Coordination between the polymerase and RNase H activity of HIV-1 reverse transcriptase

Figiel, Małgorzata; Krepl, Miroslav; Poznański, Jarosław; Gołąb, Agnieszka; Šponer, Jiřı́; Nowotny, Marcin

doi:10.1093/nar/gkx004

Cited by 22 publications

(44 citation statements)

References 44 publications

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“…Figiel et al have demonstrated that, under conditions where the DNA polymerase active site of HIV-1 RT is covalently liked to its substrate, nucleic acid can be simultaneously accessed by the RNA polymerase and RNase H active centers, indicating an important degree of coordination between its synthetic and hydrolytic activities [ 24 ]. Using this precedent, we speculated that ligand binding at the HBV RNase H domain might allosterically modulate P protein DNA polymerase activity, since we and others have demonstrated that α-HTs inhibit HBV RNase H activity in vitro, and virus replication in culture [ 25 , 26 , 27 ].…”

Section: Resultsmentioning

confidence: 99%

3,7-Dihydroxytropolones Inhibit Initiation of Hepatitis B Virus Minus-Strand DNA Synthesis

et al. 2020

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Initiation of protein-primed (-) strand DNA synthesis in hepatitis B virus (HBV) requires interaction of the viral reverse transcriptase with epsilon (ε), a cis-acting regulatory signal located at the 5’ terminus of pre-genomic RNA (pgRNA), and several host-encoded chaperone proteins. Binding of the viral polymerase (P protein) to ε is necessary for pgRNA encapsidation and synthesis of a short primer covalently attached to its terminal domain. Although we identified small molecules that recognize HBV ε RNA, these failed to inhibit protein-primed DNA synthesis. However, since initiation of HBV (-) strand DNA synthesis occurs within a complex of viral and host components (e.g., Hsp90, DDX3 and APOBEC3G), we considered an alternative therapeutic strategy of allosteric inhibition by disrupting the initiation complex or modifying its topology. To this end, we show here that 3,7-dihydroxytropolones (3,7-dHTs) can inhibit HBV protein-primed DNA synthesis. Since DNA polymerase activity of a ribonuclease (RNase H)-deficient HBV reverse transcriptase that otherwise retains DNA polymerase function is also abrogated, this eliminates direct involvement of RNase (ribonuclease) H activity of HBV reverse transcriptase and supports the notion that the HBV initiation complex might be therapeutically targeted. Modeling studies also provide a rationale for preferential activity of 3,7-dHTs over structurally related α-hydroxytropolones (α-HTs).

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Section: Resultsmentioning

confidence: 99%

3,7-Dihydroxytropolones Inhibit Initiation of Hepatitis B Virus Minus-Strand DNA Synthesis

et al. 2020

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“…Whether DNA synthesis and RNA degradation can occur simultaneously has been a topic of debate (20,21). It is well established that DNA synthesis occurs more frequently and at a much faster rate than RNA hydrolysis during reverse transcription (22).…”

Section: Resultsmentioning

confidence: 99%

“…4). This may explain why RNA is still hydrolyzed by RNase H when the DNA end is cross-linked to the thumb or polymerase active site (21).…”

Section: Discussionmentioning

confidence: 99%

Structure of HIV-1 reverse transcriptase cleaving RNA in an RNA/DNA hybrid

Tian

Kim

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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HIV-1 reverse transcriptase (RT) contains both DNA polymerase and RNase H activities to convert the viral genomic RNA to dsDNA in infected host cells. Here we report the 2.65-Å resolution structure of HIV-1 RT engaging in cleaving RNA in an RNA/DNA hybrid. A preferred substrate sequence is absolutely required to enable the RNA/DNA hybrid to adopt the distorted conformation needed to interact properly with the RNase H active site in RT. Substituting two nucleotides 4 bp upstream from the cleavage site results in scissile-phosphate displacement by 4 Å. We also have determined the structure of HIV-1 RT complexed with an RNase H-resistant polypurine tract sequence, which adopts a rigid structure and is accommodated outside of the nuclease active site. Based on this newly gained structural information and a virtual drug screen, we have identified an inhibitor specific for the viral RNase H but not for its cellular homologs.

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“…Our R7 substrate is sufficiently long to simultaneously bind both the HIV-1 polymerase and RNase H domains and is efficiently cleaved in our experiments, indicating that the constant dsDNA part of the R7 substrate binds the polymerase domain, even though a RNA–DNA heteroduplex have been shown to bind with higher affinity to the polymerase domain than dsDNA ( 53 ). Possibly, the preference for G or C in positions −2, −4 and −5 may be related to the requirement of substrate untwisting in order to reach the RNase H active site while simultaneously binding the polymerase domain ( 54 , 55 ).…”

Section: Discussionmentioning

confidence: 99%

RNase H sequence preferences influence antisense oligonucleotide efficiency

Kielpinski

Hagedorn

Lindow

et al. 2017

Nucleic Acids Research

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RNase H cleaves RNA in RNA–DNA duplexes. It is present in all domains of life as well as in multiple viruses and is essential for mammalian development and for human immunodeficiency virus replication. Here, we developed a sequencing-based method to measure the cleavage of thousands of different RNA–DNA duplexes and thereby comprehensively characterized the sequence preferences of HIV-1, human and Escherichia coli RNase H enzymes. We find that the catalytic domains of E. coli and human RNase H have nearly identical sequence preferences, which correlate with the efficiency of RNase H-recruiting antisense oligonucleotides. The sequences preferred by HIV-1 RNase H are distributed in the HIV genome in a way suggesting selection for efficient RNA cleavage during replication. Our findings can be used to improve the design of RNase H-recruiting antisense oligonucleotides and show that sequence preferences of HIV-1 RNase H may have shaped evolution of the viral genome and contributed to the use of tRNA-Lys3 as primer during viral replication.

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Coordination between the polymerase and RNase H activity of HIV-1 reverse transcriptase

Cited by 22 publications

References 44 publications

3,7-Dihydroxytropolones Inhibit Initiation of Hepatitis B Virus Minus-Strand DNA Synthesis

3,7-Dihydroxytropolones Inhibit Initiation of Hepatitis B Virus Minus-Strand DNA Synthesis

Structure of HIV-1 reverse transcriptase cleaving RNA in an RNA/DNA hybrid

RNase H sequence preferences influence antisense oligonucleotide efficiency

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