Human immunodeficiency virus reverse transcriptase ribonuclease H: specificity of tRNALys3-primer excision

Furfine, Eric S.; Reardon, John E.

doi:10.1021/bi00243a001

Cited by 79 publications

(84 citation statements)

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“…The RNase H cleavages that remove the tRNA primer, and the cleavages that generate and remove the polypurine tract (ppt) primer define the ends of the unintegrated linear viral DNA that is the substrate for the integration reaction. HIV-1 RT removes the tRNA one base from the RNA-DNA junction (15)(16)(17)(18); other retroviral RTs remove the entire tRNA (2).…”

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confidence: 99%

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Mutations in the RNase H domain of HIV-1 reverse transcriptase affect the initiation of DNA synthesis and the specificity of RNase H cleavage in vivo

Julias

McWilliams

Sarafianos

et al. 2002

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

112

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Retroviral reverse transcriptases contain a DNA polymerase activity that can copy an RNA or DNA template and an RNase H activity that degrades the viral RNA genome during reverse transcription. RNase H makes both specific and nonspecific cleavages; specific cleavages are used to generate and remove the polypurine tract primer used for plus-strand DNA synthesis and to remove the tRNA primer used for minus-strand DNA synthesis. We generated mutations in an HIV-1-based vector to change amino acids in the RNase H domain that contact either the RNA and DNA strands. Some of these mutations affected the initiation of DNA synthesis, demonstrating an interdependence of the polymerase and RNase H activities of HIV-1 reverse transcription during viral DNA synthesis. The ends of the linear DNA form of the HIV-1 genome are defined by the specific RNase H cleavages that remove the plus-and minus-strand primers; these ends can be joined to form two-longterminal repeat circles. Analysis of two-long-terminal repeat circle junctions showed that mutations in the RNase H domain affect the specificity of RNase H cleavage.H IV-1 reverse transcriptase (RT) is the virally encoded enzyme that converts the single-stranded RNA genome found in virions into double-stranded DNA. The conversion of the RNA genome into DNA is accomplished through the collaboration of the two enzymatic activities of RT: a DNA polymerase that can use either RNA or DNA as a template and an RNase H that cleaves RNA if (and only if) it is present in an RNA⅐DNA duplex (reviewed in refs. 1-3). Both polymerase and RNase H activities are required for the conversion of the RNA genome into double-stranded DNA; mutations that inactivate either the polymerase or RNase H block viral replication (4-7).In vitro assays showed that polymerase and RNase H activities of HIV-1 RT are interdependent (8-13). Both the polymerase and RNase H domains of HIV-1 RT simultaneously contact the nucleic acid substrate and contribute to the binding (and proper positioning) of the nucleic acid. In in vitro assays, mutations in the RNase H domain can affect polymerase activity (and vice versa). We found that reducing RNase H activity by cotransfecting a mixture of wild-type HIV-1 vector DNA and DNA encoding HIV-1 vectors with mutations in the RNase H active site reduced the efficiency of initiation of DNA synthesis (14). On the basis of these observations, we wanted to determine whether mutating amino acids that make specific contacts with the nucleic acid substrate in the vicinity of the RNase H active site would also affect the initiation of DNA synthesis and͞or the specificity of RNase H cleavage.Reverse transcription requires that RNase H make both specific and nonspecific cleavages. The process of reverse transcription is shown schematically in Fig. 1 (reviewed in refs. 1 and 3). The RNase H cleavages that remove the tRNA primer, and the cleavages that generate and remove the polypurine tract (ppt) primer define the ends of the unintegrated linear viral DNA that is the substrate for the int...

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confidence: 99%

“…The ribonucleotide (rA) at the 5Ј end of the minus strand is derived from the tRNA. HIV-1 RT removes the tRNA one base from the RNA-DNA junction (15)(16)(17)(18); other retroviral RTs remove the entire tRNA (2). (vii) The structure of full-length viral DNA.…”

mentioning

confidence: 99%

Mutations in the RNase H domain of HIV-1 reverse transcriptase affect the initiation of DNA synthesis and the specificity of RNase H cleavage in vivo

Julias

McWilliams

Sarafianos

et al. 2002

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

112

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“…In addition, (ϩ), or second-strand DNA synthesis requires highly specific RNase H cleavage to release the 3Ј OH of the polypurine tract (PPT) primer (3)(4)(5). Finally, the (ϩ) and (Ϫ) strand RNA primers (the PPT and a host-derived tRNA, respectively) must be precisely removed from nascent DNA to preserve the integrity of the 5Ј and 3Ј long terminal repeat termini of the provirus for recognition by the retroviral integration machinery (3,6,7). Each of these events can be considered candidates for therapeutic intervention by antiviral agents in the ongoing effort to stem the progression of human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome.…”

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confidence: 99%

Substituting a Conserved Residue of the Ribonuclease H Domain Alters Substrate Hydrolysis by Retroviral Reverse Transcriptase

Rausch

Grice

1997

Journal of Biological Chemistry

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Alterations to the highly conserved Asp 549 of the retroviral ribonuclease H (RNase H) domain were evaluated in the heterodimeric (p66/p51) reverse transcriptases of human immunodeficiency and equine infectious anemia viruses. In addition to the polymerization-dependent and -independent modes of template hydrolysis, mutants were evaluated via their ability to select and extend the 3 polypurine tract (PPT) primers of these two lentiviruses into (؉) strand DNA. Concerted and two-step reactions were designed to evaluate (؉) strand priming, the latter of which allows discrimination between selection end extension events. In contrast to enzyme mutated at the highly conserved Glu 478 , substitution of Asp 549 with Asn or Ala reduces, rather than completely eliminates, RNase H activity. When the requirement for RNase H function becomes more stringent, differences in activity are readily evident, most notably in the cleavage events liberating the 5 terminus of the PPT primer. PPT selection thus appears to represent a specialized form of RNase H activity that is more sensitive to minor structural alterations within this domain and may provide a novel therapeutic target.

