The Glutamine Side Chain at Position 91 on the β5a−β5b Loop of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Is Required for Stabilizing the dNTP Binding Pocket

Pandey, Nootan; Mishra, Chaturbhuj A.; Manvar, Dinesh; Upadhyay, Anand; Talele, Tanaji T.; Comollo, Thomas; Kaushik‐Basu, Neerja; Pandey, Virendra N.

doi:10.1021/bi200815e

Cited by 1 publication

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“…This approach would allow "dissecting" between the clamp function and all other RT activities. Therefore, one of the suspected residues, Gln91, was not tested here, since it was already found that the Q91N HIV-1 RT mutation leads to a loss of Ͼ90% of the DNA polymerase activity (30). As far as we know, none of the other HIV-1 RT mutants investigated here had been studied before, except for the E89G mutant.…”

Section: Resultsmentioning

confidence: 99%

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

Herzig

Voronin

Kucherenko

et al. 2015

J Virol

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The 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 the in vitro data. 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 the in vitro data. 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. R everse transcription (RTN) is a critical step in the life cycle of all retroviruses and the related long terminal repeat (LTR) retrotransposons. This complex multistep process is performed by a single enzyme, the retroviral reverse transcriptase (RT) that copies the viral plus strand RNA into integration competent double-stranded viral DNA (1-3). To perform RTN, RTs have two activities. These are the DNA polymerase activity, which co...

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Section: Resultsmentioning

confidence: 99%