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J. Biol. Chem., Vol. 281, Issue 40, 30269-30278, October 6, 2006

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Tracking and Elucidating Alphavirus-Host Protein Interactions^*

Ileana M. Cristea, John-William N. Carroll, Michael P. Rout^¶, Charles M. Rice, Brian T. Chait¹, and Margaret R. MacDonald²

From the Mass Spectrometry and Gaseous Ion Chemistry, Virology and Infectious Disease, and ^¶Cellular and Structural Biology, The Rockefeller University, New York, New York 10021

Viral infections cause profound alterations in host cells. Here, we explore the interactions between proteins of the Alphavirus Sindbis and host factors during the course of mammalian cell infection. Using a mutant virus expressing the viral nsP3 protein tagged with green fluorescent protein (GFP) we directly observed nsP3 localization and isolated nsP3-interacting proteins at various times after infection. These results revealed that host factor recruitment to nsP3-containing complexes was time dependent, with a specific early and persistent recruitment of G3BP and a later recruitment of 14-3-3 proteins. Expression of GFP-tagged G3BP allowed reciprocal isolation of nsP3 in Sindbis infected cells, as well as the identification of novel G3BP-interacting proteins in both uninfected and infected cells. Note-worthy interactions include nuclear pore complex components whose interactions with G3BP were reduced upon Sindbis infection. This suggests that G3BP is a nuclear transport factor, as hypothesized previously, and that viral infection may alter RNA transport. Immunoelectron microscopy showed that a portion of Sindbis nsP3 is localized at the nuclear envelope, suggesting a possible site of G3BP recruitment to nsP3-containing complexes. Our results demonstrate the utility of using a standard GFP tag to both track viral protein localization and elucidate specific viral-host interactions over time in infected mammalian cells.

Received for publication, April 26, 2006 , and in revised form, July 10, 2006.

^* This work was supported by National Institutes of Health Grants AI063233 (to M. R. M.), AI24134 (to C. M. R.), RR00862 (to B. T. C.), RR022220 (to M. P. R. and B. T. C.), and GM062427 (to M. P. R.); by The Rockefeller University Women and Science Fellowship CEN5300379 (to I. M. C.); and by the Greenberg Medical Research Institute. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S11-S14 and Tables S1 and S2.

¹ To whom correspondence may be addressed: The Rockefeller University, 1230 York Ave., New York, NY 10021. Tel.: 212-327-8849; Fax: 212-327-7547; E-mail: chait{at}rockefeller.edu.

² To whom correspondence may be addressed: The Rockefeller University, 1230 York Ave., New York, NY 10021. Tel.: 212-327-7078; Fax: 212-327-7048; E-mail: macdonm{at}rockefeller.edu.

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