scholarly journals Expression of an Mg2+-Dependent HIV-1 RNase H Construct for Drug Screening

2011 ◽  
Vol 55 (10) ◽  
pp. 4735-4741 ◽  
Author(s):  
Richard V. Farias ◽  
Deborah A. Vargas ◽  
Andres E. Castillo ◽  
Beatriz Valenzuela ◽  
Marie L. Coté ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Rnase H ◽  
Transfer Reactions ◽  
Strand Transfer ◽  
Wild Type ◽  
Content Type ◽  
Dependent Activity ◽  
Rnase Hi ◽  
Single Polypeptide ◽  
Hiv 1

ABSTRACTA single polypeptide of the HIV-1 reverse transcriptase that reconstituted Mg2+-dependent RNase H activity has been made. Using molecular modeling, the construct was designed to encode the p51 subunit joined by a linker to the thumb (T), connection (C), and RNase H (R) domains of p66. This p51-G-TCR construct was purified from the soluble fraction of anEscherichia colistrain, MIC2067(DE3), lacking endogenous RNase HI and HII. The p51-G-TCR RNase H construct displayed Mg2+-dependent activity using a fluorescent nonspecific assay and showed the same cleavage pattern as HIV-1 reverse transcriptase (RT) on substrates that mimic the tRNA removal required for second-strand transfer reactions. The mutant E706Q (E478Q in RT) was purified under similar conditions and was not active. The RNase H of the p51-G-TCR RNase H construct and wild type HIV-1 RT had similarKms for an RNA-DNA hybrid substrate and showed similar inhibition kinetics to two known inhibitors of the HIV-1 RT RNase H.

scholarly journals Developing and Evaluating Inhibitors against the RNase H Active Site of HIV-1 Reverse Transcriptase

2018 ◽  
Vol 92 (13) ◽  
Author(s):  
Paul L. Boyer ◽  
Steven J. Smith ◽  
Xue Zhi Zhao ◽  
Kalyan Das ◽  
Kevin Gruber ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Active Site ◽  
Nucleoside Analogs ◽  
Polymerase Activity ◽  
Rnase H ◽  
Strand Transfer ◽  
Hiv Replication ◽  
Content Type ◽  
Rnase Hi ◽  
Hiv 1

ABSTRACT We tested three compounds for their ability to inhibit the RNase H (RH) and polymerase activities of HIV-1 reverse transcriptase (RT). A high-resolution crystal structure (2.2 Å) of one of the compounds showed that it chelates the two magnesium ions at the RH active site; this prevents the RH active site from interacting with, and cleaving, the RNA strand of an RNA-DNA heteroduplex. The compounds were tested using a variety of substrates: all three compounds inhibited the polymerase-independent RH activity of HIV-1 RT. Time-of-addition experiments showed that the compounds were more potent if they were bound to RT before the nucleic acid substrate was added. The compounds significantly inhibited the site-specific cleavage required to generate the polypurine tract (PPT) RNA primer that initiates the second strand of viral DNA synthesis. The compounds also reduced the polymerase activity of RT; this ability was a result of the compounds binding to the RH active site. These compounds appear to be relatively specific; they do not inhibit either Escherichia coli RNase HI or human RNase H2. The compounds inhibit the replication of an HIV-1-based vector in a one-round assay, and their potencies were only modestly decreased by mutations that confer resistance to integrase strand transfer inhibitors (INSTIs), nucleoside analogs, or nonnucleoside RT inhibitors (NNRTIs), suggesting that their ability to block HIV replication is related to their ability to block RH cleavage. These compounds appear to be useful leads that can be used to develop more potent and specific compounds. IMPORTANCE Despite advances in HIV-1 treatment, drug resistance is still a problem. Of the four enzymatic activities found in HIV-1 proteins (protease, RT polymerase, RT RNase H, and integrase), only RNase H has no approved therapeutics directed against it. This new target could be used to design and develop new classes of inhibitors that would suppress the replication of the drug-resistant variants that have been selected by the current therapeutics.

