Mutations in the RNase H primer grip domain of murine leukemia virus reverse transcriptase decrease efficiency and accuracy of plus-strand DNA transfer.
The RNase H primer grip of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) contacts the DNA primer strand and positions the template strand near the RNase H active site, influencing RNase H cleavage efficiency and specificity. Sequence alignments show that 6 of the 11 residues that constitute the RNase H primer grip have functional equivalents in murine leukemia virus (MLV) RT. We previously showed that a Y586F substitution in the MLV RNase H primer grip resulted in a 17-fold increase in substitutions within 18 nucleotides of adenine-thymine tracts, which are associated with a bent DNA conformation. To further determine the effects of the MLV RNase H primer grip on replication fidelity and viral replication, we performed additional mutational analysis. Using either beta-galactosidase (lacZ) or green fluorescent protein (GFP) reporter genes, we found that S557A, A558V, and Q559L substitutions resulted in statistically significant increases in viral mutation rates, ranging from 2.1- to 3.8-fold. DNA sequencing analysis of nonfluorescent GFP clones indicated that the mutations in RNase H primer grip significantly increased the frequency of deletions between the primer-binding site (PBS) and sequences downstream of the PBS. In addition, quantitative real-time PCR analysis of reverse transcription products revealed that the mutant RTs were substantially inefficient in plus-strand DNA transfer relative to the wild-type control. These results indicate that the MLV RNase H primer grip is an important determinant of in vivo fidelity of DNA synthesis and suggest that the mutant RT was unable to copy through the DNA-RNA junction of the minus-strand DNA and the tRNA because of its bent conformation resulting in error-prone plus-strand DNA transfer.
Mbisa
Jean L
JL
HIV Drug Resistance Program, National Cancer Institute at Frederick, Frederick, Maryland 21702-1201, USA.
Nikolenko
Galina N
GN
Pathak
Vinay K
VK
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D017931:DNA Primers; D004279:DNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D004285:Dogs; D017353:Gene Deletion; D006801:Humans; D009052:Leukemia Virus, Murine / Q000201:enzymology* / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D012367:RNA, Viral / Q000096:biosynthesis; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016914:Ribonuclease H / Q000235:genetics*; D013329:Structure-Activity Relationship; D014779:Virus Replication
D017931:DNA Primers; D004279:DNA, Viral; D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H