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Identification of FAM111A as an SV40 host range restriction and adenovirus helper factor.

Fine DA, Rozenblatt-Rosen O, Padi M, Korkhin A, James RL, Adelmant G, Yoon R, Guo L, Berrios C, Zhang Y, Calderwood MA, Velmurgan S, Cheng J, Marto JA, Hill DE, Cusick ME, Vidal M, Florens L, Washburn MP, Litovchick L, DeCaprio JA - PLoS Pathog. (2012)

Bottom Line: The SV40 large T antigen (LT) contains several discrete functional domains including the LXCXE or RB-binding motif, the DNA binding and helicase domains that contribute to the viral life cycle.To understand how LT affects the host cell to facilitate viral replication, we expressed full-length or functional domains of LT in cells, identified interacting host proteins and carried out expression profiling.FAM111A functions as a host range restriction factor that is specifically targeted by SV40 LT.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.

ABSTRACT
The small genome of polyomaviruses encodes a limited number of proteins that are highly dependent on interactions with host cell proteins for efficient viral replication. The SV40 large T antigen (LT) contains several discrete functional domains including the LXCXE or RB-binding motif, the DNA binding and helicase domains that contribute to the viral life cycle. In addition, the LT C-terminal region contains the host range and adenovirus helper functions required for lytic infection in certain restrictive cell types. To understand how LT affects the host cell to facilitate viral replication, we expressed full-length or functional domains of LT in cells, identified interacting host proteins and carried out expression profiling. LT perturbed the expression of p53 target genes and subsets of cell-cycle dependent genes regulated by the DREAM and the B-Myb-MuvB complexes. Affinity purification of LT followed by mass spectrometry revealed a specific interaction between the LT C-terminal region and FAM111A, a previously uncharacterized protein. Depletion of FAM111A recapitulated the effects of heterologous expression of the LT C-terminal region, including increased viral gene expression and lytic infection of SV40 host range mutants and adenovirus replication in restrictive cells. FAM111A functions as a host range restriction factor that is specifically targeted by SV40 LT.

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FAM111A is an SV40 LT binding protein.(A) Immunoprecipitations for FAM111A and LT with lysates from U-2 OS cells stably expressing full-length LT (LT) or vector control (V). Whole cell lysate of the U-2 OS cell line stably expressing shRNA-2 (sh) against FAM111A was used as a control for FAM111A antibody specificity and normal rabbit serum (IgG) as an immunoprecipitation control. The indicated proteins were detected by western blot analysis. (B) Immunoprecipitations for FAM111A with lysates from U-2 OS cells stably expressing the LT C-terminus (CT) or vector control (V). (C) Immunoprecipitations of FAM111A and FAM111B in U-2 OS cells expressing LT or mock (M). Levels of FAM111A, FAM111B, LT, and vinculin (VIN) were determined by western blot. (D) Immunoprecipitations of FAM111A on U-2OS, BSC40, and CV-1P cells 48 hours post-infection with wild type SV40 (SV) or mock infected (M). (E) Immunoprecipitation of FAM111A in U-2 OS cells transfected with viral DNA encoding wild type SV40 (WT), K697R acetylation mutant (KR), T701A phosphorylation mutant (TA) and host range mutants HR684 (HR) and dl1066 (dl).
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ppat-1002949-g005: FAM111A is an SV40 LT binding protein.(A) Immunoprecipitations for FAM111A and LT with lysates from U-2 OS cells stably expressing full-length LT (LT) or vector control (V). Whole cell lysate of the U-2 OS cell line stably expressing shRNA-2 (sh) against FAM111A was used as a control for FAM111A antibody specificity and normal rabbit serum (IgG) as an immunoprecipitation control. The indicated proteins were detected by western blot analysis. (B) Immunoprecipitations for FAM111A with lysates from U-2 OS cells stably expressing the LT C-terminus (CT) or vector control (V). (C) Immunoprecipitations of FAM111A and FAM111B in U-2 OS cells expressing LT or mock (M). Levels of FAM111A, FAM111B, LT, and vinculin (VIN) were determined by western blot. (D) Immunoprecipitations of FAM111A on U-2OS, BSC40, and CV-1P cells 48 hours post-infection with wild type SV40 (SV) or mock infected (M). (E) Immunoprecipitation of FAM111A in U-2 OS cells transfected with viral DNA encoding wild type SV40 (WT), K697R acetylation mutant (KR), T701A phosphorylation mutant (TA) and host range mutants HR684 (HR) and dl1066 (dl).

Mentions: Homologs of FAM111A exist in several mammalian species including mouse, rat, and rhesus monkey. FAM111A is also highly similar to its paralog human FAM111B (Gene ID: 374393) with 43% identity in the C-terminal 330 residues encompassing the peptidase domain and trypsin-like catalytic triad. To confirm that LT could bind to FAM111A in human U-2 OS cells, we performed immunoprecipitations with antibodies specific for FAM111A or FAM111B. An antibody for FAM111A detected a 70 kDa band that was reduced upon shRNA-mediated knockdown of FAM111A (Figure 5A). An immunoprecipitation for LT co-precipitated FAM111A and the reciprocal immunoprecipitation for FAM111A co-precipitated LT (Figure 5A). FAM111A also co-precipitated the LT fragment C-TERM (Figure 5B). Given the similarity between FAM111A and FAM111B we tested if LT could bind to FAM111B. However, we were unable to detect co-precipitation of FAM111B by LT in U-2 OS cells (Figure 5C). This result is consistent with the MudPIT and iTRAQ analyses that only detected FAM111A and not FAM111B in association with LT.


