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

We have constructed a plasmid that, when introduced into Escherichia coli, induces the synthesis of large quantities of a polypeptide with an apparent molecular weight of 68 kDa. The HIV-2 reverse transcriptase (RT) made in E. coli is soluble in bacterial extracts and possesses both RNA-dependent DNA polymerase and ribonuclease H (RNase H) activities typical of retroviral RTs. The HIV-2 RT expression clone was used to generate mutations in HIV-2 RT. There is a strong correlation between the effects of individual mutations on the DNA polymerase and RNase H activities. Mutations that profoundly affect the two catalytic functions are not clustered in any particular region of the polypeptide. Those few mutations that selectively affect either the RNase H or the DNA polymerase suggest that, like other retroviral RTs, the DNA polymerase is associated with the amino-terminal portion of HIV-2 RT and the RNase H with the carboxy-terminal portion. Genetically, the HIV-2 RT resembles the HIV-1 RT more closely than it resembles Moloney murine leukemia virus RT. The two catalytic functions of Moloney murine leukemia virus RT can be separately expressed in active form by molecular cloning; those of HIV-1 and HIV-2 RT cannot.
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PMID:Mutational analysis of the DNA polymerase and ribonuclease H activities of human immunodeficiency virus type 2 reverse transcriptase expressed in Escherichia coli. 170 48

2-Chloro-2'-deoxyadenosine 5'-triphosphate (CldATP) was compared with dATP as a substrate for DNA synthesis by bacterial and viral DNA polymerases in vitro. Lengths of chain extension and DNA synthesis pause sites were determined by comparison with products generated by dideoxynucleotide sequencing methods on the same end-labeled primer/template duplex after high-resolution polyacrylamide gel electrophoresis. Reverse transcriptase (RT) from human immunodeficiency virus (HIV-1) and avian myeloblastosis virus (AMV) incorporated CldATP efficiently. DNA strand elongation continued past most chloroadenine (ClA) insertion sites but resulted in shorter chains than when dATP was inserted. Phage T4 DNA polymerase incorporated CldATP least efficiently; Klenow fragment of Escherichia coli DNA polymerase I and modified T7 DNA polymerase (Sequenase) showed intermediate ability to utilize the analogue. Incorporation of several consecutive ClA residues into the replicating strand dramatically reduced the ability of Sequenase, Klenow fragment, and T4 DNA polymerases to continue strand elongation. In the absence of the corresponding normal deoxyribonucleoside triphosphate during DNA synthesis, ClA was frequently misincorporated as thymine, cytosine, or guanine by both AMV RT and HIV-1 RT but rarely, if at all, by Klenow fragment, Sequenase, and T4 DNA polymerase. Except T4, for most DNA polymerases, CldATP at 10-20-fold molar excess over dATP was not a strong competitive inhibitor of dATP, as judged by the amount of strand extension and polymerase pause sites during DNA synthetic reactions. Our results indicate that the degree of strand extension in the presence of CldATP, the number and location of polymerase pause sites, and the amount of misincorporation of the analogue are both polymerase- and sequence-dependent.
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PMID:Effects of 2-chloro-2'-deoxyadenosine 5'-triphosphate on DNA synthesis in vitro by purified bacterial and viral DNA polymerases. 170 19

Two constituent protein domains of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase were expressed separately and purified to homogeneity. The N-terminal domain (p51) behaves as a monomeric protein exhibiting salt-sensitive DNA polymerase activity. The C-terminal domain (p15) on its own has no detectable RNase H activity. However, the combination of both isolated p51 and p15 in vitro leads to reconstitution of RNase H activity on a defined substrate. These results demonstrate that domains of HIV-1 reverse transcriptase are functionally interdependent to a much higher degree than in the case of reverse transcriptase from Moloney murine leukemia virus.
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PMID:Reconstitution in vitro of RNase H activity by using purified N-terminal and C-terminal domains of human immunodeficiency virus type 1 reverse transcriptase. 170 27

The DNA polymerase and RNase H activities of HIV reverse transcriptase are both essential for HIV replication. Although the two activities are both catalyzed by a single polypeptide, they are physically separate; i.e., the DNA polymerase resides in the N-terminal domain whereas the RNase H is localized in the C-terminal domain. The present study was undertaken to characterize the enzymatic properties of these two activities and to determine whether the two catalytic sites are also functionally distinct. We have observed that EGTA specifically stimulates, whereas CaCl2 selectively inhibits, the RNA-dependent DNA polymerase activity but that neither compound has any effect on the RNase H activity of a recombinant HIV reverse transcriptase. The stimulation of the DNA polymerase activity by EGTA is dependent on the Mg2+ concentration; the greatest stimulation is observed at low Mg2+ concentrations. Similarly, the inhibition of DNA polymerase activity by Ca2+ is influenced by Mg2+ concentration. Ca2+ inhibition can be reversed by increasing Mg2+ concentrations, suggesting the possibility that CaCl2 inhibits the reverse transcriptase activity by competing for a metal-binding site on the enzyme. The pyrophosphate analogue phosphonoformate selectively inhibits the polymerase activity but not the RNase H activity of HIV reverse transcriptase. In contrast, the RNase H activity can be selectively inhibited by deoxyadenosine 5'-monophosphate, whereas the DNA polymerase activity is not inhibited. These results suggest that the DNA polymerase and RNase activities are not only physically separate but that they are also functionally distinct.
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PMID:Functional characterization of RNA-dependent DNA polymerase and RNase H activities of a recombinant HIV reverse transcriptase. 170 16

