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PubMed2001

Analysis of the proliferative responses to peptides in individuals with vigorous Gag protein-specific proliferation.

Proliferative responses to recombinant HIV proteins in infected individuals may represent a correlate of protection from disease progression. In this study, the proliferative responses to HIV p24, p55 and gp120 were evaluated in infected subjects. Whereas, vigorous proliferative responses directed at the Gag proteins were detected in several individuals, Env-specific proliferation was observed in only one subject. Epitope mapping using overlapping peptides demonstrated proliferative responses of PBMC to Gag peptides. Responses were broadly directed at multiple peptides in some subjects. Although several of the peptides that induced proliferative responses also contain CTL epitopes potentially relevant in the particular individuals, many additional Gag T cell epitopes were present in each subject. This finding may be relevant for the design and testing of HIV candidate vaccines.

AIDS Vaccines↗
PubMed2002

Inclusion of full length human immunodeficiency virus type 1 (HIV-1) gag sequences in viral recombinants applied to drug susceptibility phenotyping.

Drug susceptibility phenotyping of recombinant clinical human immunodeficiency virus type 1 (HIV-1) isolates has been used widely to quantitatively assess viral resistance to antiretroviral agents. A novel method is described for HIV-1 drug susceptibility phenotyping. Recombinant virus that contains the entire HIV-1 Gag, protease (PR) and reverse transcriptase (RT) coding regions is generated from plasma of HIV-1 infected subjects, thus allowing the in vitro investigation of effects caused by all protein-coding sequence elements upstream from the drug targets on: (i) drug susceptibility; and (ii) viral replicative capacity. Mutations known to cause retarded viral growth kinetics (RT M184V and PR I50V) were introduced and analyzed in parallel using both the new Five Prime HIV assay (FPH) and a standard recombinant virus assay (RVA). The M184V and I50V mutants produced up to 4.8- and 5.9-fold higher p24 antigen levels, respectively, with the FPH when compared to the cultures containing RVA-derived viruses. The reduced number of homologous recombination events necessary to generate replication-competent provirus with the FPH is the most likely explanation for these findings. Long range RT-PCR products were generated from plasma of HIV-1 infected subjects and HIV-1 LTR sequences were added using one-step PCR-mediated recombination. FPH-recombinants generated from two patients with previous HIV PR and RT inhibitor therapy showed lower drug susceptibilities than mutants established in parallel by RVA, and relative in vitro replication of the FPH recombinant derived from one of these subjects was enhanced compared to the corresponding RVA mutant. Although there were changes from the HIV-1 subtype B consensus sequence in amino acids flanking the Gag p17/p24, p24/p2 or p2/p7 PR cleavage sites, none were within the 10 amino acids immediately flanking the sites. These data suggest that determinants of drug susceptibility may be encoded in Gag upstream of the p7/p1 and p1/p6 regions, and that some phenotyping assays may therefore be underdetermining the reduction of drug susceptibility in some viral isolates.

Acquired Immunodeficiency Syndrome↗
PubMed1998

Expression of the human immunodeficiency virus gag gene products by a replication-incompetent herpes simplex virus vector.

A recombinant replication-incompetent herpes simplex virus vector (vd120/Gag) that expressed the human immunodeficiency virus 1 gag gene and part of the pol gene that encodes the HIV-1 protease was constructed. Examination of cells infected with vd120/Gag revealed the presence of the Gag polyprotein Pr55gag by 12 h post-infection, as well as abundant levels of the proteolytically processed 24-kDa capsid protein. Analysis of vector-infected cells and culture supernatant indicated that the majority of the 24-kDa protein remained cell-associated. Although the HIV-1 Gag polyprotein was produced in vd120/Gag-infected cells, there was no evidence of HIV virus-like particle production upon examination of vector-infected cells by transmission electron microscopy.

Animals↗
PubMed2001

Enhancement of mucosal immune response against HIV-1 Gag by DNA immunization.

