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Identification of Staufen in the Human Immunodeficiency Virus Type 1 Gag Ribonucleoprotein Complex and a Role in Generating Infectious Viral Particles

Lady Davis Institute for Medical Research-Sir Mortimer B. Davis Jewish General Hospital,¹ Departments of Biochemistry,² Microbiology and Immunology, Université de Montréal,³ Departments of Microbiology and Immunology,⁴ Medicine,⁵ Division of Experimental Medicine, McGill University, Montréal, Québec, Canada⁶

Received 1 December 2003/ Accepted 7 January 2004

Staufen is a host protein that is selectively incorporated into human immunodeficiency virus type 1 (HIV-1) particles in a poorly defined process that involves the selection of HIV-1 genomic RNA for encapsidation and the activity of its third double-stranded RNA-binding domain (dsRBD3). To better understand this, we characterized its interactions with pr55^Gag, the principal mediator of HIV-1 genomic RNA encapsidation. Chimeric proviruses harboring wild-type or mutant forms of Staufen were expressed in 293T cells. Cell fractionation analyses demonstrated that Staufen cosedimented with pr55^Gag within detergent-resistant, trypsin-sensitive complexes that excluded mature capsid and matrix proteins. Coimmunoprecipitation and bioluminescence resonance energy transfer assays demonstrated a specific and direct interaction between Staufen and the nucleocapsid domain of pr55^Gag in vitro and in live cells. This interaction is shown here to be mediated by Staufen's dsRBD3, with a contribution from its C-terminal domain. Immunoprecipitation and reverse transcription-PCR analyses showed that the 9-kb genomic RNA was found within Staufen-containing immune complexes. Spliced HIV-1 RNAs were not detected in these Staufen complexes, indicating a preferential association of Staufen with the 9-kb species. These results substantiate that Staufen and pr55^Gag interact directly during HIV-1 expression. Knockdown of Staufen expression by small interfering RNAs in HIV-1-expressing cells demonstrated that this cellular protein was important for the generation of infectious virus. These data show that Staufen, pr55^Gag, and genomic RNA are part of the same intracellular complex and support a role for Staufen in pr55^Gag function in viral assembly, genomic RNA encapsidation, and the generation of infectious viral particles.

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