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Journal of Virology, August 2002, p. 8360-8373, Vol. 76, No. 16
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.16.8360-8373.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Mutations of the RNase H C Helix of the Moloney Murine Leukemia Virus Reverse Transcriptase Reveal Defects in Polypurine Tract Recognition

David Lim,1 Marianna Orlova,2 and Stephen P. Goff1,2*

Integrated Program in Cellular, Molecular and Biophysical Studies,1 Howard Hughes Medical Institute, and Department of Biochemistry and Molecular Biophysics, College of Physicians and Surgeons, Columbia University, New York, New York 100322

Received 22 January 2002/ Accepted 8 May 2002

Both the RNase H domain of Moloney murine leukemia virus (Mo-MLV) reverse transcriptase (RT) and Escherichia coli RNase H possess a positively charged {alpha}-helix (C helix) and a loop that are not present in the RNase H domains of human immunodeficiency virus (HIV) RT or avian sarcoma virus RT. Although a mutant Mo-MLV RT lacking the C helix ({Delta}C RT) retains DNA polymerase activity on homopolymeric substrates and partial RNase H activity, reverse transcription of the viral RNA genome in vivo is defective. To identify the essential features of the C helix, a panel of Mo-MLV RT mutants was generated. Analyses of these mutant viruses revealed the importance of residues H594, I597, R601, and G602. The mutants were tested for their ability to synthesize viral DNA after acute infections and to form proper 5' and 3' viral DNA ends. The mutant RTs were tested in vitro for exogenous RT activity, minus-strand strong-stop DNA synthesis in endogenous RT reactions, nonspecific RNase H activity, and finally, proper cleavage at the polypurine tract-U3 junction. The R601A mutant was the most defective mutant both in vivo and in vitro and possessed very little RNase H activity. The H594A, I597A, and G602A mutants had significant reductions in RNase H activity and in their rates of viral replication. Many of the mutants formed improper viral DNA ends and were less efficient in PPT-U3 recognition and cleavage in vitro. The data show that the C helix plays a crucial role for overall RNase H cleavage activity. The data also suggest that the C helix may play an important role in polypurine tract recognition and proper formation of the plus-strand DNA's 5' end.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biophysics and Howard Hughes Medical Institute, College of Physicians and Surgeons, Columbia University, New York, NY 10032. Phone: (212) 305-3794. Fax (212) 305-8692. E-mail: goff{at}cancercenter.columbia.edu.

Journal of Virology, August 2002, p. 8360-8373, Vol. 76, No. 16
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.16.8360-8373.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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