Recombinant HIV-1 Nucleocapsid Protein Accelerates HIV-1 Reverse Transcriptase Catalyzed DNA Strand Transfer Reactions and Modulates RNase H Activity

Biochemistry ◽  
1994 ◽  
Vol 33 (46) ◽  
pp. 13817-13823 ◽  
Author(s):  
James A. Peliska ◽  
Shankar Balasubramanian ◽  
David P. Giedroc ◽  
Stephen J. Benkovic
Keyword(s):  
Reverse Transcriptase ◽  
Nucleocapsid Protein ◽  
Rnase H ◽  
Transfer Reactions ◽  
Strand Transfer ◽  
Dna Strand ◽  
Hiv 1

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.

Fidelity of in vitro DNA Strand Transfer Reactions Catalyzed by HIV-1 Reverse Transcriptase

Biochemistry ◽  
1994 ◽  
Vol 33 (13) ◽  
pp. 3890-3895 ◽  
Author(s):  
James A. Peliska ◽  
Stephen J. Benkovic
Keyword(s):  
Reverse Transcriptase ◽  
Transfer Reactions ◽  
Strand Transfer ◽  
Dna Strand ◽  
Hiv 1

Inhibitors of DNA Strand Transfer Reactions Catalyzed by HIV-1 Reverse Transcriptase†

Biochemistry ◽  
1999 ◽  
Vol 38 (40) ◽  
pp. 13070-13076 ◽  
Author(s):  
Sam Gabbara ◽  
Wendolyn R. Davis ◽  
Lynn Hupe ◽  
Donald Hupe ◽  
James A. Peliska
Keyword(s):  
Reverse Transcriptase ◽  
Transfer Reactions ◽  
Strand Transfer ◽  
Dna Strand ◽  
Hiv 1

scholarly journals Novel Method for Simultaneous Quantification of Phenotypic Resistance to Maturation, Protease, Reverse Transcriptase, and Integrase HIV Inhibitors Based on 3′Gag(p2/p7/p1/p6)/PR/RT/INT-Recombinant Viruses: a Useful Tool in the Multitarget Era of Antiretroviral Therapy

2011 ◽  
Vol 55 (8) ◽  
pp. 3729-3742 ◽  
Author(s):  
Jan Weber ◽  
Ana C. Vazquez ◽  
Dane Winner ◽  
Justine D. Rose ◽  
Doug Wylie ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Standardized Test ◽  
New Technology ◽  
Drug Susceptibility ◽  
Antiretroviral Drugs ◽  
Rnase H ◽  
Strand Transfer ◽  
Phenotypic Resistance ◽  
Recombinant Viruses ◽  
Hiv 1

ABSTRACTTwenty-six antiretroviral drugs (ARVs), targeting five different steps in the life cycle of the human immunodeficiency virus type 1 (HIV-1), have been approved for the treatment of HIV-1 infection. Accordingly, HIV-1 phenotypic assays based on common cloning technology currently employ three, or possibly four, different recombinant viruses. Here, we describe a system to assess HIV-1 resistance to all drugs targeting the three viral enzymes as well as viral assembly using a single patient-derived, chimeric virus. Patient-derived p2-INT (gag-p2/NCp7/p1/p6/pol-PR/RT/IN) products were PCR amplified as a single fragment (3,428 bp) or two overlapping fragments (1,657 bp and 2,002 bp) and then recombined into a vector containing a near-full-length HIV-1 genome with theSaccharomyces cerevisiaeuracil biosynthesis gene (URA3) replacing the 3,428 bp p2-INT segment (Dudley et al., Biotechniques 46:458–467, 2009). P2-INT-recombinant viruses were employed in drug susceptibility assays to test the activity of protease (PI), nucleoside/nucleotide reverse transcriptase (NRTI), nonnucleoside reverse transcriptase (NNRTI), and integrase strand-transfer (INSTI) inhibitors. Using a single standardized test (ViralARTS HIV), this new technology permits the rapid and automated quantification of phenotypic resistance for all known and candidate antiretroviral drugs targeting all viral enzymes (PR, RT, including polymerase and RNase H activities, and IN), some of the current and potential assembly inhibitors, and any drug targeting Pol or Gag precursor cleavage sites (relevant for PI and maturation inhibitors) This novel assay may be instrumental (i) in the development and clinical assessment of novel ARV drugs and (ii) to monitor patients failing prior complex treatment regimens.

