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. These results illustrate the power of the selective evolution of ligands by exponential enrichment in vitro selection procedure approach to select specific short DNA aptamers able to inhibit HCV NS5B by different mechanisms. We also determined that, in addition to an in vitro inhibitory effect on RNA synthesis, aptamer 27v was able to interfere with the multiplication of HCV JFH1 in Huh7 cells. The efficient cellular entry of these short DNAs and the inhibitory effect observed on human cells infected with HCV indicate that aptamers are useful tools for the study of HCV RNA synthesis, and their use should become a very attractive and alternative approach to therapy for HCV infection.

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. Under certain conditions in the absence of ODNs IN existed only as monomers. IN preincubation with specific ODNs led mainly to formation of dimers, the relative amount of which correlated well with the increase in the enzyme activity in the 3'-processing reaction. Under these conditions, tetramers were scarce. Non-specific ODNs stimulated formation of catalytically inactive dimers and tetramers. Complexes of monomeric, dimeric and tetrameric forms of IN with specific and non-specific ODNs had varying radii of gyration (R(g)), suggesting that the specific sequence-dependent formation of IN tetramers can probably occur by dimerization of two dimers of different structure. From our data we can conclude that the DNA-induced oligomerization of HIV-1 IN is probably of importance to provide substrate specificity and to increase the enzyme activity.

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. To investigate whether the interaction with G-rich oligonucleotides (ODNs) was a characteristic of these enzymes, a second in vitro selection was performed with an isolated RNase H domain of HIV-1 RT (p15) as a target and a new DNA library. In this work we found that the second SELEX led again to the isolation of G-rich aptamers. But in contrast to the first selection, these latter ODNs were not able to inhibit the RNase H activity of either the p15 domain or the RNase H embedded in the complete RT. On the other hand, the aptamers from the first SELEX that were inhibitors of the RT-associated RNase H did not inhibit the activity of the isolated p15 domain. This suggests that the active conformation of both RNase H domains is different according to the presence or absence of the DNA polymerase domain. HIV-1 RNase H and integrase both belong to the phosphotransferase family and share structural similarities. An interesting result was obtained when the DNA aptamers initially raised against p15 RNase H were assayed against HIV-1 integrase. In contrast to RNase H, the HIV-1 integrase was inhibited by these aptamers. Our results point out that prototype structures can be exploited to develop inhibitors of two related enzymes.

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. IN migrated as a single band in SDS-PAGE and did not contain IN degradation products. The enzyme was about twofold more active than the enzyme purified from E. coli and was free of nucleases. Using the yeast system, the substitution of the putative catalytic amino acid Asp184 by alanine was also analysed. The mutated enzyme was inactive in the in vitro assays. This is the first direct demonstration that mutation of Asp184 inactivates M-MuLV IN. Finally, S. cerevisiae was used as a model to assess the ability of M-MuLV IN to interact with eukaryotic protein partners. The expression of an active M-MuLV IN in yeast strains deficient in RAD52 induced a lethal effect. This phenotype could be attributed to cellular damage, as suggested by the viability of cells expressing inactive D184A IN. Furthermore, when active IN was expressed in a yeast strain lacking the ySNF5 transcription factor, the lethal effect was abolished, suggesting the involvement of ySNF5 in the cellular damage induced by IN. These results indicate that S. cerevisiae could be a useful model to study the interaction of IN with cellular components in order to identify potential counterparts of the natural host.

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. We found that if two inhibitors bind either to different sites on the RT or to the same site but to different mechanistic forms, it is always possible to find conditions in which their combination results in synergistic inhibition of DNA polymerase activity. Though these analyses are interesting from a biochemical point of view, this kind of synergy is unlikely to play any role in vivo, since this positive interaction is lost under the conditions present in viral replication. Here we describe that the synergy found for combinations of NNRTI with AZT is due not to the inhibition of the DNA polymerase activity but to the inhibition of the RT-catalyzed phosphorolysis by the NNRTI. While phosphorolytical removal of the AZT-terminated primer has been related to the mechanism of resistance toward AZT, our data suggest that a basal phosphorolysis occurs even with the wild-type enzyme, and that the inhibition of this activity could explain the synergy found in antiviral assays.

