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Journal of Virology, May 2004, p. 5056-5067, Vol. 78, No. 10
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.10.5056-5067.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Interaction between Human Immunodeficiency Virus Type 1 Reverse Transcriptase and Integrase Proteins

Eric A. Hehl,1,{dagger} Pheroze Joshi,1 Ganjam V. Kalpana,2* and Vinayaka R. Prasad1*

Departments of Microbiology and Immunology,1 Molecular Genetics, Albert Einstein College of Medicine, Bronx, New York2

Received 19 July 2003/ Accepted 22 January 2004

Reverse transcriptase (RT) and integrase (IN) are two key catalytic enzymes encoded by all retroviruses. It has been shown that a specific interaction occurs between the human immunodeficiency virus type 1 (HIV-1) RT and IN proteins (X. Wu, H. Liu, H. Xiao, J. A. Conway, E. Hehl, G. V. Kalpana, V. R. Prasad, and J. C. Kappes, J. Virol. 73:2126-2135, 1999). We have now further examined this interaction to map the binding domains and to determine the effects of interaction on enzyme function. Using recombinant purified proteins, we have found that both a HIV-1 RT heterodimer (p66/p51) and its individual subunits, p51 and p66, are able to bind to HIV-1 IN. An oligomerization-defective mutant of IN, V260E, retained the ability to bind to RT, showing that IN oligomerization may not be required for interaction. Furthermore, we report that the C-terminal domain of IN, but not the N-terminal zinc-binding domain or the catalytic core domain, was able to bind to heterodimeric RT. Deletion analysis to map the IN-binding domain on RT revealed two separate IN-interacting domains: the fingers-palm domain and the carboxy-terminal half of the connection subdomain. The carboxy-terminal domain of IN alone retained its interaction with both the fingers-palm and the connection-RNase H fragments of RT, but not with the half connection-RNase H fragment. This interaction was not bridged by nucleic acids, as shown by micrococcal nuclease treatment of the proteins prior to the binding reaction. The influences of IN and RT on each other's activities were investigated by performing RT processivity and IN-mediated 3' processing and joining reactions in the presence of both proteins. Our results suggest that, while IN had no influence on RT processivity, RT stimulated the IN-mediated strand transfer reaction in a dose-dependent manner up to 155-fold. Thus, a functional interaction between these two viral enzymes may occur during viral replication.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Phone for Vinayaka R. Prasad: (718) 430-2517. Fax: (718) 430-8976. E-mail: prasad{at}aecom.yu.edu. Phone for Ganjam V. Kalpana: (718) 430-2354. Fax: (718) 430-8778. E-mail: kalpana{at}aecom.yu.edu.

{dagger} Present address: University of Maryland University College, College Park, MD 20742.

Journal of Virology, May 2004, p. 5056-5067, Vol. 78, No. 10
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.10.5056-5067.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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