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At least 991 records · Page 55Linked to original sources
PubMed2001

Impairment of Gag-specific CD8(+) T-cell function in mucosal and systemic compartments of simian immunodeficiency virus mac251- and simian-human immunodeficiency virus KU2-infected macaques.

The identification of several simian immunodeficiency virus mac251 (SIV(mac251)) cytotoxic T-lymphocyte epitopes recognized by CD8(+) T cells of infected rhesus macaques carrying the Mamu-A*01 molecule and the use of peptide-major histocompatibility complex tetrameric complexes enable the study of the frequency, breadth, functionality, and distribution of virus-specific CD8(+) T cells in the body. To begin to address these issues, we have performed a pilot study to measure the virus-specific CD8(+) and CD4(+) T-cell response in the blood, lymph nodes, spleen, and gastrointestinal lymphoid tissues of eight Mamu-A*01-positive macaques, six of those infected with SIV(mac251) and two infected with the pathogenic simian-human immunodeficiency virus KU2. We focused on the analysis of the response to peptide p11C, C-M (Gag 181), since it was predominant in most tissues of all macaques. Five macaques restricted viral replication effectively, whereas the remaining three failed to control viremia and experienced a progressive loss of CD4(+) T cells. The frequency of the Gag 181 (p11C, C-->M) immunodominant response varied among different tissues of the same animal and in the same tissues from different animals. We found that the functionality of this virus-specific CD8(+) T-cell population could not be assumed based on the ability to specifically bind to the Gag 181 tetramer, particularly in the mucosal tissues of some of the macaques infected by SIV(mac251) that were progressing to disease. Overall, the functionality of CD8(+) tetramer-binding T cells in tissues assessed by either measurement of cytolytic activity or the ability of these cells to produce gamma interferon or tumor necrosis factor alpha was low and was even lower in the mucosal tissue than in blood or spleen of some SIV(mac251)-infected animals that failed to control viremia. The data obtained in this pilot study lead to the hypothesis that disease progression may be associated with loss of virus-specific CD8(+) T-cell function.

Animals↗
PubMed2001

Partial restoration of replication of simian immunodeficiency virus by point mutations in either the dimerization initiation site (DIS) or Gag region after deletion mutagenesis within the DIS.

We used the simian immunodeficiency virus (SIV) molecular clone SIVmac239 to generate a deletion construct, termed SD2, in which we eliminated 22 nucleotides at positions +398 to +418 within the putative dimerization initiation site (DIS) stem. This SD2 deletion severely impaired viral replication, due to adverse effects on the packaging of viral genomic RNA, the processing of Gag proteins, and viral protein patterns. However, long-term culture of SD2 in either C8166 or CEMx174 cells resulted in restoration of replication capacity, due to two different sets of three compensatory point mutations, located within both the DIS and Gag regions. In the case of C8166 cells, both a K197R and a E49K mutation were identified within the capsid (CA) protein and the p6 protein of Gag, respectively, while the other point mutation (A423G) was found within the putative DIS loop. In the case of CEMx174 cells, two compensatory mutations were present within the viral nucleocapsid (NC) protein, E18G and Q31K, in addition to the same A423G substitution as observed with C8166 cells. A set of all three mutations was required in each case for restoration of replication capacity, and either set of mutations could be substituted for the other in both the C8166 and CEMx174 cell lines.

Base Sequence↗
PubMed2002

Nelfinavir-resistant, amprenavir-hypersusceptible strains of human immunodeficiency virus type 1 carrying an N88S mutation in protease have reduced infectivity, reduced replication capacity, and reduced fitness and process the Gag polyprotein precursor aberrantly.

