UMLS:C0019693 - FACTA Search

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Query: UMLS:C0019693 (HIV)
170,526 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The transmembrane glycoprotein (gp41) of human immunodeficiency virus type-1 (HIV-1) has a long cytoplasmic domain of unknown functional significance. To investigate the role of the carboxy-terminal (C-terminal) portion of the HIV-1 envelope protein in viral replication, infectivity, and cytopathogenicity, we examined the properties of a panel of mutants with variable deletions in the 3'-env region. Deletion of the C-terminal 76 amino acids did not abolish production of reverse transcriptase upon transfection of COS-1 cells. Deletion of the C-terminal 6-14 amino acids appeared sufficient to alter the replication pattern, infectivity, and cytopathogenicity of some clones. The data suggest that conformational determinants or specific sequences are responsible for the observed changes, rather than simply the length of the gp41 cytoplasmic tail.
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PMID:Role of the carboxy-terminal portion of the HIV-1 transmembrane protein in viral transmission and cytopathogenicity. 278 44

We have used a panel of polyclonal and monoclonal antibodies against gp120 and gp160, the envelope glycoproteins of human immunodeficiency virus type 1, to create rapid, simple, and sensitive twin-site sandwich ELISA specific for gp120 and gp160 or for gp160 alone. These assays can detect 500 COS cells in a population transiently transfected with a construct encoding gp120 and gp160, or 50 pg of recombinant gp160. We estimate that the mean amount of gp120 + gp160 in the transfected population is equivalent to 2.5 x 10(6) molecules per cell, 40-50% of which can be recovered from the culture medium as gp120 after 24 hours. The ELISA can be adapted to assess whether gp120 is detectable in the sera of HIV-1-infected persons: we show that gp120/gp160 is completely stable in normal human serum for at least 24 hours at 37 degrees C.
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PMID:Sensitive ELISA for the gp120 and gp160 surface glycoproteins of HIV-1. 284 57

The initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription occurs by the extension of a tRNA(3Lys) primer bound near the 5' end of the genomic RNA at a position termed the primer binding site (PBS). The PBS is an 18-nucleotide sequence of the HIV-1 genome which is complementary to the 3'-terminal 18 nucleotides of the tRNA(3Lys). To investigate the sequence specificity of the interaction between tRNA(3Lys) and the PBS, we have constructed proviral genomes containing mutations in the PBS region. A mutant PBS was constructed in which the 18 nucleotides complementary to tRNA(3Lys) were substituted with 18 nucleotides predicted to be complementary to the 3'-terminal bases of a tRNA(Phe) molecule [pHXB2PBS(phe)]. A second proviral genome was constructed in which the PBS complementary to tRNA(Phe) was changed such that the first six nucleotides correspond to the wild-type PBS [pHXB2PBS(pheC)]. In all models of reverse transcription, the complementarity between the minus- and plus-strand PBS DNA facilitates the template switch and elongation of plus-strand DNA, resulting in a complete proviral genome. To test this model, we have inserted a five-nucleotide sequence 6 bp 3' of the mutant PBSs, which corresponds to the last five nucleotides of the wild-type PBSs [pHXB2PBS(phe+5) and pHXB2PBS(pheC+5)]. Transfection of plasmids containing the wild-type or mutant proviral genomes into COS-1 cells resulted in similar levels of intracellular expression of HIV-1 gag and env gene products as determined by immunoprecipitation with sera from AIDS patients and release of virus as determined by p24 assay. Transfection of pHXB2PBS(phe) or pHXB2PBS(phe+5) did not result in the production of infectious virus, while replication-competent viruses from cells transfected with pHXB2PBS(pheC) were detected very infrequently. Transfection of pHXB2PBS(pheC+5), however, consistently resulted in the production of infectious virus, although the appearance of the virus was delayed compared with those from cells transfected with pHXB2(wild type). Reinfection of SupT1 cells with equal amounts of p24 antigen resulted in similar kinetics of replication. PCR was used to amplify the PBS, and individual DNA products were subcloned into M13mp18. Sequence analysis of the PBS region of integrated proviruses derived from transfection of pHXB2PBS(pheC+5) revealed that the 18-nucleotide PBS complementary to tRNA(3Lys) was regenerated with a deletion of 6 bp 3' to the PBS region in all phage clones examined.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Minimal sequence requirements of a functional human immunodeficiency virus type 1 primer binding site. 750 99

