Functional characterization of RNA-dependent DNA polymerase and RNase H activities of a recombinant HIV reverse transcriptase

Biochemistry ◽  
1991 ◽  
Vol 30 (10) ◽  
pp. 2651-2655 ◽  
Author(s):  
Cheng Keat Tan ◽  
Jian Zhang ◽  
Zhao Yan Li ◽  
W. Gary Tarpley ◽  
Kathleen M. Downey ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Dna Polymerase ◽  
Functional Characterization ◽  
Rnase H ◽  
Hiv Reverse Transcriptase

Inhibition of HIV-1 RNase H Activity by Nucleotide Dimers and Monomers

1996 ◽  
Vol 7 (1) ◽  
pp. 37-45 ◽  
Author(s):  
S. J. W. Allen ◽  
S. H. Krawczyk ◽  
L. R. McGee ◽  
N. Bischofberger ◽  
A. S. Mulato ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Biochemical Characterization ◽  
Rnase H ◽  
Hiv Reverse Transcriptase ◽  
Immunodeficiency Virus ◽  
Group A ◽  
Hiv Rt ◽  
Group B

Nucleotide dimers and monomers were shown to inhibit human immunodeficiency virus type 1 (HIV) RNase H activity. Several effective inhibitors were identified and placed into three general groups based on biochemical characterization of their inhibition, The first group (group A) inhibited HIV RNase H and the closely related feline immunodeficiency virus (FIV) RNase H, but did not inhibit less related retroviral or cellular RNases H or HIV reverse transcriptase (RT). The second group (group B) inhibited the RNase H activity of several retroviruses as well as the reverse transcriptase function of HIV RT. The third group (group C) inhibited RNases H from retroviral and cellular sources but did not inhibit HIV RT. Kinetic analyses of HIV RNase H inhibition were conducted and all three types of inhibitors exhibited a competitive mode of inhibition with regard to substrate. The small nucleotides described here represent the most potent (Ki values from 0.57 to 16 μM) and selective inhibitors of HIV RNase H reported to date. Further structure - function analyses of these molecules may lead to the discovery of unique, potent antiretroviral therapeutics.

Structural characterization of HIV reverse transcriptase: a target for the design of specific virus inhibitors

1989 ◽  
Vol 23 (suppl A) ◽  
pp. 47-54 ◽  
Author(s):  
M. Tisdale ◽  
B. A. Larder ◽  
D. M. Lowe ◽  
D. K. Stammers ◽  
D. J. M. Purifoy ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Structural Characterization ◽  
Hiv Reverse Transcriptase ◽  
Specific Virus

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 Effects of the W153L Substitution in HIV Reverse Transcriptase on Viral Replication and Drug Resistance to Multiple Categories of Reverse Transcriptase Inhibitors

2014 ◽  
Vol 58 (8) ◽  
pp. 4515-4526 ◽  
Author(s):  
Hong-Tao Xu ◽  
Susan P. Colby-Germinario ◽  
Maureen Oliveira ◽  
Daniel Rajotte ◽  
Richard Bethell ◽  
...  
Keyword(s):  
Viral Replication ◽  
Reverse Transcriptase ◽  
Rnase H ◽  
Hiv Reverse Transcriptase ◽  
Compound A ◽  
Enzymatic Function ◽  
Biochemical Assays ◽  
Rt Inhibitors ◽  
The Impact ◽  
Hiv 1

