Inhibition of the integrases of human immunodeficiency viruses type 1 and type 2 by reverse transcriptases

2002 ◽  
Vol 361 (3) ◽  
pp. 557-566 ◽  
Author(s):  
Iris OZ ◽  
Orna AVIDAN ◽  
Amnon HIZI
Keyword(s):  
Protein Structures ◽  
Three Dimensional ◽  
Rnase H ◽  
Strand Transfer ◽  
Leukaemia Virus ◽  
Reverse Transcriptases ◽  
Show Evidence ◽  
Human Immunodeficiency Viruses ◽  
Hiv 1

We present evidence that the integrases (INs) of HIV types 1 and 2 are inhibited in vitro by the reverse transcriptases (RTs) of HIV-1, HIV-2 and murine leukaemia virus. Both 3′-end processing and 3′-end joining (strand transfer) activities of IN were affected by the RTs. Full inhibitions were accomplished with most RT and IN combinations tested at around equimolar RT/IN ratios. The disintegration activity of IN was also inhibited by RTs. Neither DNA synthesis nor the ribonuclease H (RNase H) domain of RT were involved in IN inhibition, since specific DNA polymerase inhibitors did not affect the level of IN inhibition, and the p51 isoform of HIV-1 RT (which lacks the RNase H domain) is as effective in inhibiting IN as the heterodimeric p66/p51 isoform. On the other hand, the catalytic activities of HIV RTs were not affected by the INs, showing that RTs can inhibit IN activities, whereas INs do not inhibit RTs. We postulate that sequences and/or three-dimensional protein structures common to RTs interact with INs and inhibit their activities. We show evidence for this hypothesis and discuss the possible sites of IN involved in this interaction.

scholarly journals A Novel Leu92 Mutant of HIV-1 Reverse Transcriptase with a Selective Deficiency in Strand Transfer Causes a Loss of Viral Replication

2015 ◽  
Vol 89 (16) ◽  
pp. 8119-8129 ◽  
Author(s):  
Eytan Herzig ◽  
Nickolay Voronin ◽  
Nataly Kucherenko ◽  
Amnon Hizi
Keyword(s):  
Life Cycle ◽  
Viral Replication ◽  
Reverse Transcriptase ◽  
Dna Polymerase ◽  
Reverse Transcription ◽  
Polymerase Activity ◽  
Rnase H ◽  
Strand Transfer ◽  
Hiv 1

ABSTRACTThe process of reverse transcription (RTN) in retroviruses is essential to the viral life cycle. This key process is catalyzed exclusively by the viral reverse transcriptase (RT) that copies the viral RNA into DNA by its DNA polymerase activity, while concomitantly removing the original RNA template by its RNase H activity. During RTN, the combination between DNA synthesis and RNA hydrolysis leads to strand transfers (or template switches) that are critical for the completion of RTN. The balance between these RT-driven activities was considered to be the sole reason for strand transfers. Nevertheless, we show here that a specific mutation in HIV-1 RT (L92P) that does not affect the DNA polymerase and RNase H activities abolishes strand transfer. There is also a good correlation between this complete loss of the RT's strand transfer to the loss of the DNA clamp activity of the RT, discovered recently by us. This finding indicates a mechanistic linkage between these two functions and that they are both direct and unique functions of the RT (apart from DNA synthesis and RNA degradation). Furthermore, when the RT's L92P mutant was introduced into an infectious HIV-1 clone, it lost viral replication, due to inefficient intracellular strand transfers during RTN, thus supporting thein vitrodata. As far as we know, this is the first report on RT mutants that specifically and directly impair RT-associated strand transfers. Therefore, targeting residue Leu92 may be helpful in selectively blocking this RT activity and consequently HIV-1 infectivity and pathogenesis.IMPORTANCEReverse transcription in retroviruses is essential for the viral life cycle. This multistep process is catalyzed by viral reverse transcriptase, which copies the viral RNA into DNA by its DNA polymerase activity (while concomitantly removing the RNA template by its RNase H activity). The combination and balance between synthesis and hydrolysis lead to strand transfers that are critical for reverse transcription completion. We show here for the first time that a single mutation in HIV-1 reverse transcriptase (L92P) selectively abolishes strand transfers without affecting the enzyme's DNA polymerase and RNase H functions. When this mutation was introduced into an infectious HIV-1 clone, viral replication was lost due to an impaired intracellular strand transfer, thus supporting thein vitrodata. Therefore, finding novel drugs that target HIV-1 reverse transcriptase Leu92 may be beneficial for developing new potent and selective inhibitors of retroviral reverse transcription that will obstruct HIV-1 infectivity.