The evolution of human immunodeficiency virus type 1 (HIV-1) strains with reduced susceptibility to protease inhibitors (PIs) is a major cause of PI treatment failure. A subset of subjects failing a therapy regimen containing the PI nelfinavir developed mutations at position 88 in the protease region. The N88S mutation occurring in some of these subjects induces amprenavir hypersusceptibility and a reduction of fitness and replication capacity. Here we demonstrate that substitutions L63P and V77I in protease, in combination, partially compensate for the loss of fitness, loss of replication capacity, loss of specific infectivity, and aberrant Gag processing induced by the N88S mutation. In addition, these mutations partially ablate amprenavir hypersusceptibility. Addition of mutation M46L to a strain harboring mutations L63P, V77I, and N88S resulted in a reduction of fitness and infectivity without changing Gag-processing efficiency, while amprenavir hypersusceptibility was further diminished. The ratio of reverse transcriptase activity to p24 protein was reduced in this strain compared to that in the other variants, suggesting that the M46L effect on fitness occurred through a mechanism different from a Gag-processing defect. We utilized these mutant strains to undertake a systematic comparison of indirect, single, cycle-based measures of fitness with direct, replication-based fitness assays and demonstrated that both yield consistent results. However, we observed that the magnitude of the fitness loss for one of the mutants varied depending on the assay used.

Carbamates↗
PubMed2002

Sustained peptide-specific gamma interferon T-cell response in rhesus macaques immunized with human immunodeficiency virus gag DNA vaccines.

We examined the influence of dose and method of antigen delivery on the dynamics and durability of T-cell responses to candidate human immunodeficiency virus (HIV) vaccines. Codon-optimized sequences from the HIV gag gene were inserted into alternative DNA vaccine vectors to express the coding sequence with or without the tissue plasminogen activator leader sequence. We delivered the vaccines by intramuscular injection as plasmid DNA without adjuvant or as plasmid DNA formulated with a novel block copolymer adjuvant (CRL8623) and then monitored the ensuing T-cell responses by using a gamma interferon enzyme-linked immunospot assay. We demonstrated persistence of the cell-mediated immune (CMI) response in rhesus macaques for at least 18 months following a four-dose vaccination regimen. The plasmid vaccine, with or without CRL8623, was immunogenic in macaques; however, the form coadministered with adjuvant exhibited improved T-cell responses, with a bias toward more antigen-specific CD8(+) T cells. Finally, we examined the fine specificity of the T-cell response to the gag vaccines by testing the response of 23 vaccinated macaques to individual Gag 20-mer peptides. Collectively, the monkeys responded to 25 epitopes, and, on average, each monkey recognized a minimum of 2.7 epitopes. The results indicate that a broad and durable CMI response to HIV DNA vaccines can be induced in a relevant nonhuman primate model.

AIDS Vaccines↗
PubMed2003

The differential ability of HLA B*5701+ long-term nonprogressors and progressors to restrict human immunodeficiency virus replication is not caused by loss of recognition of autologous viral gag sequences.

Although the HLA B(*)5701 class I allele is highly overrepresented among human immunodeficiency virus (HIV)-infected long-term nonprogressors (LTNPs), it is also present at the expected frequency (11%) in patients with progressive HIV infection. Whether B57(+) progressors lack restriction of viral replication because of escape from recognition of highly immunodominant B57-restricted gag epitopes by CD8(+) T cells remains unknown. In this report, we investigate the association between restriction of virus replication and recognition of autologous virus sequences in 27 B(*)57(+) patients (10 LTNPs and 17 progressors). Amplification and direct sequencing of single molecules of viral cDNA or proviral DNA revealed low frequencies of genetic variations in these regions of gag. Furthermore, CD8(+) T-cell recognition of autologous viral variants was preserved in most cases. In two patients, responses to autologous viral variants were not demonstrable at one epitope. By using a novel technique to isolate primary CD4(+) T cells expressing autologous viral gene products, it was found that 1 to 13% of CD8(+) T cells were able to respond to these cells by gamma interferon production. In conclusion, escape-conferring mutations occur infrequently within immunodominant B57-restricted gag epitopes and are not the primary mechanism of virus evasion from immune control in B(*)5701(+) HIV-infected patients. Qualitative features of the virus-specific CD8(+) T-cell response not measured by current assays remain the most likely determinants of the differential abilities of HLA B(*)5701(+) LTNPs and progressors to restrict virus replication.

Amino Acid Sequence↗
PubMed2003

Involvement of the matrix and nucleocapsid domains of the bovine leukemia virus Gag polyprotein precursor in viral RNA packaging.

