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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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Related in: MedlinePlus

Depletion of FAM111A renders CV-1P cells permissive for Adenovirus 5 infection.(A) CV-1P cell lines stably expressing shRNA against FAM111A or vector control were mock infected or infected with Ad5 at the indicated dilutions. Ad5 infected cells were visualized using Adeno-X Rapid Titer (hexon protein, brown color). Results from a representative experiment, and quantification of integrated density across two biological replicates are shown (B). CV-1P cells stably expressing two different shRNAs against FAM111A or vector control were infected with Ad5 and assayed for lytic infection by plaque assay. Results from a representative experiment and a graph showing the average number of plaques in three biological replicates are shown.
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ppat-1002949-g008: Depletion of FAM111A renders CV-1P cells permissive for Adenovirus 5 infection.(A) CV-1P cell lines stably expressing shRNA against FAM111A or vector control were mock infected or infected with Ad5 at the indicated dilutions. Ad5 infected cells were visualized using Adeno-X Rapid Titer (hexon protein, brown color). Results from a representative experiment, and quantification of integrated density across two biological replicates are shown (B). CV-1P cells stably expressing two different shRNAs against FAM111A or vector control were infected with Ad5 and assayed for lytic infection by plaque assay. Results from a representative experiment and a graph showing the average number of plaques in three biological replicates are shown.

Mentions: AGMK cells can support human adenovirus replication only when co-infected with SV40 [28]. It has been long recognized that the C-terminal region of LT contains a helper function that permits human adenovirus infection of monkey cells [12], [29]. Since depletion of FAM111A or expression of the C-terminal region of LT can overcome the host range restriction in CV-1P cells, we investigated the role of FAM111A in adenovirus infection. Knockdown of FAM111A supported Adenovirus 5 (Ad5) replication as measured by increased amounts of the adenoviral hexon protein in FAM111A-depleted CV-1P cells (Figure 8A). Infection with Ad5 led to plaque formation in the FAM111A-depleted but not in the control CV-1P cell lines (Figure 8B). These results indicate a critical role for FAM111A in restriction of SV40 and adenovirus replication.


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)

Depletion of FAM111A renders CV-1P cells permissive for Adenovirus 5 infection.(A) CV-1P cell lines stably expressing shRNA against FAM111A or vector control were mock infected or infected with Ad5 at the indicated dilutions. Ad5 infected cells were visualized using Adeno-X Rapid Titer (hexon protein, brown color). Results from a representative experiment, and quantification of integrated density across two biological replicates are shown (B). CV-1P cells stably expressing two different shRNAs against FAM111A or vector control were infected with Ad5 and assayed for lytic infection by plaque assay. Results from a representative experiment and a graph showing the average number of plaques in three biological replicates are shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3475652&req=5

ppat-1002949-g008: Depletion of FAM111A renders CV-1P cells permissive for Adenovirus 5 infection.(A) CV-1P cell lines stably expressing shRNA against FAM111A or vector control were mock infected or infected with Ad5 at the indicated dilutions. Ad5 infected cells were visualized using Adeno-X Rapid Titer (hexon protein, brown color). Results from a representative experiment, and quantification of integrated density across two biological replicates are shown (B). CV-1P cells stably expressing two different shRNAs against FAM111A or vector control were infected with Ad5 and assayed for lytic infection by plaque assay. Results from a representative experiment and a graph showing the average number of plaques in three biological replicates are shown.
Mentions: AGMK cells can support human adenovirus replication only when co-infected with SV40 [28]. It has been long recognized that the C-terminal region of LT contains a helper function that permits human adenovirus infection of monkey cells [12], [29]. Since depletion of FAM111A or expression of the C-terminal region of LT can overcome the host range restriction in CV-1P cells, we investigated the role of FAM111A in adenovirus infection. Knockdown of FAM111A supported Adenovirus 5 (Ad5) replication as measured by increased amounts of the adenoviral hexon protein in FAM111A-depleted CV-1P cells (Figure 8A). Infection with Ad5 led to plaque formation in the FAM111A-depleted but not in the control CV-1P cell lines (Figure 8B). These results indicate a critical role for FAM111A in restriction of SV40 and adenovirus replication.

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