scholarly journals Peptides Derived from the Reverse Transcriptase of Human Immunodeficiency Virus Type 1 as Novel Inhibitors of the Viral Integrase

2005 ◽  
Vol 280 (23) ◽  
pp. 21987-21996 ◽  
Author(s):  
Iris Oz Gleenberg ◽  
Orna Avidan ◽  
Yehuda Goldgur ◽  
Alon Herschhorn ◽  
Amnon Hizi
Keyword(s):  
Reverse Transcriptase ◽  
Catalytic Domain ◽  
Three Dimensional ◽  
Specific Protein ◽  
Strand Transfer ◽  
Catalytic Core ◽  
Core Domain ◽  
Inhibitory Peptides ◽  
Hiv 1

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.

scholarly journals RNase H Requirements for the Second Strand Transfer Reaction of Human Immunodeficiency Virus Type 1 Reverse Transcription

1999 ◽  
Vol 73 (8) ◽  
pp. 6573-6581 ◽  
Author(s):  
Christine M. Smith ◽  
Jeffrey S. Smith ◽  
Monica J. Roth
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcription ◽  
Transfer Reaction ◽  
Rnase H ◽  
Strand Transfer ◽  
Transfer Event ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Retroviral reverse transcriptase (RT) enzymes are responsible for transcribing viral RNA into double-stranded DNA. An in vitro assay to analyze the second strand transfer event during human immunodeficiency virus type 1 (HIV-1) reverse transcription has been developed. Model substrates were constructed which mimic the viral intermediate found during plus-strand strong-stop synthesis. Utilizing wild-type HIV-1 RT and a mutant E478Q RT, the requirement for RNase H activity in this strand transfer event was analyzed. In the presence of Mg2+, HIV-1 RT was able to fully support the second strand transfer reaction in vitro. However, in the presence of Mg2+, the E478Q RT mutant had no detectable RNase H activity and was unable to support strand transfer. In the presence of Mn2+, the E478Q RT yields the initial endoribonucleolytic cleavage at the penultimate C residue of the tRNA primer yet does not support strand transfer. This suggests that subsequent degradation of the RNA primer by the RNase H domain was required for strand transfer. In reactions in which the E478Q RT was complemented with exogenous RNase H enzymes, strand transfer was supported. Additionally, we have shown that HIV-1 RT is capable of supporting strand transfer with substrates that mimic tRNAHis as well as the authentic tRNA3 Lys.

scholarly journals RNase H Cleavage of the 5′ End of the Human Immunodeficiency Virus Type 1 Genome

2001 ◽  
Vol 75 (23) ◽  
pp. 11874-11880 ◽  
Author(s):  
Hong-Qiang Gao ◽  
Stefan G. Sarafianos ◽  
Edward Arnold ◽  
Stephen H. Hughes
Keyword(s):  
Human Immunodeficiency Virus ◽  
Dna Synthesis ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Rnase H ◽  
Strand Transfer ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The synthesis of retroviral DNA is initiated near the 5′ end of the RNA. DNA synthesis is transferred from the 5′ end to the 3′ end of viral RNA in an RNase H-dependent step. In the case of human immunodeficiency virus type 1 (HIV-1) (and certain other retroviruses that have complex secondary structures at the ends of the viral RNA), there is the possibility that DNA synthesis can lead to a self-priming event that would block viral replication. The extent of RNase H cleavage must be sufficient to allow the strand transfer reaction to occur, but not so extensive that self-priming occurs. We have used a series of model RNA substrates, with and without a 5′ cap, to investigate the rules governing RNase H cleavage at the 5′ end of the HIV-1 genome. These in vitro RNase H cleavage reactions produce an RNA fragment of the size needed to block self-priming but still allow strand transfer. The cleavages seen in vitro can be understood in light of the structure of HIV-1 reverse transcriptase in a complex with an RNA/DNA substrate.

