Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: UMLS:C0021051 (immunodeficiency)
71,517 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Genetic recombination contributes to the genomic heterogeneity of human immunodeficiency virus type 1 (HIV-1). In the present study, we demonstrate that HIV-1 readily develops resistance to two classes of anti-HIV-1 drugs through in vitro genetic recombination involving large segments of the viral genome. Co-transfection of COS-7 cells with an HIV-1 plasmid (pSUM13) carrying five mutations in the reverse transcriptase (RT)-encoding region (A62V, V75I, F77L, F116Y, Q151M), conferring resistance to multiple dideoxynucleoside analogs (ddNs), and another HIV-1 plasmid (pSUM431) carrying five mutations in the protease-encoding region (V321, L33F, K451, 184V, L89M), conferring resistance to protease inhibitors such as KNI-272, readily produced HIV-1 carrying both sets of mutations when propagated in MT-2 cells in the presence of azidothymidine (AZT) and KNI-272. The resultant HIV-1 variant was highly resistant to both ddNs and KNI-272. Co-infection of MT-2 cells with HIV-1SUM13 carrying the RT mutations and HIV-1SUM431 carrying the mutations in the protease also generated HIV-1 with both sets of mutations when cultured with AZT and KNI-272. We also report here that the problematic artifactual recombination occurring during genetic analyses of heterogeneous nucleic acid sequences using polymerase chain reaction can be successfully obviated.
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PMID:HIV-1 acquires resistance to two classes of antiviral drugs through homologous recombination. 947 18

Prokaryotic and eukaryotic cells incorporate the unusual amino acid selenocysteine at a UGA codon, which conventionally serves as a termination signal. Translation of eukaryotic selenoprotein mRNA requires a nucleotide selenocysteine insertion sequence in the 3'-untranslated region. We report the molecular cloning of the binding protein that recognizes the selenocysteine insertion sequence element in human cellular glutathione peroxidase gene (GPX1) transcripts and its identification as DNA-binding protein B, a member of the EFIA/dbpB/YB-1 family. The predicted amino acid sequence contains four arginine-rich RNA-binding motifs, and one segment shows strong homology to the human immunodeficiency virus Tat domain. Recombinant DNA-binding protein B binds the selenocysteine insertion sequence elements from the GPX1 and type I iodothyronine 5'-deiodinase genes in RNA electrophoretic mobility shift assays and competes with endogenous GPX1 selenocysteine insertion sequence binding activity in COS-1 cytosol extracts. Addition of antibody to DNA-binding protein B to COS-1 electromobility shift assays produces a slowly migrating "supershift" band. The molecular cloning and identification of DNA-binding protein B as the first eukaryotic selenocysteine insertion sequence-binding protein opens the way to the elucidation of the entire complex necessary for the alternative reading of the genetic code that permits translation of selenoproteins.
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PMID:Identification and molecular cloning of a human selenocysteine insertion sequence-binding protein. A bifunctional role for DNA-binding protein B. 948 64

The presence of a polyadenylation signal in the repeat (R) region of the HIV-1 genome, which is located at both the 5' and 3' ends of the viral transcripts, requires differential regulation of polyadenylation. The HIV-1 poly(A) site can fold in a stable stem-loop structure that is well-conserved among different human and simian immunodeficiency viruses. In this study, we tested the effect of this hairpin on polyadenylation by introducing mutations that either stabilize or destabilize the RNA structure. The HIV-1 sequences were inserted into the pSV2CAT reporter plasmid upstream of the SV40 early poly(A) site. These constructs were transfected into COS cells and transcripts were analyzed for the usage of the HIV-1 versus SV40 poly(A) site. The wild-type HIV-1 poly(A) site was used efficiently in this context and destabilization of the poly(A) hairpin did not affect the polyadenylation efficiency. In contrast, further stabilization of the hairpin severely inhibited HIV-1 polyadenylation. Additional mutations that repair the thermodynamic stability of this mutant hairpin restored the polyadenylation activity. These results indicate that the mechanism of polyadenylation can be repressed by stable RNA structure encompassing the poly(A) signal. Experiments performed at reduced temperatures also suggest an inverse correlation between the stability of the RNA structure and the efficiency of polyadenylation.
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PMID:Inhibition of polyadenylation by stable RNA secondary structure. 951 78