COS-7 cells transfected with human immunodeficiency virus type 1 (HIV-1) proviral DNA produce virus in which three tRNA species are most abundant in the viral tRNA population. These tRNAs have been identified through RNA sequencing techniques as tRNA(3Lys) the primer tRNA in HIV-1, and members of the tRNA(1,2Lys) isoacceptor family. These RNAs represent 60% of the low-molecular-weight RNA isolated from virus particles, while they represent only 6% of the low-molecular-weight RNA isolated from the COS cell cytoplasm. Thus, tRNA(Lys) is selectively incorporated into HIV-1 particles. We have measured the ratio of tRNA(3Lys) molecules to copies of genomic RNA in viral RNA samples and have calculated that HIV-1 contains approximately eight molecules of tRNA(3Lys) per two copies of genomic RNA. We have also obtained evidence that the Pr160gag-pol precursor is involved in primer tRNA(3Lys) incorporation into virus. First, selective tRNA(Lys) incorporation and wild-type amounts of tRNA(3Lys) were maintained in a protease-negative virus unable to process Pr55gag and Pr160gag-pol precursors, indicating that precursor processing was not required for primer tRNA incorporation. Second, viral particles containing only unprocessed Pr55gag protein did not selectively incorporate tRNA(Lys), while virions containing both unprocessed Pr55gag and Pr160gag-pol proteins demonstrated select tRNA(3Lys) packaging. Third, studies with a proviral mutant containing a deletion of most of the reverse transcriptase sequences and approximately one-third of the integrase sequence in the Pr160gag-pol precursor resulted in the loss of selective tRNA incorporation and an eightfold decrease in the amount of tRNA(3Lys) per two copies of genomic RNA. We have also confirmed herein finding of a previous study which indicated that the primer binding site is not required for the selective incorporation of tRNA(Lys).
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PMID:Role of Pr160gag-pol in mediating the selective incorporation of tRNA(Lys) into human immunodeficiency virus type 1 particles. 751 Nov 67

Proportional expression of retroviral genes requires that splicing of the viral primary transcript be an inefficient process. Much of our current knowledge about retroviral suboptimal splicing comes from studies with Rous sarcoma virus. In this report, we describe the use of chimeric introns composed of human beta-globin and human immunodeficiency virus type 1 (HIV-1) splice sites to establish the basis for inefficient splicing of the intron which comprises most of the HIV-1 env coding sequences (referred to as the tat/rev intron). S1 RNA analysis of transfected COS-7 cells revealed that the 3' splice site (3' ss) of this region was significantly less efficient than the 3' ss of the first intron of beta-globin. Deletion of sequences flanking the tat/rev intron 3' ss demonstrated that the requirements for its inefficiency reside within the region that is expected to comprise the essential signals for splicing (i.e., the branchpoint region, the polypyrimidine tract, and the AG dinucleotide). Introduction of an exact copy of the efficient beta-globin branchpoint sequence within a highly conserved region rendered the tat/rev intron 3' ss highly efficient. Improvement of the polypyrimidine tract also increased the splicing efficiency, but to a degree slightly less than that obtained with the branchpoint mutation. Subsequent examination of the tat/rev intron 5' splice site in a heterologous context revealed that it is efficiently utilized. These results indicate that both a poor branchpoint region and a poor polypyrimidine tract are responsible for the low splicing efficiency of the HIV-1 tat/rev intron. It is of fundamental interest to establish the basis for inefficient splicing of the HIV-1 tat/rev intron since it may provide the key to understanding why nuclear export of mRNAs encoding HIV-1 structural proteins is Rev dependent.
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PMID:The tat/rev intron of human immunodeficiency virus type 1 is inefficiently spliced because of suboptimal signals in the 3' splice site. 751 59