A phosphorothioate homocytidine 10-mer containing a cholesteryl moiety covalently linked to the 5'-end (Chol-SdC10) inhibited syncytium formation in susceptible T cells induced by HIV-1 and HIV-2. The syncytium inhibition effect was minimal with unmodified cytidine homopolymer of the same net charge. Chol-SdC10 was shown to protect CEM cells against infection by cell-free HIV-1 particles without any apparent toxicity to the growth of CD4+ T cells. The DNA polymerase activity of the purified reverse transcriptase (RT) of HIV-1 was markedly inhibited by Chol-SdC10 but the effect on the RNase H activity of RT was minimal. Analysis of the kinetics of reverse transcriptase inhibition mediated by the drug revealed that the inhibition at a higher concentration was competitive with respect to template primer binding and noncompetitive at lower concentrations. Chol-SdC10 also partially blocked the binding of gp120 to CD4 in a solid-phase ELISA. These results confirm that the anti-HIV activity of phosphorothioate cytidine homopolymers increases markedly by covalent modification with the cholesteryl moiety at the 5'-end and demonstrates that the cytoprotective effect is manifested at multiple steps in the virus life cycle. These steps include inhibition of retroviral replication activity as well as the binding and fusion of HIV with CD4+ T cells.
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PMID:Mode of action of 5'-linked cholesteryl phosphorothioate oligodeoxynucleotides in inhibiting syncytia formation and infection by HIV-1 and HIV-2 in vitro. 170 17

The pyrophosphate analogue, foscarnet, selectively inhibits the DNA polymerase of human herpes viruses, including cytomegalovirus, and the reverse transcriptase of HIV. Viral replication is therefore prevented, but resumes when the drug is cleared from infected cells. In vitro, the combination of foscarnet and zidovudine (azidothymidine) has an additive effect against cytomegalovirus and acts synergistically against HIV. An improvement in cytomegalovirus retinitis is obtained in over 85% of affected AIDS patients during foscarnet induction therapy, but relapse usually occurs within a month of ceasing treatment. There is a similar duration of remission during maintenance therapy given for 5 days each week, but this can be extended 4- to 5-fold with daily administration of higher doses. In allograft recipients, progression of retinitis can be halted by foscarnet until immune function recovers and eradicates the virus. The incidence of acute renal failure, which is common during foscarnet therapy, may be reduced by dosage adjustment and adequate prehydration. Anaemia, phlebitis, nausea and vomiting, and disturbances in serum calcium and phosphate levels, perhaps resulting from uptake of foscarnet into bone or chelation with ionised calcium, have also been associated with administration of the drug. Cytomegalovirus retinitis is difficult to treat, with few therapeutic options available. Although treatment with foscarnet produces some severe adverse effects, with care these can be minimised, and the drug produces clinical improvement in a large proportion of patients; this is a highly encouraging finding at this stage in its development. Preliminary comparative data indicate that foscarnet and ganciclovir are similarly effective, but foscarnet may have some theoretical advantages in AIDS patients since it can be used in combination with zidovudine without potentiating myelosuppression.
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PMID:Foscarnet. A review of its antiviral activity, pharmacokinetic properties and therapeutic use in immunocompromised patients with cytomegalovirus retinitis. 170 82

The crystal structure of the ribonuclease (RNase) H domain of HIV-1 reverse transcriptase (RT) has been determined at a resolution of 2.4 A and refined to a crystallographic R factor of 0.20. The protein folds into a five-stranded mixed beta sheet flanked by an asymmetric distribution of four alpha helices. Two divalent metal cations bind in the active site surrounded by a cluster of four conserved acidic amino acid residues. The overall structure is similar in most respects to the RNase H from Escherichia coli. Structural features characteristic of the retroviral protein suggest how it may interface with the DNA polymerase domain of p66 in the mature RT heterodimer. These features also offer insights into why the isolated RNase H domain is catalytically inactive but when combined in vitro with the isolated p51 domain of RT RNase H activity can be reconstituted. Surprisingly, the peptide bond cleaved by HIV-1 protease near the polymerase-RNase H junction of p66 is completely inaccessible to solvent in the structure reported here. This suggests that the homodimeric p66-p66 precursor of mature RT is asymmetric with one of the two RNase H domains at least partially unfolded.
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PMID:Crystal structure of the ribonuclease H domain of HIV-1 reverse transcriptase. 184 17

