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PubMed2002

Changes in human immunodeficiency virus type 1 Gag at positions L449 and P453 are linked to I50V protease mutants in vivo and cause reduction of sensitivity to amprenavir and improved viral fitness in vitro.

Human immunodeficiency virus type 1 (HIV-1) Gag protease cleavage sites (CS) undergo sequence changes during the development of resistance to several protease inhibitors (PIs). We have analyzed the association of sequence variation at the p7/p1 and p1/p6 CS in conjunction with amprenavir (APV)-specific protease mutations following PI combination therapy with APV. Querying a central resistance data repository resulted in the detection of significant associations (P < 0.001) between the presence of APV protease signature mutations and Gag L449F (p1/p6 LP1'F) and P453L (p1/p6 PP5'L) CS changes. In population-based sequence analyses the I50V mutant was invariably linked to either L449F or P453L. Clonal analysis revealed that both CS mutations were never present in the same genome. Sequential plasma samples from one patient revealed a transition from I50V M46L P453L viruses at early time points to I50V M46I L449F viruses in later samples. Various combinations of the protease and Gag mutations were introduced into the HXB2 laboratory strain of HIV-1. In both single- and multiple-cycle assay systems and in the context of I50V, the L449F and P453L changes consistently increased the 50% inhibitory concentration of APV, while the CS changes alone had no measurable effect on inhibitor sensitivity. The decreased in vitro fitness of the I50V mutant was only partially improved by addition of either CS change (I50V M46I L449F mutant replicative capacity approximately 16% of that of wild-type virus). Western blot analysis of Pr55 Gag precursor cleavage products from infected-cell cultures indicated accumulation of uncleaved Gag p1-p6 in all I50V viruses without coexisting CS changes. Purified I50V protease catalyzed cleavage of decapeptides incorporating the L449F or P453L change 10-fold and 22-fold more efficiently than cleavage of the wild-type substrate, respectively. HIV-1 protease CS changes are selected during PI therapy and can have effects on both viral fitness and phenotypic resistance to PIs.

Binding Sites↗

PubMed2002

Analysis of bovine leukemia virus gag membrane targeting and late domain function.

Assembly of retrovirus-like particles only requires the expression of the Gag polyprotein precursor. We have exploited this in the development of a model system for studying the virus particle assembly pathway for bovine leukemia virus (BLV). BLV is closely related to the human T-cell leukemia viruses (HTLVs), and all are members of the Deltaretrovirus genus of the Retroviridae family. Overexpression of a BLV Gag polyprotein containing a carboxy-terminal influenza virus hemagglutinin (HA) epitope tag in mammalian cells led to the robust production of virus-like particles (VLPs). Site-directed mutations were introduced into HA-tagged Gag to test the usefulness of this model system for studying certain aspects of the virus assembly pathway. First, mutations that disrupted the amino-terminal glycine residue that is important for Gag myristylation led to a drastic reduction in VLP production. Predictably, the nature of the VLP production defect was correlated to Gag membrane localization. Second, mutation of the PPPY motif (located in the MA domain) greatly reduced VLP production in the absence of the viral protease. This reduction in VLP production was more severe in the presence of an active viral protease. Examination of particles by electron microscopy revealed an abundance of particles that began to pinch off from the plasma membrane but were not completely released from the cell surface, indicating that the PPPY motif functions as a late domain (L domain).

Amino Acid Sequence↗

PubMed2002

Replacement of the P1 amino acid of human immunodeficiency virus type 1 Gag processing sites can inhibit or enhance the rate of cleavage by the viral protease.

