Mutagenesis of Cysteine 280 of the Reverse Transcriptase of Human Immunodeficiency Virus Type-1: The Effects on the Ribonuclease H Activity

Sevilya, Ziv; Loya, Shoshana; Duvshani, Amit; Adir, Noam; Hizi, Amnon

doi:10.1016/s0022-2836(03)00052-4

Cited by 13 publications

(14 citation statements)

References 27 publications

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“…The change in cleavage specificity seen between full-length and truncated PPT virus-infected samples could be due to an effect on the positioning of either the polymerase and/or RNase H active sites of RT relative to the nucleic acid substrate. Mutations in the polymerase domain of RT can influence the activity of the RNase H domain, including cleavage specificity, and in a similar sense mutations in RNase H can affect the activity of the polymerase (6,25,27,31).…”

Section: Discussionmentioning

confidence: 99%

Alternate Polypurine Tracts (PPTs) Affect the Rous Sarcoma Virus RNase H Cleavage Specificity and Reveal a Preferential Cleavage following a GA Dinucleotide Sequence at the PPT-U3 Junction

Chang

Julias

Alvord

et al. 2005

J Virol

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Reverse transcription is catalyzed by the viral enzyme reverse transcriptase (RT). Retroviral RTs have two activities, a DNA polymerase that can copy both RNA and DNA templates and an RNase H that digests RNA if, and only if, it is hybridized to DNA. The RT of avian sarcoma leukosis viruses, of which Rous sarcoma virus (RSV) is a member, is found predominantly as a heterodimeric complex comprised of ␣ and ␤ subunits. The ␣ subunit (63 kDa) contains the polymerase and RNase H domains, and the ␤ subunit (95 kDa) contains both the polymerase and RNase H domains as well as the viral integrase. In the ␣␤ heterodimeric complex, both the polymerase and the RNase H active sites are found in the ␣ subunit (28).During reverse transcription, cleavage of the RNA genome by RNase H is generally nonspecific; however, RNase H does make several specific cleavages involving the plus-strand and minus-strand RNA primers. One specific cleavage removes the tRNA primer used for minus-strand DNA synthesis, and this cleavage ultimately defines the right end of the linear viral DNA. Specific cleavages also generate (and remove) the polypurine tract (PPT) primer used for plus-strand DNA synthesis. Removal of the PPT primer defines the left end of the linear viral DNA. Thus, proper cleavage of the RNA primers generates the ends of the linear viral DNA that are the substrates for integration of the viral DNA into the host genome. More complete descriptions of reverse transcription and integration are given in the following reviews: references 20, 26, and 29.The cleavage and the generation of the PPT plus-strand DNA primer by RNase H have been studied extensively. In order for the PPT primer to be specifically cleaved, the RNase H domain must be able to solve several problems. First, it must be able to distinguish an RNA-DNA duplex from an RNA-RNA and a DNA-DNA duplex and degrade the RNA only when it is in a complex with DNA. RT can make this distinction because RNA-RNA, RNA-DNA, and DNA-DNA duplexes differ in structure. RNA-RNA duplexes preferentially adopt an A-form structure in solution, while DNA-DNA duplexes preferentially adopt a B-form structure. The RNA-DNA duplex adopts a structure that is intermediate between the A and B forms, called the H-form (5).The second problem encountered by the RT is how to distinguish the PPT (which should not be cleaved) from the rest of the RNA genome. RNase H does not recognize a specific sequence per se; rather, it appears that the structure of the RT/PPT complex allows RT to cleave the PPT specifically. Crystallographic studies of the structure of the human immunodeficiency virus type 1 (HIV-1) PPT (RNA/DNA) in a complex with RT provide a valuable framework for interpreting the biochemical data on RNase H cleavage. The HIV-1 PPT (5Ј-AAAAGAAAAGGGGGG-3Ј) has two stretches of adenine residues (A-tracts) at the 5Ј end and a G-tract at the 3Ј end.

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Section: Discussionmentioning

confidence: 99%

Alternate Polypurine Tracts (PPTs) Affect the Rous Sarcoma Virus RNase H Cleavage Specificity and Reveal a Preferential Cleavage following a GA Dinucleotide Sequence at the PPT-U3 Junction

Chang

Julias

Alvord

et al. 2005

J Virol

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“…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%

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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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“…In HIV-2 RT (Km = 49.9 M), this position is occupied by a glutamine while HIV-1 RT (Km = 5.4 M) is a proline. Mutations between these two enzymes (Q294P and P294Q, respectively) reveal an enhancement of the RNaseH activity of HIV-2 RT in almost 10-fold and a decrease of ~70% for HIV-1 RT [115]. The RNaseH activity of the Rod strain of HIV-2 RT is about tenfold lower than that of HIV-1 RT, while the DNA polymerase activity of these RTs is similar.…”

Section: Comparing Hiv-1 Rt With Hiv-2 Rt and Other Counterpartsmentioning

confidence: 97%

HIV-1 Reverse Transcriptase: A Therapeutical Target in the Spotlight

Castro¹,

Loureiro²,

Pujol-Luz³

et al. 2006

CMC

104

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Human Immunodeficiency Virus type 1 Reverse Transcriptase (HIV-1 RT) is one of the most important targets for treatment of Acquired Immune Deficiency Syndrome (AIDS). It catalyzes the reverse transcription of HIV-RNA into a double stranded DNA, and the knowledge of its substrate specificity and catalytic mechanism has guided the development of several inhibitors widely used on current HIV/AIDS therapy. However, mutations in HIV-1 RT structure can lead to the emergence of drug-resistant virus strains. The goal of this review is to summarize relevant structural features of HIV-1 RT and its inhibitors in such a way that this cost-effective target in the development of new antiretroviral drugs is particularly highlighted.

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Mutagenesis of Cysteine 280 of the Reverse Transcriptase of Human Immunodeficiency Virus Type-1: The Effects on the Ribonuclease H Activity

Cited by 13 publications

References 27 publications

Alternate Polypurine Tracts (PPTs) Affect the Rous Sarcoma Virus RNase H Cleavage Specificity and Reveal a Preferential Cleavage following a GA Dinucleotide Sequence at the PPT-U3 Junction

Alternate Polypurine Tracts (PPTs) Affect the Rous Sarcoma Virus RNase H Cleavage Specificity and Reveal a Preferential Cleavage following a GA Dinucleotide Sequence at the PPT-U3 Junction

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

HIV-1 Reverse Transcriptase: A Therapeutical Target in the Spotlight

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