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“…Several groups have studied the coordination of DNA polymerase and RNase H activities (Oyama ef a/., 1989;Luo and Taylor, 1990;Wöhrl and Moelling, 1990;Furfine and Reardon, 1991 a;Furfine and Reardon, 1991b; Gopalakrishnan ef a/., 1992; Kati ef a/., 1992). The two activity site are separated by 10 to 19 nucleotides (Gopalakrishnan ef a/., 1992; Kati ef a/., 1992).…”

Section: Rnase Hmentioning

confidence: 99%

“…Although there is considerable support for coupling of DNA polymerase and RNAse H activities, there are both biochemical (Huber ef a/., 1989; DeStefano ef a/., 1991b) and genetical data(Telesnitsky and Goff, 1993c) that suggested uncoupled activities. One way to rationalize these results is to imagine that RNase H can function in two modes, one dependent upon and the other independent of polymerization (Furfine and Reardon, 1991b;Gopalakrishnan ef a/., 1992). Genetic studies indicated that the uncoupled mode may be sufficient for low level viral replication (Telesnitsky and Goff, 1993c).…”

Section: Rnase Hmentioning

confidence: 99%

Review

1996

Biological Chemistry Hoppe-Seyler

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Proteolytic enzymes have many physiological functions in plants, bacteria, viruses, protozoa and mammals. They play a role in processes such as food digestion, complement activation or blood coagulation. The action of proteolytic enzymes is biologically controlled by proteinase inhibitors and increasing attention is being paid to the physiological significance of these natural inhibitors in pathological processes. The reason for this growing interest is that uncontrolled proteolysis can lead to irreversible damage e.g. in chronic inflammation or tumor metastasis. This review focusses on the possible role of the cystatins, natural and specific inhibitors of the cysteine proteinases, in pathological processes. Key words: Cystatins / Cysteine proteinases / Inflammation / Inhibitors / Pathological processes. Cysteine ProteinasesCysteine Proteinases, synonymous with thiol proteinases, are small proteins with molecular masses varying from 23 to 24 kDa and with two catalytic residues, Cys25 and His159, which are involved in the hydrolytic reaction. Cysteine proteinases catalyze the hydrolysis of various polypeptide substrates and are most active under reducing and mildly acidic conditions (pH 5 -6.5). They have been detected in and isolated from a large number of biological sources including plants, bacteria, animals and humans. Bacterial cysteine proteinases are produced by Clostridium histolyticum, named clostripain, by hemolytic group A streptococci and by the pathogenic anaerobic Porphyromonas gingivalis, a bacterium associated with periodontitis. Bacterial cysteine proteinases probably play a role in the penetration of normal tissues by the bacteria and in the food digestion. Viral cysteine proteinases from picornaviruses, e.g. the poliovirus and rhinovirus type I, are involved in proteolytic cleavage of precursor proteins for virus replication and for the production of new virus particles. A virus-coded cysteine proteinase is involved in this process (Kay and Dunn, 1990; Körant et a/., 1985;. HIV-I also needs proteolytic processing by cysteine proteinases to regulate the expression of viral proteins (Guy ef a/., 1991). Protozoal cysteine proteinases are produced by a number of protozoan parasites to facilitate host invasion, metabolize host proteins, to degrade the host immune molecules or to use them for intracellular replication. Cysteine proteinases are produced by e.g. Trypanosoma cruzi, the causative agent of American trypanosomiasis or by Leishmania species, causing one of the six major parasitic diseases. Furthermore, human malarial parasites produce a broad scala of proteinases including cysteine proteinase which are involved in erythrocyte invasion and rupture (McKerrow, 1993). Mammalian cysteine proteinases are present in the lysosomes of cells. Lysosomes contain different cysteine proteinases, the most important being the cathepsins B, H, L and S Kirschke, 1981, Barrett et a/., 1988). These cathepsins have common ancestors and are related to papain. Cathepsin B has been detected in every tissue or cel...

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Human immunodeficiency virus reverse transcriptase ribonuclease H: specificity of tRNALys3-primer excision

Cited by 79 publications

References 23 publications

Mutations in the RNase H domain of HIV-1 reverse transcriptase affect the initiation of DNA synthesis and the specificity of RNase H cleavage in vivo

Mutations in the RNase H domain of HIV-1 reverse transcriptase affect the initiation of DNA synthesis and the specificity of RNase H cleavage in vivo

Substituting a Conserved Residue of the Ribonuclease H Domain Alters Substrate Hydrolysis by Retroviral Reverse Transcriptase

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