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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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Depletion of FAM111A increases viral gene expression and renders CV-1P cells permissive for host range mutant viruses.(A) U-2 OS cells were co-transfected with host range viral DNA (HR684) and control siRNA (black bars), siRNA targeting FAM111A (white bars) or an expression vector for the C-terminus of LT (grey bars). Quantitative RT-PCR was performed 72 hours post-transfection to determine the expression levels of LT, VP1 and FAM111A (latter not shown) mRNA relative to actin. Error bars represent standard deviation from the mean. U-2 OS (B) and CV-1P (C) cell lines stably expressing two different shRNAs against FAM111A or vector control were generated and the amount of FAM111A RNA remaining (% FAM111A RNA) was confirmed by quantitative RT-PCR. Viral DNA encoding HR684 was transfected into the indicated cell lines and whole cell lysates were harvested at 48 and 96 hours post transfection. (D). CV-1P cells stably expressing two different shRNAs against FAM111A or vector control were transfected with viral DNA and assayed for lytic infection by plaque assay. Plaques were counted 8 days after transfection. Results shown are the average of three independent experiments with standard deviation from the mean denoted by +/−. (E) Control or FAM111A shRNA depleted CV-1P cells were infected at a multiplicity of infection of three with either wild-type SV40 virus or the host range mutant dl1066 virus. Cells were freeze thawed at the indicated time points to extract virus and the viral titer was determined in BSC40 cells. Results shown are the average of three independent experiments with standard deviation from the mean indicated.
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ppat-1002949-g007: Depletion of FAM111A increases viral gene expression and renders CV-1P cells permissive for host range mutant viruses.(A) U-2 OS cells were co-transfected with host range viral DNA (HR684) and control siRNA (black bars), siRNA targeting FAM111A (white bars) or an expression vector for the C-terminus of LT (grey bars). Quantitative RT-PCR was performed 72 hours post-transfection to determine the expression levels of LT, VP1 and FAM111A (latter not shown) mRNA relative to actin. Error bars represent standard deviation from the mean. U-2 OS (B) and CV-1P (C) cell lines stably expressing two different shRNAs against FAM111A or vector control were generated and the amount of FAM111A RNA remaining (% FAM111A RNA) was confirmed by quantitative RT-PCR. Viral DNA encoding HR684 was transfected into the indicated cell lines and whole cell lysates were harvested at 48 and 96 hours post transfection. (D). CV-1P cells stably expressing two different shRNAs against FAM111A or vector control were transfected with viral DNA and assayed for lytic infection by plaque assay. Plaques were counted 8 days after transfection. Results shown are the average of three independent experiments with standard deviation from the mean denoted by +/−. (E) Control or FAM111A shRNA depleted CV-1P cells were infected at a multiplicity of infection of three with either wild-type SV40 virus or the host range mutant dl1066 virus. Cells were freeze thawed at the indicated time points to extract virus and the viral titer was determined in BSC40 cells. Results shown are the average of three independent experiments with standard deviation from the mean indicated.

Mentions: Binding of LT to p53 and RB serves to inactivate their growth suppressing functions. By analogy, LT binding to FAM111A might serve to inactivate the host range restriction function of FAM111A, thereby promoting increased and sustained viral gene expression. If so, then expression of the SV40 LT C-terminal region should have the same effect on virus replication as reduced FAM111A expression. Cells expressing LT C-TERM showed eight-to-ten fold increases of early (LT) and late (VP1) viral transcripts from the HR684 viral DNA relative to cells without LT C-TERM (Figure 7A). Knockdown of FAM111A also resulted in an eight-to-ten-fold increase in early (LT) and late (VP1) viral mRNA expression compared to non-targeting siRNA controls (Figure 7A).


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 increases viral gene expression and renders CV-1P cells permissive for host range mutant viruses.(A) U-2 OS cells were co-transfected with host range viral DNA (HR684) and control siRNA (black bars), siRNA targeting FAM111A (white bars) or an expression vector for the C-terminus of LT (grey bars). Quantitative RT-PCR was performed 72 hours post-transfection to determine the expression levels of LT, VP1 and FAM111A (latter not shown) mRNA relative to actin. Error bars represent standard deviation from the mean. U-2 OS (B) and CV-1P (C) cell lines stably expressing two different shRNAs against FAM111A or vector control were generated and the amount of FAM111A RNA remaining (% FAM111A RNA) was confirmed by quantitative RT-PCR. Viral DNA encoding HR684 was transfected into the indicated cell lines and whole cell lysates were harvested at 48 and 96 hours post transfection. (D). CV-1P cells stably expressing two different shRNAs against FAM111A or vector control were transfected with viral DNA and assayed for lytic infection by plaque assay. Plaques were counted 8 days after transfection. Results shown are the average of three independent experiments with standard deviation from the mean denoted by +/−. (E) Control or FAM111A shRNA depleted CV-1P cells were infected at a multiplicity of infection of three with either wild-type SV40 virus or the host range mutant dl1066 virus. Cells were freeze thawed at the indicated time points to extract virus and the viral titer was determined in BSC40 cells. Results shown are the average of three independent experiments with standard deviation from the mean indicated.
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ppat-1002949-g007: Depletion of FAM111A increases viral gene expression and renders CV-1P cells permissive for host range mutant viruses.(A) U-2 OS cells were co-transfected with host range viral DNA (HR684) and control siRNA (black bars), siRNA targeting FAM111A (white bars) or an expression vector for the C-terminus of LT (grey bars). Quantitative RT-PCR was performed 72 hours post-transfection to determine the expression levels of LT, VP1 and FAM111A (latter not shown) mRNA relative to actin. Error bars represent standard deviation from the mean. U-2 OS (B) and CV-1P (C) cell lines stably expressing two different shRNAs against FAM111A or vector control were generated and the amount of FAM111A RNA remaining (% FAM111A RNA) was confirmed by quantitative RT-PCR. Viral DNA encoding HR684 was transfected into the indicated cell lines and whole cell lysates were harvested at 48 and 96 hours post transfection. (D). CV-1P cells stably expressing two different shRNAs against FAM111A or vector control were transfected with viral DNA and assayed for lytic infection by plaque assay. Plaques were counted 8 days after transfection. Results shown are the average of three independent experiments with standard deviation from the mean denoted by +/−. (E) Control or FAM111A shRNA depleted CV-1P cells were infected at a multiplicity of infection of three with either wild-type SV40 virus or the host range mutant dl1066 virus. Cells were freeze thawed at the indicated time points to extract virus and the viral titer was determined in BSC40 cells. Results shown are the average of three independent experiments with standard deviation from the mean indicated.
Mentions: Binding of LT to p53 and RB serves to inactivate their growth suppressing functions. By analogy, LT binding to FAM111A might serve to inactivate the host range restriction function of FAM111A, thereby promoting increased and sustained viral gene expression. If so, then expression of the SV40 LT C-terminal region should have the same effect on virus replication as reduced FAM111A expression. Cells expressing LT C-TERM showed eight-to-ten fold increases of early (LT) and late (VP1) viral transcripts from the HR684 viral DNA relative to cells without LT C-TERM (Figure 7A). Knockdown of FAM111A also resulted in an eight-to-ten-fold increase in early (LT) and late (VP1) viral mRNA expression compared to non-targeting siRNA controls (Figure 7A).

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