The Role of Adaptor Protein Grb2 in Ecotropic Retrovirus Entry and Receptor Trafficking
For retroviruses such as HIV-1 and murine leukemia virus (MLV) active receptor recruitment and trafficking occur during viral entry. However, the underlying mechanisms and cellular factors involved in the process are largely uncharacterized. The viral receptor for ecotropic murine leukemia virus (eMLV), a classical model for retrovirus infection mechanism and pathogenesis, is the mouse cationic amino acid transporter 1 (mCAT1). Using a siRNA screen to detect host cell components important for virus infection, we identified growth factor receptor-bound protein 2 (Grb2), as a key protein for eMLV infection. Grb2 is an adaptor that has been shown to couple cell surface receptors such as the epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (Met) to intracellular signaling events. RNAi-mediated suppression of endogenous Grb2 resulted in a significant reduction of virus binding and membrane fusion. The binding between eMLV and cells promoted increased GRB2-mCAT-1 interactions, as detected by bi-directional immunoprecipitation and confocal microscopy. This association was time dependent and parallelled the kinetics of cell-virus membrane fusion. Expression of the dominant-negative Grb2 mutant (R86K) resulted in the removal of mCAT1 from the cell surface into intracellular vesicles. Consistent with this observation, the inhibition of endogenous Grb2 also lead to a reduction in surface mCAT1, which was detected by immunoprecipitation. I found that unlike the canonical binding pattern between Grb2 and growth factor receptors, Grb2-mCAT-1 binding was not phosphotyrosine-dependent. The binding of eMLV to the cells also leads to the recruitment of E3 ubiquitin ligase Cbl to mCAT-1 and the translocation of dynamin 2, which are both binding partners of Grb2. Taken together, these findings suggest an essential role for Grb2 in ecotropic MLV entry and infection by facilitating mCAT1 trafficking. My work not only elucidates an important aspect of the molecular mechanism of receptor trafficking during retrovirus entry and infection, but also reveals a previously unknown potential mechanism by which mCAT-1 can participate in signaling pathways by its interaction with Grb2. CAT-1 proteins are implicated in heart and renal disease, diabetes and Alzheimer’s disease. Our finding could contribute to better understanding of molecular mechanisms of how CAT-1 involved in these pathological conditions and useful for development of new therapeutic strategies.