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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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Mapping the LT and FAM111A interacting domains.(A) LT fragments were tested for binding to full-length FAM111A by yeast-two-hybrid (Y2H) in pairwise fashion. (B) Fragments of FAM111A were tested in pair wise Y2H analysis with full-length LT (T1). Numbers indicate residue position in human FAM111A.
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ppat-1002949-g004: Mapping the LT and FAM111A interacting domains.(A) LT fragments were tested for binding to full-length FAM111A by yeast-two-hybrid (Y2H) in pairwise fashion. (B) Fragments of FAM111A were tested in pair wise Y2H analysis with full-length LT (T1). Numbers indicate residue position in human FAM111A.

Mentions: We tested FAM111A binding to LT in a yeast two-hybrid (Y2H) assay. The LT constructs T1, T16 and C-TERM bound to FAM111A either as bait or prey in Y2H, while T8 could not (Figure 4A) consistent with the mass spectrometry analyses. To determine where LT bound to FAM111A, we generated fifty N- and C-terminal deletion constructs of FAM111A and tested them as bait or prey by Y2H against full-length LT (Figure S4 in Text S1). The C-terminal half of FAM111A (residues 336 to 611) was necessary and sufficient for interaction with LT (Figure 4B). This region of FAM111A contains a trypsin-like serine peptidase domain including the conserved catalytic triad of histidine, aspartate, and serine residues [27].


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)

Mapping the LT and FAM111A interacting domains.(A) LT fragments were tested for binding to full-length FAM111A by yeast-two-hybrid (Y2H) in pairwise fashion. (B) Fragments of FAM111A were tested in pair wise Y2H analysis with full-length LT (T1). Numbers indicate residue position in human FAM111A.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3475652&req=5

ppat-1002949-g004: Mapping the LT and FAM111A interacting domains.(A) LT fragments were tested for binding to full-length FAM111A by yeast-two-hybrid (Y2H) in pairwise fashion. (B) Fragments of FAM111A were tested in pair wise Y2H analysis with full-length LT (T1). Numbers indicate residue position in human FAM111A.
Mentions: We tested FAM111A binding to LT in a yeast two-hybrid (Y2H) assay. The LT constructs T1, T16 and C-TERM bound to FAM111A either as bait or prey in Y2H, while T8 could not (Figure 4A) consistent with the mass spectrometry analyses. To determine where LT bound to FAM111A, we generated fifty N- and C-terminal deletion constructs of FAM111A and tested them as bait or prey by Y2H against full-length LT (Figure S4 in Text S1). The C-terminal half of FAM111A (residues 336 to 611) was necessary and sufficient for interaction with LT (Figure 4B). This region of FAM111A contains a trypsin-like serine peptidase domain including the conserved catalytic triad of histidine, aspartate, and serine residues [27].

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