Identification of FAM111A as an SV40 host range restriction and adenovirus helper factor.

Fine DA, Rozenblatt-Rosen O, Padi M, Korkhin A, James RL, Adelmant G, Yoon R, Guo L, Berrios C, Zhang Y, Calderwood MA, Velmurgan S, Cheng J, Marto JA, Hill DE, Cusick ME, Vidal M, Florens L, Washburn MP, Litovchick L, DeCaprio JA - PLoS Pathog. (2012)

FAM111A is an SV40 LT binding protein.(A) Immunoprecipitations for FAM111A and LT with lysates from U-2 OS cells stably expressing full-length LT (LT) or vector control (V). Whole cell lysate of the U-2 OS cell line stably expressing shRNA-2 (sh) against FAM111A was used as a control for FAM111A antibody specificity and normal rabbit serum (IgG) as an immunoprecipitation control. The indicated proteins were detected by western blot analysis. (B) Immunoprecipitations for FAM111A with lysates from U-2 OS cells stably expressing the LT C-terminus (CT) or vector control (V). (C) Immunoprecipitations of FAM111A and FAM111B in U-2 OS cells expressing LT or mock (M). Levels of FAM111A, FAM111B, LT, and vinculin (VIN) were determined by western blot. (D) Immunoprecipitations of FAM111A on U-2OS, BSC40, and CV-1P cells 48 hours post-infection with wild type SV40 (SV) or mock infected (M). (E) Immunoprecipitation of FAM111A in U-2 OS cells transfected with viral DNA encoding wild type SV40 (WT), K697R acetylation mutant (KR), T701A phosphorylation mutant (TA) and host range mutants HR684 (HR) and dl1066 (dl).
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ppat-1002949-g005: FAM111A is an SV40 LT binding protein.(A) Immunoprecipitations for FAM111A and LT with lysates from U-2 OS cells stably expressing full-length LT (LT) or vector control (V). Whole cell lysate of the U-2 OS cell line stably expressing shRNA-2 (sh) against FAM111A was used as a control for FAM111A antibody specificity and normal rabbit serum (IgG) as an immunoprecipitation control. The indicated proteins were detected by western blot analysis. (B) Immunoprecipitations for FAM111A with lysates from U-2 OS cells stably expressing the LT C-terminus (CT) or vector control (V). (C) Immunoprecipitations of FAM111A and FAM111B in U-2 OS cells expressing LT or mock (M). Levels of FAM111A, FAM111B, LT, and vinculin (VIN) were determined by western blot. (D) Immunoprecipitations of FAM111A on U-2OS, BSC40, and CV-1P cells 48 hours post-infection with wild type SV40 (SV) or mock infected (M). (E) Immunoprecipitation of FAM111A in U-2 OS cells transfected with viral DNA encoding wild type SV40 (WT), K697R acetylation mutant (KR), T701A phosphorylation mutant (TA) and host range mutants HR684 (HR) and dl1066 (dl).
Mentions: Homologs of FAM111A exist in several mammalian species including mouse, rat, and rhesus monkey. FAM111A is also highly similar to its paralog human FAM111B (Gene ID: 374393) with 43% identity in the C-terminal 330 residues encompassing the peptidase domain and trypsin-like catalytic triad. To confirm that LT could bind to FAM111A in human U-2 OS cells, we performed immunoprecipitations with antibodies specific for FAM111A or FAM111B. An antibody for FAM111A detected a 70 kDa band that was reduced upon shRNA-mediated knockdown of FAM111A (Figure 5A). An immunoprecipitation for LT co-precipitated FAM111A and the reciprocal immunoprecipitation for FAM111A co-precipitated LT (Figure 5A). FAM111A also co-precipitated the LT fragment C-TERM (Figure 5B). Given the similarity between FAM111A and FAM111B we tested if LT could bind to FAM111B. However, we were unable to detect co-precipitation of FAM111B by LT in U-2 OS cells (Figure 5C). This result is consistent with the MudPIT and iTRAQ analyses that only detected FAM111A and not FAM111B in association with LT.

Bottom Line: The SV40 large T antigen (LT) contains several discrete functional domains including the LXCXE or RB-binding motif, the DNA binding and helicase domains that contribute to the viral life cycle.To understand how LT affects the host cell to facilitate viral replication, we expressed full-length or functional domains of LT in cells, identified interacting host proteins and carried out expression profiling.FAM111A functions as a host range restriction factor that is specifically targeted by SV40 LT.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.

ABSTRACT
The small genome of polyomaviruses encodes a limited number of proteins that are highly dependent on interactions with host cell proteins for efficient viral replication. The SV40 large T antigen (LT) contains several discrete functional domains including the LXCXE or RB-binding motif, the DNA binding and helicase domains that contribute to the viral life cycle. In addition, the LT C-terminal region contains the host range and adenovirus helper functions required for lytic infection in certain restrictive cell types. To understand how LT affects the host cell to facilitate viral replication, we expressed full-length or functional domains of LT in cells, identified interacting host proteins and carried out expression profiling. LT perturbed the expression of p53 target genes and subsets of cell-cycle dependent genes regulated by the DREAM and the B-Myb-MuvB complexes. Affinity purification of LT followed by mass spectrometry revealed a specific interaction between the LT C-terminal region and FAM111A, a previously uncharacterized protein. Depletion of FAM111A recapitulated the effects of heterologous expression of the LT C-terminal region, including increased viral gene expression and lytic infection of SV40 host range mutants and adenovirus replication in restrictive cells. FAM111A functions as a host range restriction factor that is specifically targeted by SV40 LT.

Show MeSH
Related in: MedlinePlus