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

Abstract

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.

We thank Kristi Bangs for originally identifying efficient siRNA targets for Staufen, Sylvie Bannwarth for performing the TRBP gradient analysis, Damian Purcell for DNA constructs and for providing advice on the RT-PCR analysis, éric A. Cohen for generous supply of materials and antibodies, Michel Tremblay for ELISA reagents and advice, Michel Bouvier and Billy Breton for constructs and advice on BRET assays, and the NIH AIDS Reference and Reagent Program and Spearman and Jacques Corbeil for the anti-MA antibody and CEM-GFP cell line, respectively. We thank Johanne Mercier for technical contributions to some of these studies and Hugo Dilhuydy for confocal imaging analyses.

J.-F.C. and C.M. were supported by studentships from the Fonds pour la recherche en santé du Québec (FRSQ) and the Natural Sciences and Engineering Research Council of Canada (NSERC). A.J.M. is a New Investigator of the Canadian Institutes of Health Research (CIHR) and a Scholar of the FRSQ. This work was supported by grants from the CIHR, the Canadian Foundation for Innovation, and the Canadian Foundation for AIDS Research to A.J.M. and from NSERC to L.D.