Identification of FAM111A as a novel simian virus 40 large T antigen binding protein that affects viral host range.

摘要

The host range function of SV40 large T antigen (T Ag) has been described as the inability of viruses lacking the C-terminus of T Ag to form plaques on certain types of African green monkey kidney cells. Host range mutant viruses lacking the C-terminus of T Ag fail to form plaques on CV-1P cells, but can form plaques on BSC40 cells. Co-expression of the C-terminus in trans rescues the cytopathic effect of host range virus on CV-1P cells. The host range virus has been shown to be severely defective for early and late viral gene expression at both the protein and mRNA level and that this defect can be rescued by co-expression of the C-terminus of T Ag in trans in both human and monkey cell lines.Immunoprecipitation of a C-terminal fragment of T Ag expressed in the human cell line U-2OS followed by mass spectrometric identification of associated proteins revealed a previously uncharacterized protein called FAM111A that bound specifically to T Ag. FAM111A binding maps to the C-terminal host range region of T Ag. The interaction of endogenous FAM111A with untagged T Ag was confirmed by immunoprecipitation and western blotting. Depletion of FAM111A by siRNA in U-2OS cells increases early and late viral gene expression eight-to-ten-fold. Stable depletion of FAM111A in restrictive CV-1P cells rescues plaque formation of the host range mutant virus, indicating that FAM111A fulfills the criteria for a host range restriction factor. Our data supports a model in which FAM111A acts a repressor of viral gene expression that is inactivated by the C-terminus of T Ag and the T Ag C-terminus-mediated inactivation of FAM111A is required for plaque formation in the restrictive CV-1P cell line.