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Volume 272, Number 13, Issue of March 28, 1997 pp. 8602-8610
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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Substituting a Conserved Residue of the Ribonuclease H Domain Alters Substrate Hydrolysis by Retroviral Reverse Transcriptase

(Received for publication, October 9, 1996, and in revised form, December 20, 1996)

Jason W. Rausch ^§ and Stuart F. J. Le Grice ^§¶

From the  Department of Biochemistry, ^§ Division of Infectious Diseases, and ^¶ Center for AIDS Research, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4984

Alterations to the highly conserved Asp⁵⁴⁹ of the retroviral ribonuclease H (RNase H) domain were evaluated in the heterodimeric (p66/p51) reverse transcriptases of human immunodeficiency and equine infectious anemia viruses. In addition to the polymerization-dependent and -independent modes of template hydrolysis, mutants were evaluated via their ability to select and extend the 3 polypurine tract (PPT) primers of these two lentiviruses into (+) strand DNA. Concerted and two-step reactions were designed to evaluate (+) strand priming, the latter of which allows discrimination between selection end extension events. In contrast to enzyme mutated at the highly conserved Glu⁴⁷⁸, substitution of Asp⁵⁴⁹ with Asn or Ala reduces, rather than completely eliminates, RNase H activity. When the requirement for RNase H function becomes more stringent, differences in activity are readily evident, most notably in the cleavage events liberating the 5 terminus of the PPT primer. PPT selection thus appears to represent a specialized form of RNase H activity that is more sensitive to minor structural alterations within this domain and may provide a novel therapeutic target.

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