scholarly journals The Base Component of 3′-Azido-2′,3′-Dideoxynucleosides Influences Resistance Mutations Selected in HIV-1 Reverse Transcriptase

2011 ◽  
Vol 55 (8) ◽  
pp. 3758-3764 ◽  
Author(s):  
Jeffrey D. Meteer ◽  
Dianna Koontz ◽  
Ghazia Asif ◽  
Hong-wang Zhang ◽  
Mervi Detorio ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Nucleoside Analogs ◽  
Rnase H ◽  
Major Determinant ◽  
Site Directed Mutagenesis ◽  
Wild Type Virus ◽  
Wild Type ◽  
Resistance Mutations ◽  
Hiv 1

ABSTRACTWe recently reported that HIV-1 resistant to 3′-azido-3′-deoxythymidine (AZT) is not cross-resistant to 3′-azido-2′,3′-dideoxypurines. This finding suggested that the nucleoside base is a major determinant of HIV-1 resistance to nucleoside analogs. To further explore this hypothesis, we conductedin vitroselection experiments by serial passage of HIV-1LAIin MT-2 cells in increasing concentrations of 3′-azido-2′,3′-dideoxyguanosine (3′-azido-ddG), 3′-azido-2′,3′-dideoxycytidine (3′-azido-ddC), or 3′-azido-2′,3′-dideoxyadenosine (3′-azido-ddA). 3′-Azido-ddG selected for virus that was 5.3-fold resistant to 3′-azido-ddG compared to wild-type HIV-1LAIpassaged in the absence of drug. Population sequencing of the entire reverse transcriptase (RT) gene identified L74V, F77L, and L214F mutations in the polymerase domain and K476N and V518I mutations in the RNase H domain. However, when introduced into HIV-1 by site-directed mutagenesis, these 5 mutations only conferred ∼2.0-fold resistance. Single-genome sequencing analyses of the selected virus revealed a complex population of mutants that all contained L74V and L214F linked to other mutations, including ones not identified during population sequencing. Recombinant HIV-1 clones containing RT derived from single sequences exhibited 3.2- to 4.0-fold 3′-azido-ddG resistance. In contrast to 3′-azido-ddG, 3′-azido-ddC selected for the V75I mutation in HIV-1 RT that conferred 5.9-fold resistance, compared to the wild-type virus. Interestingly, we were unable to select HIV-1 that was resistant to 3′-azido-ddA, even at concentrations of 3′-azido-ddA that yielded high intracellular levels of 3′-azido-ddA-5′-triphosphate. Taken together, these findings show that the nucleoside base is a major determinant of HIV-1 resistance mechanisms that can be exploited in the design of novel nucleoside RT inhibitors.

scholarly journals A Novel Molecular Mechanism of Dual Resistance to Nucleoside and Nonnucleoside Reverse Transcriptase Inhibitors

2010 ◽  
Vol 84 (10) ◽  
pp. 5238-5249 ◽  
Author(s):  
Galina N. Nikolenko ◽  
Krista A. Delviks-Frankenberry ◽  
Vinay K. Pathak
Keyword(s):  
Reverse Transcriptase ◽  
Binding Pocket ◽  
Rnase H ◽  
Wild Type ◽  
Reverse Transcriptase Inhibitors ◽  
Nnrti Resistance ◽  
Nonnucleoside Reverse Transcriptase Inhibitors ◽  
Resistance Model ◽  
Hiv 1