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

2008 ◽  
Vol 412 (1) ◽  
pp. 163-170 ◽  
Author(s):  
Alon Herschhorn ◽  
Iris Oz-Gleenberg ◽  
Amnon Hizi
Keyword(s):  
Conformational Changes ◽  
Protein Interactions ◽  
Molecular Mechanisms ◽  
Reaction Model ◽  
Protein Protein Interactions ◽  
Common Site ◽  
Leukaemia Virus ◽  
Reverse Transcriptases ◽  
Interaction Kinetics ◽  
Hiv 1

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.

scholarly journals Altered error specificity of RNase H-deficient HIV-1 reverse transcriptases during DNA-dependent DNA synthesis

2013 ◽  
Vol 41 (8) ◽  
pp. 4601-4612 ◽  
Author(s):  
Mar Álvarez ◽  
Verónica Barrioluengo ◽  
Raquel N. Afonso-Lehmann ◽  
Luis Menéndez-Arias
Keyword(s):  
Dna Synthesis ◽  
Rnase H ◽  
Reverse Transcriptases ◽  
Hiv 1

scholarly journals Preclinical Profile of BI 224436, a Novel HIV-1 Non-Catalytic-Site Integrase Inhibitor

2014 ◽  
Vol 58 (6) ◽  
pp. 3233-3244 ◽  
Author(s):  
Craig Fenwick ◽  
Ma'an Amad ◽  
Murray D. Bailey ◽  
Richard Bethell ◽  
Michael Bös ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Phase 1 ◽  
Integrase Inhibitor ◽  
Parallel Synthesis ◽  
Cell Permeability ◽  
Strand Transfer ◽  
Primary Resistance ◽  
Catalytic Core ◽  
Hiv 1

ABSTRACTBI 224436 is an HIV-1 integrase inhibitor with effective antiviral activity that acts through a mechanism that is distinct from that of integrase strand transfer inhibitors (INSTIs). This 3-quinolineacetic acid derivative series was identified using an enzymatic integrase long terminal repeat (LTR) DNA 3′-processing assay. A combination of medicinal chemistry, parallel synthesis, and structure-guided drug design led to the identification of BI 224436 as a candidate for preclinical profiling. It has antiviral 50% effective concentrations (EC50s) of <15 nM against different HIV-1 laboratory strains and cellular cytotoxicity of >90 μM. BI 224436 also has a low, ∼2.1-fold decrease in antiviral potency in the presence of 50% human serum and, by virtue of a steep dose-response curve slope, exhibits serum-shifted EC95values ranging between 22 and 75 nM. Passage of virus in the presence of inhibitor selected for either A128T, A128N, or L102F primary resistance substitutions, all mapping to a conserved allosteric pocket on the catalytic core of integrase. BI 224436 also retains full antiviral activity against recombinant viruses encoding INSTI resistance substitutions N155S, Q148H, and E92Q. In drug combination studies performed in cellular antiviral assays, BI 224436 displays an additive effect in combination with most approved antiretrovirals, including INSTIs. BI 224436 has drug-likein vitroabsorption, distribution, metabolism, and excretion (ADME) properties, including Caco-2 cell permeability, solubility, and low cytochrome P450 inhibition. It exhibited excellent pharmacokinetic profiles in rat (clearance as a percentage of hepatic flow [CL], 0.7%; bioavailability [F], 54%), monkey (CL, 23%;F, 82%), and dog (CL, 8%;F, 81%). Based on the excellent biological and pharmacokinetic profile, BI 224436 was advanced into phase 1 clinical trials.