In order to examine the feasibility of Gag-expression DNA as a potential candidate for HIV vaccine using a mouse model, we injected DNA into mice either intramuscularly or by using a gene gun. Both methods induced a low level of antibody production. However, after booster immunization with p24 protein emulsified with complete Freund's adjuvant via a footpad, we found that only the preceding intramuscular DNA immunization induced an anti-Gag Th1-type (IgG(2a)) antibody response, in addition to the enhancement of a Th2-type (IgG(1)) antibody response. Importantly, when mice were boosted intranasally with p24 and cholera toxin, intramuscular DNA injection was found to enhance both systemic and mucosal Gag-specific immune responses. These results indicate that intramuscular DNA immunization confers the inducibility of memory cells, which circulate around various mucosal tissues. Therefore, intramuscular DNA priming, followed by a mucosal booster immunization, could be considered as a regimen applicable to HIV vaccine.

3T3 Cells↗
PubMedDate not supplied

Induction of feline immunodeficiency virus specific antibodies in cats with an attenuated Salmonella strain expressing the Gag protein.

Salmonella typhimurium aroA strains (SL3261), expressing high levels of the Gag protein of feline immunodeficiency virus (FIV) fused with maltose binding protein (SL3261-MFG), were constructed using an invertible promoter system that allows the stable expression of heterologous antigens at levels toxic for bacteria. A SL3261 strain expressing the B subunit of cholera toxin by a similar system (SL3261-CtxB) served as a control in FIV-immunization experiments. Cats immunized once orally or intraperitoneally with SL3261-MFG or SL3261-CtxB all developed serum antibodies to SL3261 lipopolysaccharide and against maltose binding protein or the B subunit of cholera toxin, respectively. Two intraperitoneal immunizations with SL3261-MFG also resulted in the development of Gag specific serum antibodies. Two oral immunizations with SL3261-MFG primed for a Gag specific response, which was demonstrated upon FIV challenge. All challenged cats became infected and no significant differences in viral loads were found between SL3261-MFG and SL3261-CtxB immunized cats.

Animals↗
PubMed1997

Developmental expression analysis of the 1731 retrotransposon reveals an enhancement of Gag-Pol frameshifting in males of Drosophila melanogaster.

Extensive analyses of Drosophila melanogaster retrotransposon transcriptions in cultured cells or during development have been reported, but little is known about their translation during the development of the fly. Analysis of the translational products of the 1731 Drosophila melanogaster retrotransposon in Kc Drosophila cultured cells has been reported, showing the existence of primary products (Gag and Pol) and of processed polypeptides of various sizes. Study of 1731 retrotransposon expression at both levels of transcription and translation during the development of Drosophila melanogaster, is presented. 1731 transcripts were detected by in situ hybridization and 1731 proteins were detected by immunostaining and immunoblotting in embryos and in adult gonads. 1731 transcripts and proteins were detected in the mesoderm and central nervous system during embryonic development, in nurse cells and follicle cells in adult ovaries and in primary spermatocytes in adult testes. Moreover, Western blot analysis of the 1731 proteins with anti-Gag or anti-Pol antibodies in gonads revealed that the 1731 mRNA could be translated differentially according to the expressing tissue: essentially, ovarian translation and/or processing of 1731 products is different from that operating in testes, where the Gag-Pol fusion polyprotein is the most prominent product. Our results indicate that expression of the 1731 mobile element is regulated not only at the transcriptional level but also at the translational level, and that this regulation is different in the two sexes.

Animals↗
PubMed1998

Comparison of four tests to evaluate the reactivity of rabbit sera against envelope or Gag-related proteins of bovine leukemia virus (BLV).

Bovine leukemia virus (BLV) has a long latency period during which animals are inapparently infected, may spread the disease, and are only detected by serological techniques or by the most cumbersome molecular biology techniques. We have compared techniques for detecting either total antibodies (ELISA), anti-p24 and Gag-related proteins (Western blot), or anti-gp51 (agar gel immunodiffusion, AGID, and syncytia inhibition, SI) in rabbits inoculated experimentally with inocula of variable immunogenicity. The two tests to detect antibodies to gp51 correlated well in sera clearly positive or clearly negative by either one, but correlation was poor in the intermediate groups. All sera positive by AGID were also positive by ELISA, but results did not agree in sera negative by AGID, ELISA proving to be more sensitive. Western blot was a good technique for detecting antibodies against Gag-related proteins. However, no band was identified to clearly correspond to anti-Env-related proteins. As for other retroviruses, testing of animals for infection with BLV should include the detection of antibodies anti-Gag and anti-Env proteins.