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. Accurate concerted integration by IN tetramers was confirmed by cloning and sequencing. Kinetic studies of DNA-integrase complexes led us to propose a model explaining the formation of an active complex. Our data suggest that the tetrameric IN bound to the viral DNA ends is the minimal complex involved in the concerted integration of both LTRs and should be the oligomeric form targeted by future inhibitors.

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. Several phage clones were identified that specifically bound IN. Two of the selected peptides (FHNHGKQ and HLEHLLF) exhibited a high affinity for IN and were chemically synthesized. High affinity was confirmed by a displacement assay which showed that these two synthetic peptides were able to compete with the phages expressing the corresponding peptide. These agents were assayed on the in vitro IN activities. While none of them inhibited the 3'-processing reaction, the FHNHGKQ peptide was found to be an inhibitor of the strand transfer reaction. Despite its high affinity for IN, the HLEHLLF peptide selected and assayed under the same conditions was unable to inhibit this reaction. We showed that the FHNHGKQ peptide inhibits specifically the strand transfer activity by competing with the target DNA for binding to IN. These IN-binding agents could be used as a base for developing new anti-integrase compounds as well as for structural studies of the still unknown three-dimensional structure of the entire integrase molecule.

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. This non-sequence-specific endonuclease activity was further characterized. Although the enzyme was active on DNA substrates devoid of viral long terminal repeat (LTR) sequences, the presence of LTR regions stimulated significantly this activity. Genetic experiments were designed to show that both the mutagenic effect and the level of recombination events were affected in cells expressing the active retroviral enzyme, while expression of the mutated inactive IN D116A has no significant effect. A close interaction was demonstrated between integrase activity and in vivo/in vitro recombination process, suggesting that retroviral integration and recombination mechanism are linked in the infected cell. Our results show that the yeast system is a powerful cellular model to study the non-sequence-specific endonucleolytic activity of IN. Its characterization is essential since this activity might represent a very important step in the retroviral infectious cycle and would provide further insights into the function of IN. Indeed, effectors of this activity should be sought as potential antiviral agents since stimulation of this enzymatic activity would induce the destruction of early synthesized proviral DNA.

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. The introduction of a 2'-O-methyl modification increased the inhibitor activity 11-fold (IC(50) = 50 nM) in just 1 (ODN7) of the 4 inhibitory ODNs. ODNs did not inhibit RNA synthesis by interfering with the elongation process as no short RNAs products were detected. We also show that ODN7 did not prevent binding of NS5B to the template or cause polymerase trapping by the duplex RNA/ODN. Our data demonstrate that ODN7 inhibits the initiation process, most probably by modifying structural features present at the 3'-end of the minus-strand RNA.

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. We found that only mutations in the p66 subunit were responsible for this ability. We also found that three structurally divergent non-nucleoside reverse transcriptase inhibitor (NNRTI), nevirapine, TIBO, and a 4-arylmethylpyridinone derivative, were able to inhibit the phosphorolytic activity of the enzyme, rendering the AZT-resistant RT sensitive to AZTTP. The 4-arylmethylpyridinone derivative proved to be about 1000-fold more potent in inhibiting phosphorolysis than nevirapine or TIBO. Moreover, combinations of AZTTP with NNRTIs exhibited an exceptionally high degree of synergy in the inhibition of AZT-resistant enzyme only when ATP or PPi were present, indicating that inhibition of phosphorolysis was responsible for the synergy found in the combination. Our results not only demonstrate the importance of phosphorolysis concerning HIV-1 RT resistance to AZT but also point to the implication of this activity in the strong synergy found in some combinations of NNRTIs with AZT.