The RNA packaging process for retroviruses involves a recognition event of the genome-length viral RNA by the viral Gag polyprotein precursor (PrGag), an important step in particle morphogenesis. The mechanism underlying this genome recognition event for most retroviruses is thought to involve an interaction between the nucleocapsid (NC) domain of PrGag and stable RNA secondary structures that form the RNA packaging signal. Presently, there is limited information regarding PrGag-RNA interactions involved in RNA packaging for the deltaretroviruses, which include bovine leukemia virus (BLV) and human T-cell leukemia virus types 1 and 2 (HTLV-1 and -2, respectively). To address this, alanine-scanning mutagenesis of BLV PrGag was done with a virus-like particle (VLP) system. As predicted, mutagenesis of conserved basic residues as well as residues of the zinc finger domains in the BLV NC domain of PrGag revealed residues that led to a reduction in viral RNA packaging. Interestingly, when conserved basic residues in the BLV MA domain of PrGag were mutated to alanine or glycine, but not when mutated to another basic residue, reductions in viral RNA packaging were also observed. The ability of PrGag to be targeted to the cell membrane was not affected by these mutations in MA, indicating that PrGag membrane targeting was not associated with the reduction in RNA packaging. These observations indicate that these basic residues in the MA domain of PrGag influence RNA packaging, without influencing Gag membrane localization. It was further observed that (i) a MA/NC double mutant had a more severe RNA packaging defect than either mutant alone, and (ii) RNA packaging was not found to be associated with transient localization of Gag in the nucleus. In summary, this report provides the first direct evidence for the involvement of both the BLV MA and NC domains of PrGag in viral RNA packaging.

Amino Acid Sequence↗
PubMed2005

Identification of domains in gag important for prototypic foamy virus egress.

Sequence motifs (L domains) have been described in viral structural proteins. Mutations in these lead to a defect at a late stage in virus assembly and budding. For several viruses, recruitment of an endosomal sorting complexes required for transport 1 subunit (Tsg101), a component of the class E vacuolar protein sorting (EVPS) machinery, is a prerequisite for virion budding. To effect this, Tsg101 interacts with the PT/SAP L domain. We have identified candidate L-domain motifs, PSAP, PPPI, and YEIL, in the prototypic foamy virus (PFV) Gag protein, based on their homology to known viral L domains. Mutation of the PSAP and PPPI motifs individually reduced PFV egress, and their combined mutation had an additive effect. When PSAP was mutated, residual infectious PFV release was unaffected by dominant negative Vps4 (an ATPase involved in the final stages of budding), and sensitivity to dominant negative Tsg101 was dramatically reduced, suggesting that the PSAP motif functions as a conventional class E VPS-dependent L domain. Consistent with this notion, yeast two-hybrid analysis showed a PSAP motif-dependent interaction between PFV Gag and Tsg101. Surprisingly, PFV release which is dependent on the PPPI motif was Vps4-independent and was partially inhibited by dominant negative Tsg101, suggesting that PPPI functions by an unconventional mechanism to facilitate PFV egress. Mutation of the YEIL sequence completely abolished particle formation and also reduced the rate of Gag processing by the viral protease, suggesting that the integrity of YEIL is required at an assembly step prior to budding and YEIL is not acting as an L domain.

Amino Acid Sequence↗
PubMed2005

Characterization of prototype foamy virus gag late assembly domain motifs and their role in particle egress and infectivity.

Foamy viruses (FV) are unusual among retroviruses since they require both Gag and Env structural proteins for particle egress. Recently significant progress has been made towards the mechanistic understanding of the viral release process, in particular that of retroviruses, and the viral domains and cellular pathways involved. However little is currently known about domains of FV structural proteins and cellular proteins engaged in this process. By mutational analysis of sequence motifs in prototype FV (PFV) Gag, bearing homology to known late assembly (L) domains, a PSAP motif with L domain function that was functionally interchangeable by heterologous L domains was identified. In contrast the inactivation of a PPPI motif had no significant influence on PFV particle release, although mutant viral particles displayed reduced infectivity. Similarly mutation of an evolutionary conserved YXXL motif revealed no classical L-domain function but resulted in release of noninfectious viruslike particles. Biochemical and electron microscopy analysis demonstrated that these mutant particles incorporated all viral structural proteins but contained aberrantly capsid structures, suggesting a role in capsid assembly for this PFV Gag sequence motif. In line with the mutational analysis, overexpression of dominant negative (DN) mutants and wild-type TSG101 but not the DN mutant of AIP-1/ALIX reduced PFV particle release and infectivity. Furthermore, DN mutants of Vps4A, Vps4B, and CHMP3 inhibited PFV egress and infectivity. Taken together these results demonstrate that PFV, like other viruses, requires components of the vacuolar protein sorting (VPS) machinery for egress and enters the VPS pathway through interaction with TSG101.