ABSTRACT Recently, mutations in the connection subdomain (CN) and RNase H domain of HIV-1 reverse transcriptase (RT) were observed to exhibit dual resistance to nucleoside and nonnucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs). To elucidate the mechanism by which CN and RH mutations confer resistance to NNRTIs, we hypothesized that these mutations reduce RNase H cleavage and provide more time for the NNRTI to dissociate from the RT, resulting in the resumption of DNA synthesis and enhanced NNRTI resistance. We observed that the effect of the reduction in RNase H cleavage on NNRTI resistance is dependent upon the affinity of each NNRTI to the RT and further influenced by the presence of NNRTI-binding pocket (BP) mutants. D549N, Q475A, and Y501A mutants, which reduce RNase H cleavage, enhance resistance to nevirapine (NVP) and delavirdine (DLV), but not to efavirenz (EFV) and etravirine (ETR), consistent with their increase in affinity for RT. Combining the D549N mutant with NNRTI BP mutants further increases NNRTI resistance from 3- to 30-fold, supporting the role of NNRTI-RT affinity in our NNRTI resistance model. We also demonstrated that CNs from treatment-experienced patients, previously reported to enhance NRTI resistance, also reduce RNase H cleavage and enhance NNRTI resistance in the context of the patient RT pol domain or a wild-type pol domain. Together, these results confirm key predictions of our NNRTI resistance model and provide support for a unifying mechanism by which CN and RH mutations can exhibit dual NRTI and NNRTI resistance.

scholarly journals Mutants of Human Immunodeficiency Virus Type 1 (HIV-1) Reverse Transcriptase Resistant to Nonnucleoside Reverse Transcriptase Inhibitors Demonstrate Altered Rates of RNase H Cleavage That Correlate with HIV-1 Replication Fitness in Cell Culture

2000 ◽  
Vol 74 (18) ◽  
pp. 8390-8401 ◽  
Author(s):  
Richard H. Archer ◽  
Carrie Dykes ◽  
Peter Gerondelis ◽  
Amanda Lloyd ◽  
Philip Fay ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Rnase H ◽  
Wild Type ◽  
Reverse Transcriptase Inhibitors ◽  
Nonnucleoside Reverse Transcriptase Inhibitors ◽  
Immunodeficiency Virus ◽  
Replication Fitness ◽  
Hiv 1

ABSTRACT Three mutants of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (V106A, V179D, and Y181C), which occur in clinical isolates and confer resistance to nonnucleoside reverse transcriptase inhibitors (NNRTIs), were analyzed for RNA- and DNA-dependent DNA polymerization and RNase H cleavage. All mutants demonstrated processivities of polymerization that were indistinguishable from wild-type enzyme under conditions in which deoxynucleoside triphosphates were not limiting. The V106A reverse transcriptase demonstrated a three- to fourfold slowing of both DNA 3′-end-directed and RNA 5′-end-directed RNase H cleavage relative to both wild-type and V179D enzymes, similar to what was observed for P236L in a previously published study (P. Gerondelis et al., J. Virol. 73:5803–5813, 1999). In contrast, the Y181C reverse transcriptase demonstrated a selective acceleration of the secondary RNase H cleavage step during both modes of RNase H cleavage. The relative replication fitness of these mutants in H9 cells was assessed in parallel infections as well as in growth competition experiments. Of the NNRTI-resistant mutants, V179D was more fit than Y181C, and both of these mutants were more fit than V106A, which demonstrated the greatest reduction in RNase H cleavage. These findings, in combination with results from previous work, suggest that abnormalities in RNase H cleavage are a common characteristic of HIV-1 mutants resistant to NNRTIs and that combined reductions in the rates of DNA 3′-end- and RNA 5′-end-directed cleavages are associated with significant reductions in the replication fitness of HIV-1.

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