title: Tandem immunoprecipitation approach to identify HIV-1 Gag associated host factors.
abstract: HIV-1 Gag by itself is able to assemble and release from host cells and thus serves as a simplified model to identify host factors involved in this stage of the HIV-1 life cycle. In this study, a tandem immunoprecipitation approach is taken to immunoprecipitate Gag-interacting host proteins from transfected 293T cells. It is demonstrated that with the tandem immunoprecipitation method Gag-interacting host factors can be precipitated more efficiently than by single-step immunoprecipitation. Gag proteins are found to interact with multiple RNA-binding proteins such as hnRNPs, nucleolin, EF1a and ribosomal proteins. Such interactions are mediated by cellular RNAs and the Gag Nuclear Capsid (NC) domain. Deletion of the NC domain results in removal of most of the RNA-binding proteins, as well as a reduction of the Gag releasing capability, which can be restored by replacing the deleted NC domain with another multimerization motif. Importantly, interactions between Gag and host factors are relevant functionally, as evidenced by significantly increased nucleolin protein in the cytoplasm where it is recruited into the Gag complex, and enhanced Gag release when nucleolin is over-expressed.
authors.affiliation: Liaoning Medical University, First Affiliated Hospital, Jinzhou, Liaoning 121001, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Gao
authors.affiliation: Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China.
authors.forename: Min
authors.initials: M
authors.lastname: Li
authors.affiliation: Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China. Electronic address: zhang_jingxin@hotmail.com.
authors.forename: Jingxin
authors.initials: J
authors.lastname: Zhang
mesh_terms: D002460:Cell Line; D004252:DNA Mutational Analysis; D015497:HIV-1 / Q000502:physiology*; D054884:Host-Pathogen Interactions; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D016601:RNA-Binding Proteins / Q000302:isolation & purification; D012269:Ribosomal Proteins / Q000302:isolation & purification; D014773:Virology / Q000379:methods*; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000378:metabolism*
chemical_list: D016601:RNA-Binding Proteins; D012269:Ribosomal Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus
keywords: Assembly; FPLC; Gag; HIV; Tandem immunoprecipitation