Inhibition of hepatitis C virus (HCV) RNA polymerase by DNA aptamers: mechanism of inhibition of in vitro RNA synthesis and effect on HCV-infected cells.

We describe here the further characterization of two DNA aptamers that specifically bind to hepatitis C virus (HCV) RNA polymerase (NS5B) and inhibit its polymerase activity in vitro. Although they were obtained from the same selection procedure and contain an 11-nucleotide consensus sequence, our results indicate that aptamers 27v and 127v use different mechanisms to inhibit HCV polymerase. While aptamer 27v was able to compete with the RNA template for binding to the enzyme and blocked both the initiation and the elongation of RNA synthesis, aptamer 127v competed poorly and exclusively inhibited initiation and postinitiation events.

Small-angle X-ray characterization of the nucleoprotein complexes resulting from DNA-induced oligomerization of HIV-1 integrase.

HIV-1 integrase (IN) catalyses integration of a DNA copy of the viral genome into the host genome. Specific interactions between retroviral IN and long terminal repeats (LTR) are required for this insertion. To characterize quantitatively the influence of the determinants of DNA substrate specificity on the oligomerization status of IN, we used the small-angle X-ray scattering (SAXS) technique.

Targeting HIV-1 integrase with aptamers selected against the purified RNase H domain of HIV-1 RT.

Several in vitro strategies have been developed to selectively screen for nucleic acid sequences that bind to specific proteins. We previously used the SELEX procedure to search for aptamers against HIV-1 RNase H activity associated with reverse transcriptase (RT) and human RNase H1. Aptamers containing G-rich sequences were selected in both cases.

Yeast system as a model to study Moloney murine leukemia virus integrase: expression, mutagenesis and search for eukaryotic partners.

Moloney murine leukemia virus (M-MuLV) integrase (IN) catalyses the insertion of the viral genome into the host chromosomal DNA. The limited solubility of the recombinant protein produced in Escherichia coli led the authors to explore the use of Saccharomyces cerevisiae for expression of M-MuLV IN. IN was expressed in yeast and purified by chromatography on nickel-NTA agarose.

Inhibition of phosphorolysis catalyzed by HIV-1 reverse transcriptase is responsible for the synergy found in combinations of 3'-azido-3'-deoxythymidine with nonnucleoside inhibitors.

In spite of the growing attention to the combined chemotherapy in the treatment of AIDS, the molecular mechanisms underlying the antiviral synergy of combinations of reverse transcriptase (RT) inhibitors are in most cases unknown. Most combinations of nonnucleoside inhibitors (NNRTI) with nucleoside analogues synergistically inhibit HIV-1 replication in cell culture, though they fail to show synergy in enzymatic assays. In this work we have examined the mechanisms mediating the synergy in combinations of AZTTP with NNRTIs on HIV-1 RT and their possible relevance in antiretroviral therapy.

HIV-1 integrase crosslinked oligomers are active in vitro.

The oligomeric state of active human immunodeficiency virus type 1 (HIV-1) integrase (IN) has not been clearly elucidated. We analyzed the activity of the different purified oligomeric forms of recombinant IN obtained after stabilization by platinum crosslinking. The crosslinked tetramer isolated by gel chromatography was able to catalyze the full-site integration of the two viral LTR ends into a target DNA in vitro, whereas the isolated dimeric form of the enzyme was involved in the processing and integration of only one viral end.

Identification by phage display selection of a short peptide able to inhibit only the strand transfer reaction catalyzed by human immunodeficiency virus type 1 integrase.

Human immunodeficiency virus type 1 integrase catalyzes the integration of proviral DNA into the infected cell genome, so it is an important potential target for antiviral drug design. In an attempt to search for peptides that specifically interact with integrase (IN) and inhibit its function, we used an in vitro selection procedure, the phage display technique. A phage display library of random heptapeptides was used to screen for potential peptide ligands of HIV-1 IN.

Selection of DNA aptamers that bind the RNA-dependent RNA polymerase of hepatitis C virus and inhibit viral RNA synthesis in vitro.

