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Query: UNIPROT:Q9NRP7 (fused)
58,367 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The host immune response of cell-mediated immunity, particularly that of cytotoxic T lymphocytes (CTLs), is a major immune defence mechanism which may provide resistance to a human immunodeficiency virus type 1 (HIV-1) spread leading to acquired immune deficiency syndrome (AIDS). To prevent the accompanying activity of HIV-1 proteins responsible for the loss of helper T-lymphocyte function, it is crucial to develop a live attenuated recombinant vaccine expressing only T- or both T- and B-cell epitopes. Here, we examined the expression of the HIV-1 Env protein V3 region (15 amino acids from Arg315 to Lys329) in Mycobacterium bovis BCG as a fused form with an extracellular alpha antigen of Mycobacterium kansasii. Balb/c mice inoculated with this recombinant BCG (rBCG), rapidly induced V3 peptide-specific CTLs. Target cell lysis was restricted to the murine class I major histocompatibility complex, H-2d. A similar CTL response was also elicited after Balb/c mice were immunized with the same rBCG even when pre-inoculated with non-recombinant BCG. Thus, the rapid induction of HIV-1-specific CTLs indicates that this vaccine may be a therapeutic approach to preventing progression to AIDS.
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PMID:Cytotoxic T lymphocyte response in mice induced by a recombinant BCG vaccination which produces an extracellular alpha antigen that fused with the human immunodeficiency virus type 1 envelope immunodominant domain in the V3 loop. 814 98

Replication of human immunodeficiency virus type 1 requires expression of the viral trans activator Rev. Rev binds to a highly structured RNA, the Rev response element, which is present in singly spliced and unspliced genomic viral RNAs. Although Rev helps to transport these transcripts from the nucleus to the cytoplasm, the mechanism(s) involved is not fully understood. Using the yeast two-hybrid system, we isolated a murine protein (YL2) that interacts with the basic domain of Rev, which is essential for the function of Rev in vivo and for the inhibitory splicing activity of Rev in vitro. YL2 has 92% identity to a human 32-kDa protein (p32), which copurifies with alternative splicing factor SF2/ASF. Furthermore, we found that whereas expression of YL2 greatly potentiated the activity of Rev, antisense YL2 transcripts blocked the effects of Rev in mammalian cells. YL2 also increased the activities of Rex on the Rex response element and of hybrid Rev proteins fused to Tat and the coat protein of bacteriophage MS2 on their respective RNAs. Thus, YL2 or p32 is a cellular protein that modulates the function of human immunodeficiency virus type 1 Rev.
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PMID:Cellular protein modulates effects of human immunodeficiency virus type 1 Rev. 818 22

There has been significant progress on several candidate vaccines against HIV infection over the past few years, i.e., live recombinant virus vaccines, inactivated virus vaccines, and purified antigens (subunit vaccines). There have been many phase I studies in healthy volunteers uninfected with HIV, as well as phase II trials with high-risk uninfected volunteers. Most of them focused on productions of neutralizing antibodies and cytotoxic T lymphocytes (CTLs) against HIV Env proteins. In addition, host immune responses, particularly cell-mediated immunity, were shown to be a major immune defense mechanism which may provide resistance to HIV spread leading to AIDS, indicating the possibility to develop prophylactic vaccine which may prevent the onset of AIDS in HIV carriers. However, various studies have implicated the concurrent activities of several HIV proteins, such as Env gp120, Env gp41, Tat, and Nef, in the induction of immunodeficiency. Therefore, the role of HIV-1 proteins in inducing immunodeficiency after HIV vaccination should be considered. Here, I introduce a recombinant BCG (rBCG) vaccine which expresses the immunodominant gp120 epitope. The tuberculosis vaccine strain Mycobacterium bovis BCG is a widely used vaccines with a low rate of serious complications. To develop a vaccine expressing an immunodominant epitope in the gp120 V3-loop, we used our system to express and secrete an epitope which was fused with an extracellular alpha antigen of Mycobacterium kansasii. Balb/c mice inoculated with this rBCG, rapidly induced HIV-specific CTLs. Target cell lysis was restricted to the murine class I major histocompatibility complex, H-2d.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[HIV vaccine trials]. 822 64

The product of the vpr open reading frame of human immunodeficiency virus type 1 (HIV-1) is a 15-kDa, arginine-rich protein that is present in virions in molar quantities equivalent to that of Gag. We report here the results of our investigations into the mechanism by which Vpr is incorporated into virions during assembly in infected cells. For these studies we used an expression vector encoding a Vpr molecule fused at its amino terminus to a nine-amino-acid peptide from influenza virus hemagglutinin. The tagged Vpr expression vector and a vpr mutant HIV-1 provirus were used to cotransfect COS cells, and the resulting virions were tested for the presence of the tagged protein on immunoblots probed with monoclonal antibody against the hemagglutinin peptide. The COS-produced virions were found to contain readily detectable amounts of tagged Vpr and smaller amounts of a putative tagged Vpr dimer. Infectivity of the particles was not altered by incorporation of tagged Vpr. Our results using this system in combination with mutant HIV-1 proviruses suggested that incorporation of Vpr into virions requires the carboxy-terminal Gag protein of HIV-1 (p6) but not gp160, Pol, or genomic viral RNA. In addition, analysis of mutated, tagged Vpr molecules suggested that amino acids near the carboxy terminus (amino acids 84 to 94) are required for incorporation of Vpr into HIV-1 virions. The single cysteine residue near the carboxy terminus was required for production of a stable protein. Arginine residues tested were not important for incorporation or stability of tagged Vpr. These results suggested a novel strategy for blocking HIV-1 replication.
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PMID:Incorporation of Vpr into human immunodeficiency virus type 1 virions: requirement for the p6 region of gag and mutational analysis. 823 Apr 45