The presence of pre-S1 proteins in peripheral blood mononuclear cell (PBMC) samples from 115 patients with different forms of hepatitis B virus (HBV) infection was investigated by Western blot. Among 67 chronic HBsAg carriers, HBV antigens were detected in the PBMC in 80% for HBsAg, 27% for HBc/e Ag and 34% for pre-S1 proteins. The detection of pre-S1 proteins in PBMC was significantly associated with the presence of serum markers of HBV replication (HBV DNA and/or DNA polymerase). In the group of 48 consecutive patients negative for serum HBsAg, but positive for anti-HBc with or without anti-HBs, HBsAg and pre-S1 proteins could be detected in PBMC. This finding was more frequent among anti-HIV-positive patients (77 and 23% of the cases, respectively) than in the negative ones (23 and 4% of the cases, respectively). The detection of HBV DNA and polyadenylated RNA in some of the PBMC samples positive for HBV proteins suggests that these proteins may be expressed in PBMC, especially during intense HBV replication. In patients negative for serum HBsAg, PBMC may constitute a reservoir of HBV.
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PMID:Detection of pre-S1 proteins in peripheral blood mononuclear cells from patients with HBV infection. 171 Oct 65

A new class of compounds, 9-[(2RS)-3-fluoro-2-phosphonylmethoxypropyl] [(RS)-FPMP] derivatives of purines, is described that has selective activity against a broad spectrum of retroviruses [including human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2)] but not other RNA or DNA viruses. This activity spectrum is completely different from that of the parental compounds, 9-[(2S)-3-hydroxy-2-phosphonylmethoxypropyl] [(S)-HPMP] derivatives of purines, which are active against a broad range of DNA viruses. The racemic (RS)-FPMP derivatives of adenine and 2,6-diaminopurine, termed (RS)-FPMPA and (RS)-FPMPDAP, respectively, are markedly more selective as in vitro antiretroviral agents than their 9-(2-phosphonylmethoxyethyl) (PME) counterparts, PMEA and PMEDAP. Also, (RS)-FPMPA and (RS)-FPMPDAP have a substantially higher therapeutic index in mice in inhibiting Moloney murine sarcoma virus-induced tumor formation and associated death and are markedly less inhibitory to human bone marrow cells than PMEA and PMEDAP. The diphosphate derivative of (RS)-FPMPA [(RS)-FPMPApp] is a potent and selective inhibitor of HIV-1 reverse transcriptase but not of HSV-1 DNA polymerase or DNA polymerase alpha. (RS)-FPMPApp, akin to PMEA diphosphate (PMEApp), acts as a DNA chain terminator. The DNA chain-terminating properties of PMEApp and (RS)-FPMPApp seem to be a prerequisite for acyclic nucleoside phosphonates to exhibit antiretrovirus (i.e., anti-HIV) activity.
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PMID:9-[(2RS)-3-fluoro-2-phosphonylmethoxypropyl] derivatives of purines: a class of highly selective antiretroviral agents in vitro and in vivo. 171 Dec 14

The human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) is a protein of 66 kDa, p66, which contains two domains, an amino-terminal DNA polymerase and an RNase H at the carboxy terminus of the molecule. In order to characterize the mode of action of the RNase H, two previously described mutant enzymes were used, with substitutions in the highly conserved histidine 539, which was mutated to the neutral amino acid asparagine and to the negatively charged aspartate. The purified wild-type (wt) and mutant (mt) enzyme activities are analyzed here using RNA-DNA hybrids consisting of in vitro transcribed RNA that harbors the polypurine tract (PPT) from HIV-1 and DNA oligonucleotides complementary to the PPT or to other regions of the RNA. Analysis of the radioactively labeled RNA of these model hybrids after RNase H treatment indicates that both, wt and mt enzymes, are capable of cleaving the RNA in an endonucleolytic manner. The mt enzymes exhibit a severely reduced exonuclease activity. They are more sensitive towards salt and competition with excess of unlabeled hybrid, suggesting a reduced substrate binding affinity. DNA elongation by the RT is coupled with RNA hydrolysis by the 3'-5' exonuclease of the wt RNase H. The RNase Hmt of the mt enzymes, however, does not exhibit such processive 3'-5' exonuclease activity during DNA synthesis but gives rise to sporadic endonucleolytic cuts, whereas the RT is not affected. The endonuclease activities of the RNase H mt enzymes exhibit cleavage preferences in the absence or presence of DNA synthesis different from those of the wt enzyme. They cannot recognize specific sequences required to generate a PPT-primer and therefore cannot initiate plus-strand DNA synthesis in vitro at the 3' end of the PPT, which is essential for viral replication.
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PMID:Mutations of a conserved residue within HIV-1 ribonuclease H affect its exo- and endonuclease activities. 171 5


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