Live cell visualization of the interactions between HIV-1 Gag and the cellular RNA-binding protein Staufen1.
BACKGROUND
Human immunodeficiency virus type 1 (HIV-1) uses cellular proteins and machinery to ensure transmission to uninfected cells. Although the host proteins involved in the transport of viral components toward the plasma membrane have been investigated, the dynamics of this process remain incompletely described. Previously we showed that the double-stranded (ds)RNA-binding protein, Staufen1 is found in the HIV-1 ribonucleoprotein (RNP) that contains the HIV-1 genomic RNA (vRNA), Gag and other host RNA-binding proteins in HIV-1-producing cells. Staufen1 interacts with the nucleocapsid domain (NC) domain of Gag and regulates Gag multimerization on membranes thereby modulating HIV-1 assembly. The formation of the HIV-1 RNP is dynamic and likely central to the fate of the vRNA during the late phase of the HIV-1 replication cycle.


RESULTS
Detailed molecular imaging of both the intracellular trafficking of virus components and of virus-host protein complexes is critical to enhance our understanding of factors that contribute to HIV-1 pathogenesis. In this work, we visualized the interactions between Gag and host proteins using bimolecular and trimolecular fluorescence complementation (BiFC and TriFC) analyses. These methods allow for the direct visualization of the localization of protein-protein and protein-protein-RNA interactions in live cells. We identified where the virus-host interactions between Gag and Staufen1 and Gag and IMP1 (also known as VICKZ1, IGF2BP1 and ZBP1) occur in cells. These virus-host interactions were not only detected in the cytoplasm, but were also found at cholesterol-enriched GM1-containing lipid raft plasma membrane domains. Importantly, Gag specifically recruited Staufen1 to the detergent insoluble membranes supporting a key function for this host factor during virus assembly. Notably, the TriFC experiments showed that Gag and Staufen1 actively recruited protein partners when tethered to mRNA.


CONCLUSIONS
The present work characterizes the interaction sites of key components of the HIV-1 RNP (Gag, Staufen1 and IMP1), thereby bringing to light where HIV-1 recruits and co-opts RNA-binding proteins during virus assembly.
Milev
Miroslav P
MP
HIV-1 RNA Trafficking Laboratory, Lady Davis Institute for Medical Research-Sir Mortimer B, Davis Jewish General Hospital, 3755 Côte-Ste-Catherine Road, Montréal, H3T 1E2 Québec, Canada.
Brown
Chris M
CM
Mouland
Andrew J
AJ
D002462:Cell Membrane / Q000737:chemistry; D003584:Cytological Techniques / Q000379:methods*; D003593:Cytoplasm / Q000737:chemistry; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D015497:HIV-1 / Q000502:physiology*; D006367:HeLa Cells; D054884:Host-Pathogen Interactions; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods; D019169:Jurkat Cells; D008856:Microscopy, Fluorescence / Q000379:methods; D011485:Protein Binding; D025941:Protein Interaction Mapping; D016601:RNA-Binding Proteins / Q000378:metabolism*; D019065:Virus Assembly; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000378:metabolism*
D003598:Cytoskeletal Proteins; C406144:IGF2BP1 protein, human; D016601:RNA-Binding Proteins; C492185:STAU1 protein, human; D054301:gag Gene Products, Human Immunodeficiency Virus
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