Amino Acid Motifs↗
PubMed2006

A mutation in the human immunodeficiency virus type 1 Gag protein destabilizes the interaction of the envelope protein subunits gp120 and gp41.

The Gag protein of human immunodeficiency virus type 1 (HIV-1) associates with the envelope protein complex during virus assembly. The available evidence indicates that this interaction involves recognition of the gp41 cytoplasmic tail (CT) by the matrix protein (MA) region of Pr55(Gag). Here we show that substitution of Asp for Leu at position 49 (L49D) in MA results in a specific reduction in particle-associated gp120 without affecting the levels of gp41. Mutant virions were markedly reduced in single-cycle infectivity despite a relatively modest defect in fusion with target cells. Studies with HIV-1 particles containing decreased levels of envelope proteins suggested that the L49D mutation also inhibits a postentry step in infection. Truncation of the gp41 tail, or pseudotyping by vesicular stomatitis virus glycoprotein, restored both the fusion and infectivity of L49D mutant virions to wild-type levels. Truncation of gp41 also resulted in equivalent levels of gp120 on particles with and without the MA mutation and enhanced the replication of the L49D mutant virus in T cells. The impaired fusion and infectivity of L49D mutant particles were also complemented by a single point mutation in the gp41 CT that disrupted the tyrosine-containing endocytic motif. Our results suggest that an altered interaction between the MA domain of Gag and the gp41 cytoplasmic tail leads to dissociation of gp120 from gp41 during HIV-1 particle assembly, thus resulting in impaired fusion and infectivity.

Amino Acid Motifs↗
PubMed1992

Inhibitory effect of myristylation on transrepression by FBR (Gag-Fos) protein.

The myristylated v-fos product, FBR murine sarcoma virus (Gag-Fos) protein, exhibits a lower level of transrepression of the serum response element (SRE) than does c-fos protein (Fos). Mutation of the N-terminal myristylation site in FBR protein restored SRE transrepression. Replacement of N-terminal viral Gag sequences with the Fos N terminus also restored this activity, providing additional evidence that myristylation inhibits transrepression by FBR protein. However, the myristylated Gag domain did not inhibit SRE transrepression when fused to Fos, indicating that myristylation of a fos protein is not by itself sufficient to prevent SRE transrepression and that C-terminal mutation is necessary to inhibit transrepression by N myristylation. Comparison of transfection results with Fos C-terminal deletion mutants and the Fos/FBR chimeric mutant revealed that the FBR C terminus retained the potential for transrepression despite deletion of the normal Fos C terminus, whereas similar Fos deletion mutants did not. These results indicate that both N- and C-terminal mutations are required to inhibit transrepression by FBR protein and that multiple structural mutations accompanied by posttranslational protein modification alter gene regulation by FBR protein.

3T3 Cells↗
PubMed1994

His-154 is involved in the linkage of the Saccharomyces cerevisiae L-A double-stranded RNA virus Gag protein to the cap structure of mRNAs and is essential for M1 satellite virus expression.

The coat protein (Gag) of the double-stranded RNA virus L-A was previously shown to form a covalent bond with the cap structure of eukaryotic mRNAs. Here, we identify the linkage as a phosphoroimidazole bond between the alpha phosphate of the cap structure and a nitrogen in the Gag protein His-154 imidazole side chain. Mutations of His-154 abrogate the ability of Gag to bind to the cap structure, without affecting cap recognition, in vivo virus particle formation from an L-A cDNA clone, or in vitro specific binding and replication of plus-stranded single-stranded RNA. However, genetic analyses demonstrate that His-154 is essential for M1 satellite virus expression.

