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Journal of Virology, May 2003, p. 5837-5845, Vol. 77, No. 10
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.10.5837-5845.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Mutations Proximal to the Minor Groove-Binding Track of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Differentially Affect Utilization of RNA versus DNA as Template

Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461,¹ National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709²

Received 3 December 2002/ Accepted 7 February 2003

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), like all retroviral RTs, is a versatile DNA polymerase that can copy both RNA and DNA templates. In spite of extensive investigations into the structure-function of this enzyme, the structural basis for this dual template specificity is poorly understood. Biochemical studies with two mutations in HIV-1 RT that affect residues contacting the template-primer now provide some insight into this specialized property. The mutations are N255D and N265D, both adjoining the minor groove-binding track, in the thumb region. The N265D substitution led to a loss of processive polymerization on DNA but not on RNA, whereas N255D drastically reduced processive synthesis on both templates. This differential template usage was accompanied by a rapid dissociation of the N265D variant on DNA but not RNA templates, whereas the N255D variant rapidly dissociated from both templates. Molecular dynamics modeling suggested that N265D leads to a loss of template strand-specific hydrogen bonding, indicating that this is a key determinant of the differential template affinity. The N255D substitution caused local changes in conformation and a consequent loss of interaction with the primer, leading to a loss of processive synthesis with both templates. We conclude that N265 is part of a subset of template-enzyme contacts that enable RT to utilize DNA templates in addition to RNA templates and that such residues play an important role in facilitating processive DNA synthesis on both RNA and DNA templates.

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