ABSTRACTA W153L substitution in HIV-1 reverse transcriptase (RT) was recently identified by selection with a novel nucleotide-competing RT inhibitor (NcRTI) termed compound A that is a member of the benzo[4,5]furo[3,2,d]pyrimidin-2-one NcRTI family of drugs. To investigate the impact of W153L, alone or in combination with the clinically relevant RT resistance substitutions K65R (change of Lys to Arg at position 65), M184I, K101E, K103N, E138K, and Y181C, on HIV-1 phenotypic susceptibility, viral replication, and RT enzymatic function, we generated recombinant RT enzymes and viruses containing each of these substitutions or various combinations of them. We found that W153L-containing viruses were impaired in viral replicative capacity and were hypersusceptible to tenofovir (TFV) while retaining susceptibility to most nonnucleoside RT inhibitors. The nucleoside 3TC retained potency against W153L-containing viruses but not when the M184I substitution was also present. W153L was also able to reverse the effects of the K65R substitution on resistance to TFV, and K65R conferred hypersusceptibility to compound A. Biochemical assays demonstrated that W153L alone or in combination with K65R, M184I, K101E, K103N, E138K, and Y181C impaired enzyme processivity and polymerization efficiency but did not diminish RNase H activity, providing mechanistic insights into the low replicative fitness associated with these substitutions. We show that the mechanism of the TFV hypersusceptibility conferred by W153L is mainly due to increased efficiency of TFV-diphosphate incorporation. These results demonstrate that compound A and/or derivatives thereof have the potential to be important antiretroviral agents that may be combined with tenofovir to achieve synergistic results.

scholarly journals Preparation and characterization of the RNase H domain of Moloney murine leukemia virus reverse transcriptase

2015 ◽  
Vol 113 ◽  
pp. 44-50 ◽  
Author(s):  
Kosaku Nishimura ◽  
Kanta Yokokawa ◽  
Tetsuro Hisayoshi ◽  
Kosuke Fukatsu ◽  
Ikumi Kuze ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Rnase H ◽  
Moloney Murine Leukemia Virus ◽  
Murine Leukemia

scholarly journals HIV-1 Reverse Transcriptase Can Simultaneously Engage Its DNA/RNA Substrate at Both DNA Polymerase and RNase H Active Sites: Implications for RNase H Inhibition

2009 ◽  
Vol 388 (3) ◽  
pp. 462-474 ◽  
Author(s):  
Greg L. Beilhartz ◽  
Michaela Wendeler ◽  
Noel Baichoo ◽  
Jason Rausch ◽  
Stuart Le Grice ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Dna Polymerase ◽  
Active Sites ◽  
Rnase H ◽  
Hiv 1

scholarly journals Biochemical and functional characterization of Plasmodium falciparum DNA polymerase δ

2016 ◽  
Vol 15 (1) ◽  
Author(s):  
Jitlada Vasuvat ◽  
Atcha Montree ◽  
Sangduen Moonsom ◽  
Ubolsree Leartsakulpanich ◽  
Songsak Petmitr ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Dna Polymerase ◽  
Functional Characterization ◽  
Dna Polymerase Δ

scholarly journals Structural and functional characterization of mouse U7 small nuclear RNA active in 3' processing of histone pre-mRNA.

1988 ◽  
Vol 8 (4) ◽  
pp. 1518-1524 ◽  
Author(s):  
D Soldati ◽  
D Schümperli
Keyword(s):  
Sea Urchin ◽  
Functional Characterization ◽  
Rnase H ◽  
Primer Extension ◽  
Small Nuclear Rna ◽  
Nuclear Rna ◽  
Processing Signal ◽  
Insight Into

Oligonucleotides derived from the spacer element of the histone RNA 3' processing signal were used to characterize mouse U7 small nuclear RNA (snRNA), i.e., the snRNA component active in 3' processing of histone pre-mRNA. Under RNase H conditions, such oligonucleotides inhibited the processing reaction, indicating the formation of a DNA-RNA hybrid with a functional ribonucleoprotein component. Moreover, these oligonucleotides hybridized to a single nuclear RNA species of approximately 65 nucleotides. The sequence of this RNA was determined by primer extension experiments and was found to bear several structural similarities with sea urchin U7 snRNA. The comparison of mouse and sea urchin U7 snRNA structures yields some further insight into the mechanism of histone RNA 3' processing.

Sign in / Sign up

Export Citation Format

Share Document