Defects in Primer-Template Binding, Processive DNA Synthesis, and RNase H Activity Associated with Chimeric Reverse Transcriptases Having the Murine Leukemia Virus Polymerase Domain Joined to Escherichia coli RNase H

Biochemistry ◽  
1995 ◽  
Vol 34 (15) ◽  
pp. 5018-5029 ◽  
Author(s):  
Jianhui Guo ◽  
Weixing Wu ◽  
Zhong Yi Yuan ◽  
Klara Post ◽  
Robert J. Crouch ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Synthesis ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Rnase H ◽  
Reverse Transcriptases ◽  
Polymerase Domain ◽  
Murine Leukemia

scholarly journals Specific Cleavages by RNase H Facilitate Initiation of Plus-Strand RNA Synthesis by Moloney Murine Leukemia Virus

2003 ◽  
Vol 77 (9) ◽  
pp. 5275-5285 ◽  
Author(s):  
Sharon J. Schultz ◽  
Miaohua Zhang ◽  
James J. Champoux
Keyword(s):  
Dna Synthesis ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Rnase H ◽  
Primer Extension ◽  
Moloney Murine Leukemia Virus ◽  
Cleavage Sites ◽  
Reverse Transcriptases ◽  
Hybrid Substrates ◽  
Murine Leukemia

ABSTRACT Successful generation, extension, and removal of the plus-strand primer is integral to reverse transcription. For Moloney murine leukemia virus, primer removal at the RNA/DNA junction leaves the 3′ terminus of the plus-strand primer abutting the downstream plus-strand DNA, but this 3′ terminus is not efficiently reutilized for another round of extension. The RNase H cleavage to create the plus-strand primer might similarly result in the 3′ terminus of this primer abutting downstream RNA, yet efficient initiation must occur to synthesize the plus-strand DNA. We hypothesized that displacement synthesis, RNase H activity, or both must participate to initiate plus-strand DNA synthesis. Using model hybrid substrates and RNase H-deficient reverse transcriptases, we found that displacement synthesis alone did not efficiently extend the plus-strand primer at a nick with downstream RNA. However, specific cleavage sites for RNase H were identified in the sequence immediately following the 3′ end of the plus-strand primer. During generation of the plus-strand primer, cleavage at these sites generated a gap. When representative gaps separated the 3′ terminus of the plus-strand primer from downstream RNA, primer extension significantly improved. The contribution of RNase H to the initiation of plus-strand DNA synthesis was confirmed by comparing the effects of downstream RNA versus DNA on plus-strand primer extension by wild-type reverse transcriptase. These data suggest a model in which efficient initiation of plus-strand synthesis requires the generation of a gap immediately following the plus-strand primer 3′ terminus.

scholarly journals Development of an In Vivo Assay To Identify Structural Determinants in Murine Leukemia Virus Reverse Transcriptase Important for Fidelity

2000 ◽  
Vol 74 (1) ◽  
pp. 312-319 ◽  
Author(s):  
Elias K. Halvas ◽  
Evguenia S. Svarovskaia ◽  
Vinay K. Pathak
Keyword(s):  
Dna Synthesis ◽  
Murine Leukemia Virus ◽  
Gene Transfection ◽  
Leukemia Virus ◽  
Rnase H ◽  
Wild Type ◽  
Structural Determinants ◽  
In Vivo Assay ◽  
Murine Leukemia

ABSTRACT Error-prone DNA synthesis by retroviral reverse transcriptases (RTs) is a major contributor to variation in retroviral populations. Structural features of retroviral RTs that are important for accuracy of DNA synthesis in vivo are not known. To identify structural elements of murine leukemia virus (MLV) RT important for fidelity in vivo, we developed a D17-based encapsidating cell line (ANGIE P) which is designed to express the amphotropic MLV envelope. ANGIE P also contains an MLV-based retroviral vector (GA-1) which encodes a wild-type bacterial β-galactosidase gene (lacZ) and a neomycin phosphotransferase gene. Transfection of ANGIE P cells with wild-type or mutated MLV gag-pol expression constructs generated GA-1 virus that was able to undergo only one cycle of viral replication upon infection of D17 cells. The infected D17 cell clones were characterized by staining with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal), and the frequencies of inactivating mutations in lacZ were quantified. Three mutations in the YVDD motif (V223M, V223S, and V223A) and two mutations in the RNase H domain (S526A and R657S) exhibited frequencies of lacZ inactivation 1.2- to 2.3-fold higher than that for the wild-type MLV RT (P < 0.005). Two mutations (V223I and Y598V) did not affect the frequency oflacZ inactivation. These results establish a sensitive in vivo assay for identification of structural determinants important for accuracy of DNA synthesis and indicate that several structural determinants may have an effect on the in vivo fidelity of MLV RT.

Expression of the murine leukemia virus protease in fusion with maltose-binding protein in Escherichia coli

2004 ◽  
Vol 35 (1) ◽  
pp. 62-68 ◽  
Author(s):  
Anita Fehér ◽  
Péter Boross ◽  
Tamás Sperka ◽  
Stephen Oroszlan ◽  
József Tözsér
Keyword(s):  
Escherichia Coli ◽  
Murine Leukemia Virus ◽  
Binding Protein ◽  
Leukemia Virus ◽  
Maltose Binding Protein ◽  
Murine Leukemia
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