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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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Transcriptome perturbations induced by LT.(A) Schematic representation of the SV40 LT protein. Functional domains including the J domain, the LXCXE or RB binding motif, the nuclear localization signal (NLS), the DNA binding domain (DBD), the bipartite p53 binding domain contained within the helicase domain, and the C-terminal host range (HR)/adeno-helper (AH) domain are depicted. Residue numbers indicate limits for LT functional domains (B) Gene clusters that showed functional enrichment upon expression of full-length LT (T1) or LT fragments. The heatmap shows the expression of these genes in U-2 OS cells expressing T1 or T16 and IMR-90 cells expressing T1 relative to vector or GFP controls, respectively. Replicates were collapsed and genes hierarchically clustered (rows, genes; columns, experiments; red, induced from baseline; blue, repressed from baseline; white, unchanged from baseline). Enriched GO terms are listed adjacent to the numbered expression clusters and next to them are listed enriched gene sets in the cluster. In cluster C14 all transcripts are histones (C) GSEA plots determining whether the expression of the defined gene sets (DREAM, B-Myb-MuvB, or p53) show statistically significant, concordant differences between two biological states (T1 or T16 and vector control).
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ppat-1002949-g002: Transcriptome perturbations induced by LT.(A) Schematic representation of the SV40 LT protein. Functional domains including the J domain, the LXCXE or RB binding motif, the nuclear localization signal (NLS), the DNA binding domain (DBD), the bipartite p53 binding domain contained within the helicase domain, and the C-terminal host range (HR)/adeno-helper (AH) domain are depicted. Residue numbers indicate limits for LT functional domains (B) Gene clusters that showed functional enrichment upon expression of full-length LT (T1) or LT fragments. The heatmap shows the expression of these genes in U-2 OS cells expressing T1 or T16 and IMR-90 cells expressing T1 relative to vector or GFP controls, respectively. Replicates were collapsed and genes hierarchically clustered (rows, genes; columns, experiments; red, induced from baseline; blue, repressed from baseline; white, unchanged from baseline). Enriched GO terms are listed adjacent to the numbered expression clusters and next to them are listed enriched gene sets in the cluster. In cluster C14 all transcripts are histones (C) GSEA plots determining whether the expression of the defined gene sets (DREAM, B-Myb-MuvB, or p53) show statistically significant, concordant differences between two biological states (T1 or T16 and vector control).

Mentions: Discrete functional domains within the SV40 LT protein bind to diverse host cell proteins (Figure 2A). We generated LT expression constructs encoding epitope-tagged fusions of full-length LT as well as fragments corresponding to computationally- and functionally-defined domains. Full-length LT (T1), the LT N-terminal region encoded by residues 1 to 135 (T6 fragment) or residues 1 to 350 (T8 fragment), and the LT C-terminal region between residues 260 and 708 (T16 fragment) were stably expressed in U-2 OS cells (Figure S2 in Text S1).


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)

Transcriptome perturbations induced by LT.(A) Schematic representation of the SV40 LT protein. Functional domains including the J domain, the LXCXE or RB binding motif, the nuclear localization signal (NLS), the DNA binding domain (DBD), the bipartite p53 binding domain contained within the helicase domain, and the C-terminal host range (HR)/adeno-helper (AH) domain are depicted. Residue numbers indicate limits for LT functional domains (B) Gene clusters that showed functional enrichment upon expression of full-length LT (T1) or LT fragments. The heatmap shows the expression of these genes in U-2 OS cells expressing T1 or T16 and IMR-90 cells expressing T1 relative to vector or GFP controls, respectively. Replicates were collapsed and genes hierarchically clustered (rows, genes; columns, experiments; red, induced from baseline; blue, repressed from baseline; white, unchanged from baseline). Enriched GO terms are listed adjacent to the numbered expression clusters and next to them are listed enriched gene sets in the cluster. In cluster C14 all transcripts are histones (C) GSEA plots determining whether the expression of the defined gene sets (DREAM, B-Myb-MuvB, or p53) show statistically significant, concordant differences between two biological states (T1 or T16 and vector control).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3475652&req=5

ppat-1002949-g002: Transcriptome perturbations induced by LT.(A) Schematic representation of the SV40 LT protein. Functional domains including the J domain, the LXCXE or RB binding motif, the nuclear localization signal (NLS), the DNA binding domain (DBD), the bipartite p53 binding domain contained within the helicase domain, and the C-terminal host range (HR)/adeno-helper (AH) domain are depicted. Residue numbers indicate limits for LT functional domains (B) Gene clusters that showed functional enrichment upon expression of full-length LT (T1) or LT fragments. The heatmap shows the expression of these genes in U-2 OS cells expressing T1 or T16 and IMR-90 cells expressing T1 relative to vector or GFP controls, respectively. Replicates were collapsed and genes hierarchically clustered (rows, genes; columns, experiments; red, induced from baseline; blue, repressed from baseline; white, unchanged from baseline). Enriched GO terms are listed adjacent to the numbered expression clusters and next to them are listed enriched gene sets in the cluster. In cluster C14 all transcripts are histones (C) GSEA plots determining whether the expression of the defined gene sets (DREAM, B-Myb-MuvB, or p53) show statistically significant, concordant differences between two biological states (T1 or T16 and vector control).
Mentions: Discrete functional domains within the SV40 LT protein bind to diverse host cell proteins (Figure 2A). We generated LT expression constructs encoding epitope-tagged fusions of full-length LT as well as fragments corresponding to computationally- and functionally-defined domains. Full-length LT (T1), the LT N-terminal region encoded by residues 1 to 135 (T6 fragment) or residues 1 to 350 (T8 fragment), and the LT C-terminal region between residues 260 and 708 (T16 fragment) were stably expressed in U-2 OS cells (Figure S2 in Text S1).

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