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

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.

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Inhibition of HIV-1 Reverse Transcriptase-Catalyzed DNA Strand Transfer Reactions by 4-Chlorophenylhydrazone of Mesoxalic Acid†

Biochemistry ◽

10.1021/bi0015764 ◽

2000 ◽

Vol 39 (46) ◽

pp. 14279-14291 ◽

Cited By ~ 28

Author(s):

Wendolyn R. Davis ◽

John Tomsho ◽

Savita Nikam ◽

Elizabeth M. Cook ◽

David Somand ◽

...

Keyword(s):

Reverse Transcriptase ◽

Transfer Reactions ◽

Strand Transfer ◽

Dna Strand ◽

Hiv 1

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

Journal of Virology ◽

10.1128/jvi.00809-15 ◽

2015 ◽

Vol 89 (16) ◽

pp. 8119-8129 ◽

Cited By ~ 4

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.

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Nucleocapsid Protein Precursors NCp9 and NCp15 Suppress ATP-Mediated Rescue of AZT-Terminated Primers by HIV-1 Reverse Transcriptase

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00958-20 ◽

2020 ◽

Vol 64 (10) ◽

Author(s):

Moisés A. Árquez ◽

Samara Martín-Alonso ◽

Robert J. Gorelick ◽

Walter A. Scott ◽

Antonio J. Acosta-Hoyos ◽

...

Keyword(s):

Reverse Transcriptase ◽

Nucleocapsid Protein ◽

Inhibitory Effect ◽

Rnase H ◽

Resistance Mutations ◽

Site Mutation ◽

Development Of Resistance ◽

Transcription Process ◽

Dna Primers ◽

Hiv 1

ABSTRACT In HIV-1, development of resistance to AZT (3′-azido-3′-deoxythymidine) is mediated by the acquisition of thymidine analogue resistance mutations (TAMs) (i.e., M41L, D67N, K70R, L210W, T215F/Y, and K219E/Q) in the viral reverse transcriptase (RT). Clinically relevant combinations of TAMs, such as M41L/T215Y or D67N/K70R/T215F/K219Q, enhance the ATP-mediated excision of AZT monophosphate (AZTMP) from the 3′ end of the primer, allowing DNA synthesis to continue. Additionally, during HIV-1 maturation, the Gag polyprotein is cleaved to release a mature nucleocapsid protein (NCp7) and two intermediate precursors (NCp9 and NCp15). NC proteins interact with the viral genome and facilitate the reverse transcription process. Using wild-type and TAM-containing RTs, we showed that both NCp9 and NCp15 inhibited ATP-mediated rescue of AZTMP-terminated primers annealed to RNA templates but not DNA templates, while NCp7 had no effect on rescue activity. RNase H inactivation by introducing the active-site mutation E478Q led to the loss of the inhibitory effect shown by NCp9. NCp15 had a stimulatory effect on the RT’s RNase H activity not observed with NCp7 and NCp9. However, analysis of RNase H cleavage patterns revealed that in the presence of NCp9, RNA/DNA complexes containing duplexes of 12 bp had reduced stability in comparison with those obtained in the absence of NC or with NCp7 or NCp15. These effects are expected to have a strong influence on the inhibitory action of NCp9 and NCp15 by affecting the efficiency of RNA-dependent DNA polymerization after unblocking DNA primers terminated with AZTMP and other nucleotide analogues.

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Mutations in HIV reverse transcriptase which alter RNase H activity and decrease strand transfer efficiency are suppressed by HIV nucleocapsid protein

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.94.13.6700 ◽

1997 ◽

Vol 94 (13) ◽

pp. 6700-6705 ◽

Cited By ~ 63

Author(s):

C. E. Cameron ◽

M. Ghosh ◽

S. F. J. Le Grice ◽

S. J. Benkovic

Keyword(s):

Reverse Transcriptase ◽

Nucleocapsid Protein ◽

Rnase H ◽

Transfer Efficiency ◽

Strand Transfer ◽

Hiv Reverse Transcriptase

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

Journal of Virology ◽

10.1128/jvi.02203-17 ◽

2018 ◽

Vol 92 (13) ◽

Cited By ~ 15

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.

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