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J. Biol. Chem., Vol. 280, Issue 34, 30083-30090, August 26, 2005

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Isolation of TAK-779-resistant HIV-1 from an R5 HIV-1 GP120 V3 Loop Library^*

Keisuke Yusa, Yosuke Maeda, Aki Fujioka, Kazuaki Monde, and Shinji Harada

From the Department of Medical Virology, Graduate School of Medical Sciences, Kumamoto University, 2-1-1 Honjo, Kumamoto 860-8556, Japan

The human immunodeficiency virus (HIV-1) envelope glycoprotein (GP) 120 interacts with CD4 and the CCR5 coreceptor for viral entry. The V3 loop in GP120 is a crucial region for determining coreceptor usage during viral entry, and a variety of amino acid substitutions has been observed in clinical isolates. To construct an HIV-1 V3 loop library, we chose 10 amino acid positions in the V3 loop and incorporated random combinations (27,648 possibilities) of the amino acid substitutions derived from 31 R5 viruses into the V3 loop of HIV-1_JR-FL proviral DNA. The constructed HIV-1 library contained 6.6 x 10⁶ independent clones containing a set of 0–10 amino acid substitutions in the V3 loop. To address whether restricted steric alteration in the V3 loop could confer resistance to an entry inhibitor, TAK-779, we selected entry inhibitor-resistant HIV-1 by increasing the concentration of TAK-779 from 0.10 to 0.30 µM in PM1-CCR5 cells with high expression of CCR5. The selected viruses at passage 8 contained five amino acid substitutions in the V3 loop without any other mutations in GP120 and showed 15-fold resistance compared with the parental virus. These results indicated that a certain structure of the V3 loop containing amino acid substitutions derived from 31 R5 viruses can contribute to the acquisition of resistance to entry inhibitors binding to CCR5. Taken together, this type of HIV-1 V3 loop library is useful for isolating and analyzing the specific biological features of HIV-1 with respect to alterations of the V3 loop structure.

Received for publication, December 21, 2004 , and in revised form, June 27, 2005.

^* This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology and the Ministry of Health, Labor, and Welfare, Japan. 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.

To whom correspondence should be addressed: Dept. of Medical Virology, Graduate School of Medical Sciences, Kumamoto University 2-1-1 Honjo, Kumamoto 860-8556, Japan. Tel.: 81-96-373-5129; Fax: 81-96-373-5132; E-mail: yusak{at}kaiju.medic.kumamoto-u.ac.jp.

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