Animals↗
PubMed2003

Determinants of HIV-specific CD8 T-cell responses in HIV-infected pediatric patients and enhancement of HIV-gag-specific responses with exogenous IL-15.

Cellular immune responses play a central role in controlling HIV-1 infection. HIV-specific IFN-gamma production by CD8 T cells was evaluated in 17 HLA-A2+ HIV-infected pediatric patients (age range 1 month to 16 years) in an ELISPOT assay. Most patients (15/17) exhibited responses to HIV-gag, followed by responses to envelope gp120, gp41, and V3 loop. Only 7 patients responded to all four antigenic peptides. Treatment-related immune reconstitution of CD4 T cells was associated with increase in gag-specific responses, but these declined with prolonged viral suppression. Exogenous IL-15 resulted in augmentation of HIV-gag-specific response in 71% of patients, while IL-2 and IL-7 had variable effects, augmenting responses in 25% patients. Thus, HIV-specific CD8 T-cell responses are dependent on both CD4 T-cell help and antigenic stimulation. The cytokine IL-15 may be a useful modality as adjunctive therapy to augment HIV-specific memory CD8 T cells.

Adolescent↗
PubMed1998

A single amino-acid substitution in gag p19 protein (MA) of Rous sarcoma virus suppresses virus production from infected cells.

Gag p19 protein (MA) of the transformation-defective Rous sarcoma virus mutant, tdPH2010, has a point mutation at nucleotide 376 (G to A) that results in an amino-acid change at residue 126 of p19 (Glu to Lys). This single amino-acid change is the cause of the aberrantly fast migration of this protein on SDS-polyacrylamide gels. To study the biological significance of the mutation, we introduced this mutation into a transformation-defective derivative of the molecularly cloned Rous sarcoma virus, SRA2, and examined its effect on virus replication. The virus possessing the mutation in its gag p19 gene had 50% slower replication as measured by the amount of reverse transcriptase as well as gag p27 protein (CA) in the culture media. Glu at position 126 appears to be important for efficient production of Rous sarcoma virus in vitro.

Amino Acid Substitution↗
PubMed1988

HIV-1 gag-specific cytotoxic T lymphocytes defined with recombinant vaccinia virus and synthetic peptides.

Current candidate vaccines fail to protect primates against challenge with human immunodeficiency virus (HIV) in the presence of antibody responses; this underlines the importance of studying cell-mediated immunity to HIV and identifying specific epitopes that stimulate cytotoxic T lymphocytes (CTL). Using a recombinant vaccinia virus to express the gag protein of HIV-1 we found HLA class-I-restricted gag-specific CTL in thirteen out of fifteen healthy HIV seropositive patients. We then used short synthetic peptides in the lysis assay to screen for gag CTL epitopes. In one patient we have identified a peptide in p24 that is recognized by CTL in association with HLA-B27. This peptide, and further peptide sequences defined by these methods, could be incorporated in vaccines designed to induce cell-mediated immunity against HIV.

Acquired Immunodeficiency Syndrome↗
PubMed1985

Bacterial expression of the acquired immunodeficiency syndrome retrovirus p24 gag protein and its use as a diagnostic reagent.

A retrovirus [lymphoadenopathy-associated virus, human T-cell leukemia virus type III, acquired immunodeficiency syndrome (AIDS)-related virus] suspected of causing AIDS has been isolated recently. The detection of exposure to this retrovirus in donors of various blood products is important to prevent transmission of the disease from these donors to recipients. In the majority of cases, the detection of antibodies directed against either the viral core protein, a Mr approximately equal to 24,000 protein termed p24 gag, or the viral envelope antigen is proof of previous viral infection. Thus, we have expressed the p24 gag antigen in Escherichia coli in order to produce a diagnostic reagent for the detection of virus exposure. The bacterially synthesized antigen reacts with human and rabbit antisera directed against the native p24 gag protein in both electrophoretic transfer blot assay and ELISA. In addition, the use of bacterially produced antigens for ELISAs gave results that were comparable to those obtained by using antigens isolated from the virus.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Identification of amino acids inserted during suppression of UAA and UGA termination codons at the gag-pol junction of Moloney murine leukemia virus.