Three-dimensional structure of the RNase H domain of HIV-1 reverse transcriptase at 2. 4 Å resolution

Peptides ◽  
1992 ◽  
pp. 682-684
Author(s):  
David A. Matthews ◽  
Jay F. Davies ◽  
Zuzana Hostomska ◽  
Zdenek Hostomsky ◽  
Steven R. Jordan
Keyword(s):  
Reverse Transcriptase ◽  
Three Dimensional ◽  
Rnase H ◽  
Dimensional Structure ◽  
Three Dimensional Structure ◽  
Hiv 1

scholarly journals Actinomycin D Inhibits Human Immunodeficiency Virus Type 1 Minus-Strand Transfer in In Vitro and Endogenous Reverse Transcriptase Assays

1998 ◽  
Vol 72 (8) ◽  
pp. 6716-6724 ◽  
Author(s):  
Jianhui Guo ◽  
Tiyun Wu ◽  
Julian Bess ◽  
Louis E. Henderson ◽  
Judith G. Levin
Keyword(s):  
Human Immunodeficiency Virus ◽  
Nucleic Acid ◽  
Actinomycin D ◽  
Strand Transfer ◽  
Minus Strand ◽  
Nucleic Acid Chaperone ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT In this report we demonstrate that human immunodeficiency virus type 1 (HIV-1) minus-strand transfer, assayed in vitro and in endogenous reactions, is greatly inhibited by actinomycin D. Previously we showed that HIV-1 nucleocapsid (NC) protein (a nucleic acid chaperone catalyzing nucleic acid rearrangements which lead to more thermodynamically stable conformations) dramatically stimulates HIV-1 minus-strand transfer by preventing TAR-dependent self-priming from minus-strand strong-stop DNA [(−) SSDNA]. Despite this potent activity, the addition of NC to in vitro reactions with actinomycin D results in only a modest increase in the 50% inhibitory concentration (IC50) for the drug. PCR analysis of HIV-1 endogenous reactions indicates that minus-strand transfer is inhibited by the drug with an IC50 similar to that observed when NC is present in the in vitro system. Taken together, these results demonstrate that NC cannot overcome the inhibitory effect of actinomycin D on minus-strand transfer. Other experiments reveal that at actinomycin D concentrations which severely curtail minus-strand transfer, neither the synthesis of (−) SSDNA nor RNase H degradation of donor RNA is affected; however, the annealing of (−) SSDNA to acceptor RNA is significantly reduced. Thus, inhibition of the annealing reaction is responsible for actinomycin D-mediated inhibition of strand transfer. Since NC (but not reverse transcriptase) is required for efficient annealing, we conclude that actinomycin D inhibits minus-strand transfer by blocking the nucleic acid chaperone activity of NC. Our findings also suggest that actinomycin D, already approved for treatment of certain tumors, might be useful in combination therapy for AIDS.

Synthesis and Evaluation of 3-(1,3-dioxoisoindolin-2-yl)-N-substituted Phenyl Benzamide Analogues as HIV Integrase Strand Transfer Inhibitors

2019 ◽  
Vol 17 (2) ◽  
pp. 105-114
Author(s):  
Pankaj Wadhwa ◽  
Priti Jain ◽  
Arpit Patel ◽  
Shantanu Shinde ◽  
Hemant R. Jadhav
Keyword(s):  
Structural Features ◽  
Docking Studies ◽  
Significant Inhibition ◽  
Strand Transfer ◽  
Hiv Integrase ◽  
In Silico Docking ◽  
Immunodeficiency Virus ◽  
Hiv 1

<P>Background: A series of novel 3-(1,3-dioxoisoindolin-2-yl)-N-substituted phenyl benzamide derivatives was synthesized and tested in vitro against human immunodeficiency virus type-1 Integrase (HIV-1 IN). Methods: Out of the 18 analogues, six (compounds 16c, 16h, 16i, 16m, 16n and 16r) showed significant inhibition of strand transfer by HIV-1 integrase. For these six compounds. IC50 was below 5.0 µM. In silico docking studies revealed that the presence of 2-phenyl isoindoline-1,3-dione motif was essential as it was found to interact with active site magnesium. Results: To further confirm the results, cell-based HIV-1 and HIV-2 inhibitory assay was carried out. Conclusion: These compounds possess structural features not seen in previously reported HIV-1 integrase inhibitors and thus can help further optimization of anti-HIV-1 integrase activity.</P>

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