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)

We have investigated the ability of pyridoxal-5'-phosphate to inhibit a recombinant deletion mutant of human immunodeficiency virus type 1(HIV-1) reverse transcriptase (RT) which is missing the last 23 amino acids of the C-terminus. This mutant reverse transcriptase is characterized by normal polymerase activity as compared with full-length enzyme; however, it has no RNase H activity. Inhibition studies with pyridoxal-5'-phosphate showed several differences as compared with inhibition of full-length enzyme: (1) Inhibition of mutant reverse transcriptase was independent of divalent cation, (2) Either substrate alone could protect mutant reverse transcriptase from inactivation by pyridoxal-5'-phosphate, and (3) stoichiometry of pyridoxal-5'-phosphate binding to mutant reverse transcriptase was 2 mol/mol under the same conditions in which 1 mol/mol bound to full-length enzyme. Furthermore, in the presence of either substrate alone, the stoichiometry of pyridoxal-5'-phosphate binding to the mutant was reduced to 1 mol/mol. These results indicate that the second binding site for pyridoxal-5'-phosphate seen in the mutant reverse transcriptase is at or near the primer-template binding site of the enzyme. They also suggest that the RNase H domain of HIV RT plays a functional role in substrate binding at the polymerase domain.
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PMID:Pyridoxal-5'-phosphate inhibits the polymerase activity of a recombinant RNAase H-deficient mutant of HIV-1 reverse transcriptase. 138 Dec 4

In a search for compounds active against human immunodeficiency virus type 1 (HIV-1), it was found that the novel low-molecular weight immunoenhancer ammonium trichloro(dioxyethylene-O,O'-) tellurate (AS101) suppresses production of HIV-1 in vitro. Treatment of HIV-1-infected peripheral blood mononuclear cells (PBMC) with increasing concentrations of AS101 resulted in substantial inhibition of virus production as measured by both reverse transcriptase (RT) activity and antigen presence in supernatants of treated cells. AS101 had no effect on PBMC viability, growth, or morphology up to a concentration of 15 microM for 14 days. To elucidate a possible mechanism for the inhibition of AS101, we have analyzed the effect of the drug on the catalytic functions associated with HIV RT, namely the RDDP, DDDP, and RNase H activities. RDDP and DDDP activities were impaired by the drug with calculated IC50 value of about 4 microM. On the other hand, the RNase H activity was less sensitive to AS101, with an apparent IC50 value of about 30 microM. The anti-HIV-1 activity of AS101 as reflected by inhibition of the different catalytic functions associated with viral RT, in the absence of drug-related toxicity to lymphocytes, together with its immunomodulating activity strongly argues in favor of its evaluation, as a therapeutic agent for patients with HIV infection.
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PMID:Inhibition of the reverse transcriptase activity and replication of human immunodeficiency virus type 1 by AS 101 in vitro. 138 Dec 5

We have generated by site-directed mutagenesis plasmids that induce the synthesis of specific mutants of the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). These recombinant mutants of HIV-1 RT, designed on the basis of our previous studies of HIV-1 and HIV-2 RTs, were analyzed for structure-function relationship by assessing their RNA-dependent and DNA-dependent DNA polymerase as well as the ribonuclease H activities. Three groups of mutants were studied. 1) We have investigated the importance of the only two sets of highly conserved double prolines found in the sequence of HIV-1 RT. The results indicate that the conversion of either one or both prolines (at positions 225 and 226) to threonines have no significant effect on all catalytic activities of the enzyme. The mutants in which prolines 419 and 420 were individually modified to threonines exhibit full activities, whereas the double proline 419/420 mutant lost most of its RNase H activity (although the DNA polymerase function was fully retained). 2) We have deleted phenylalanine 346 from HIV-1 RT, which is absent in wild type HIV-2 RT. This mutant of HIV-1 RT lost practically all catalytic activities. 3) A mutant of HIV-1 RT in which a cysteine residue substituted for alanine 446, was found to be slightly hyperactive for both DNA polymerase and RNase H activities.
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PMID:Functional analysis of novel selective mutants of the reverse transcriptase of human immunodeficiency virus type 1. 138 52

