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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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Expression of SV40 LT C-terminus increases early and late viral gene levels.(A) CV-1P or U-2 OS cells were transfected with vector control (M), viral DNA encoding wild type SV40 (WT), host range mutant HR684 (HR), or HR plus HA-tagged LT C-TERM (HR+C). Ninety-six hours post-transfection, cell lysates were western blotted for LT, VP1, LT C-TERM (HA) and vinculin (VIN). (B) U-2 OS cells stably expressing vector only or LT C-TERM were transfected with HR684 viral DNA. Lysates were prepared at the indicated time points (hours) and blotted as in (A).
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ppat-1002949-g001: Expression of SV40 LT C-terminus increases early and late viral gene levels.(A) CV-1P or U-2 OS cells were transfected with vector control (M), viral DNA encoding wild type SV40 (WT), host range mutant HR684 (HR), or HR plus HA-tagged LT C-TERM (HR+C). Ninety-six hours post-transfection, cell lysates were western blotted for LT, VP1, LT C-TERM (HA) and vinculin (VIN). (B) U-2 OS cells stably expressing vector only or LT C-TERM were transfected with HR684 viral DNA. Lysates were prepared at the indicated time points (hours) and blotted as in (A).

Mentions: The C-terminal region of LT is required for efficient viral gene expression and replication in the African green monkey kidney (AGMK) CV-1P cell line [8], [9]. The SV40 host range mutant virus HR684 lacks the C-terminal 24 residues of LT and has significantly reduced expression of early (LT) and late viral (VP1) genes compared to wild type virus in CV-1P cells (Figure 1A; [10]). Heterologous expression of the LT C-terminal 82 residues (C-TERM; residues 627 to 708) markedly increased levels of HR684 LT and VP1 in these cells [10]. Since LT C-TERM could support increased viral gene expression in trans, we suspected that this LT fragment could bind to a specific host cell factor and thereby increase viral gene expression.


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)

Expression of SV40 LT C-terminus increases early and late viral gene levels.(A) CV-1P or U-2 OS cells were transfected with vector control (M), viral DNA encoding wild type SV40 (WT), host range mutant HR684 (HR), or HR plus HA-tagged LT C-TERM (HR+C). Ninety-six hours post-transfection, cell lysates were western blotted for LT, VP1, LT C-TERM (HA) and vinculin (VIN). (B) U-2 OS cells stably expressing vector only or LT C-TERM were transfected with HR684 viral DNA. Lysates were prepared at the indicated time points (hours) and blotted as in (A).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3475652&req=5

ppat-1002949-g001: Expression of SV40 LT C-terminus increases early and late viral gene levels.(A) CV-1P or U-2 OS cells were transfected with vector control (M), viral DNA encoding wild type SV40 (WT), host range mutant HR684 (HR), or HR plus HA-tagged LT C-TERM (HR+C). Ninety-six hours post-transfection, cell lysates were western blotted for LT, VP1, LT C-TERM (HA) and vinculin (VIN). (B) U-2 OS cells stably expressing vector only or LT C-TERM were transfected with HR684 viral DNA. Lysates were prepared at the indicated time points (hours) and blotted as in (A).
Mentions: The C-terminal region of LT is required for efficient viral gene expression and replication in the African green monkey kidney (AGMK) CV-1P cell line [8], [9]. The SV40 host range mutant virus HR684 lacks the C-terminal 24 residues of LT and has significantly reduced expression of early (LT) and late viral (VP1) genes compared to wild type virus in CV-1P cells (Figure 1A; [10]). Heterologous expression of the LT C-terminal 82 residues (C-TERM; residues 627 to 708) markedly increased levels of HR684 LT and VP1 in these cells [10]. Since LT C-TERM could support increased viral gene expression in trans, we suspected that this LT fragment could bind to a specific host cell factor and thereby increase viral gene expression.

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