Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0019693 (HIV)
 170,526 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human immunodeficiency virus type 1 (HIV-1) incorporates the cellular peptidyl-prolyl cis-trans isomerase cyclophilin A (CyPA), the cytosolic receptor for the immunosuppressant cyclosporin A (CsA). CsA inhibits the incorporation of CyPA and reduces HIV-1 virion infectivity but is inactive against closely related primate lentiviruses that do not interact with CyPA. The incorporation of CyPA into HIV-1 virions is mediated by a specific interaction with a proline-containing, solvent-exposed loop in the capsid (CA) domain of the Gag polyprotein. CsA, which disrupts the interaction with CA, binds at the active site of CyPA. To test whether active-site residues are also involved in the interaction with HIV-1 CA, we used a panel of previously characterized active-site mutants of human CyPA. Expression vectors for epitope-tagged wild-type and mutant CyPA were transfected into COS-gamma cells along with HIV-1 proviral DNA, and the virions produced were analyzed for the presence of tagged proteins. Cotransfection of the wild-type expression vector led to the incorporation of readily detectable amounts of epitope-tagged CyPA into HIV-1 virions. One CyPA mutant with a substantially decreased sensitivity to CsA was incorporated with wild-type efficiency, demonstrating that the requirements for binding to CsA and to HIV-1 CA are not identical. The remaining six CyPA mutants were incorporated with markedly reduced efficiency, providing in vivo evidence that HIV-1 CA interacts with the active site of CyPA.
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PMID:Active-site residues of cyclophilin A are crucial for its incorporation into human immunodeficiency virus type 1 virions. 926 45

Complementary DNA encoding a human Gal(beta1-3)GalNAc alpha2,3-sialyltransferase type II (hST3Gal II) was cloned from a CEM T-cell cDNA library using a 23-base oligonucleotide probe. The sequence of this probe was established on the basis of a slightly divergent sialylmotif L that was obtained by polymerase chain reaction with degenerate oligonucleotide primers based on the conserved sialylmotif L of mammalian Gal(beta1-3)GalNAc alpha2,3-sialyltransferases. It was thus confirmed that a short oligonucleotide probe may be sensitive and highly specific. The nucleotide and amino acid sequences of hST3Gal II show, respectively, 56.3% and 49.3% similarity to hST3Gal I [Kitagawa, H. & Paulson, J. C. (1994) J. Biol. Chem. 269, 17872-17878] and 88.1% and 93.7% similarity to murine ST3Gal II [Lee, Y. C., Kojima, N., Wada, E., Kurosawa, N., Nakaoka, T., Hamamoto, T. & Tsuji, S. (1994) J. Biol. Chem. 269, 10028-10033]. hST3Gal II mRNA was highly expressed in heart, liver, skeletal muscle and various lymphoid tissues but not in brain and kidney. A soluble form of hST3Gal II expressed in COS-7 cells was tested in vitro for substrate specificity and kinetic properties. Asialofetuin and asialo-bovine submaxillary mucin appeared better substrates for hST3Gal II than for its murine counterpart as previously reported [Kojima, N., Lee, Y.-C., Hamamoto, T., Kurosawa, N. & Tsuji, S. (1994) Biochemistry 33, 5772-5776]. In previous studies, we have shown hyposialylation of O-glycans attached to two major lymphocyte CD43 and CD45 cell surface molecules in human-immunodeficiency-virus-1(HIV-1)-infected T-cell lines. Since comparable levels of hST3Gal I and hST3Gal II mRNA and enzymatic activity were observed in parental and HIV-1-infected CEM T-cell lysates, the sialylation defect associated with HIV infection of this cell line is probably due to a mechanism different from a simple altered catalytic activity of these sialyltransferases.
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PMID:Cloning and expression of cDNA for a human Gal(beta1-3)GalNAc alpha2,3-sialyltransferase from the CEM T-cell line. 926 97

Human immunodeficiency virus type-1 (HIV-1) Rev overcomes negative elements within viral RNAs to allow expression of gag, pol, and env. The effect of Rev on protein and RNA expression of HIV-1 protease (PR)-containing constructs was investigated utilizing transient transfection of COS cells. Rev, through the Rev response element (RRE), resulted in a large increase in proteolytic activity and cytoplasmic RNA accumulation. Furthermore, Rev increased the level of total RNA produced by a PR-containing construct. The increase in cytoplasmic RNA accumulation in the presence of Rev indicated the presence of cis-acting repressor sequences (CRS) within the RNA produced by this construct. Therefore, components of the construct were analyzed for CRS activity. PR sequences in both sense and antisense orientations exhibited CRS activity. RRE sequences alone conferred a small CRS effect. Additional CRS activity was present within an unspliced RNA containing only nef and LTR sequences. These results indicate a novel form of cis-acting repressor activity within HIV-1 PR; this activity is exerted regardless of the orientation of PR and appears to function at the level of cytoplasmic or nuclear RNA stability.
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PMID:Identification of cis-acting repressor activity within human immunodeficiency virus type 1 protease sequences. 926 56

