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J. Biol. Chem., Vol. 282, Issue 6, 3847-3855, February 9, 2007

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An Alix Fragment Potently Inhibits HIV-1 Budding

CHARACTERIZATION OF BINDING TO RETROVIRAL YPXL LATE DOMAINS^*

Utpal M. Munshi¹, Jaewon Kim¹², Kunio Nagashima^¶, James H. Hurley³, and Eric O. Freed⁴

From the Virus-Cell Interaction Section, HIV Drug Resistance Program, National Cancer Institute-Frederick, Frederick, Maryland 21702-1201, the Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892, and the ^¶Image Analysis Laboratory, Research Technology Program, SAIC-Frederick, NCI-Frederick, Frederick, Maryland 21702-1201

The retroviral structural protein, Gag, contains small peptide motifs known as late domains that promote efficient virus release from the infected cell. In addition to the well characterized PTAP late domain, the p6 region of HIV-1 Gag contains a binding site for the host cell protein Alix. To better understand the functional role of the Gag/Alix interaction, we overexpressed an Alix fragment composed of residues 364–716 (Alix 364–716) and examined the effect on release of wild type (WT) and Alix binding site mutant HIV-1. We observed that Alix 364–716 expression significantly inhibited WT virus release and Gag processing and that mutation of the Alix binding site largely relieved this inhibition. Furthermore, Alix 364–716 expression induced a severe defect on WT but not mutant particle morphology. Intriguingly, the impact of Alix 364–716 expression on HIV-1 release and Gag processing was markedly different from that induced by mutation of the Alix binding site in p6. The association of Alix 364–716 with HIV-1 and equine infectious anemia virus late domains was quantitatively evaluated by isothermal titration calorimetry and surface plasmon resonance techniques, and the effects of mutations in these viral sequences on Alix 364–716 binding was determined. This study identifies a novel Alix-derived dominant negative inhibitor of HIV-1 release and Gag processing and provides quantitative information on the interaction between Alix and viral late domains.

Received for publication, August 7, 2006 , and in revised form, October 27, 2006.

^* This work was supported in part by the Intramural Research Program of the NCI, National Institutes of Health (NIH), Center for Cancer Research (to E. O. F.) and the NIDDK, NIH (to J. H. H.). This work was also supported by the Intramural AIDS Targeted Antiviral Program (to E. O. F. and J. H. H.) and NCI, NIH, under Contract N01-CO-12400 (to K. N.). 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 These two authors contributed equally to this work.

² Present address: Amgen, Inc., 1201 Amgen Court W., Seattle, WA 98119-3105.

³ To whom correspondence may be addressed: Laboratory of Molecular Biology, MSC 0580, Bldg. 50, Rm. 4517, NIDDK, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892-0580. Tel.: 301-402-4703; Fax: 301-480-0639; E-mail: hurley{at}helix.nih.gov. ⁴ To whom correspondence may be addressed: Virus-Cell Interaction Section, HIV Drug Resistance Program, NCI-Frederick, Bldg. 535, Rm. 108, Frederick, MD 21702-1201. Tel.: 301-846-6223; Fax: 301-846-6777; E-mail: efreed{at}nih.gov.

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