Mutagenesis of Human Immunodeficiency Virus Reverse Transcriptase p51 Subunit Defines Residues Contributing to Vinylogous Urea Inhibition of Ribonuclease H Activity

Chung, Shinjae; Miller, Jennifer T.; Johnson, Barry C.; Hughes, Stephen H.; Grice, Stuart F. J. Le

doi:10.1074/jbc.m111.314781

Cited by 24 publications

(46 citation statements)

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“…Since the RNase H function is essential for viral replication (5), it has been explored as a drug target, and a number of RNase H inhibitors (RHIs) have been reported (6)(7)(8). RHIs can be classified based on their binding sites, i.e., (i) RHIs that coordinate the two Mg 2ϩ catalytic cofactors at the RNase H active site, such as N-hydroxyimides (9), hydroxytropolones (10), hydroxypyrimidines (11), naphthyridinones (12), nitrofuran-2-carboxylic acid carbamoylmethyl esters (13), hydroxyquinolinones (14), and thiocarbamates and triazoles (15), or (ii) allosteric RHIs, such as vinylogous ureas (16), thienopyrimidinones (17), hydrazones (18), anthraquinones (19), isatines (20,21), and propenones (22).…”

mentioning

confidence: 99%

Identification of Highly Conserved Residues Involved in Inhibition of HIV-1 RNase H Function by Diketo Acid Derivatives

Corona

Leva

Thierry

et al. 2014

Antimicrob Agents Chemother

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confidence: 99%

Identification of Highly Conserved Residues Involved in Inhibition of HIV-1 RNase H Function by Diketo Acid Derivatives

Corona

Leva

Thierry

et al. 2014

Antimicrob Agents Chemother

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“…Such observations suggest that removing what has been described as disordered residues from the p51 C terminus may improve packing at the dimer interface, a notion forwarded by Bauman et al (27) as a means of crystal engineering. However, our previous work (21,22,24), and data presented herein, suggest that such stabilization of the RT heterodimer may remove a significant degree of flexibility, which can have important consequences on substrate positioning and RNase H activity. Thus, the observed Tyr-427 and Asn-348 hydrogen bond may correlate with an activated state for RNA degradation.…”

Section: C-terminal P51 Truncations Stabilize the Hiv-1 Rtmentioning

confidence: 79%

“…Multiple structures of the RT apo enzyme and cocrystals containing nucleic acid support the notion that although p66, which contains both the DNA polymerase and the RNase H catalytic centers, undergoes large scale motions, the p51 subunit is essentially rigid (3)(4)(5)(6)(7)(8)(9)(10)(11)(12). However, observations that (a) the p51 mutation N348I confers resistance to nucleoside and non-nucleoside RT inhibitors (NRTIs 2 and NNRTIs, respectively (13-16)), (b) p51 residues Lys-395 and Glu-396 contribute to the architecture of the RNase H primer grip/phosphate binding pocket (9,(17)(18)(19)(20), and (c) p51 C-terminal deletions induce alterations in both RNase H activity (21) and RNase H inhibitor sensitivity (22) convincingly demonstrate that p51 of the p66/ p51 RT heterodimer is not inert.…”

mentioning

confidence: 99%

Examining the Role of the HIV-1 Reverse Transcriptase p51 Subunit in Positioning and Hydrolysis of RNA/DNA Hybrids

Chung

Miller

Lapkouski

et al. 2013

Journal of Biological Chemistry

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Background: New crystallographic data for HIV-1 RT containing an RNA/DNA hybrid show important interactions involving the p51 C terminus. Results: Altered hydrogen bonding at the p51 C terminus affects both NNRTI sensitivity and enzyme activity. Conclusion: An intact p51 subunit is necessary to faithfully recapitulate activities of the RT heterodimer. Significance: The extreme C terminus of p51 plays an important role in RNA/DNA hybrid positioning and hydrolysis.

