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Journal of Virology, January 2005, p. 419-427, Vol. 79, No. 1
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.1.419-427.2005

Mutations in the RNase H Primer Grip Domain of Murine Leukemia Virus Reverse Transcriptase Decrease Efficiency and Accuracy of Plus-Strand DNA Transfer

Jean L. Mbisa,¹ Galina N. Nikolenko, and Vinay K. Pathak^*

HIV Drug Resistance Program, National Cancer Institute at Frederick, Frederick, Maryland

Received 20 May 2004/ Accepted 19 August 2004

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 ß-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.

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* Corresponding author. Mailing address: HIV Drug Resistance Program, NCI-Frederick, P.O. Box B, Bldg. 535, Rm. 334, Frederick, MD 21702-1201. Phone: (301) 846-1710. Fax: (301) 846-6013. E-mail: vpathak{at}ncifcrf.gov.

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Journal of Virology, January 2005, p. 419-427, Vol. 79, No. 1
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.1.419-427.2005

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