclear cells as early as 14 days post-EIAV inoculation, and frequencies of tetramer-positive cells ranged from 0.4% to 6.7% of nonstimulated peripheral blood CD8+ cells during the 127-day study period. Although both horses terminated the initial viremic peak, only horse A2171 effectively controlled viral load. Neutralizing antibody was present during the initial control of viral load in both horses, but the ability to maintain control correlated with Gag-GW12-specific CD8+ cells in A2171. Despite Env-RW12 dominance, Env-RW12 escape viral variants were identified in both horses and there was no correlation between Env-RW12-specific CD8+ cells and control of viral load. Although Gag-GW12 CTL escape did not occur, a Gag-GW12 epitope variant arose in A2164 that was recognized less efficiently than the original epitope. These data indicate that tetramers are useful for identification and quantitation of CTL responses in horses, and suggest that the observed control of EIAV replication and clinical disease was associated with sustained CTL recognition of Gag-specific epitopes.

Amino Acid Sequence↗

PubMed2005

HTLV-1 Gag protein associates with CD82 tetraspanin microdomains at the plasma membrane.

We examined the association of HTLV-1 Gag with tetraspanin-enriched microdomains in the plasma membrane. Immunofluorescent staining and confocal image analysis showed that HTLV-1 Gag protein colocalized with CD82 and other tetraspanins at the plasma membrane of T cells. HTLV-1 Gag, which is associated with the inner surface of the plasma membrane, was concentrated to the patches formed by antibody-mediated cross-linking of CD82 on the cell surface. Also, CD82 and HTLV-1 Gag rapidly segregated to the immune synapse that is formed between Raji B cells and Jurkat T cells in the presence of bacterial superantigen. CD82, which was immunoprecipitated from cell extracts prepared in Brij97 detergent conditions, was associated with the matrix (MA) protein. Stable interaction of MA and CD82 in Brij97-disrupted cell extracts required Gag multimerization and proteolytic processing. The form of MA that coimmunoprecipitated with CD82 was a cysteine-linked homodimer. The viral envelope glycoprotein was not required for the association of Gag with CD82-enriched membrane regions. In contrast to HTLV-1, HIV-1 Gag did not colocalize, cosegregate, or coimmunoprecipitate with CD82. Our data suggest that once at the plasma membrane, HTLV-1 virion components associate with CD82-containing microdomains, which may facilitate the mobilization of nascent virions to sites of intercellular adhesion.

Cell Line↗

PubMed2006

Capsid is an important determinant for functional complementation of murine leukemia virus and spleen necrosis virus Gag proteins.

In this report, we examined the abilities and requirements of heterologous Gag proteins to functionally complement each other to support viral replication. Two distantly related gammaretroviruses, murine leukemia virus (MLV) and spleen necrosis virus (SNV), were used as a model system because SNV proteins can support MLV vector replication. Using chimeric or mutant Gag proteins that could not efficiently support MLV vector replication, we determined that a homologous capsid (CA) domain was necessary for the functional complementation of MLV and SNV Gag proteins. Findings from the bimolecular fluorescence complementation assay revealed that MLV and SNV Gag proteins were capable of colocalizing and interacting in cells. Taken together, our results indicated that MLV and SNV Gag proteins can interact in cells; however, a homologous CA domain is needed for functional complementation of MLV and SNV Gag proteins to complete virus replication. This requirement of homologous Gag most likely occurs at a postassembly step(s) of the viral replication.

Animals↗

PubMed2006

Implications for viral capsid assembly from crystal structures of HIV-1 Gag(1-278) and CA(N)(133-278).