We have studied the effect of mutations in the human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) sequence on tRNA(3Lys) genomic placement, i.e., the in vivo placement of primer tRNA(3Lys) on the HIV-1 primer binding site (PBS). HIV-1 produced from COS cells transfected with wild-type or mutant proviral DNA was used in this study. We have found that mutations in the amino acid sequences flanking the first Cys-His box in the NC sequence produce the maximum inhibition of genomic placement. A similar finding was obtained when the NC-facilitated annealing of primer tRNA(3Lys) to the HIV PBS in vitro was studied. However, since the genomic placement of tRNA(3Lys) occurs independently of precursor protein processing, the NC mutations studied here have probably exerted their effect through one or both of the precursor proteins, Pr55gag and/or Pr160(gag-pol). One mutation in the linker region between the two Cys-His boxes, P31L, prevented packaging of both Pr160(gag-pol) and tRNA(3Lys) and prevented the genomic placement of tRNA(3Lys). Both packaging and genomic placement were rescued by cotransfection with a plasmid coding for wild-type Pr160(gag-pol). For other linker mutations [R7R10K11 S, R32G, and S3(32-34)], packaging of Pr160(gag-pol) and tRNA(3Lys) was not affected, but genomic placement was, and placement could not be rescued by cotransfection with plasmids coding for either Pr55gag or Pr160(gag-pol). After placement, the initiation of reverse transcription within extracellular virions is characterized by a 2-base DNA extension of the placed tRNA(3Lys). This process requires precursor processing, and those NC mutations which showed the most inhibition of initiation were in either of the two NC Cys-His boxes. Destabilization of a U5 stem-A-rich loop immediately upstream of the PBS (through deletion of four consecutive A's in the loop) did not affect the in vivo genomic placement of tRNA(3Lys) but resulted in the presence in the extracellular virus of longer cDNA extensions of tRNA(3Lys), with a corresponding decrease in the presence of unextended and 2-base-extended tRNA(3Lys).
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PMID:The role of nucleocapsid and U5 stem/A-rich loop sequences in tRNA(3Lys) genomic placement and initiation of reverse transcription in human immunodeficiency virus type 1. 955 76

Large deletions of the upstream U3 sequences in the long terminal repeats (LTRs) of human immunodeficiency virus and simian immunodeficiency virus (SIV) accumulate in vivo in the absence of an intact nef gene. In the SIV U3 region, about 65 bp just upstream of the single NF-kappaB binding site always remained intact, and some evidence for a novel enhancer element in this region exists. We analyzed the transcriptional and replicative capacities of SIVmac239 mutants containing deletions or mutations in these upstream U3 sequences and/or the NF-kappaB and Sp1 binding sites. Even in the absence of 400 bp of upstream U3 sequences, the NF-kappaB site and all four Sp1 binding sites, the SIV promoter maintained about 15% of the wild-type LTR activity and was fully responsive to Tat activation in transient reporter assays. The effects of these deletions on virus production after transfection of COS-1 cells with full-length proviral constructs were much greater. Deletion of the upstream U3 sequences had no significant influence on viral replication when either the single NF-kappaB site or the Sp1 binding sites were intact. In contrast, the 26 bp of sequence located immediately upstream of the NF-kappaB site was essential for efficient replication when all core enhancer elements were deleted. A purine-rich site in this region binds specifically to the transcription factor Elf-1, a member of the ets proto-oncogene-encoded family. Our results indicate a high degree of functional redundancy in the SIVmac U3 region. Furthermore, we defined a novel regulatory element located immediately upstream of the NF-kappaB binding site that allows efficient viral replication in the absence of the entire core enhancer region.
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PMID:Sequences just upstream of the simian immunodeficiency virus core enhancer allow efficient replication in the absence of NF-kappaB and Sp1 binding elements. 962 Oct 17

The molecular chaperone cyclophilin A (Cyp A) modulates human immunodeficiency virus type 1 (HIV-1) infectivity through its interactions with Gag structural proteins. The molecular mechanism for CypA in HIV-1 replication is not known. We studied chaperone effects on Gag precursor processing using cyclosporin A (CsA) to bind CypA and prevent its interaction with p55Gag. CsA treatment inhibited p55Gag processing in extracellular virus-like particles produced from COS cells. We confirmed the effect of CsA on Gag processing by examining virions produced from CEMx174 cells infected with HIV-1LAI. Particles accumulated in the presence of CsA displayed mostly immature virion morphology and lacked condensed capsids. CsA has a direct effect on HIV-1 Gag processing that implicates CypA as having an important role in the maturation of HIV-1 particles.
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PMID:Cyclophilin a modulates processing of human immunodeficiency virus type 1 p55Gag: mechanism for antiviral effects of cyclosporin A. 963 59

Infection of T lymphocytes by the human immunodeficiency virus causes drastic alterations in the intracellular cation content of the infected cells. The human immunodeficiency virus type 1 genome encodes several accessory proteins, including Vpu, an integral membrane protein that forms ion channels in planar lipid bilayers. The effect of Vpu on the permeability of the plasma membrane to several molecules has been analyzed. Expression of vpu in Escherichia coli cells increases membrane permeability to a number of molecules such as 2-nitrophenyl beta-D-galactopyranoside, uridine, the impermeable translation inhibitor hygromycin B, and lysozyme. In addition, transient expression of Vpu in eukaryotic COS cells enhances entry of charged molecules such as hygromycin B and neurobiotin into these cells. The effect of Vpu on cell membrane permeability resembles that reported for other membrane-active proteins from different animal viruses, including influenza M2, Semliki Forest virus 6K, and poliovirus 2B and 3A proteins.
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PMID:The human immunodeficiency virus type 1 Vpu protein enhances membrane permeability. 975 59

