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Journal of Virology, July 2004, p. 7089-7096, Vol. 78, No. 13
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.13.7089-7096.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Subunit-Specific Analysis of the Human Immunodeficiency Virus Type 1 Reverse Transcriptase In Vivo

Alok Mulky,1 Stefan G. Sarafianos,2 Edward Arnold,2 Xiaoyun Wu,3 and John C. Kappes1,3,4*

Departments of Microbiology,1 Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294,3 Center for Advanced Biotechnology and Medicine and Rutgers University Chemistry Department, Piscataway, New Jersey 08854-5638,2 Research Service, Birmingham Veterans Affairs Medical Center, Birmingham, Alabama 352334

Received 3 December 2003/ Accepted 18 February 2004

The human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a heterodimer comprised of two structurally distinct subunits (p51 and p66). Since p51 and p66 are derived from the same coding region, subunit-specific structure-function studies of RT have been conducted exclusively by in vitro biochemical approaches. To study RT subunit function in the context of infectious virus, we constructed an LTR-vpr-p51-IRES-p66 expression cassette in which the HIV-1 vpr gene was fused in frame with p51, followed by an internal ribosome entry site (IRES) sequence and the p66 coding region. By coexpression with RT-deficient proviral DNA, we demonstrated that the p66 subunit is specifically and selectively packaged into virions as a Vpr-p51/p66 complex. Our analysis showed that cleavage by the viral protease liberates Vpr and generates functional heterodimeric RT (p51/p66) that supports HIV-1 reverse transcription and virus infection. By exploiting this novel trans-complementation approach, we demonstrated, for the first time with infectious virions, that the YMDD aspartates of p66 are both required and sufficient for RT polymerase function. Mutational analyses of the p51 YMDD aspartates indicated that they play an important structural role in p51 folding and subunit interactions that are required for the formation of an active RT heterodimer within infected cells. Understanding the role of the individual RT subunits in RNA- and DNA-dependent DNA synthesis is integral to our understanding of RT function. Our findings will lead to important new insights into the role of the p51 and p66 subunits in HIV-1 reverse transcription.

* Corresponding author. Mailing address: Department of Medicine, University of Alabama at Birmingham, LHRB 613, 701 South 19th St., Birmingham, AL 35294. Phone: (205) 934-0051. Fax: (205) 975-7300. E-mail: kappesjc{at}uab.edu.

Journal of Virology, July 2004, p. 7089-7096, Vol. 78, No. 13
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.13.7089-7096.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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