Improvement of Moloney murine leukemia virus reverse transcriptase thermostability by introducing a disulfide bridge in the ribonuclease H region

2021 ◽  
Vol 34 ◽  
Author(s):  
Yutaro Narukawa ◽  
Mako Kandabashi ◽  
Tongyang Li ◽  
Misato Baba ◽  
Haruka Hara ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Disulfide Bridge ◽  
Rnase H ◽  
Site Directed Mutagenesis ◽  
Moloney Murine Leukemia Virus ◽  
Ribonuclease H ◽  
Cdna Synthesis ◽  
Murine Leukemia

Abstract Moloney murine leukemia virus (MMLV) reverse transcriptase (RT) is widely used in research and clinical diagnosis. Improvement of MMLV RT thermostability has been an important topic of research for increasing the efficiency of cDNA synthesis. In this study, we attempted to increase MMLV RT thermostability by introducing a disulfide bridge in its RNase H region using site-directed mutagenesis. Five variants were designed, focusing on the distance between the two residues to be mutated into cysteine. The variants were expressed in Escherichia coli and purified. A551C/T662C was determined to be the most thermostable variant.

scholarly journals Preparation and characterization of the RNase H domain of Moloney murine leukemia virus reverse transcriptase

2015 ◽  
Vol 113 ◽  
pp. 44-50 ◽  
Author(s):  
Kosaku Nishimura ◽  
Kanta Yokokawa ◽  
Tetsuro Hisayoshi ◽  
Kosuke Fukatsu ◽  
Ikumi Kuze ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Rnase H ◽  
Moloney Murine Leukemia Virus ◽  
Murine Leukemia

scholarly journals Small Dumbbell Oligonucleotides Inhibitors of RNase H Activity of MOMULV Reverse Transcriptase

2011 ◽  
Vol 8 (2) ◽  
pp. 629-634
Author(s):  
Ajay Kumar
Keyword(s):  
Reverse Transcriptase ◽  
Murine Leukemia Virus ◽  
Dissociation Constants ◽  
Leukemia Virus ◽  
Rnase H ◽  
Moloney Murine Leukemia Virus ◽  
Mobility Shift ◽  
Hiv 1 ◽  
Murine Leukemia

The small dumbbell oligonucleotides containing loops of phosphodiester (OL-1), two trimethylene, C3moieties in each loop (OL-2) and phosphorothioate (OL-3) linkages were synthesized. Incubation of OL-1 and OL-2 with S-1 nuclease generated break down products whereas incubation of OL-3 did not result in significant cleavage. Their binding to moloney murine leukemia virus reverse transcriptase was evaluated by PAGE band mobility shift assays. The OL-3 bound more strongly to the reverse transcriptase than OL-1 and OL-2. The dissociation constants evaluated using PAGE band mobility shift assays were of the order of 10-7. Investigation of inhibition of RNase H activity of reverse transcriptase showed that the OL-3 is a better inhibitor of the retroviral RNase H activity than both OL-1 and OL-2. Thus OL-3 may be used as RNase H inhibitor. Our studies demonstrated that this particularly designed oligonucleotide (OL-3) displays an IC50of 25 nM in its inhibition on the reverse transcriptase RNase H activity, a magnitude lower than that of first nucleotide reverse transcriptase of HIV-1, tenofovir, introduced by Gilead Science in the market.

scholarly journals RNase H Activity Is Required for High-Frequency Repeat Deletion during Moloney Murine Leukemia Virus Replication

2002 ◽  
Vol 76 (1) ◽  
pp. 88-95 ◽  
Author(s):  
Jennifer L. Brincat ◽  
Julie K. Pfeiffer ◽  
Alice Telesnitsky
Keyword(s):  
Reverse Transcriptase ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Direct Repeat ◽  
Rnase H ◽  
Moloney Murine Leukemia Virus ◽  
Functional Dna ◽  
Mutant Reverse Transcriptase ◽  
Murine Leukemia ◽  
Deletion Frequency

ABSTRACT It has been postulated that retroviral recombination, like strong stop template switching, requires the RNase H activity of reverse transcriptase. To address this hypothesis, Moloney murine leukemia virus-based vectors, which were designed to test the recombination-related property of direct repeat deletion, were encapsidated in virions engineered to contain phenotypic mixtures of wild-type and RNase H catalytic site point mutant reverse transcriptase. Integrated provirus titers per milliliter were determined for these phenotypically mixed virions, and vector proviruses were screened to determine what percentage contained repeat deletions. The results revealed a steady decline in direct repeat deletion frequency that correlated with decreases in functional RNase H, with greater than fourfold decreases in repeat deletion frequency observed when 95% of virion reverse transcriptase was RNase H defective. Parallel experiments were performed to address effects of molar excesses of RNase H relative to functional DNA polymerase. These experiments demonstrated that increasing the stoichiometry of RNase H relative to the amount of functional DNA polymerase had minimal effects on direct repeat deletion frequency. DNA synthesis was error prone when directed principally by RNase H mutant reverse transcriptase, suggesting a role for RNase H catalytic integrity in the fidelity of intracellular reverse transcription.

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