Although the HIV gp120 binding site of CD4 is well characterized, its interaction site with HLA class II molecules is still controversial. Sixty-seven mutations within the four extracellular domains of CD4 were examined for their effects on cell surface expression and conformational changes and for adhesion of HLA class II-expressing B lymphocytes and HIV gp120 binding to CD4-transfected COS cells. Mutations within the gp120 binding site affected both assays similarly, indicating that the two sites fully overlap. A few additional substitutions of residues mapping on the same face of domains 1 and 2 induced decreased rosette formation. Molecular modeling studies indicated that this is likely to be the consequence of conformational changes induced by the mutations. Thus, CD4 appears to interact with HLA class II molecules mainly through the HIV gp120 binding site and possibly through a second minor interaction site mapping on the same face of the molecule.
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PMID:HLA class II antigens and the HIV envelope glycoprotein gp120 bind to the same face of CD4. 751 97

HIV-1, in contrast to animal retroviruses, is not lysed by human complement, but is readily inactivated by the sera from different animal species. To identify a possible species-specific protection mechanism. HIV-1 was expressed in cells of non-human origin. Recombinant HIV-1 virions that could encode the chloramphenicol acetyltransferase (CAT) protein were produced in African green monkey COS-1 cells, mink cells and, as a control, in human HEp-2 cells and were then used to infect CD4-positive target cells. Analysis of the CAT activity of the target cells revealed that fresh HIV-1-negative human serum reduced the infectivity of HIV-1 derived from monkey and mink cells five- to tenfold, but had no effect on HIV-1 produced in human cells. In addition, human serum efficiently lysed HIV-1 produced in non-human cells in contrast to HIV-1 originating from human cells, suggesting lysis as an important mechanism of virus inactivation. Mammalian cells are protected against lysis by homologous complement by membrane-bound regulatory molecules. Two of these complement inhibitors, namely decay-accelerating factor (DAF) and, to a lesser extent, CD59 were found on the surface of HIV-1 virions by means of a virus capture assay. Antibodies against DAF, but not against other host cell molecules found on the viral surface, efficiently blocked the resistance of HIV-1 produced in human cells to human complement. These results suggest that the acquisition of DAF during the budding process from human cells protects HIV-1 in a species-specific way against the attack of human complement.
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PMID:Decay-accelerating factor (CD55) protects human immunodeficiency virus type 1 from inactivation by human complement. 753 Nov 47

The human immunodeficiency virus 1 (HIV-1) Rev transactivator protein plays a critical role in the regulation of expression of structural proteins by controlling the pathway of mRNA transport. The Rev protein is located predominantly in the nucleoli of HIV-1 infected or Rev-expressing cells. Previous studies demonstrated that the Rev protein forms a specific complex in vitro with protein B23 which is suggested to be a nucleolar receptor and/or carrier for the Rev protein. To study the role of the nucleolus and nucleolar proteins in Rev function, transfected COS-7 or transformed CMT3 cells expressing the Rev protein were examined for subcellular locations of Rev and other proteins using indirect immunofluorescence and immunoelectron microscopy. One day after transfection the Rev protein was found in most cells only in the nucleolar dense fibrillar and granular components where it colocalized with protein B23. These were designated class 1 cells. In a second class of cells Rev and B23 accumulated in the nucleoplasm as well as in nucleoli. Treatment of class 1 cells with actinomycin D (AMD) under conditions that blocked only RNA polymerase I transcription caused Rev to completely redistribute from nucleoli to the cytoplasm. Simultaneously, protein B23 was partially released from nucleoli, mostly into the nucleoplasm, with detectable amounts in the cytoplasm. In cells recovering from AMD treatment in the presence of cycloheximide Rev and B23 showed coincident relocation to nucleoli. Class 2 cells were resistant to AMD-induced Rev redistribution. Selective inhibition of RNA polymerase II transcription by alpha-amanitin or by DRB did not cause Rev to be released into the cytoplasm suggesting that active preribosomal RNA transcription is required for the nucleolar location of Rev. However, treatment with either of the latter two drugs at higher doses and for longer times caused partial disruption of nucleoli accompanied by translocation of the Rev protein to the cytoplasm. These results suggest that the nucleolar location of Rev depends on continuous preribosomal RNA transcription and a substantially intact nucleolar structure.
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PMID:The roles of nucleolar structure and function in the subcellular location of the HIV-1 Rev protein. 759 22