Processing of the human immunodeficiency virus type 1 (HIV-1) Gag precursor is highly regulated, with differential rates of cleavage at the five major processing sites to give characteristic processing intermediates. We examined the role of the P1 amino acid in determining the rate of cleavage at each of these five sites by using libraries of mutants generated by site-directed mutagenesis. Between 12 and 17 substitution mutants were tested at each P1 position in Gag, using recombinant HIV-1 protease (PR) in an in vitro processing reaction of radiolabeled Gag substrate. There were three sites in Gag (MA/CA, CA/p2, NC/p1) where one or more substitutions mediated enhanced rates of cleavage, with an enhancement greater than 60-fold in the case of NC/p1. For the other two sites (p2/NC, p1/p6), the wild-type amino acid conferred optimal cleavage. The order of the relative rates of cleavage with the P1 amino acids Tyr, Met, and Leu suggests that processing sites can be placed into two groups and that the two groups are defined by the size of the P1' amino acid. These results point to a trans effect between the P1 and P1' amino acids that is likely to be a major determinant of the rate of cleavage at the individual sites and therefore also a determinant of the ordered cleavage of the Gag precursor.

Binding Sites↗

PubMed2003

Barrier-to-autointegration factor BAF binds p55 Gag and matrix and is a host component of human immunodeficiency virus type 1 virions.

Barrier-to-autointegration factor (BAF) is a conserved human chromatin protein exploited by retroviruses. Previous investigators showed that BAF binds double-stranded DNA nonspecifically and is a host component of preintegration complexes (PICs) isolated from cells infected with human immunodeficiency virus type 1 (HIV-1) or Moloney murine leukemia virus. BAF protects PIC structure and stimulates the integration of salt-stripped PICs into target DNA in vitro. PICs are thought to acquire BAF from the cytoplasm during infection. However, we identified two human tissues (of 16 tested) in which BAF mRNA was not detected: thymus and peripheral blood leukocytes, which are enriched in CD4(+) T lymphocytes and macrophage precursors, respectively. BAF protein was detected in activated but not resting CD4(+) T lymphocytes; thus, if BAF were essential for PIC function, we hypothesized that virions might "bring their own BAF." Supporting this model, BAF copurified with HIV-1 virions that were digested with subtilisin to remove microvesicle contaminants, and BAF was present in approximately zero to three copies per virion. In three independent assays, BAF bound directly to both p55 Gag (the structural precursor of HIV-1 virions) and its cleaved product, matrix. Using lysates from cells overexpressing Gag, endogenous BAF and Gag were coimmunoprecipitated by antibodies against Gag. Purified recombinant BAF had low micromolar affinities (1.1 to 1.4 micro M) for recombinant Gag and matrix. We conclude that BAF is present at low levels in incoming virions, in addition to being acquired from the cytoplasm of newly infected cells. We further conclude that BAF might contribute to the assembly or activity of HIV-1 PICs through direct binding to matrix, as well as DNA.

CD4-Positive T-Lymphocytes↗

PubMed2004

Human immunodeficiency virus type 1 matrix inhibits and confers cooperativity on gag precursor-membrane interactions.

Human immunodeficiency virus type 1 (HIV-1) Gag multimerization and membrane binding are required for particle formation. However, it is unclear what constitutes a minimal plasma membrane-specific targeting signal and what role the matrix (MA) globular head and other Gag domains play in membrane targeting. Here, we use membrane flotation and microscopic analysis of Gag deletion mutants to demonstrate that the HIV-1 MA globular head inhibits a plasma membrane-specific targeting signal contained within the six amino-terminal MA residues. MA-mediated inhibition is relieved by concentration-dependent Gag multimerization and imparts a high degree of cooperativity on Gag-membrane association. This cooperativity may confer temporal and spatial regulation on HIV-1 assembly.

Allosteric Regulation↗

PubMed2005

Cooperative effect of gag proteins p12 and capsid during early events of murine leukemia virus replication.