Expression of the murine leukemia virus pol gene occurs by translational readthrough of an in-frame UAG codon between the gag and pol coding regions. In a previous study, we mutated the UAG codon to UAA or UGA and demonstrated that both of these termination codons could be suppressed in reticulocyte lysates and in infected cells with the same efficiency as UAG. We now report the identity of the amino acids inserted in vitro in response to UAA and UGA in fusion products containing the gag-pol junction region. The results show that UAA, like UAG, directs the incorporation of glutamine, whereas UGA directs the incorporation of three amino acids, arginine, cysteine, and tryptophan. To our knowledge, this is the first report indicating misreading of UAA as glutamine and UGA as arginine and cysteine in higher eukaryotes. Interestingly, although our protein synthesis system presumably contains other known UAG and UGA suppressors, these tRNAs did not suppress the termination codons in our experiments. Thus, it seems possible that the sequence surrounding the gag-pol junction not only promotes suppression but also helps determine which tRNAs function in suppression.

Amino Acid Sequence↗
PubMed1994

A Rev-inducible mutant gag gene stably transferred into T lymphocytes: an approach to gene therapy against human immunodeficiency virus type 1 infection.

One strategy for somatic gene therapy for human immunodeficiency virus type 1 (HIV-1) infection is based on the regulated expression of dominant negative mutants of the HIV-1 gag gene. To limit expression of the mutant Gag polypeptide to HIV-1-infected cells, we have constructed a replication-defective retroviral vector that contains a Rev-responsive element. By using this construct we have obviated problems that can be associated with constitutive expression of an exogenous gene, an important step toward developing a human therapy. In uncloned T lymphocytes infected (transduced) with this retroviral construct, HIV-1 replication was inhibited by 94% with a concomitant decrease in the cytopathic effects of the virus. In addition, simian immunodeficiency virus (SIV) replication was also shown to be significantly inhibited, suggesting that this mutant Gag protein may have antiviral efficacy against a broad range of primate lentiviruses and that an SIV/macaque model can be used for further in vivo studies. These results have important implications in assessing the potential of somatic gene therapy in the treatment of HIV-1 infection.

Amino Acid Sequence↗
PubMed1998

Recombinant modified vaccinia virus Ankara-simian immunodeficiency virus gag pol elicits cytotoxic T lymphocytes in rhesus monkeys detected by a major histocompatibility complex class I/peptide tetramer.

The utility of modified vaccinia virus Ankara (MVA) as a vector for eliciting AIDS virus-specific cytotoxic T lymphocytes (CTL) was explored in the simian immunodeficiency virus (SIV)/rhesus monkey model. After two intramuscular immunizations with recombinant MVA-SIVSM gag pol, the monkeys developed a Gag epitope-specific CTL response readily detected in peripheral blood lymphocytes by using a functional killing assay. Moreover, those immunizations also elicited a population of CD8+ T lymphocytes in the peripheral blood that bound a specific major histocompatibility complex class I/peptide tetramer. These Gag epitope-specific CD8+ T lymphocytes also were demonstrated by using both functional and tetramer-binding assays in lymph nodes of the immunized monkeys. These observations suggest that MVA may prove a useful vector for an HIV-1 vaccine. They also suggest that tetramer staining may be a useful technology for monitoring CTL generation in vaccine trials in nonhuman primates and in humans.

AIDS Vaccines↗
PubMed2002

A novel long distance base-pairing interaction in human immunodeficiency virus type 1 RNA occludes the Gag start codon.

The 5'-untranslated region (5'-UTR) is the most conserved part of the HIV-1 RNA genome, and it contains regulatory motifs that mediate various steps in the viral life cycle. Previous work showed that the 5'-terminal 290 nucleotides of HIV-1 RNA adopt two mutually exclusive secondary structures, long distance interaction (LDI) and branched multiple hairpin (BMH). BMH has multiple hairpins, including the dimer initiation signal (DIS) hairpin that mediates RNA dimerization. LDI contains a long distance base-pairing interaction that occludes the DIS region. Consequently, the two conformations differ in their ability to form RNA dimers. In this study, we have presented evidence that the full-length 5'-UTR also adopts the LDI and BMH conformations. The downstream 290-352 region, including the Gag start codon, folds differently in the context of the LDI and BMH structures. These nucleotides form an extended hairpin structure in the LDI conformation, but the same sequences create a novel long distance interaction with upstream U5 sequences in the BMH conformation. The presence of this U5-AUG duplex was confirmed by computer-assisted RNA structure prediction, biochemical analyses, and a phylogenetic survey of different virus isolates. The U5-AUG duplex may influence translation of the Gag protein because it occludes the start codon of the Gag open reading frame.

