Substrate variations that affect the nucleic acid clamp activity of reverse transcriptases.

We have recently shown that reverse transcriptases (RTs) perform template switches when there is a very short (two-nucleotide) complementarity between the 3' ends of the primer (donor) strand and the DNA or RNA template acceptor strands [Oz-Gleenberg et al. (2011) Nucleic Acids Res 39, 1042-1053]. These dinucleotide pairs are stabilized by RTs that are capable of 'clamping' together the otherwise unstable duplexes. This RT-driven stabilization of the micro-homology sequence promotes efficient DNA synthesis. In the present study, we have examined several factors associated with the sequence and structure of the DNA substrate that are critical for the clamp activity of RTs from human immunodeficiency virus type 1 (HIV-1), murine leukemia virus (MLV), bovine immunodeficiency virus (BIV) and the long terminal repeat retrotransposon Tf1. The parameters studied were the minimal complementarity length between the primer and functional template termini that sustains stable clamps, the effects of gaps between the two template strands on the clamp activity of the tested RTs, the effects of template end phosphorylations on the RT-associated clamp activities, and clamp activity with a long 'hairpin' double-stranded primer comprising both the primer and the complementary non-functional template strands. The results show that the substrate conditions for clamp activity of HIV-1 and MLV RTs are more stringent, while Tf1 and BIV RTs show clamp activity under less rigorous substrate conditions. These differences shed light on the dissimilarities in catalytic activities of RTs, and suggest that clamp activity may be a potential new target for anti-retroviral drugs.

Template-independent DNA synthesis activity associated with the reverse transcriptase of the long terminal repeat retrotransposon Tf1.

Reverse transcriptases (RTs) possess a non-templated addition (NTA) activity while synthesizing DNA with blunt-ended DNA primer/templates. Interestingly, the RT of the long terminal repeat retrotransposon Tf1 has an NTA activity that is substantially higher than that of HIV-1 or murine leukemia virus RTs. By performing steady state kinetics, we found that the differences between the NTA activities of Tf1 and HIV-1 RTs can be explained by the substantially lower K(M) value for the incoming dNTP of Tf1 RT (while the differences between the apparent k(cat) values of these two RTs are relatively small). Furthermore, the K(M) values, calculated for both RTs with the same dNTP, are much lower for the template-dependent synthesis (TDS) than those of NTA. However, TDS of HIV-1 RT is higher than that of Tf1 RT. The overall relative order of the apparent k(cat)/K(M) values for dATP is: HIV-1 RT (TDS) > Tf1 RT (TDS) >> Tf1 RT (NTA) > HIV-1 RT (NTA). Under the employed conditions, Tf1 RT can add up to seven nucleotides to the blunt-ended substrate, while the other RTs add mostly a single nucleotide. The NTA activity of Tf1 RT is restricted to DNA primers. Furthermore, the NTA activity of Tf1 and HIV-1 RTs is suppressed by ATP, as it competes with the incoming dATP (although ATP is not incorporated by the NTA activity of the RTs). The unusually high NTA activity of Tf1 RT can explain why, after completing cDNA synthesis, the in vivo generated Tf1 cDNA has relatively long extra sequences beyond the highly conserved CA at its 3'-ends.

Strand selections resulting from the combined template-independent DNA synthesis and clamp activities of HIV-1 reverse transcriptase.

The reverse transcriptase (RT) of HIV-1 has a non-templated addition (NTA) activity and can perform template switches with a very short (even two nucleotides) complementarity between the 3'-ends of the primer donor strand and the template acceptor strands. We have studied how the combination of several pivotal parameters can all lead to strand switches during DNA synthesis by HIV-1 RT. These include dNTP bias in the NTA step, the availability of acceptor strands with 3'-end sequences complementary to the de novo-generated primer tails and the stabilities of the clamped duplexes formed between these primer tails and the acceptor strands.