Gag, the major structural protein of retroviruses such as HIV-1, comprises a series of domains connected by flexible linkers. These domains drive viral assembly by mediating multiple interactions between adjacent Gag molecules and by binding to viral genomic RNA and host cell membranes. Upon viral budding, Gag is processed by the viral protease to liberate distinct domains as separate proteins. The first two regions of Gag are MA, a membrane-binding module, and CA, which is a two-domain protein that makes important Gag-Gag interactions, forms the cone-shaped outer shell of the core (the capsid) in the mature HIV-1 particle, and makes an important interaction with the cellular protein cyclophilin A (CypA). Here, we report crystal structures of the mature CA N-terminal domain (CA(N)(133-278)) and a MA-CA(N) fusion (Gag(1-278)) at resolutions/R(free) values of 1.9 A/25.7% and 2.2 A/25.8%, respectively. Consistent with earlier studies, a comparison of these structures indicates that processing at the MA-CA junction causes CA to adopt an N-terminal beta-hairpin conformation that seems to be required for capsid morphology and viral infectivity. In contrast with an NMR study (Tang, C., et al. (2002) Nat. Struct. Biol. 9, 537-543), structural overlap reveals only small relative displacements for helix 6, which is located between the beta-hairpin and the CypA-binding loop. These observations argue against the proposal that CypA binding is coupled with beta-hairpin formation and support an earlier surface plasmon resonance study (Yoo, S., et al. (1997) J. Mol. Biol. 269, 780-795), which concluded that beta-hairpin formation and CypA-binding are energetically independent events.

Amino Acid Sequence↗

PubMed1996

The major homology region of the HIV-1 gag precursor influences membrane affinity.

Assembly of retroviruses, including HIV-1, involves movement of newly synthesized viral proteins and RNA to the plasma membranes of host cells. The major homology region (MHR, aa 285-304), a highly conserved sequence in the capsid domain of the HIV-1 Gag precursor polyprotein, plays a critical, but unknown, role in infectious particle assembly. Mutations of invariant residues in the sequence have pleiotropic effects: Mutation of Gln287 blocks viral assembly while mutation of Arg299 permits assembly, but blocks formation of infectious particles. In this report, we demonstrate that Gag proteins lacking the entire MHR accumulated in the cytoplasm of transfected COS-1 cells, as did the wild-type protein, but were processed in a defective manner at the cellular membrane resulting in impaired particle assembly. To further examine the role of the MHR in membrane association, membrane binding of unmyristylated recombinant Gag proteins with alterations in the MHR was investigated in vitro. The wild-type Gag precursor bound to acidic phospholipid vesicles highly efficiently, as determined by fluorescence spectroscopy or velocity sedimentation. In contrast, deletion of the entire MHR reduced membrane affinity an average of approximately 3-fold or greater. Mutation of the invariant Gln287 residue disrupted membrane affinity approximately 6-fold relative to the wild-type, which was similar to the level of inhibition obtained by deletion of a membrane-binding signal previously identified in the matrix domain of the Gag precursor. Mutation of the invariant Arg299 residue reduced the affinity to a lesser extent. The results indicate that correct membrane binding is determined not only by signals in the MA domain of the precursor but also by sequences in the CA domain of Gag. We speculate that defects in the highly conserved MHR affect a Gag conformation that is required for productive interactions at the membrane assembly site.

Amino Acid Sequence↗

PubMed2000

Rev-dependent association of the intron-containing HIV-1 gag mRNA with the nuclear actin bundles and the inhibition of its nucleocytoplasmic transport by latrunculin-B.

BACKGROUND: A hallmark of HIV-1 gene expression is that unspliced genomic RNA, which also acts as mRNA for the expression of Gag/Pol, is exported to the cytoplasm. Rev directs this transport through the nuclear export signal (NES). RESULTS: Fluorescence in situ hybridization and immunocytochemistry demonstrated that gag mRNA, Rev, and its NES receptor, CRM1, and RanGTPase formed nuclear tracks which were congruent with underlying beta-actin bundles. Actin bundle formation was confirmed electron-microscopically. These bundles were observed upon Rev-containing gag RNP formation. The loss of bundles was associated with the nuclear retention of gag mRNA. Reverse transcription-polymerase chain reaction analysis of both cytoplasmic and nuclear gag mRNAs demonstrated that disruption of nuclear actin filament formation by latrunculin-B (LAT-B), an F-actin depolymerizing compound, resulted in the dose-dependent inhibition of gag mRNA export. The differential subtyping of the mRNA-positive cells confirmed morphologically the effect of LAT-B treatment. The export inhibition was specific to gag mRNA and export of fully spliced HIV-1 tat/rev mRNAs as well as cellular GAPDH mRNA was not affected by the compound. CONCLUSIONS: Nuclear beta-actin bundles are suggested to be functionally involved in the Rev-dependent nucleocytoplasmic transport of intron-containing HIV-1 gag mRNA.

