title: Host cell interactome of HIV-1 Rev includes RNA helicases involved in multiple facets of virus production.
abstract: The HIV-1 Rev protein plays a key role in the late phase of virus replication. It binds to the Rev Response Element found in underspliced HIV mRNAs, and drives their nuclear export by the CRM1 receptor pathway. Moreover, mounting evidence suggests that Rev has additional functions in viral replication. Here we employed proteomics and statistical analysis to identify candidate host cell factors that interact with Rev. For this we studied Rev complexes assembled in vitro with nuclear or cytosolic extracts under conditions emulating various intracellular environments of Rev. We ranked the protein-protein interactions by combining several statistical features derived from pairwise comparison of conditions in which the abundance of the binding partners changed. As a validation set, we selected the eight DEAD/H box proteins of the RNA helicase family from the top-ranking 5% of the proteins. These proteins all associate with ectopically expressed Rev in immunoprecipitates of cultured cells. From gene knockdown approaches, our work in combination with previous studies indicates that six of the eight DEAD/H proteins are linked to HIV production in our cell model. In a more detailed analysis of infected cells where either DDX3X, DDX5, DDX17, or DDX21 was silenced, we observed distinctive phenotypes for multiple replication features, variously involving virus particle release, the levels of unspliced and spliced HIV mRNAs, and the nuclear and cytoplasmic concentrations of these transcripts. Altogether the work indicates that our top-scoring data set is enriched in Rev-interacting proteins relevant to HIV replication. Our more detailed analysis of several Rev-interacting DEAD proteins suggests a complex set of functions for the helicases in regulation of HIV mRNAs. The strategy used here for identifying Rev interaction partners should prove effective for analyzing other viral and cellular proteins.
authors.affiliation: Departments of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: Souad
authors.initials: S
authors.lastname: Naji
authors.forename: Géza
authors.initials: G
authors.lastname: Ambrus
authors.forename: Peter
authors.initials: P
authors.lastname: Cimermančič
authors.forename: Jason R
authors.initials: JR
authors.lastname: Reyes
authors.forename: Jeffrey R
authors.initials: JR
authors.lastname: Johnson
authors.forename: Rebecca
authors.initials: R
authors.lastname: Filbrandt
authors.forename: Michael D
authors.initials: MD
authors.lastname: Huber
authors.forename: Paul
authors.initials: P
authors.lastname: Vesely
authors.forename: Nevan J
authors.initials: NJ
authors.lastname: Krogan
authors.forename: John R
authors.initials: JR
authors.lastname: Yates
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Saphire
authors.forename: Larry
authors.initials: L
authors.lastname: Gerace
mesh_terms: D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015658:HIV Infections / Q000378:metabolism; D015497:HIV-1 / Q000378:metabolism / Q000472:pathogenicity*; D006367:HeLa Cells; D054884:Host-Pathogen Interactions; D006801:Humans; D040901:Proteomics; D034741:RNA, Small Interfering / Q000235:genetics; D014779:Virus Replication / Q000502:physiology*; D054321:rev Gene Products, Human Immunodeficiency Virus / Q000378:metabolism*
chemical_list: D034741:RNA, Small Interfering; D054321:rev Gene Products, Human Immunodeficiency Virus; C514860:rev protein, Human Immunodeficiency Virus-1; D053487:DEAD-box RNA Helicases