Reverse transcriptases can clamp together nucleic acids strands with two complementary bases at their 3'-termini for initiating DNA synthesis.

We present evidence that the reverse transcriptase (RT) of human immunodeficiency virus type-1 stabilizes in vitro very short (2-nt) duplexes of 3'-overhangs of the primer strand that are annealed to complementary dinucleotides tails of DNA or RNA template strands, provided that these sequences contain at least one C or G. This RT-induced strand 'clamping' activity promotes RT-directed DNA synthesis. This function is achieved only when the functional template strand is adjacent to a second DNA or RNA segment, annealed upstream to most of the primer (without gaps). The combined clamp/polymerase activity is typical to RTs, as it was found in different RTs from diverse retroviral groups, whereas cellular DNA-polymerases (devoid of 3'→5' exonucleolytic activity) showed no clamp activity. The clamp-associated DNA-binding activity is markedly stabilized by dGTP, even when dGTP is not incorporated into the nascent DNA strand. The hereby-described function can help RTs in bridging over nicks in the copied RNA or DNA templates, encountered during reverse transcription. Moreover, the template-independent blunt-end synthesis of RTs can allow strand transfers onto compatible acceptor strands while synthesizing DNA. These RT properties can shed light on potentially-new roles of RTs in the reverse-transcription process and define new targets for anti-retroviral drugs.

Quantitative analysis of the interactions between HIV-1 integrase and retroviral reverse transcriptases.

The RT (reverse transcriptase) of HIV-1 interacts with HIV-1 IN (integrase) and inhibits its enzymatic activities. However, the molecular mechanisms underling these interactions are not well understood. In order to study these mechanisms, we have analysed the interactions of HIV-1 IN with HIV-1 RT and with two other related RTs: those of HIV-2 and MLV (murine-leukaemia virus). All three RTs inhibited HIV-1 IN, albeit to a different extent, suggesting a common site of binding that could be slightly modified for each one of the studied RTs. Using surface plasmon resonance technology, which monitors direct protein-protein interactions, we performed kinetic analyses of the binding of HIV-1 IN to these three RTs and observed interesting binding patterns. The interaction of HIV-1 RT with HIV-1 IN was unique and followed a two-state reaction model. According to this model, the initial IN-RT complex formation was followed by a conformational change in the complex that led to an elevation of the total affinity between these two proteins. In contrast, HIV-2 and MLV RTs interacted with IN in a simple bi-molecular manner, without any apparent secondary conformational changes. Interestingly, HIV-1 and HIV-2 RTs were the most efficient inhibitors of HIV-1 IN activity, whereas HIV-1 and MLV RTs showed the highest affinity towards HIV-1 IN. These modes of direct protein interactions, along with the apparent rate constants calculated and the correlations of the interaction kinetics with the capacity of the RTs to inhibit IN activities, are all discussed.

Mechanism of inhibition of HIV-1 reverse transcriptase by the novel broad-range DNA polymerase inhibitor N-{2-[4-(aminosulfonyl)phenyl]ethyl}-2-(2-thienyl)acetamide.

Employing a novel strategy, we have virtually screened a large library of compounds to identify novel inhibitors of the reverse transcriptase (RT) of HIV-1. Fifty-six top scored compounds were tested in vitro, and two of them inhibited efficiently the DNA polymerase activity of RT. The most effective compound, N-{2-[4-(aminosulfonyl)phenyl]ethyl}-2-(2-thienyl)acetamide (NAPETA), inhibited both RNA-dependent and DNA-dependent DNA polymerase activities, with apparent IC50 values of 1.2 and 2.1 microM, respectively. This inhibition was specific to the RT-associated polymerase activity and did not affect the RNase H activity. NAPETA also inhibited two drug-resistant HIV-1 RT mutants as well as HIV-2 RT and other DNA polymerases. Kinetic analysis of RT inhibition indicated that the DNA polymerase activity of HIV-1 RT was inhibited in a classic noncompetitive manner with respect to dTTP, demonstrating a Ki value of 1.2 microM. In contrast, the inhibition with respect to the RNA.DNA template was a mixed linear type with a Ki value of 0.12 microM and was not affected by the order in which the template.primer and inhibitor were added to the reaction mixture. Gel shift and surface plasmon resonance analyses confirmed that NAPETA interfered with the formation of the RT.DNA complex (that is crucial for the polymerization activity) by reducing the affinity of RT for DNA, accounting at least partially for the inhibition. It is likely that NAPETA inhibited RT via a mechanism that is different from that of the classic non-nucleoside RT inhibitors used for treating AIDS/HIV patients and, thus, may serve as a lead compound for the development of novel anti-HIV drugs.