Actins↗

PubMed2001

Efficient assembly of an HIV-1/MLV Gag-chimeric virus in murine cells.

In human cells infected by HIV type 1 (HIV-1), the viral Gag protein directs the assembly of nascent viral particles at the plasma membrane. In murine cells, HIV-1 Gag fails to reach the plasma membrane and instead forms nonfunctional intracellular aggregates. The viral determinants of this species incompatibility are previously undefined. To address this problem, we replaced a region of HIV-1 Gag known to direct its localization, the matrix (MA) domain, with functionally homologous regions from Moloney murine leukemia virus (MLV), a murine retrovirus. An HIV-1 clone carrying such a chimeric Gag protein, designated murine HIV (MHIV), assembled more efficiently than nonchimeric HIV-1 and restored plasma membrane localization of Gag in murine cells. Increased efficiency of viral assembly in murine cells was observed from MHIV constructs carrying MLV MA in place of HIV-1 MA. Efficient processing of the HIV-1 capsid protein from the chimeric Gag polyprotein and subsequent infectivity of MHIV required the presence of MLV p12 in addition to MLV MA. These findings strongly suggest that the HIV-1 MA domain of HIV-1 Gag is responsible for the assembly defect in mouse cells. Although these MHIV do not recruit native HIV-1 Env efficiently, they are capable of single-round infection when produced by high-efficiency transfection of human 293 cells and provided with an HIV-1 Env lacking its cytoplasmic tail. With further adaptation, this chimeric MHIV approach may provide the basis for creating an infectious mouse model for HIV/AIDS.

Animals↗

PubMed1983

Construction and characterization of Moloney murine leukemia virus mutants unable to synthesize glycosylated gag polyprotein.

Murine leukemia virus (MuLV) encodes two independent pathways for expression of the gag gene. One pathway results in processing and cleavage of the precursor Pr65gag to yield the internal capsid proteins of the virion and is analogous to gag polyprotein precursors for all classes of retroviruses. The other pathway, which is not encoded by several other classes of retroviruses, begins with a glycosylated polyprotein gPr80gag . gPr80gag is synthesized independently of Pr65gag; it contains Pr65gag peptides and additional amino-terminal protein. It is modified by further addition of carbohydrate, exported to the cell surface, and released from the cell but does not appear in virus particles. To investigate the role of glycosylated gag in MuLV infection, two mutants of Moloney MuLV (M-MuLV) deficient for synthesis of gPr80gag but able to synthesize Pr65gag were constructed. The mutants were obtained by substitution into a molecular clone of M-MuLV DNA by DNA from two acutely transforming viruses, Ableson MuLV (Ab-MuLV) and Moloney murine sarcoma virus (M-MSV). Both Ab-MuLV and M-MSV are derived from M-MuLV and they express M-MuLV gag sequences, but some strains do not synthesize glycosylated gag protein. For Ab-MuLV, a 177-base-pair Pst I fragment from the P90 strain containing the initiation codon for Pr65gag was substituted for the equivalent fragment in M-MuLV DNA. For M-MSV, 1.5 kilobases at the 5' end of the genome was substituted. Transfection of the recombined DNAs onto NIH-3T3 cells produced infectious M-MuLV, although the infected cells did not produce gPr80gag. Therefore glycosylated gag is not absolutely required for MuLV replication. Deletion of the glycosylated gag pathway did not significantly reduce the level of virus production, although a minor difference in XC plaque morphology was observed.

Animals↗

PubMed1985

Mutagenesis of avian carcinoma virus MH2: only one of two potential transforming genes (delta gag-myc) transforms fibroblasts.