The Gag polyprotein of murine leukemia virus (MLV) is processed into matrix (MA), p12, capsid (CA), and nucleocapsid (NC) proteins. p12 affects early events of virus replication and contains a PPPY motif important for virus release. To probe the functions of p12 in the early steps of MLV replication, we tested whether p12 can be replaced by spleen necrosis virus (SNV) p18, human immunodeficiency virus type 1 p6, or Rous sarcoma virus p2b. Analyses revealed that all chimeras generated virions at levels similar to that of MLV gag-pol; however, none of them could support MLV vector replication, and all of them exhibited severely reduced DNA synthesis upon virus infection. Because a previously reported SNV gag-MLV pol chimera, but not the MLV hybrid with SNV p18, can support replication of an MLV vector, we hypothesized that other Gag proteins act cooperatively with p12 during the early phase of virus replication. To test this hypothesis, we generated three more MLV-based chimeras containing SNV CA, p18-CA, or p18-CA-NC. We found that the MLV chimera containing SNV p18-CA or p18-CA-NC could support MLV vector replication, but the chimera containing SNV CA could not. Furthermore, viruses derived from the MLV chimera with SNV CA could synthesize viral DNA upon infection but were blocked at a post-reverse-transcription step and generated very little two long terminal repeat circle DNA, thereby producing a phenotype similar to that of the provirus formation-defective p12 mutants. Taken together, our data indicate that when p12/p18 or CA was from different viruses, despite abundant virus production and proper Gag processing, the resulting viruses were not infectious. However, when p12/p18 and CA were from the same virus, even though they were from SNV and not MLV, the resulting viruses were infectious. Therefore, these results suggest a cooperative effect of p12 and CA during the early events of MLV replication.

Capsid Proteins↗

PubMed1999

Host cell-dependent replication of HIV-1 gag MA, CA, and NC mutants are independent of the functions of Vif and Vpu.

We have previously shown that some gag gene mutants of human immunodeficiency virus type 1 (HIV-1) display a replication-defect in a cell-dependent manner. We and others have also demonstrated that the requirement of vif and vpu genes for HIV-1 replication is cell-dependent. To determine whether the cell-dependent growth of the HIV-1 gag mutants is related to the functions of Vif and Vpu, double mutants of gag-vif and gag-vpu were constructed, and monitored for their replication in various cell lines. The results obtained showed that the mutations in gag do not affect the cell-dependent functions of Vif and Vpu.

Capsid↗

PubMed2004

Novel and promiscuous CTL epitopes in conserved regions of Gag targeted by individuals with early subtype C HIV type 1 infection from southern Africa.

Characterization of optimal CTL epitopes in Gag can provide crucial information for evaluation of candidate vaccines in populations at the epicenter of the HIV-1 epidemic. We screened 38 individuals with recent subtype C HIV-1 infection using overlapping consensus C Gag peptides and hypothesized that unique HLA-restricting alleles in the southern African population would determine novel epitope identity. Seventy-four percent of individuals recognized at least one Gag peptide pool. Ten epitopic regions were identified across p17, p24, and p2p7p1p6, and greater than two-thirds of targeted regions were directed at: TGTEELRSLYNTVATLY (p17, 35%); GPKEPFRDYVDRFFKTLRAEQATQDV (p24, 19%); and RGGKLDKWEKIRLRPGGKKHYMLKHL (p17, 15%). After alignment of these epitopic regions with consensus M and a consensus subtype C sequence from the cohort, it was evident that the regions targeted were highly conserved. Fine epitope mapping revealed that five of nine identified optimal Gag epitopes were novel: HLVWASREL, LVWASRELERF, LYNTVATLY, PFRDYVDRFF, and TLRAEQATQD, and were restricted by unique HLA-Cw*08, HLA-A*30/B*57, HLA-A*29/B*44, and HLA-Cw*03 alleles, respectively. Notably, three of the mapped epitopes were restricted by more than one HLA allele. Although these epitopes were novel and restricted by unique HLA, they overlapped or were embedded within previously described CTL epitopes from subtype B HIV-1 infection. These data emphasize the promiscuous nature of epitope binding and support our hypothesis that HLA diversity between populations can shape fine epitope identity, but may not represent a constraint for universal recognition of Gag in highly conserved domains.

Africa, Southern↗

PubMed2003

Codon usage-mediated inhibition of HIV-1 gag expression in mammalian cells occurs independently of translation.