Ile178 of HIV-1 reverse transcriptase is critical for inhibiting the viral integrase.

HIV-1 reverse transcriptase (RT) was shown to inhibit in vitro the viral integrase (IN). We have reported previously that an RT-derived 20-residue peptide binds IN and inhibits its enzymatic activities. In this peptide, Leu168, Phe171, Gln174, and Ile178 were predicted to be involved in IN inhibition. In the presented study, these residues were mutagenized and the resulting peptides were tested for binding and inhibiting IN activities. Ile178 was found to be the major contributor to IN inhibition, probably by interacting with IN residue Gly149. As Gly149 is a key IN residue, this inhibition probably results from a steric hindrance of the IN active site.

Inhibition of human immunodeficiency virus type-1 reverse transcriptase by a novel peptide derived from the viral integrase.

Previous studies show that the reverse transcriptase (RT) of human immunodeficiency virus type-1 (HIV-1) and RT-derived peptides interact with and inhibit the viral integrase (IN). In the present study, we have performed the complementary study by screening a complete library of HIV-1 IN-derived peptides for their effects on the RT. We have identified a 20-residues long peptide, derived from the IN (residues 46-65) that binds the RT and inhibits its DNA-polymerase activities (without affecting the ribonuclease-H activity). The full 20-residues sequence is required for maximal inhibition. This inhibition is non-competitive and probably results from obstructing the formation of RT-DNA complexes by the peptide. The data and the molecular docking model presented suggest that this inhibition is probably caused by a steric hindrance or conformational changes of the RT. These results can facilitate the development of novel and specific peptide-based HIV-1 RT inhibitors that might help in the fight against AIDS.

Peptides derived from the reverse transcriptase of human immunodeficiency virus type 1 as novel inhibitors of the viral integrase.

Recent studies have shown that the integrase (IN) of HIV-1 is inhibited in vitro by HIV-1 reverse transcriptase (RT). We further investigated the specific protein sequences of RT that were involved in this inhibition by screening a complete library of RT-derived peptides for their inhibition of IN activities. Two 20-residue peptides, peptide 4286, derived from the RT DNA polymerase domain, and the one designated 4321, from the RT ribonuclease H domain, inhibit the enzymatic activities of IN in vitro. The former peptide inhibits all three IN-associated activities (3'-end processing, strand transfer, and disintegration), whereas the latter one inhibits primarily the first two functions. We showed the importance of the sequences and peptide length for the effective inhibition of IN activities. Binding assays of the peptides to IN (with no DNA substrate present) indicated that the two inhibitory peptides (as well as several non-inhibitory peptides) interact directly with IN. Moreover, the isolated catalytic core domain of IN also interacted directly with the two inhibitory peptides. Nevertheless, only peptide 4286 can inhibit the disintegration activity associated with the IN core domain, because this activity is the only one exhibited by this domain. This result was expected from the lack of inhibition of disintegration of full-length IN by peptide 4321. The data and the three-dimensional models presented suggested that the inhibition resulted from steric hindrance of the catalytic domain of IN. This information can substantially facilitate the development of novel drugs against HIV INs and thus contribute to the fight against AIDS.