Recombinant DNA technology has been widely used for the production of proteins in recent years. In this paper, we describe the expression and the purification of two specific peptides corresponding to parts of the human immunodeficiency virus Rev protein. The strategy of this method relies on the chemical synthesis of a pair of two complementary oligodeoxynucleotides corresponding to the coding region of the peptide of interest and the subsequent cloning into a prokaryotic expression vector. Transformation of Escherichia coli with these synthetic gene constructs yielded high production levels of recombinant protein in the bacteria. The recombinant protein was composed of two moieties, one corresponding to an "affinity handle" and the second corresponding to the peptide. Chemical cleavage of the fused protein followed by a combination of affinity chromatography and rp-HPLC led to rapid and convenient peptide purification. Peptide fused to the affinity handle as well as cleaved peptide were fully characterized by N-terminal microsequencing and mass spectrometry. The data presented demonstrate that although the major recombinant products had the expected amino acid composition, we detected unexpected processing such as alternative cleavage within the signal peptide, modified cysteines, and deamidations. These results emphasize the importance of the complete characterization of recombinant products by efficient analytical tools such as N-terminal microsequencing and mass spectrometry.
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PMID:Recombinant technology as an alternative to chemical peptide synthesis: expression and characterization of HIV-1 Rev recombinant peptides. 823 80

The human immunodeficiency virus type 1 (HIV-1) proteinase (PR) and its flanking sequences have been fused in frame between the DNA-binding domain and the transcription-activation domain of the yeast protein, GAL4. As has been shown before with the 3C proteinase of Coxsackie virus B3 (CVB3) [Das Mahapatra et al., Proc. Natl. Acad. Sci. USA 89 (1992) 4159-4162], the GAL4::PR fusion protein retains its GAL4 function, providing the PR is inactive. When PR is active, its autocatalytic activity in the hybrid protein is shown to inactivate the transactivation function of GAL4. This provides a simple assay to monitor PR activity. A dose-dependent effect of a potent PR-specific inhibitor is demonstrated in this system and illustrates the sensitivity of the assay. The assay is used for high throughput screening to identify novel inhibitors of the viral PR, and provides a method to generate and analyze mutants and revertants of the PR.
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PMID:Inactivation of a yeast transactivator by the fused HIV-1 proteinase: a simple assay for inhibitors of the viral enzyme activity. 824 23

We report here that the transcriptional activity of early mouse embryos is affected by their manipulation and culture in vitro, using transgenic embryos that express the reporter gene lacZ. We examined the pattern of expression of the lacZ gene fused to the human immunodeficiency virus type 1 long terminal repeat during the preimplantation stages. Transgene expression is induced as early as the two-cell stage in embryos developed in vitro, while there is no constitutive expression at the same stage in embryos developed in vivo. We have established a relation between this inducible expression occurring in vitro and an oxidative stress phenomenon. Indeed, when the culture medium is supplemented with antioxidants such N-acetyl-cysteine or CuZn-superoxide dismutase the transgene expression is markedly reduced. We also present evidence that the transgene expression in vitro coincides with the onset of the embryonic genome activation as attested by the synthesis of the 70 x 10(3) M(r) protein complex. Therefore, this transgene expression could prove to be a useful tool in our understanding of the molecular mechanisms involved in this crucial developmental event.
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PMID:In vitro manipulation of early mouse embryos induces HIV1-LTRlacZ transgene expression. 830 88

