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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 expression is cell cycle dependent.(A) U-2 OS cells were fractionated and equal amounts of cytoplasmic (C) and nuclear (N) lysates were blotted with FAM111A antibodies. Tubulin and lamin serve as cytoplasmic and nuclear markers, respectively. (B) Box plot depicting the average expression of 79 genes in T98G cells with profiles similar to that of FAM111A (Pearson correlation coefficient R>0.9). FAM111A expression is denoted by the red dot. (C) RNA was extracted from asynchronously (Async) growing T98G cells or that were serum starved (SS) for 24, 48 or 72 (G0) hours then stimulated to enter the cell cycle by addition of serum for indicated hours. Heatmap depicting the expression of genes with expression pattern was similar to FAM111A. (D) Whole cell lysates prepared from T98G cells at the indicated hours after serum starvation and release were immunoprecipitated with FAM111A antibodies and immunoblotted with FAM111A antibodies. Negative control included immunoprecipitation with normal rabbit serum (IgG) from asynchronous T98G cells. The whole cell lysates that were used for immunoprecipitations were also probed with the indicated antibodies to mark cell cycle progression. Time points after release form G0 and cell cycle stage are depicted.
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ppat-1002949-g006: FAM111A expression is cell cycle dependent.(A) U-2 OS cells were fractionated and equal amounts of cytoplasmic (C) and nuclear (N) lysates were blotted with FAM111A antibodies. Tubulin and lamin serve as cytoplasmic and nuclear markers, respectively. (B) Box plot depicting the average expression of 79 genes in T98G cells with profiles similar to that of FAM111A (Pearson correlation coefficient R>0.9). FAM111A expression is denoted by the red dot. (C) RNA was extracted from asynchronously (Async) growing T98G cells or that were serum starved (SS) for 24, 48 or 72 (G0) hours then stimulated to enter the cell cycle by addition of serum for indicated hours. Heatmap depicting the expression of genes with expression pattern was similar to FAM111A. (D) Whole cell lysates prepared from T98G cells at the indicated hours after serum starvation and release were immunoprecipitated with FAM111A antibodies and immunoblotted with FAM111A antibodies. Negative control included immunoprecipitation with normal rabbit serum (IgG) from asynchronous T98G cells. The whole cell lysates that were used for immunoprecipitations were also probed with the indicated antibodies to mark cell cycle progression. Time points after release form G0 and cell cycle stage are depicted.

Mentions: We sought to characterize FAM111A expression. Differential cellular extraction revealed that FAM111A was present in the nuclear and cytoplasmic fractions of U-2 OS cells (Figure 6A). Prior work revealed that the FAM111A promoter was bound by the DREAM complex in G0 or quiescent T98G cells [14]. Given that expression of DREAM target genes are regulated in a cell cycle-dependent manner, we examined mRNA expression profiles of cell cycle synchronized T98G cells. FAM111A levels were reduced in serum-starved G0 cells and increased 20 hr after serum addition when cells were enriched for S phase (Figure 6B and Figure S5 in Text S1). We identified 79 genes that exhibited cell cycle expression profiles similar to that of FAM111A (Pearson correlation coefficient R>0.9). This FAM111A gene set was significantly enriched for the GO term “M phase of mitotic cell cycle” (Figure 6C).


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 expression is cell cycle dependent.(A) U-2 OS cells were fractionated and equal amounts of cytoplasmic (C) and nuclear (N) lysates were blotted with FAM111A antibodies. Tubulin and lamin serve as cytoplasmic and nuclear markers, respectively. (B) Box plot depicting the average expression of 79 genes in T98G cells with profiles similar to that of FAM111A (Pearson correlation coefficient R>0.9). FAM111A expression is denoted by the red dot. (C) RNA was extracted from asynchronously (Async) growing T98G cells or that were serum starved (SS) for 24, 48 or 72 (G0) hours then stimulated to enter the cell cycle by addition of serum for indicated hours. Heatmap depicting the expression of genes with expression pattern was similar to FAM111A. (D) Whole cell lysates prepared from T98G cells at the indicated hours after serum starvation and release were immunoprecipitated with FAM111A antibodies and immunoblotted with FAM111A antibodies. Negative control included immunoprecipitation with normal rabbit serum (IgG) from asynchronous T98G cells. The whole cell lysates that were used for immunoprecipitations were also probed with the indicated antibodies to mark cell cycle progression. Time points after release form G0 and cell cycle stage are depicted.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3475652&req=5

ppat-1002949-g006: FAM111A expression is cell cycle dependent.(A) U-2 OS cells were fractionated and equal amounts of cytoplasmic (C) and nuclear (N) lysates were blotted with FAM111A antibodies. Tubulin and lamin serve as cytoplasmic and nuclear markers, respectively. (B) Box plot depicting the average expression of 79 genes in T98G cells with profiles similar to that of FAM111A (Pearson correlation coefficient R>0.9). FAM111A expression is denoted by the red dot. (C) RNA was extracted from asynchronously (Async) growing T98G cells or that were serum starved (SS) for 24, 48 or 72 (G0) hours then stimulated to enter the cell cycle by addition of serum for indicated hours. Heatmap depicting the expression of genes with expression pattern was similar to FAM111A. (D) Whole cell lysates prepared from T98G cells at the indicated hours after serum starvation and release were immunoprecipitated with FAM111A antibodies and immunoblotted with FAM111A antibodies. Negative control included immunoprecipitation with normal rabbit serum (IgG) from asynchronous T98G cells. The whole cell lysates that were used for immunoprecipitations were also probed with the indicated antibodies to mark cell cycle progression. Time points after release form G0 and cell cycle stage are depicted.
Mentions: We sought to characterize FAM111A expression. Differential cellular extraction revealed that FAM111A was present in the nuclear and cytoplasmic fractions of U-2 OS cells (Figure 6A). Prior work revealed that the FAM111A promoter was bound by the DREAM complex in G0 or quiescent T98G cells [14]. Given that expression of DREAM target genes are regulated in a cell cycle-dependent manner, we examined mRNA expression profiles of cell cycle synchronized T98G cells. FAM111A levels were reduced in serum-starved G0 cells and increased 20 hr after serum addition when cells were enriched for S phase (Figure 6B and Figure S5 in Text S1). We identified 79 genes that exhibited cell cycle expression profiles similar to that of FAM111A (Pearson correlation coefficient R>0.9). This FAM111A gene set was significantly enriched for the GO term “M phase of mitotic cell cycle” (Figure 6C).

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