5' Untranslated Regions↗
PubMed2005

"Hypnopuncture"--a dental-emergency treatment concept for patients with a distinctive gag reflex.

The present case report describes a newly developed dental treatment concept for patients with a distinctive gag reflex. "Hypnopuncture" is a combination therapy of hypnosis and acupuncture. Its simple, fast, and effective application autonomous of the cause makes it a valuable tool for dental-emergency treatment procedures. Physiologic and psychological aspects of gagging are influenced at the same time. The protocol is illustrated in the case of a 76-year-old patient with a severe gag reflex who was successfully treated by this combination approach. Necessary and effective therapeutic measures from both acupuncture and hypnosis are portrayed.

Acupuncture Therapy↗
PubMed2005

A long-term therapeutic treatment for patients with a severe gag reflex.

"Hypnopuncture," a combination treatment of hypnosis and acupuncture, provides a therapeutic treatment plan for long-term therapy for patients with a distinctive gag reflex. The treatment is applied independently of the cause. In cases of emergency treatment in dentistry, the immediate compliance of a patient is of utmost importance. The long-term goal of any therapeutic measure is control of the gag reflex. A new treatment protocol is illustrated in the case of a 50-year-old patient with a severe gag reflex. After only 5 visits, dental treatment could be conducted without any auxiliary means. Hypnosis is applied in the form of hypnosedation (not as psychotherapy), while stereognosis occupies a central position for desensitization.

Acupuncture Therapy↗
PubMed1998

Analysis of Gag-specific cytotoxic T lymphocytes in simian immunodeficiency virus-infected rhesus monkeys by cell staining with a tetrameric major histocompatibility complex class I-peptide complex.

A tetrameric recombinant major histocompatibility complex (MHC) class I-peptide complex was used as a staining reagent in flow cytometric analyses to quantitate and define the phenotype of Gag-specific cytotoxic T lymphocytes (CTLs) in the peripheral blood of simian immunodeficiency virus macaque (SIVmac)-infected rhesus monkeys. The heavy chain of the rhesus monkey MHC class I molecule Mamu-A*01 and beta2-microglobulin were refolded in the presence of an SIVmac Gag synthetic peptide (p11C, C-M) representing the optimal nine-amino acid peptide of Mamu-A*01-restricted predominant CTL epitope to create a tetrameric Mamu-A*01/p11C, C-M complex. Tetrameric Mamu-A*01/p11C, C-M complex bound to T cells of SIVmac-infected, Mamu-A*01(+), but not uninfected, Mamu-A*01(+), or infected, Mamu-A*01(-) rhesus monkeys. Specific staining of peripheral blood mononuclear cells (PBMC) from SIVmac-infected, Mamu-A*01(+) rhesus monkeys was only found in the cluster of differentiation (CD)8alpha/beta+ T lymphocyte subset and the percentage of CD8alpha/beta+ T cells in the peripheral blood of four SIVmac-infected, Mamu-A*01+ rhesus monkeys staining with this complex ranged from 0.7 to 10.3%. Importantly, functional SIVmac Gag p11C-specific CTL activity was seen in sorted and expanded tetrameric Mamu-A*01/p11C, C-M complex-binding, but not nonbinding, CD8alpha/beta+ T cells. Furthermore, the percentage of CD8alpha/beta+ T cells binding this tetrameric Mamu-A*01/p11C, C-M complex correlated well with p11C-specific cytotoxic activity as measured in both bulk and limiting dilution effector frequency assays. Finally, phenotypic characterization of the cells binding this tetrameric complex indicated that this lymphocyte population is heterogeneous. These studies indicate the power of this approach for examining virus-specific CTLs in in vivo settings.

Animals↗