Ek. Halvas et al., Development of an in vivo assay to identify structural determinants in murine leukemia virus reverse transcriptase important for fidelity, J VIROLOGY, 74(1), 2000, pp. 312-319
Error-prone DNA synthesis by retroviral reverse transcriptases (RTs) is a m
ajor contributor to variation in retroviral populations. Structural feature
s of retroviral RTs that are important fur accuracy of DNA Synthesis in viv
o are not known. To identify structural elements of murine leukemia virus (
MLV) RT important for fidelity in vivo, we developed a D17-based encapsidat
ing cell line (ANGIE P) which is designed to express the amphotropic MLV en
velope. ANGIE: P also contains an MLV-based retroviral vector (GA-1) which
encodes a wild-type bacterial beta-galactosidase gene (lacZ) and a neomycin
phosphotransferase gene. Transfection of ANGIE P cells with wild-type or m
utated 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-beta-D-galactopyranoside (X-Gal), and the frequencies of
inactivating mutations in lacZ were quantified. Three mutations in the YVD
D 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-fol
d higher than that for the wild-type MLV RT (P < 0.005). Two mutations (V22
3I and Y598V) did not affect the frequency of lacZ inactivation. These resu
lts establish a sensitive in vivo assay for identification of structural de
terminants important for accuracy of DNA synthesis and indicate that severa
l structural determinants may have an effect on the in vivo fidelity of MLV
RT.