The RNA-dependent RNA polymerase (NS5B) of the hepatitis C virus (HCV) plays a key role in the life cycle of the virus. In order to find inhibitors of the HCV polymerase, we screened a library of 81 nucleotide (nt)-long synthetic DNA containing 35 random nucleotides by the Systematic Evolution of Ligands by Exponential enrichment (SELEX) approach. Thirty ligands selected for their binding affinity to the NS5B were classified into four groups on the basis of their sequence homologies.

The lethal phenotype observed after HIV-1 integrase expression in yeast cells is related to DNA repair and recombination events.

Human immunodeficiency virus type 1 (HIV-1) integrase (IN) catalyzes the insertion of the viral genome into the host cell DNA, an essential reaction during the retroviral cycle. We described previously that expression of HIV-1 IN in some yeast strains may lead to the emergence of a lethal phenotype which was not observed when the catalytically crucial residues D, D, (35)E were mutated. The lethal effect in yeast seems to be related to the mutagenic effect of the recombinant HIV-1 IN, most probably via the non-sequence-specific endonucleolytic activity carried by this enzyme.

An oligonucleotide complementary to the SL-B1 domain in the 3'-end of the minus-strand RNA of the hepatitis C virus inhibits in vitro initiation of RNA synthesis by the viral polymerase.

We describe oligonucleotides (ODNs) that inhibit hepatitis C virus (HCV) RNA synthesis in vitro. From a series of 13 ODNs complementary to the 3'-end of the minus-strand HCV RNA, only 4 inhibited RNA synthesis with IC(50) values lower than 1 microM. The inhibition was sequence-specific, since no effect was observed when the ODNs were used with a noncomplementary template.

Non-nucleoside inhibitors of HIV-1 reverse transcriptase inhibit phosphorolysis and resensitize the 3'-azido-3'-deoxythymidine (AZT)-resistant polymerase to AZT-5'-triphosphate.

Removal of 3'-azido-3'deoxythymidine (AZT) 3'-azido-3'-deoxythymidine 5'-monophosphate (AZTMP) from the terminated primer mediated by the human HIV-1 reverse transcriptase (RT) has been proposed as a relevant mechanism for the resistance of HIV to AZT. Here we compared wild type and AZT-resistant (D67N/K70R/T215Y/K219Q) RTs for their ability to unblock the AZTMP-terminated primer by phosphorolysis in the presence of physiological concentrations of pyrophosphate or ATP. The AZT-resistant enzyme, as it has been previously described, showed an increased ability to unblock the AZTMP-terminated primer by an ATP-dependent mechanism.

Dynamic, thermodynamic, and kinetic basis for recognition and transformation of DNA by human immunodeficiency virus type 1 integrase.

Specific interactions between retroviral integrase (IN) and long terminal repeats are required for insertion of viral DNA into the host genome. To characterize quantitatively the determinants of substrate specificity, we used a method based on a stepwise increase in ligand complexity. This allowed an estimation of the relative contributions of each nucleotide from oligonucleotides to the total affinity for IN.

HIV-1 integrase interacts with yeast microtubule-associated proteins.

The human immunodeficiency virus type 1 (HIV-1) integrase (IN) mediates the insertion of viral DNA into the human genome. In addition to IN, cellular and viral proteins are associated to proviral DNA in the so-called preintegration complex (PIC). We previously reported that the expression of HIV-1 IN in yeast leads to the emergence of a lethal phenotype.

Inhibitors of HIV-1 reverse transcriptase and integrase: classical and emerging therapeutical approaches.

The rapid spread of the AIDS epidemic has stimulated the search for new agents able to arrest the replication of the causative virus, HIV. The best strategy for AIDS treatment involves a combination therapy using inhibitors of reverse transcriptase and protease. However, the emergence of HIV-1 strains resistant to these drugs and their cytotoxicity requires the synthesis and the biochemical and cellular characterization of new antiviral drugs, as well as the development of newer strategies and viral targets.