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Journal of Virology, January 2000, p. 312-319, Vol. 74, No. 1
Mary Babb Randolph Cancer Center and
Department of Biochemistry, West Virginia University, Morgantown,
West Virginia 26506
Received 18 June 1999/Accepted 4 October 1999
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
0022-538X/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Development of an In Vivo Assay To Identify
Structural Determinants in Murine Leukemia Virus Reverse
Transcriptase Important for Fidelity
-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-
-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 of
lacZ 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.
*
Corresponding author. Mailing address: HIV Drug
Resistance Program, DBS, National Cancer Institute, FCRDC, Bldg. 535, Rm. 334, Frederick, MD 21702. Phone: (301) 846-1710. Fax: (301)
846-6013. E-mail: vpathak{at}mail.ncifcrf.gov.
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