Codon usage is considered one of the critical factors that limit the expression rate of heterologous genes. Impaired translation efficiency, specifically insufficient amount of corresponding tRNAs and changed startcodon context, are believed to account for the low translation initiation and elongation rates during the protein biosynthesis in unicellular organisms. Translational efficiency is probably not the primary factor influencing codon usage diversity in mammalian cells. However, the other possible mechanisms preventing expression of genes with low-usage such as mRNA stability, processing and nucleocytoplasmic transport, are not adequately explored. In our work, we addressed the question of whether codon usage differences affect exclusively translational efficiency of mammalian gene products. We demonstrated that the CMV-induced expression of gag-reporter in human H1299 cell line was influenced by the nucleotide composition of the mRNA, and the limitation of gag expression appeared to be inversely related to the level of codon optimization. However, cytoplasmic expression of the gag-reporter driven by vaccinia virus/T7 RNA polymerase hybrid system rescued its expression independently of HIV-1 gag mRNA nucleotide content. We concluded that impaired HIV-1 gag expression may be caused by translation-independent mechanisms, which probably play a major role in codon usage-mediated defects in heterologous gene expression in mammalian cells.

Codon↗

PubMed1991

HIV-1 gag proteins in virions and in infected cell fractions.

The relation of the initial products of the HIV-1 gag gene to the final products was determined in virus samples and cell fractions of infected H9 and Jurkat-tat cell cultures. The proteins were identified by immunoblotting with pooled sera from AIDS patients or monoclonal antibodies. The proportion in the virions of gag precursor proteins and the products of their proteolytic cleavage varied according to the maturity of the virus particles as determined by electron microscopy. The distribution of viral gag proteins in the cell fractions was determined 2, 4, and 24 h after infection. Treatment of cells with cycloheximide to block de novo protein synthesis did not significantly affect the results. Gag proteins containing the N terminus of the precursor p55 (including p55, the intermediate precursors p41(45) and p39, and mature protein p17) were found in the cell nuclei up to 24 h after infection. The major core protein p24 was located in the cytoplasmic fraction. These data strongly suggest that gag precursors from the p55 N terminus and the matrix protein p17 enter the infected cell separately from the major core protein p24, or become separated from it in the cytoplasm.

Acquired Immunodeficiency Syndrome↗

PubMed2001

Human immunodeficiency virus gag and pol-specific CD8 T cells in perinatal HIV infection.

BACKGROUND: Binding of fluorochrome-conjugated MHC class I tetramers is a powerful means to detect antigen-specific CD8 T lymphocytes. In human immunodeficiency virus (HIV) infection, cellular immune response is essential in curtailing HIV disease progression but gaps persist in our understanding of HIV-specific cells during the disease course. In this study, we evaluated tetramer binding HIV-specific CD8 T cells in HIV-infected children. METHODS: Fluorescently labeled tetramers for HIV gag and pol were utilized to quantify antigen-specific cells by flow cytometry using a whole blood labeling method in a cohort of 19 HLA-A2+ HIV-infected children (age range 1 month to 17 years). RESULTS: Fourteen children had detectable gag (median 0.4%) and pol (median 0.1%) binding CD8 T cells, three children had gag binding cells only, and two had neither. Numbers of gag and pol binding cells correlated with each other and each correlated independently with total CD8 T cells and total CD4 T cells. CONCLUSIONS: HIV gag and pol-specific CD8 T cells are maintained during the chronic phase of HIV infection in children and CD4 lymphocytes appear to be important for sustaining their levels.

Adolescent↗

PubMed1989

The GAG precursor of simian immunodeficiency virus assembles into virus-like particles.

To examine the potential role of the GAG precursor polyprotein in morphogenesis and assembly of the simian immunodeficiency virus (SIV), we have expressed the gag gene of SIVMac using a baculovirus expression vector. Infection of insect cells with recombinant virus containing the entire gag gene results in high expression of the GAG precursor protein, Pr57gag. The recombinant protein is myristylated and is released in the culture supernatant in an insoluble particulate form. A point mutation in the N-terminal glycine codon (Gly----Ala) inhibits myristylation. This mutated product is highly expressed but is not found in the culture supernatant. Electron microscopy and immunogold labelling of infected cells show that the native Pr57gag protein assembles into 100-120 nm virus-like particles that bud from the cell plasma membrane and are released in the culture supernatant. The unmyristylated protein also assembles into particulate structures which only accumulate inside the cells. These results demonstrate that the unprocessed GAG precursor of SIV can spontaneously assemble into particles in the absence of other viral proteins. Myristylation of the Pr57gag precursor is necessary for its association with the cell plasma membrane, for budding and for extracellular release.