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“…Several groups have introduced amino acid substitutions at different subdomains of RT (11,31,33,(41)(42)(43)(44)(45). Many of these mutations led to destabilization of the RT protein in the virus particle as well as a loss of infectivity.…”

Section: Figmentioning

confidence: 99%

“…The RNA-dependent DNA polymerase and RNase H activities of HIV-1 RT are mainly carried out by the p66 subunit, while p51 was thought to be enzymatically inactive and serve only a structural role (5,(7)(8)(9)(10). However, recent structural and biochemical evidence suggests that the C-terminal end of the p51 subunit is involved in hydrolysis and positioning of the RNA/DNA hybrid formed during the reverse transcription process (11)(12)(13).…”

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confidence: 99%

The Nature of the N-Terminal Amino Acid Residue of HIV-1 RNase H Is Critical for the Stability of Reverse Transcriptase in Viral Particles

Boso

Örvell

Somia

2015

J Virol

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Reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) is synthesized and packaged into the virion as a part of the GagPol polyprotein. Mature RT is released by the action of viral protease. However, unlike other viral proteins, RT is subject to an internal cleavage event leading to the formation of two subunits in the virion: a p66 subunit and a p51 subunit that lacks the RNase H domain. We have previously identified RNase H to be an HIV-1 protein that has the potential to be a substrate for the N-end rule pathway, which is an ubiquitin-dependent proteolytic system in which the identity of the N-terminal amino acid determines the half-life of a protein. Here we examined the importance of the N-terminal amino acid residue of RNase H in the early life cycle of HIV-1. We show that changing this residue to an amino acid structurally different from the conserved residue leads to the degradation of RT and, in some cases, integrase in the virus particle and this abolishes infectivity. Using intravirion complementation and in vitro protease cleavage assays, we show that degradation of RT in RNase H N-terminal mutants occurs in the absence of active viral protease in the virion. Our results also indicate the importance of the RNase H N-terminal residue in the dimerization of RT subunits. IMPORTANCEHIV-1 proteins are initially made as part of a polyprotein that is cleaved by the viral protease into the proteins that form the virus particle. We were interested in one particular protein, RNase H, that is cleaved from reverse transcriptase. In particular, we found that the first amino acid of RNase H never varied in over 1,850 isolates of HIV-1 that we compared. When we changed the first amino acid, we found that the reverse transcriptase in the virus was degraded. While other studies have implied that the viral protease can degrade mutant RT proteins, we show here that this may not be the case for our mutants. Our results suggest that the presence of active viral protease is not required for the degradation of RT in RNase H N-terminal mutants, suggesting a role for a cellular protease in this process. Like all retroviruses, human immunodeficiency virus type 1 (HIV-1), the causative agent of AIDS, synthesizes and packages its main structural and enzymatic proteins as precursor polyproteins. For HIV-1, these polyproteins are p55 (Gag) and p160 (GagPol). Gag is the most abundant polyprotein and is translated from a genome-length mRNA that contains the Gag and GagPol open reading frames. The synthesis of GagPol requires a ribosomal frameshift leading to a Gag/GagPol ratio of about 20:1 in the virus particle (1). Individual mature viral proteins are generated following viral assembly as a result of a series of proteolytic cleavage events at specific positions catalyzed by the viral protease, which is synthesized as a part of GagPol (2).One protein that is released as a result of proteolytic processing of GagPol is reverse transcriptase (RT). RT catalyzes the reaction for the conversion of viral RNA to...

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Mutagenesis of Human Immunodeficiency Virus Reverse Transcriptase p51 Subunit Defines Residues Contributing to Vinylogous Urea Inhibition of Ribonuclease H Activity

Cited by 24 publications

References 44 publications

Identification of Highly Conserved Residues Involved in Inhibition of HIV-1 RNase H Function by Diketo Acid Derivatives

Identification of Highly Conserved Residues Involved in Inhibition of HIV-1 RNase H Function by Diketo Acid Derivatives

Examining the Role of the HIV-1 Reverse Transcriptase p51 Subunit in Positioning and Hydrolysis of RNA/DNA Hybrids

The Nature of the N-Terminal Amino Acid Residue of HIV-1 RNase H Is Critical for the Stability of Reverse Transcriptase in Viral Particles

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