The precursor homodimeric p66/p66 form of human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT) possesses the DNA polymerase and RNase H activities involved in the synthesis of the double-stranded provirus DNA. Reverse transcription is initiated from tRNALys in the case of HIV-1. The present study confirmed that interactions between HIV-1 RT and tRNALys induce protein conformational changes and demonstrated that these interactions stimulate the enzymatic activities associated with the p66 subunit. Thus, the p66/p66 form of the enzyme is strongly stimulated in both DNA polymerase and RNase H activities. Preincubation of the enzyme with tRNA is an obligatory step to obtain the stimulatory effect. The affinity of template, primer, or substrate for RT p66/p66 did not change when the enzyme was preincubated with tRNALys at stimulatory concentrations; the interaction of tRNA with p66/p66 has an effect only on the maximal rate of polymerization. It is further shown that the RNase H domain of RT is much more accessible to protease attack than the DNA polymerase active site.
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PMID:Interaction of tRNALys with the p66/p66 form of HIV-1 reverse transcriptase stimulates DNA polymerase and ribonuclease H activities. 138 72

The backbone dynamics of the uniformly 15N-labeled ribonuclease H (RNase H) domain of human immunodeficiency virus (HIV-1) reverse transcriptase have been investigated using two-dimensional inverse-detected heteronuclear 15N-1HNMR spectroscopy. 15N T1, T2, and nuclear Overhauser enhancement (NOE) data were obtained for 107 out of a total of 134 backbone amide groups. The overall rotational correlation time (tau R) for the protein at 26 degrees C is 10.4 ns. The backbone N-H vectors for all the measurable residues exhibit very fast motions on a time scale of less than or equal to 20 ps. The 15N relaxation data for only 14 residues can be explained by this single internal motion alone. A further 39 residues display a second motion on a time scale ranging from 28.8 ps to 3.9 ns, while another 15 residues are characterized by an additional motion on the 170-ns to 2.25-ms time scale resulting in 15N T2 exchange line broadening. There are 39 residues that exhibit both the additional 15N T2 exchange line broadening and the slow (28.8 ps-3.9 ns) internal motion. Thus, the RNase H domain experiences extensive mobility throughout its structure as evidenced by the 93 residues which exhibit multiple modes of motion. Distinctly mobile regions of the protein are identified by large decreases in the overall order parameter (S2) and correspond to the C-terminal residues and the loop regions between beta-strands beta 1 and beta 2 and between alpha-helix alpha B and beta-strand beta 4.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Analysis of the backbone dynamics of the ribonuclease H domain of the human immunodeficiency virus reverse transcriptase using 15N relaxation measurements. 138 87

We have examined the ability of the reverse transcriptase (RT) from human immunodeficiency virus (HIV) to carry out strand transfer synthesis (i.e., switching of the primer to a new template) from internal regions of natural-sequence RNA. A 142-nucleotide RNA template (donor) primed with a specific 20-nucleotide DNA oligonucleotide was used to initiate synthesis. DNA oligonucleotides with homology to internal regions of the donor were used as acceptors. In this system, HIV RT produced strand transfer products. An HIV RT having RNase H depleted to 3% of normal (HIV RTRD) catalyzed the transfer reaction inefficiently. An RNase H-minus deletion mutant of murine leukemia virus RT was unable to catalyze strand transfer. HIV RTRD, however, efficiently catalyzed transfer when Escherichia coli RNase H was included in the reactions, while the mutant murine leukemia virus RT was not efficiently complemented by the E. coli enzyme. Evidently, RNase H activity enhances, or is required for, internal strand transfer. Two acceptors homologous to 27-nucleotide regions of the donor, one offset from the other by 6 nucleotides, were tested. The offset eliminated a sequence homologous to a prevalent DNA synthesis pause site in the donor. Strand transfer to this acceptor was about 25% less efficient, suggesting that RT pausing can enhance strand transfer. When the deoxynucleoside triphosphates in the reactions were reduced from 50 to 0.2 microM, increasing RT pausing, the efficiency of strand transfer also increased. A model for RT-catalyzed strand transfer consistent with our results is presented.
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PMID:Requirements for strand transfer between internal regions of heteropolymer templates by human immunodeficiency virus reverse transcriptase. 138 63