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. The interaction of HIV-1 integrase with specific (containing sequences from the LTR) or nonspecific oligonucleotides was analyzed using a thermodynamic model. Integrase interacted with oligonucleotides through a superposition of weak contacts with their bases, and more importantly, with the internucleotide phosphate groups. All these structural components contributed in a combined way to the free energy of binding with the major contribution made by the conserved 3'-terminal GT, and after its removal, by the CA dinucleotide. In contrast to nonspecific oligonucleotides that inhibited the reaction catalyzed by IN, specific oligonucleotides enhanced the activity, probably owing to the effect of sequence-specific ligands on the dynamic equilibrium between the oligomeric forms of IN. However, after preactivation of IN by incubation with Mn(2+), the specific oligonucleotides were also able to inhibit the processing reaction. We found that nonspecific interactions of IN with DNA provide approximately 8 orders of magnitude in the affinity (Delta G degrees approximately equal to -10.3 kcal/mol), while the relative contribution of specific nucleotides of the substrate corresponds to approximately 1.5 orders of magnitude (Delta G degrees approximately equal to - 2.0 kcal/mol). Formation of the Michaelis complex between IN and specific DNA cannot by itself account for the major contribution of enzyme specificity, which lies in the k(cat) term; the rate is increased by more than 5 orders of magnitude upon transition from nonspecific to specific oligonucleotides.

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. Among the selected molecules, two were able to inhibit in vitro the polymerase activity of the HCV NS5B. These aptamers appeared to be specific for HCV polymerase, as no inhibition of poliovirus 3D polymerase activity was observed. The binding and inhibitory potential of one aptamer (27v) was associated with the 35 nt-long variable region. This oligonucleotide displayed an apparent dissociation constant (K(d)) in the nanomolar range. Our results showed that it was able to compete with RNA templates corresponding to the 3'-ends of the (+) and the (-) HCV RNA for binding to the polymerase. The fact that a DNA aptamer could interfere with the binding of natural templates of the enzyme could help in performing structure-function analysis of the NS5B and might constitute a basis for further structure-based drug design of this crucial enzyme of HCV replication.

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. This effect may be linked to the IN activity on infected human cells where integration requires the cleavage of genomic DNA. To isolate and characterize potential cellular partners of HIV-1 IN, we used it as a bait in a two-hybrid system with a yeast genomic library. IN interacted with proteins belonging to the microtubule network, or involved in the protein synthesis apparatus. We focused our interest on one of the selected inserts, L2, which corresponds to the C-end half of the yeast STU2p, a microtubule-associated protein (MAP). STU2p is an essential component of the yeast spindle pole body (SPB), which is able to bind microtubules in vitro. After expressing and purifying L2 as a recombinant protein, we showed its binding to IN by ELISA immunodetection. L2 was also able to inhibit IN activity in vitro. In addition, the effect of L2 was tested using the "lethal yeast phenotype". The coexpression of IN and the L2 peptide abolished the lethal phenotype, thus showing important in vivo interactions between IN and L2. The identification of components of the microtubule network associated with IN suggest a role of this complex in the transport of HIV-1 IN present in the PIC to the nucleus, as already described for other human viruses.

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. In addition to reverse transcriptase and protease, other retroviral enzymes acting in the replicative cycle of HIV-1 are potential targets for chemotherapeutic intervention. Like all retroviruses, HIV-1 requires the integration of the proviral double-stranded DNA, arising from the reverse transcription step, into the host chromosome for its efficient replication, maintenance of a stably infected state and productive infection. DNA integration is carried out by integrase so this enzyme represents a key area in developing new anti-retroviral therapy. Another novel enzymatic target concerns the RNase H activity associated with the retroviral reverse transcriptase, since a functional RNase H is essential for retroviral replication. Inhibitors against HIV-1 integrase and RNase H having potential therapeutical propeties have not yet been described. We focus this review on the properties of inhibitors of reverse transcriptase and integrase. Some of these antiviral agents have been known for several years while others are emerging as new promising strategies based on the use of oligonucleotides with special emphasis on the SELEX approach, peptides and retrovirucides.