Avian carcinoma virus MH2 contains two potential transforming genes, delta gag-mht and delta gag-myc. Thus, MH2 may be a model for two-gene carcinogenesis in which transformation depends on two synergistic genes. Most other directly oncogenic viruses contain single, autonomous transforming (onc) genes and are models for single-gene carcinogenesis. To determine which role each potential onc gene of MH2 plays in oncogenesis, we have prepared deletion and frameshift mutants of each of the two MH2 genes by in vitro mutagenesis of cloned proviral DNA and have tested transforming function and virus production in cultured primary quail cells. We have found that mht deletion mutants and wild-type virus transform primary cells and that myc deletion and frameshift mutants do not. The morphologies of cells transformed by the mht deletion mutants and by wild-type MH2 are similar yet vary considerably. Nevertheless, typical mutant transformed cells can often be distinguished from cells transformed by wild-type MH2. We conclude that the delta gag-myc gene transforms primary cells by itself, without the second potential onc gene. This myc-related gene is the smallest that has direct transforming function. delta gag-mht is without detectable transforming function but may affect transformation by delta gag-myc. Thus, MH2 behaves like a virus with a single onc gene, although it expresses two potential onc genes, and it appears not to be a model for two-gene carcinogenesis. Further work is necessary to determine whether the delta gag-mht gene possibly enhances oncogenic function of delta gag-myc or has independent oncogenic function in animals.

Animals↗

PubMed1987

Two efficient ribosomal frameshifting events are required for synthesis of mouse mammary tumor virus gag-related polyproteins.

The primary translation products of retroviral pol genes are polyproteins initiated in an upstream gene (gag). To investigate the manner in which the gag-initiated polyproteins of the mouse mammary tumor virus are produced, we determined the nucleotide sequence of a 1.8-kilobase DNA fragment that spans the region between gag and pol in the C3H strain of mouse mammary tumor virus. The sequence reveals three overlapping open reading frames: the first encodes products of gag (p27gag and p14gag); the second encodes a protein domain of unknown function (termed X) that is highly related to a similarly positioned sequence in simian type D retroviruses and the viral protease (pro); and the third encodes the reverse transcriptase. The reading frames are organized to permit uninterrupted readthrough from gag to pol if ribosomal frameshifts occur in the -1 direction within each of the two overlapping regions, one of which is 16 nucleotides in length and the other 13 nucleotides. Cell-free translation of RNA containing these overlap regions shows that fusion of the reading frames by ribosomal frameshifting occurs efficiently: about one-fourth of the ribosomes traversing the gag-X/pro overlap and one-tenth traversing the X/pro-pol overlap shift frames, generating gag-related polyproteins in ratios similar to those observed in vivo. Synthetic oligonucleotides containing either of the overlap regions inserted into novel contexts do not induce frameshifting; hence the overlapping portions of the reading frames are not sufficient to induce a frameshift event, and a larger sequence context or secondary structure may be implicated.

Amino Acid Sequence↗

PubMed2000

Proteasome inhibition interferes with gag polyprotein processing, release, and maturation of HIV-1 and HIV-2.

Retrovirus assembly and maturation involve folding and transport of viral proteins to the virus assembly site followed by subsequent proteolytic cleavage of the Gag polyprotein within the nascent virion. We report that inhibiting proteasomes severely decreases the budding, maturation, and infectivity of HIV. Although processing of the Env glycoproteins is not changed, proteasome inhibitors inhibit processing of Gag polyprotein by the viral protease without affecting the activity of the HIV-1 viral protease itself, as demonstrated by in vitro processing of HIV-1 Gag polyprotein Pr55. Furthermore, this effect occurs independently of the virus release function of the HIV-1 accessory protein Vpu and is not limited to HIV-1, as proteasome inhibitors also reduce virus release and Gag processing of HIV-2. Electron microscopy analysis revealed ultrastructural changes in budding virions similar to mutants in the late assembly domain of p6(gag), a C-terminal domain of Pr55 required for efficient virus maturation and release. Proteasome inhibition reduced the level of free ubiquitin in HIV-1-infected cells and prevented monoubiquitination of p6(gag). Consistent with this, viruses with mutations in PR or p6(gag) were resistant to detrimental effects mediated by proteasome inhibitors. These results indicate the requirement for an active proteasome/ubiquitin system in release and maturation of infectious HIV particles and provide a potential pharmaceutical strategy for interfering with retrovirus replication.

Cell Line↗