The handle region (residues 84-99) in ribonuclease HI (RNase HI) from Escherichia coli, which is rich in basic amino acid residues, was altered by alanine-scanning mutagenesis. Fifteen mutant proteins were purified to homogeneity and analyzed for the enzymatic activity. A mutation of either of 2 tryptophan residues at 85 or 90 resulted in a large increase in the Km value along with a large decrease in the Vmax value. These values probably resulted from conformational changes introduced by the mutations as indicated by the CD spectra of these mutant proteins. All other mutant enzymes had Vmax values similar to that of the wild-type enzyme. In contrast, replacement of any basic amino acid residue in the handle region, except for lysine 86, yielded proteins whose Km values were 3-5-fold higher than the wild-type enzyme. Such effects were shown to be cumulative, suggesting strongly that the cluster of positive charges in the handle region is important for the effective binding of the substrate. Interestingly, the region of human immunodeficiency virus reverse transcriptase with homology to E. coli RNase HI lacks the handle region which may account for the poor RNase H activity of the domain when separated from the polymerase domain.
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PMID:Importance of the positive charge cluster in Escherichia coli ribonuclease HI for the effective binding of the substrate. 164 12

We have constructed a series of plasmids that, when introduced into Escherichia coli, induce the expression of high levels of either wild-type or mutated forms of the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). Mutant forms of RT that had been previously analyzed for their RNA-dependent DNA polymerase activity were tested for RNase H activity using an in situ polyacrylamide gel assay. Mutations affecting the RNase H are not clustered in a single region of the 66-kDa RT molecule. With only few exceptions, mutations that affect the RNase H activity also cause a substantial decrease in the DNA polymerase function. This suggests that, unlike the RT from murine leukemia virus (MuLV), it is difficult to genetically separate the catalytic domains responsible for the RNase H and DNA polymerase functions of HIV-1 RT. Those few mutations that differentially affect the RNase H and the polymerase activities of HIV-1 RT suggest that, as in MuLV, the polymerase domain is in the amino-terminus and the RNase H domain is in the carboxy-terminus. We have also generated chimeric molecules that are composed of sequences from the RT of HIV-1 and MuLV and these hybrid RTs were analyzed for their enzymatic properties. Two of these chimeric RTs possess RNase H activity but lack detectable DNA polymerase activity.
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PMID:Mutational analysis of the ribonuclease H activity of human immunodeficiency virus 1 reverse transcriptase. 169 64

We have analyzed the processing of the RNA primer for (+) strand DNA synthesis by reverse transcriptase of the human immunodeficiency virus 1. To test for specific RNA cleavage and primer usage, we constructed a 99-base pair RNA-DNA hybrid containing the viral polypurine tract and flanking viral sequences. Although the RNase H activity of reverse transcriptase cleaves the RNA strand into multiple fragments, only two primers are extended in the presence of nucleoside triphosphates. The major RNA primer includes the entire polypurine tract except for the last adenosine and has the sequence 5'-UUUUAAAAGAAAAGGGGGG-3'. The minor primer has the same 3' end but is two nucleotides shorter. In a subsequent processing step reverse transcriptase releases the primer intact via a cleavage at the RNA-DNA junction. RNA cleavage, primer extension, and primer removal can take place in a single reaction. However, specificity does not require coupling of the three steps and is preserved in the individual reactions. The polypurine primer is generated and removed after its elongation in the absence of DNA synthesis. Furthermore, the polypurine primer is selected among the several RNA fragments available and extended by reverse transcriptase as well as by p51, a short form of reverse transcriptase lacking RNase H activity.
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PMID:Processing of the primer for plus strand DNA synthesis by human immunodeficiency virus 1 reverse transcriptase. 169 20

The sequence of the LTR-LTR circle junction of human immunodeficiency virus type 1 (HIV-1) was determined. The circle junction sequences were amplified by the polymerase chain reaction and cloned into M13 sequencing vectors. The circle junction contains 4 base pairs that are not present in the integrated provirus. We show that reverse transcription in HIV-1 initiates with the addition of a dC to the tRNA primer, suggesting that the tRNA used to initiate reverse transcription ends with the consensus CCA triplet. This indicates that the source of one of the four bases in the circle junction is probably the terminal A of the tRNA primer used to initiate reverse transcription. We propose that, in HIV-1, removal of the tRNA primer by RNase H cleavage shows an unusual specificity such that cleavage occurs between the terminal rA and the adjacent rC of the tRNA primer. These data also imply that the HIV-1 integration protein removes two bases from each end of the linear viral DNA during integration as has been described for other well-studied retroviruses.
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PMID:Sequence of the circle junction of human immunodeficiency virus type 1: implications for reverse transcription and integration. 169 9


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