Animals↗

PubMed2001

Myristoylation of human immunodeficiency virus type 1 gag protein is required for efficient env protein transportation to the surface of cells.

Highly conserved amino acids in the N-terminal region of the human immunodeficiency virus type 1 (HIV-1) Pr55(gag) are recognized to be critical for the attachment of myristic acid. We previously reported that the env protein was not detected on the cell surface by blocking of N-myristoylation of Pr55(gag) with N-myristoyl glycinal diethylacetal. Here, we constructed a mutant by substituting the N-terminal glycine of Pr55(gag) with alanine to demonstrate that N-myristoylation of Pr55(gag) is required for efficient env protein transportation to the cell surface. The expression level of the env protein on the surface of Jurkat cells transfected with the myristoylation-defective phenotype was observed to be significantly reduced by electron microscopic analyses with a gold-labeled monoclonal antibody against the env protein. In addition, Jurkat cells transfected with the myristoylation-defective phenotype lost the ability of envelope-mediated cell-to-cell fusion. The results suggest that N-myristoylation of the HIV-1 gag protein is necessary for efficient env protein transportation to the cell surface.

Amino Acid Substitution↗

PubMed1998

Further evidence for hexagonal organization of HIV gag protein in prebudding assemblies and immature virus-like particles.

The fullerene-like model for the organization of HIV gag encoded precursor pr55gag was based on the study of prebudding assemblies at the plasma membrane of cells infected with a recombinant baculovirus expressing HIV-1 gag protein. The objective of the present study was to support the model by image processing of virus-like particles (VLP). In this work we used VLP purified by density gradient centrifugation, which caused partial or occasionally complete loss of the lipid bilayer in some VLP without the use of detergent. In addition more plasma membrane-associated pr55gag protein assemblies were processed. Image processing of negatively stained specimens revealed the presence of threefold symmetry and a hexagonal network of rings with a resolution of 29 A in VLP and better than 25 A in membrane associated assemblies. The center-to-center spacing of the rings was 67 A in VLP and 70 A in membrane assemblies. Patches of gag protein oligomers at the plasma membrane were usually round and varying in size, but some of them were triangular. Indication of triangular-shaped gag protein assemblies was also seen in partly dissociated VLP. Since the hexagonal network is formed by the uncleaved gag polyprotein, we conclude that the threefold symmetry applies to all domains including p24gag. The presence of threefold symmetry and the hexagonal network in VLP are consistent with the hypothesis that immature HIV particles possess icosahedral symmetry.

Animals↗

PubMed1996

The glycosylated gag protein of MuLV is a determinant of neuroinvasiveness: analysis of second site revertants of a mutant MuLV lacking expression of this protein.

Neuroinvasiveness is a property of all neurovirulent murine retroviruses, although the factors which facilitate infection of the CNS are not understood. We previously showed that mutant MuLV which lack expression of an accessory protein, glycosylated gag, had lost neurovirulence, indicating that this protein may be involved in promoting CNS infection. The mutations were located in the "Kozak" consensus sequence of the initiation codon for this protein. Here it is shown that shortly after inoculation of mice with one of these mutant viruses, revertants emerged which had regained expression of glycosylated gag and had also regained the neuroinvasiveness and neurovirulence exhibited by the wild-type virus. The phenotypic revertants retained the mutations in the "Kozak" consensus sequence but exhibited a G-->A mutation 12 codons downstream from the mutated start site, creating a new initiation codon and a glycosylated gag protein, which was truncated at its N-terminus. Using antibodies specific for glycosylated gag it is shown that the frequency of splenic infectious centers expressing revertant virus increased progressively during the 2 months following inoculation of mutant virus until > or = 50% of the virus-producing cells in the spleen expressed revertant virus. In contrast, although phenotypic revertants were detectable at low frequency after cell-free passage in vitro in M. dunni fibroblasts, there was no evidence for selection. These results indicate that glycosylated gag facilitates virus spread within the spleen and to extra-splenic sites, such as the CNS, and suggest that the protein may function through its interaction with the host.