Calcium depletion from the endoplasmic reticulum inhibits protein synthesis and correlates with increased phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) by a mechanism that does not require ongoing protein synthesis. To elucidate whether protein synthesis inhibition requires eIF-2 alpha phosphorylation and whether eIF-2 alpha phosphorylation is mediated by the double-stranded RNA-dependent protein kinase (PKR), we studied protein synthesis in response to calcium depletion mediated by calcium ionophore A23187 in cell lines overexpressing wild-type eIF-2 alpha, a mutant eIF-2 alpha (S51A) that is resistant to phosphorylation, or a dominant negative mutant PKR (K296P in catalytic subdomain II). Expression of either mutant eIF-2 alpha or mutant PKR partially protected NIH3T3 cells from inhibition of protein synthesis upon A23187 treatment. In contrast, overexpression of wild-type PKR increased sensitivity to protein synthesis inhibition mediated by A23187 treatment. In a COS-1 monkey cell transient transfection system, increased eIF-2 alpha phosphorylation in response to A23187 treatment was inhibited by expression of the dominant negative PKR mutant. Overexpression of the PKR regulatory RNA binding domain, independent of the PKR catalytic domain, was sufficient to inhibit increased phosphorylation of eIF-2 alpha upon A23187 treatment. In addition, overexpression of the HIV TAR RNA binding protein also inhibited eIF-2 alpha phosphorylation upon A23187 treatment. Taken together, our data show that calcium depletion activates PKR to phosphorylate eIF-2 alpha, and this activation is likely mediated through the PKR RNA binding domain.
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PMID:Calcium depletion from the endoplasmic reticulum activates the double-stranded RNA-dependent protein kinase (PKR) to inhibit protein synthesis. 762 70

The 96-amino acid Vpr protein is the only virion-associated regulatory protein encoded by the human immunodeficiency virus type 1 (HIV-1). Vpr incorporation into the viral particle is most likely due to an interaction with a viral structural protein. Recent data have shown that DNA encoding for the p55 Gag precursor protein (Pr55gag) is the minimal viral genetic information necessary for Vpr incorporation. Other studies have suggested that the p6 portion of Pr55gag, which is unique to lentiviruses, is involved in Vpr incorporation. To investigate the mechanism of incorporation of Vpr into HIV-1 virions, COS-7 cells were cotransfected with ptrENV, an expression vector that encodes all of the HIV-1 regulatory proteins including Rev and Vpr, and different constructs of pIIIgagCAR, a rev-dependent Gag expression plasmid that encodes Pr55gag and the viral protease. Virions produced from gag constructs containing a premature p6 termination codon at positions Leu-1, Ser-17, Tyr-36, or Leu-44 lacked detectable Vpr. In contrast, gag constructs with double Pro-10-Pro-11 substitutions for Leu-10-Leu-11 or a premature termination codon at position Pro-49 of p6 were still able to incorporate Vpr, however, with lower efficiency than wild type. The mutations described in this study affected directly two short regions within the p6 domain, which are highly conserved among primate immunodeficiency viruses. Our results suggest that the conserved (P-T/S-A-P-P) and (L-X-S-L-F-G) motifs located near the N-terminus and C-terminus, respectively, of the p6 domain of Gag are critical for Vpr incorporation into HIV-1 virions.
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PMID:Incorporation of Vpr into human immunodeficiency virus type 1: role of conserved regions within the P6 domain of Pr55gag. 764 78

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