The bel1 gene of human spumaretrovirus (HSRV) encodes a 300-amino-acid nuclear protein termed Bel1 that is a potent activator of transcription from the cognate long terminal repeat (LTR). Bel1 can also efficiently activate the human immunodeficiency virus type 1 (HIV-1) LTR. We have previously shown that the amino-terminal 227-residue region (minimal activator region) of Bel1 can activate the HSRV LTR at low levels and that two distinct domains within the carboxy-terminal 73 residues, from residues 255 to 266 and 272 to 300, that bear little sequence homology can independently enhance the activity of the minimal activator domain (L. K. Venkatesh, C. Yang, P. A. Theodorakis, and G. Chinnadurai, J. Virol. 67:161-169, 1993). We now report on the further characterization of these two transcriptional enhancement regions. Mutational analysis of the region comprising residues 255 to 266 indicates that a cluster of leucine residues is critical to the function of this region. Also, residues 273 to 287, which are identical in sequence to a 15-amino-acid segment near the carboxy terminus of the simian foamy virus transcriptional activator Taf, can independently enhance the activity of the minimal activator region. To delineate the region(s) of Bel1 that could function autonomously as an activator domain, we tested the activity of chimeric proteins comprising either wild-type or functionally defective forms of Bel1 fused to the DNA binding domain, Gal4(1-147), of the yeast transcriptional activator Gal4 on a synthetic promoter comprising Gal4 DNA binding sites linked to the adenovirus E1B TATA box (minimal promoter). Gal4-Bel1 was found to activate basal transcription from the E1B TATA box at least 35-fold, and the region responsible for this activation function was localized to the carboxy-terminal 73 amino acids. When the transcriptional enhancement regions were tested for autonomous activator function as Gal4(1-147) chimeras, residues 272 to 300, but not 255 to 266, were found to activate transcription efficiently when targeted to the E1B TATA motif and also to HSRV and HIV-1 LTRs. The highly conserved region between amino acids 273 and 287 alone was found to activate transcription efficiently when targeted to the HSRV LTR but not to the E1B TATA box or the HIV-1 LTR. Thus, our results demonstrate that the carboxy-terminal 29-amino-acid region (residues 272 to 300) contributes to Bel1 transactivation by functioning as an autonomous activator of TATA motif-directed transcription in a manner similar to that of other modular transcriptional activators.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The carboxy-terminal transcription enhancement region of the human spumaretrovirus transactivator contains discrete determinants of the activator function. 838 9

Varicella-zoster virus (VZV) open reading frame 4 (ORF4) encodes a protein with a predicted molecular weight of 51,540 presenting amino acid sequence homology with the immediate-early regulatory protein ICP27 of herpes simplex virus type 1. To investigate the regulatory properties of the ORF4 gene product, we performed a series of transient expression assays in Vero cells, using a plasmid expressing ORF4 as effector and several VZV genes and heterologous genes as targets. The VZV target plasmids contained promoter/regulatory regions from genes belonging to the three putative VZV kinetic classes fused to the chloramphenicol acetyltransferase (CAT) gene. The heterologous target plasmids consisted of promoter/regulatory regions of human cytomegalovirus, Rous sarcoma virus, and human immunodeficiency virus type 1 fused to the reporter gene. These experiments demonstrated that the ORF4 gene product activated expression of ORF62 in a dose-dependent fashion but had no effect on the expression of the three other putative immediate-early genes (ORF4, ORF61, and ORF63). When various amounts of ORF4 were transfected in the presence of early gene promoters, dose-dependent transactivation was evidenced with the thymidine kinase gene (ORF36) and the major DNA-binding protein gene (ORF29) promoters; interestingly, little activity was detected with the promoter of the DNA polymerase gene (ORF28). No activation of late gene expression, represented by the glycoprotein I and glycoprotein II genes, was seen even over a wide range of concentrations of input ORF4 plasmid. Expression of pCMVCAT, pRSVCAT, and pHIVCAT was also stimulated by the ORF4 gene product. CAT mRNA analysis showed that activation of VZV target promoters occurs at the transcriptional and/or posttranscriptional level.
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PMID:Characterization of the regulatory functions of varicella-zoster virus open reading frame 4 gene product. 838 35

The Gag protein encoded by Rous sarcoma virus (RSV) is the only viral product required for the process of budding whereby virus particles are formed at the plasma membrane. Deletion analysis of this Gag molecule has revealed several regions (assembly domains) that are important for budding. One of these domains is located at the amino terminus and is needed for membrane binding. Another is located within the carboxy-terminal third of the protein. Though there is little sequence homology among the Gag proteins of unrelated retroviruses, it seemed possible that their assembly domains might be functionally conserved, and to explore this idea, numerous Gag chimeras were made. The results indicate that the first 10 amino acids of the human immunodeficiency virus (HIV) Gag protein can suppress the block to budding caused by deletions in the RSV MA sequence, much as described previously for the first 10 residues from the Src oncoprotein (J.W. Wills, R.C. Craven, R. A. Weldon, Jr., T. D. Nelle, and C.R. Erdie, J. Virol. 65:3804-3812, 1991). In addition, the carboxy-terminal half of the HIV Gag protein was fused to a truncated RSV Gag molecule, mutant Bg-Bs, which is unable to direct core assembly. This chimera was able to produce particles at a rate identical to that of RSV and of a density similar to that of authentic virions. Deletion analysis of the carboxy-terminal chimera revealed two small regions within the HIV NC protein that were sufficient for endowing mutant Bg-Bs with these properties. Chimeras lacking both regions produced particles of a low density, suggesting that these sequences may be involved in the tight packing of Gag molecules during assembly. In a related set of experiments, replacement of the RSV protease with that of HIV resulted in premature processing within the RSV sequence and a block to budding. Particle assembly was restored when the HIV PR activity was inactivated by mutagenesis. Collectively, the data presented here illustrate the functional similarities of Gag proteins from unrelated retroviruses.
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PMID:Functional chimeras of the Rous sarcoma virus and human immunodeficiency virus gag proteins. 841 52

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