Amino Acid Sequence↗

PubMed2002

HIV-1 gag polyprotein rescues HLA-DR intracellular transport in a human CD4+ cell line.

Major histocompatibility complex class II HLA-DR molecules are plasma-membrane integral heterodimers, constitutively expressed in antigen-presenting cells. Their expression is known to be upregulated in peripheral T lymphocytes upon cell activation and to be constitutive in T cell lines. In H78-C10.0, a subclone of the human CD4+ T cell line HUT-78, the transport of MHC class II HLA-DR molecules is blocked, resulting in their localization within internal vesicular compartments rather than at the cell surface. In this article, we show that HIV-1(HX10) infection of H78-C10.0 cells induces HLA-DR surface expression. Moreover, the produced infectious viruses harbor the heterodimer molecules in their envelopes. To define which of the viral proteins was involved in this phenomenon, we infected H78-C10.0 cells with recombinant vaccinia vectors containing either the gag-pro coding sequence or the entire env gene. Only gag expression was able to induce HLA-DR cell-surface localization in H78-C10.0 cells. RT-PCR analysis of the infected cells revealed no significant alteration in the amount of HLA-DRalpha-specific RNA compared to untreated cells. This implies that Gag acts on downstream events. When the env viral gene, coding for the precursor glycoprotein gp160, was expressed in H78-C10.0, the Env protein targeted to the cell surface was poorly processed to its final mature forms gp120 and gp41. However, coexpression of the env and gag genes led to restoration of this phenotype. Although the mechanism is unknown, the data compiled in this study strongly suggest that the viral Gag protein can interact with the cellular trafficking apparatus. Moreover, in a specific cell type as H78-C10.0 this interaction can even reverse intracellular transport defects.

Base Sequence↗

PubMed1989

Scrambled duplications in the feline leukemia virus gag gene: a putative pattern for molecular evolution.

The present study is a detailed computer-assisted analysis of the feline leukemia virus gag gene nucleotide sequence together with its flanking sequences (ST-FeLV GAG) that is compared with the aligned sectors of the Moloney strain of murine leukemia virus (Mo-MuLV GAG) and of three strains of feline sarcoma virus. It shows that perfectly matched repeated oligomers up to 13 nucleotides long are overrepresented and scattered throughout both ST-FeLV GAG and Mo-MuLV GAG, in noncoding and coding sectors, with no stringent correlation to codon usage in ST-FeLV gPr80gag. Many repeated oligomers share a core consensus that is intriguingly part of the inverted repeat at the termini of the long terminal repeat. Local scrambled repetitions of nucleotide subsequences have been found; they suggest a model of molecular evolution by slippage-like mechanisms. Thus, viral genomes could be subject to the same evolutionary mechanisms that are now known to be operating extensively in eukaryotic genomes. The data are discussed in light of putative patterns of molecular evolution.

Antigens, Viral↗

PubMed1998

Construction of a recombinant feline herpesvirus type 1 expressing Gag precursor protein of feline immunodeficiency virus.

We constructed a deletion mutant of feline herpesvirus type 1 (FHV-1) and a recombinant FHV-1. The deletion mutant is the virus with a region (367 bp) deleted from the start codon of thymidine kinase (TK) gene to the SmaI site within the TK gene, and the other is a recombinant FHV-1 expressing Gag protein of feline immunodeficiency virus (FIV), in which a cDNA encoding the Gag protein of FIV was inserted at the TK deletion site of the former deletion mutant. These viruses were designated as C7301ddlTK and C7301ddlTK-gag, respectively. Growth kinetics of these viruses in Crandell feline kidney cells was similar to that of the parent C7301 strain. By immunoblot analysis, C7301 ddlTK-gag was confirmed to express the FIV Gag precursor protein in the cells.

Alphaherpesvirinae↗