The bait region of the general protease inhibitor alpha 2-macroglobulin (alpha 2M) was mutated by introducing a recognition sequence of furin. This did not interfere with folding, S-ester formation or tetramerization of the mutant recombinant alpha 2M (r alpha 2M). Mutant r alpha 2M inhibited furin in vitro, by a similar mechanism to that used by plasma alpha 2M to inhibit high-molecular-mass proteases. The mutant alpha 2M was intracellularly active in COS-1 cells in inhibiting the endogenous processing of the soluble substrates for furin (von Willebrand factor, transforming growth factor beta1 and a soluble form of the envelope glycoprotein gp160 from HIV-1) but not the membrane-bound form of gp160. The intracellular activity of mutant alpha 2M strongly indicated that alpha 2M attains its native conformation, and thus that the unusual internal S-ester is formed, before alpha 2M passes through the cleavage compartment(s). Our results show for the first time that modulation of the bait region of alpha 2M allows the creation of an inhibitor against membrane-bound proteases. It can be expected that the use of alpha 2M-bait mutants will become important as a technique for the study of various proteolytic processes and for the identification of the proteases involved.
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PMID:Inhibition of intracellular proteolytic processing of soluble proproteins by an engineered alpha 2-macroglobulin containing a furin recognition sequence in the bait region. 929 Nov 25

To define the role of human immunodeficiency virus type 1 splice sites in the cytoplasmic accumulation of viral RNAs, sequential deletion mutagenesis on an infectious proviral clone of HIV-1 was performed. Deletion of the majority of intron sequences, containing previously identified CRS, did not attenuate CRS activity. Retention of either the first or second tat intron preserved CRS activity. RNAs containing splice donor sequences, in the absence of known downstream splice acceptor sequences, retained CRS activity. Unexpectedly, these splice donors were still utilized for splicing. These results indicate that the major HIV-1 splice donors can function as CRS and function to negatively regulate the cytoplasmic accumulation of HIV-1 RNAs in COS cells.
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PMID:Involvement of human immunodeficiency virus type-1 splice sites in the cytoplasmic accumulation of viral RNA. 929 21

We demonstrated previously that PP2A exists in many cell types as two abundant forms: (1) holoenzyme composed of two regulatory subunits, A and B, and a catalytic subunit C; and (2) core enzyme consisting of the A and C subunits. These two forms have different substrate specificities. Since published data suggested that HIV-1 transcription may be regulated by a cellular protein phosphatase, it was of interest to determine whether changing the ratio between PP2A core and holoenzyme affects HIV-1 gene expression. This question was addressed by expression in COS cells of an N-terminal mutant of the A subunit, A delta 5, which binds the C but not the B subunit. This resulted in an increase in the amount of core enzyme and a decrease in the amount of holoenzyme concomitant with the expected change in phosphatase activity. Tat-stimulated transcription from the HIV-1 LTR was inhibited 5-fold by mutant A delta 5, whereas mRNA synthesis directed by the actin promoter was not affected. Furthermore, virus production in COS, HeLa, and Jurkat T cells was inhibited 45-, 5-, and 3-fold, respectively, by mutant A delta 5. These results demonstrate that the balance between PP2A holoenzyme and core enzyme is important for HIV-1 gene expression and virus production.
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PMID:Increasing the ratio of PP2A core enzyme to holoenzyme inhibits Tat-stimulated HIV-1 transcription and virus production. 940 Jun 15

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

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

Tat is a virally expressed regulatory protein involved in the replication of HIV-1, the etiological agent of AIDS. To investigate the effect of tat inhibition on HIV replication, we constructed a retroviral vector to express an anti-tat hammerhead ribozyme as part of the 3' untranslated region of beta-galactosidase transcripts. Initial testing of this vector in tat-expressing COS-7 cells reduced tat activity by 85-95% as measured by tat-dependent CAT assays. Amphotropic and HIV-pseudotyped retroviral particles generated with this vector were used in HIV challenge experiments to determine the ability of this reagent to control HIV replication. CD4(+) peripheral blood lymphocytes (PBLs) stably transduced with this vector were subsequently challenged with HIV. These cells were able to resist HIV infection for up to 20 days as measured by cell death and reverse transcriptase activity. These data yield proof of principle that a pseudotyped retroviral vector can target and deliver a protective ribozyme to CD4(+) cells.
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PMID:Inhibition of HIV-1 replication by an anti-tat hammerhead ribozyme. 953 87

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


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