Discrimination of DNA polymerase and RNase H activities in reverse transcriptase of avian myeloblastosis virus

Biochemistry ◽  
1978 ◽  
Vol 17 (12) ◽  
pp. 2438-2442 ◽  
Author(s):  
Marian Gorecki ◽  
Amos Panet
Keyword(s):  
Reverse Transcriptase ◽  
Dna Polymerase ◽  
Rnase H ◽  
Avian Myeloblastosis Virus

scholarly journals Mn2+ Suppressor Mutations and Biochemical Communication between Ty1 Reverse Transcriptase and RNase H Domains

2007 ◽  
Vol 81 (17) ◽  
pp. 9004-9012 ◽  
Author(s):  
Robert M. Yarrington ◽  
Jichao Chen ◽  
Eric C. Bolton ◽  
Jef D. Boeke
Keyword(s):  
Reverse Transcriptase ◽  
Ion Homeostasis ◽  
Point Mutations ◽  
Rnase H ◽  
Avian Myeloblastosis Virus ◽  
Manganese Ion ◽  
Suppressor Mutations ◽  
Suppressor Mutants ◽  
Ty1 Retrotransposition

ABSTRACT Ty1 reverse transcriptase/RNase H (RT/RH) is exquisitely sensitive to manganese concentrations. Elevated intracellular free Mn2+ inhibits Ty1 retrotransposition and in vitro Ty1 RT-polymerizing activity. Furthermore, Mn2+ inhibition is not limited to the Ty1 RT, as this ion similarly inhibits the activities of both avian myeloblastosis virus and human immunodeficiency virus type 1 RTs. To further characterize Mn2+ inhibition, we generated RT/RH suppressor mutants capable of increased Ty1 transposition in pmr1Δ cells. PMR1 codes for a P-type ATPase that regulates intracellular calcium and manganese ion homeostasis, and pmr1 mutants accumulate elevated intracellular manganese levels and display 100-fold less transposition than PMR1 + cells. Mapping of these suppressor mutations revealed, surprisingly, that suppressor point mutations localize not to the RT itself but to the RH domain of the protein. Furthermore, Mn2+ inhibition of in vitro RT activity is greatly reduced in all the suppressor mutants, whereas RH activity and cleavage specificity remain largely unchanged. These intriguing results reveal that the effect of these suppressor mutations is transmitted to the polymerase domain and suggest biochemical communication between these two domains during reverse transcription.

scholarly journals Specific binding of tryptophan transfer RNA to avian myeloblastosis virus RNA-dependent DNA polymerase (reverse transcriptase).

1975 ◽  
Vol 72 (7) ◽  
pp. 2535-2539 ◽  
Author(s):  
A. Panet ◽  
W. A. Haseltine ◽  
D. Baltimore ◽  
G. Peters ◽  
F. Harada ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Dna Polymerase ◽  
Specific Binding ◽  
Transfer Rna ◽  
Avian Myeloblastosis Virus ◽  
Virus Rna

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

2015 ◽  
Vol 89 (16) ◽  
pp. 8119-8129 ◽  
Author(s):  
Eytan Herzig ◽  
Nickolay Voronin ◽  
Nataly Kucherenko ◽  
Amnon Hizi
Keyword(s):  
Life Cycle ◽  
Viral Replication ◽  
Reverse Transcriptase ◽  
Dna Polymerase ◽  
Reverse Transcription ◽  
Polymerase Activity ◽  
Rnase H ◽  
Strand Transfer ◽  
Hiv 1

ABSTRACTThe 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 thein vitrodata. 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 thein vitrodata. 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.

scholarly journals HIV-1 Reverse Transcriptase Can Simultaneously Engage Its DNA/RNA Substrate at Both DNA Polymerase and RNase H Active Sites: Implications for RNase H Inhibition

2009 ◽  
Vol 388 (3) ◽  
pp. 462-474 ◽  
Author(s):  
Greg L. Beilhartz ◽  
Michaela Wendeler ◽  
Noel Baichoo ◽  
Jason Rausch ◽  
Stuart Le Grice ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Dna Polymerase ◽  
Active Sites ◽  
Rnase H ◽  
Hiv 1

scholarly journals Apparent allosterism by avian myeloblastosis virus reverse transcriptase and E coli DNA polymerase I

1979 ◽  
Vol 6 (3) ◽  
pp. 1189-1201 ◽  
Author(s):  
Thomas L. Darling ◽  
Ted W. Reid
Keyword(s):  
Reverse Transcriptase ◽  
Dna Polymerase ◽  
Dna Polymerase I ◽  
Avian Myeloblastosis Virus ◽  
E Coli ◽  
Polymerase I

Model RNA-directed DNA synthesis by avian myeloblastosis virus DNA polymerase and its associated RNase H

Biochemistry ◽  
1979 ◽  
Vol 18 (15) ◽  
pp. 3210-3219 ◽  
Author(s):  
Kenneth F. Watson ◽  
Peter L. Schendel ◽  
Mae J. Rosok ◽  
Leonard R. Ramsey
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Rnase H ◽  
Avian Myeloblastosis Virus

Captan binding to avian myeloblastosis virus reverse transcriptase and its effect on RNase H activity

1990 ◽  
Vol 94 (1) ◽  
Author(s):  
M.J. Freeman-Wittig ◽  
RogerA. Lewis
Keyword(s):  
Reverse Transcriptase ◽  
Rnase H ◽  
Avian Myeloblastosis Virus

scholarly journals Inhibition of Human Immunodeficiency Virus Type 1 Reverse Transcriptase, RNase H, and Integrase Activities by Hydroxytropolones

2005 ◽  
Vol 49 (12) ◽  
pp. 4884-4894 ◽  
Author(s):  
Joël Didierjean ◽  
Catherine Isel ◽  
Flore Querré ◽  
Jean-François Mouscadet ◽  
Anne-Marie Aubertin ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Metal Ions ◽  
Dna Polymerase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Active Site ◽  
Rnase H ◽  
Strand Transfer ◽  
Immunodeficiency Virus

ABSTRACT Human immunodeficiency virus type I reverse transcriptase (RT) possesses distinct DNA polymerase and RNase H sites, whereas integrase (IN) uses the same active site to perform 3′-end processing and strand transfer of the proviral DNA. These four enzymatic activities are essential for viral replication and require metal ions. Two Mg2+ ions are present in the RT polymerase site, and one or two Mg2+ ions are required for the catalytic activities of RNase H and IN. We tested the possibility of inhibition of the RT polymerase and RNase H as well as the IN 3′-end processing and transfer activities of purified enzymes by a series of 3,7-dihydroxytropolones designed to target two Mg2+ ions separated by ∼3.7 Å. The RT polymerase and IN 3′ processing and strand transfer activities were inhibited at submicromolar concentrations, while the RNase H activity was inhibited in the low micromolar range. In all cases, the lack of inhibition by tropolones and O-methylated 3,7-dihydroxytropolones was consistent with the active molecules binding the metal ions in the active site. In addition, inhibition of the DNA polymerase activity was shown to depend on the Mg2+ concentration. Furthermore, selective inhibitors were identified for several of the activities tested, leaving some potential for design of improved inhibitors. However, all tested compounds exhibited cellular toxicity that presently limits their applications.

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