It is unclear whether proteolytic processing of the human immunodeficiency virus type 1 (HIV-1) Gag protein is dependent on virus assembly at the plasma membrane. Mutations that prevent myristylation of HIV-1 Gag proteins have been shown to block virus assembly and release from the plasma membrane of COS cells but do not prevent processing of Gag proteins. In contrast, in HeLa cells similar mutations abolished processing of Gag proteins as well as virus production. We have now addressed this issue with CD4(+) T cells, which are natural target cells of HIV-1. In these cells, myristylation of Gag proteins was required for proteolytic processing of Gag proteins and production of extracellular viral particles. This result was not due to a lack of expression of the viral protease in the form of a Gag-Pol precursor or a lack of interaction between unmyristylated Gag and Gag-Pol precursors. The processing defect of unmyristylated Gag was partially rescued ex vivo by coexpression with wild-type myristylated Gag proteins in HeLa cells. The cell type-dependent processing of HIV-1 Gag precursors was also observed when another part of the plasma membrane binding signal, a polybasic region in the matrix protein, was mutated. The processing of unmyristylated Gag precursors was inhibited in COS cells by HIV-1 protease inhibitors. Altogether, our findings demonstrate that the processing of HIV-1 Gag precursors in CD4(+) T cells occurs normally at the plasma membrane during viral morphogenesis. The intracellular environment of COS cells presumably allows activation of the viral protease and proteolytic processing of HIV-1 Gag proteins in the absence of plasma membrane binding.
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PMID:A bipartite membrane-binding signal in the human immunodeficiency virus type 1 matrix protein is required for the proteolytic processing of Gag precursors in a cell type-dependent manner. 976 51

We have selectively mutagenized specific residues at the junction between the protease (PR) and reverse transcriptase (RT) genes of human immunodeficiency virus type 1 (HIV-1) to study the effects of PR-RT fusion proteins in the context of a full-length, infectious proviral construct. Mutant viruses derived from COS-7 cells transfected with this construct were analyzed in regard to each of viral replication, maturation, and infectivity. Immunoblot analysis revealed that the mutation prevented cleavage between the PR and RT proteins and that both existed as a PR-RT fusion protein in each of cellular and viral lysates. Interestingly, intracellular PR that existed within the PR-RT fusion protein remained functionally active, whereby HIV-1 precursor proteins were processed efficiently. Furthermore, the RT component of the fusion protein also retained its enzymatic activity as shown in RT assays. Electron microscopy revealed that the mutant viruses containing the PR-RT fusion protein possessed wild-type morphology. These viruses also displayed wild-type sensitivities to inhibitors of each of the HIV-1 PR and RT activities. However, viruses containing the PR-RT fusion protein were 20 times less infectious than wild-type viruses. This defect was further pronounced when mutated Gag-Pol proteins were overexpressed as a consequence of an additional mutation that interfered with frameshifting. Thus, unlike cleavage site mutations at the N terminus of PR, a cleavage site mutation between PR and RT did not affect the enzymatic activities of either PR or RT and viruses containing PR-RT fusion proteins were viable.
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PMID:Characterization of human immunodeficiency virus type-1 (HIV-1) particles that express protease-reverse transcriptase fusion proteins. 981 41

Four isoforms of human fibroblast growth factor 2 (FGF-2) result from alternative initiations of translation at three CUG start codons and one AUG start codon. Here we characterize a new 34-kDa FGF-2 isoform whose expression is initiated at a fifth initiation codon. This 34-kDa FGF-2 was identified in HeLa cells by using an N-terminal directed antibody. Its initiation codon was identified by site-directed mutagenesis as being a CUG codon located at 86 nucleotides (nt) from the FGF-2 mRNA 5' end. Both in vitro translation and COS-7 cell transfection using bicistronic RNAs demonstrated that the 34-kDa FGF-2 was exclusively expressed in a cap-dependent manner. This contrasted with the expression of the other FGF-2 isoforms of 18, 22, 22.5, and 24 kDa, which is controlled by an internal ribosome entry site (IRES). Strikingly, expression of the other FGF-2 isoforms became partly cap dependent in vitro in the presence of the 5,823-nt-long 3' untranslated region of FGF-2 mRNA. Thus, the FGF-2 mRNA can be translated both by cap-dependent and IRES-driven mechanisms, the balance between these two mechanisms modulating the ratio of the different FGF-2 isoforms. The function of the new FGF-2 was also investigated. We found that the 34-kDa FGF-2, in contrast to the other isoforms, permitted NIH 3T3 cell survival in low-serum conditions. A new arginine-rich nuclear localization sequence (NLS) in the N-terminal region of the 34-kDa FGF-2 was characterized and found to be similar to the NLS of human immunodeficiency virus type 1 Rev protein. These data suggest that the function of the 34-kDa FGF-2 is mediated by nuclear targets.
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PMID:A new 34-kilodalton isoform of human fibroblast growth factor 2 is cap dependently synthesized by using a non-AUG start codon and behaves as a survival factor. 985 74


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