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Originally published In Press as doi:10.1074/jbc.M710149200 on January 24, 2008

J. Biol. Chem., Vol. 283, Issue 14, 9206-9216, April 4, 2008
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Reduced dNTP Binding Affinity of 3TC-resistant M184I HIV-1 Reverse Transcriptase Variants Responsible for Viral Infection Failure in Macrophage*

Varuni K Jamburuthugoda{ddagger}1, Jose M. Santos-Velazquez§1, Mark Skasko{ddagger}, Darwin J. Operario{ddagger}, Vandana Purohit§, Pauline Chugh{ddagger}, Erika A. Szymanski{ddagger}, Joseph E. Wedekind§, Robert A. Bambara§, and Baek Kim{ddagger}2

From the Departments of {ddagger}Microbiology and Immunology and §Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642

We characterized HIV-1 reverse transcriptase (RT) variants either with or without the (-)-2',3'-deoxy-3'-thiacytidine-resistant M184I mutation isolated from a single HIV-1 infected patient. First, unlike variants with wild-type M184, M184I RT variants displayed significantly reduced DNA polymerase activity at low dNTP concentrations, which is indicative of reduced dNTP binding affinity. Second, the M184I variant displayed a ~10- to 13-fold reduction in dNTP binding affinity, compared with the Met-184 variant. However, the kpol values of these two RTs were similar. Third, unlike HIV-1 vectors with wild-type RT, the HIV-1 vector harboring M184I RT failed to transduce cell types containing low dNTP concentrations, such as human macrophage, likely due to the reduced DNA polymerization activity of the M184I RT under low cellular dNTP concentration conditions. Finally, we compared the binary complex structures of wild-type and M184I RTs. The Ile mutation at position 184 with a longer and more rigid β-branched side chain, which was previously known to alter the RT-template interaction, also appears to deform the shape of the dNTP binding pocket. This can restrict ground state dNTP binding and lead to inefficient DNA synthesis particularly at low dNTP concentrations, ultimately contributing to viral replication failure in macrophage and instability in vivo of the M184I mutation.


Received for publication, December 12, 2007 , and in revised form, January 18, 2008.

* This work was supported by Grants AI49781 (to B. K.) and R25GM64133 (to J. M. S.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Both authors contributed equally to this work.

2 To whom correspondence should be addressed: Dept. of Microbiology and Immunology, University of Rochester Medical Center, 601 Elmwood Ave., Box 672, Rochester, NY 14642. Tel.: 585-275-6916; Fax: 585-473-9573; E-mail: baek_kim{at}urmc.rochester.edu.


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