title: Tracking and elucidating alphavirus-host protein interactions.
abstract: 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.
authors.affiliation: Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, New York 10021, USA.
authors.forename: Ileana M
authors.initials: IM
authors.lastname: Cristea
authors.forename: John-William N
authors.initials: JW
authors.lastname: Carroll
authors.forename: Michael P
authors.initials: MP
authors.lastname: Rout
authors.forename: Charles M
authors.initials: CM
authors.lastname: Rice
authors.forename: Brian T
authors.initials: BT
authors.lastname: Chait
authors.forename: Margaret R
authors.initials: MR
authors.lastname: MacDonald
mesh_terms: D000818:Animals; D002460:Cell Line; D005347:Fibroblasts / Q000737:chemistry / Q000648:ultrastructure / Q000821:virology; D006801:Humans; D025941:Protein Interaction Mapping; D051381:Rats; D012845:Sindbis Virus / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D017361:Viral Nonstructural Proteins; C089383:nsp3 protein, alphavirus