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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

Associations between T1 and host proteins.(A) Network of associations of full-length SV40 LT (hexagon) with host proteins (circles) detected in at least three of five replica affinity purification (AP)-MudPIT experiments. Host proteins reported to associate with LT in VirusMint are colored (Blue). Circle size is proportional to the number of times the association was observed. Solid lines (links) represent viral-host protein associations and dashed lines represent host-host associations reported in the BioGRID database. (B) Extracts from T98G cells transfected with HA-tagged p130 and LT were immunoprecipitated with anti-HA antibodies and the indicated proteins were detected by western blot. (C) Summary of AP-MudPIT analysis from full-length LT or LT fragments for the indicated host proteins. Relative abundance values (dNSAF as defined in the supplemental data) were averaged across 5 biological replicate analyses of T1 (full-length LT) affinity purification experiments. The number of times each host protein was identified in the biological replicates is shown. CT indicates C-TERM. (D) Summary of iTRAQ analysis. Estimates of protein stoichiometry, relative to LT were based on reconstructed ion chromatogram (RIC) intensities of the most abundant peptides assigned to each protein. Number of biological replicates for each affinity purification experiment is indicated in parentheses in header. ND indicates not detected.
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ppat-1002949-g003: Associations between T1 and host proteins.(A) Network of associations of full-length SV40 LT (hexagon) with host proteins (circles) detected in at least three of five replica affinity purification (AP)-MudPIT experiments. Host proteins reported to associate with LT in VirusMint are colored (Blue). Circle size is proportional to the number of times the association was observed. Solid lines (links) represent viral-host protein associations and dashed lines represent host-host associations reported in the BioGRID database. (B) Extracts from T98G cells transfected with HA-tagged p130 and LT were immunoprecipitated with anti-HA antibodies and the indicated proteins were detected by western blot. (C) Summary of AP-MudPIT analysis from full-length LT or LT fragments for the indicated host proteins. Relative abundance values (dNSAF as defined in the supplemental data) were averaged across 5 biological replicate analyses of T1 (full-length LT) affinity purification experiments. The number of times each host protein was identified in the biological replicates is shown. CT indicates C-TERM. (D) Summary of iTRAQ analysis. Estimates of protein stoichiometry, relative to LT were based on reconstructed ion chromatogram (RIC) intensities of the most abundant peptides assigned to each protein. Number of biological replicates for each affinity purification experiment is indicated in parentheses in header. ND indicates not detected.

Mentions: Given the effects of LT on cellular and viral gene expression, we sought to identify host proteins that bind to LT. We used multidimensional protein identification technology (MudPIT) to analyze preparative scale immunoprecipitations by mass spectrometry [18]. We pooled the results of five independent experiments from cells expressing full-length T1. In total, we identified 89 proteins that co-purified with T1 and were detected in at least three out of five independent affinity purification experiments (Figure 3A and Table S3 in Text S1). We detected several previously reported interactors of LT including FBXW7 and p53 [19]. In addition, we identified co-complexes with RB family proteins and E2F/DP1 transcription factors, but not any of the five proteins contained within the MuvB core complex (LIN9, LIN37, LIN52, LIN54 and RBBP4) that bind to p130 (RBL2) in the DREAM complex [14]. Immunoprecipitation for p130 co-precipitated the MuvB subunit LIN37 in the absence of LT but not when LT was present (Figure 3B), further supporting the conclusion that LT disrupts the DREAM complex.


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)

Associations between T1 and host proteins.(A) Network of associations of full-length SV40 LT (hexagon) with host proteins (circles) detected in at least three of five replica affinity purification (AP)-MudPIT experiments. Host proteins reported to associate with LT in VirusMint are colored (Blue). Circle size is proportional to the number of times the association was observed. Solid lines (links) represent viral-host protein associations and dashed lines represent host-host associations reported in the BioGRID database. (B) Extracts from T98G cells transfected with HA-tagged p130 and LT were immunoprecipitated with anti-HA antibodies and the indicated proteins were detected by western blot. (C) Summary of AP-MudPIT analysis from full-length LT or LT fragments for the indicated host proteins. Relative abundance values (dNSAF as defined in the supplemental data) were averaged across 5 biological replicate analyses of T1 (full-length LT) affinity purification experiments. The number of times each host protein was identified in the biological replicates is shown. CT indicates C-TERM. (D) Summary of iTRAQ analysis. Estimates of protein stoichiometry, relative to LT were based on reconstructed ion chromatogram (RIC) intensities of the most abundant peptides assigned to each protein. Number of biological replicates for each affinity purification experiment is indicated in parentheses in header. ND indicates not detected.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3475652&req=5

ppat-1002949-g003: Associations between T1 and host proteins.(A) Network of associations of full-length SV40 LT (hexagon) with host proteins (circles) detected in at least three of five replica affinity purification (AP)-MudPIT experiments. Host proteins reported to associate with LT in VirusMint are colored (Blue). Circle size is proportional to the number of times the association was observed. Solid lines (links) represent viral-host protein associations and dashed lines represent host-host associations reported in the BioGRID database. (B) Extracts from T98G cells transfected with HA-tagged p130 and LT were immunoprecipitated with anti-HA antibodies and the indicated proteins were detected by western blot. (C) Summary of AP-MudPIT analysis from full-length LT or LT fragments for the indicated host proteins. Relative abundance values (dNSAF as defined in the supplemental data) were averaged across 5 biological replicate analyses of T1 (full-length LT) affinity purification experiments. The number of times each host protein was identified in the biological replicates is shown. CT indicates C-TERM. (D) Summary of iTRAQ analysis. Estimates of protein stoichiometry, relative to LT were based on reconstructed ion chromatogram (RIC) intensities of the most abundant peptides assigned to each protein. Number of biological replicates for each affinity purification experiment is indicated in parentheses in header. ND indicates not detected.
Mentions: Given the effects of LT on cellular and viral gene expression, we sought to identify host proteins that bind to LT. We used multidimensional protein identification technology (MudPIT) to analyze preparative scale immunoprecipitations by mass spectrometry [18]. We pooled the results of five independent experiments from cells expressing full-length T1. In total, we identified 89 proteins that co-purified with T1 and were detected in at least three out of five independent affinity purification experiments (Figure 3A and Table S3 in Text S1). We detected several previously reported interactors of LT including FBXW7 and p53 [19]. In addition, we identified co-complexes with RB family proteins and E2F/DP1 transcription factors, but not any of the five proteins contained within the MuvB core complex (LIN9, LIN37, LIN52, LIN54 and RBBP4) that bind to p130 (RBL2) in the DREAM complex [14]. Immunoprecipitation for p130 co-precipitated the MuvB subunit LIN37 in the absence of LT but not when LT was present (Figure 3B), further supporting the conclusion that LT disrupts the DREAM complex.

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