Amino Acid Sequence↗
PubMed1997

Detection of bovine immunodeficiency virus antibodies in cattle by western blot assay with recombinant gag protein.

A western blot assay using purified recombinant bovine immunodeficiency virus gag protein has been developed for detection of bovine immunodeficiency virus antibodies in bovine serum samples. The test was standardized with known bovine immunodeficiency virus positive and negative bovine serum samples and the monoclonal antibody to gag protein. Both naturally and experimentally infected cattle sera demonstrated positive test results. The result of western blot assay was compared with polymerase chain reaction test results in 134 blood samples collected from Kansas. Twenty-six samples tested positive for bovine immunodeficiency virus DNA with polymerase chain reaction (18.7%) and 25 were positive for the antibody to gag protein by western blot analysis (17.9%). Of 26 cattle testing positive using the polymerase chain reaction assay, 24 were antibody-positive by western blot assay, thus establishing a strong correlation between the two tests. The sensitivity and specificity of western blot relative to polymerase chain reaction are 0.92 and 0.99, respectively. The western blot assay proved to be a specific and sensitive test.

Animals↗
PubMed2005

Processing sites in the human immunodeficiency virus type 1 (HIV-1) Gag-Pro-Pol precursor are cleaved by the viral protease at different rates.

We have examined the kinetics of processing of the HIV-1 Gag-Pro-Pol precursor in an in vitro assay with mature protease added in trans. The processing sites were cleaved at different rates to produce distinct intermediates. The initial cleavage occurred at the p2/NC site. Intermediate cleavages occurred at similar rates at the MA/CA and RT/IN sites, and to a lesser extent at sites upstream of RT. Late cleavages occurred at the sites flanking the protease (PR) domain, suggesting sequestering of these sites. We observed paired intermediates indicative of half- cleavage of RT/RH site, suggesting that the RT domain in Gag-Pro-Pol was in a dimeric form under these assay conditions. These results clarify our understanding of the processing kinetics of the Gag-Pro-Pol precursor and suggest regulated cleavage. Our results further suggest that early dimerization of the PR and RT domains may serve as a regulatory element to influence the kinetics of processing within the Pol domain.

Dimerization↗
PubMed2003

Targeting of incoming retroviral Gag to the centrosome involves a direct interaction with the dynein light chain 8.

The role of cellular proteins in the replication of retroviruses, especially during virus assembly, has been partly unraveled by recent studies. Paradoxically, little is known about the route taken by retroviruses to reach the nucleus at the early stages of infection. To get insight into this stage of virus replication, we have studied the trafficking of foamy retroviruses and have previously shown that incoming viral proteins reach the microtubule organizing center (MTOC) prior to nuclear translocation of the viral genome. Here, we show that incoming viruses concentrate around the MTOC as free and structured capsids. Interestingly, the Gag protein, the scaffold component of viral capsids, targets the pericentrosomal region in transfected cells in the absence of any other viral components but in a microtubule- and dynein/dynactin-dependent manner. Trafficking of Gag towards the centrosome requires a minimal 30 amino acid coiled-coil motif in the N-terminus of the molecule. Finally, we describe a direct interaction between Gag and dynein light chain 8 that probably accounts for the specific routing of the incoming capsids to the centrosome prior to nuclear import of the viral genome.

Amino Acid Motifs↗
PubMed2002

The management of blood phobia and a hypersensitive gag reflex by hypnotherapy: a case report.

Coping with a hypersensitive gag reflex can be a cause for concern for both the patient and the operator. This report describes a case of blood phobia directed solely towards the oral cavity, linked with the inability to tolerate dentures due to a hypersensitive gag reflex. Management by hypnotherapy using a systematic desensitization technique allowed for extraction of teeth and permanent elimination of the gagging problem.

Adaptation, Psychological↗
PubMed2002

Detailed analysis of CD4+ Th responses to envelope and Gag proteins of simian immunodeficiency virus reveals an exclusion of broadly reactive Th epitopes from the glycosylated regions of envelope.

