The ribonuclease H activity of the reverse transcriptases of human immunodeficiency viruses type 1 and type 2 is affected by the thumb subdomain of the small protein subunits11Edited by J. Karn

Sevilya, Ziv; Loya, Shoshana; Hughes, Stephen H.; Hizi, Amnon

doi:10.1006/jmbi.2001.4904

Cited by 49 publications

(90 citation statements)

References 38 publications

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“…Hizi and colleagues reported that the RNase H activity of HIV-1 RT was 10-fold higher than that of HIV-2 RT, while differences in polymerase activities were less prominent (28,29,62). On the basis of results obtained with a substrate containing 5Ј genomic RNA sequences and a series of HIV-1-HIV-2 chimeric enzymes, it was shown that the previously reported 10-fold difference in the RNase H activities of HIV-1 and HIV-2 RTs could be attributed to differences in the activities of the thumb subdomains of the small subunits (61) and, more specifically, to the identity of residue 294 (60).…”

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confidence: 99%

Human Immunodeficiency Virus Type 2 Reverse Transcriptase Activity in Model Systems That Mimic Steps in Reverse Transcription

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Guo

Howard

et al. 2003

J Virol

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Human immunodeficiency virus type 2 (HIV-2) infection is a serious problem in West Africa and Asia.However, there have been relatively few studies of HIV-2 reverse transcriptase (RT), a potential target for antiviral therapy. Detailed knowledge of HIV-2 RT activities is critical for development of specific highthroughput screening assays of potential inhibitors. Here, we have conducted a systematic evaluation of HIV-2 RT function, using assays that model specific steps in reverse transcription. Parallel studies were performed with HIV-1 RT. In general, under standard assay conditions, the polymerase and RNase H activities of the two enzymes were comparable. However, when the RT concentration was significantly reduced, HIV-2 RT was less active than the HIV-1 enzyme. HIV-2 RT was also impaired in its ability to catalyze secondary RNase H cleavage in assays that mimic tRNA primer removal during plus-strand transfer and degradation of genomic RNA fragments during minus-strand DNA synthesis. In addition, initiation of plus-strand DNA synthesis was much less efficient with HIV-2 RT than with HIV-1 RT. This may reflect architectural differences in the primer grip regions in the p66 (HIV-1) and p68 (HIV-2) palm subdomains of the two enzymes. The implications of our findings for antiviral therapy are discussed.

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confidence: 99%

Human Immunodeficiency Virus Type 2 Reverse Transcriptase Activity in Model Systems That Mimic Steps in Reverse Transcription

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Guo

Howard

et al. 2003

J Virol

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“…2A and B), it was imperative also to assess whether the novel HIV-1 RT mutants retained their full RNase H function. Under the assay conditions used (see Materials and Methods), HIV-1 RT sequentially generates two products (17,19,39,40). The primary cleavage product centers 17 to 18 nt from the 3= (28), residues closer than 7 Å to the clamp site have been marked (i.e., residues 89 to 92, 154, 157, and 158 of the p66 RT subunit are highlighted and numbered).…”

Section: Resultsmentioning

confidence: 99%

“…All RTs used in the present study were expressed in Escherichia coli. WT HIV-1 RT from the BH-10 isolate was expressed in bacteria using a plasmid (designated pHIV-1 RT), similar to that described earlier by us (17)(18)(19).…”

Section: Methodsmentioning

confidence: 99%

“…All recombinant RTs were expressed in bacteria, with a six-histidine tag attached to the carboxyl terminus of the p66 subunit in the heterodimeric p66/p51 RT, and was purified as described previously (17)(18)(19).…”

Section: Methodsmentioning

confidence: 99%

“…(iv) RNase H. This assay was performed as described earlier by us (17)(18)(19)25). Each assay contained ϳ0.1 pmol of the 5=-32 P-end-labeled RNA transcript (E11), which was annealed to 0.4 pmol of a 20mer synthetic oligonucleotide DNA (E10 in Table 1).…”

Section: Methodsmentioning

confidence: 99%

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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

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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retroviral ribonuclease H 3.1.26.13

Schomburg

2013

Class 2–3.2 Transferases, Hydrolases

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The ribonuclease H activity of the reverse transcriptases of human immunodeficiency viruses type 1 and type 2 is affected by the thumb subdomain of the small protein subunits11Edited by J. Karn

Cited by 49 publications

References 38 publications

Human Immunodeficiency Virus Type 2 Reverse Transcriptase Activity in Model Systems That Mimic Steps in Reverse Transcription

Human Immunodeficiency Virus Type 2 Reverse Transcriptase Activity in Model Systems That Mimic Steps in Reverse Transcription

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

retroviral ribonuclease H 3.1.26.13

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