Ag-specific CD4(+) Th cells play a key role in the development, maturation, and maintenance of pathogen-specific humoral and cellular immune responses. To define the fine specificity of broadly reactive Th responses associated with mature immunity in a lentiviral system, we analyzed peptide-specific Th responses in eight macaques chronically infected with a reference live attenuated SIV at 12-14 mo postinoculation. All macaques had stable immunocompetent Th cells at the time of analysis, and a unique array of Th responses to 20-mer overlapping peptides from envelope (Env) and Gag was identified for each macaque, which were then used to define a set of 31 broadly reactive peptide epitopes. Only 5 of the 31 broadly reactive Th epitope peptides mapped to the surface (SU) domain of Env. Interestingly, these were all confined to two conserved nonglycosylated regions toward the carboxyl terminus of SU, suggesting a structural influence of glycosylation on development of Th responses. Gag and the Env transmembrane proteins contained the majority of broadly reactive peptide epitopes (12 and 14 peptides, respectively), which were uniformly distributed throughout their sequence. This study defines for the first time broadly reactive Th epitope peptides of SIV Env and Gag proteins that are associated with enduring broadly protective vaccine immunity to attenuated SIV, which may be used for the design and evaluation of experimental vaccines. Moreover, the data suggest that extensive glycosylation of SU may provide yet another immune escape mechanism developed by lentiviruses to restrict the breadth of Th repertoire to SU, a major immunologically exposed protein of the virus.

Aging↗
PubMed2003

A simian replication-defective adenoviral recombinant vaccine to HIV-1 gag.

In animal models, E1-deleted human adenoviral recombinants of the serotype 5 (AdHu5) have shown high efficacy as vaccine carriers for different Ags including those of HIV-1. Humans are infected by common serotypes of human adenovirus such as AdHu5 early in life and a significant percentage has high levels of neutralizing Abs to these serotypes, which will very likely impair the efficacy of recombinant vaccines based on the homologous virus. To circumvent this problem, a novel replication-defective adenoviral vaccine carrier based on an E1-deleted recombinant of the chimpanzee adenovirus 68 (AdC68) was developed. An AdC68 construct expressing a codon-optimized, truncated form of gag of HIV-1 induces CD8(+) T cells to gag in mice which at the height of the immune response encompass nearly 20% of the entire splenic CD8(+) T cell population. The vaccine-induced immune response provides protection to challenge with a vaccinia gag recombinant virus. Induction of transgene-specific CD8(+) T cells and protection against viral challenge elicited by the AdC68 vaccines is not strongly inhibited in animals preimmune to AdHu5 virus. However, the response elicited by the AdHu5 vaccine is greatly attenuated in AdHu5 preimmune animals.

Adenovirus E1 Proteins↗
PubMed2003

Prime-boost vaccination with HIV-1 Gag protein and cytosine phosphate guanosine oligodeoxynucleotide, followed by adenovirus, induces sustained and robust humoral and cellular immune responses.

A prophylactic vaccine for HIV-1 will probably require the induction and maintenance of both humoral and cellular immunity. One current strategy to achieve such long term immune responses is a prime-boost vaccination approach using a DNA priming inoculation, followed by recombinant viral boost. In this report we use a novel prime-boost approach in which the priming injections consist of recombinant HIV-1 Gag protein mixed with cytosine phosphate guanosine oligodeoxynucleotide (CpG ODN), followed by recombinant adenoviral boost expressing HIV-1 Gag. Analysis of the immune responses indicates that HIV-1 Gag protein plus CpG ODN immunization alone induces potent humoral as well as Th1 and CD8+ T cell responses. Boosting with recombinant adenovirus strikingly enhances CD8+, but not Th1, T cell responses, resulting in CD8+ T cell responses far greater in magnitude than Th1 responses. Furthermore, the Th1 and CD8+ T cell responses following prime-boost immunization were seen in both lymphoid and peripheral mucosal organs and were sustained over several months. Together, these data suggest a new immunization approach for elicitation of long term humoral and cellular immune responses.

AIDS Vaccines↗