Identification of FAM111A as an SV40 host range restriction and adenovirus helper factor

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.

Figure 1. Expression of SV40 LT C-terminus increases early and late viral gene levels.

(A) CV-1P or U-2 OS cells were transfected with vector control (M), viral DNA encoding wild type SV40 (WT), host range mutant HR684 (HR), or HR plus HA-tagged LT C-TERM (HR+C). Ninety-six hours post-transfection, cell lysates were western blotted for LT, VP1, LT C-TERM (HA) and vinculin (VIN). (B) U-2 OS cells stably expressing vector only or LT C-TERM were transfected with HR684 viral DNA. Lysates were prepared at the indicated time points (hours) and blotted as in (A).

Figure 2. 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).

Figure 3. 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.

Figure 4. Mapping the LT and FAM111A interacting domains.

(A) LT fragments were tested for binding to full-length FAM111A by yeast-two-hybrid (Y2H) in pairwise fashion. (B) Fragments of FAM111A were tested in pair wise Y2H analysis with full-length LT (T1). Numbers indicate residue position in human FAM111A.

Figure 5. FAM111A is an SV40 LT binding protein.

(A) Immunoprecipitations for FAM111A and LT with lysates from U-2 OS cells stably expressing full-length LT (LT) or vector control (V). Whole cell lysate of the U-2 OS cell line stably expressing shRNA-2 (sh) against FAM111A was used as a control for FAM111A antibody specificity and normal rabbit serum (IgG) as an immunoprecipitation control. The indicated proteins were detected by western blot analysis. (B) Immunoprecipitations for FAM111A with lysates from U-2 OS cells stably expressing the LT C-terminus (CT) or vector control (V). (C) Immunoprecipitations of FAM111A and FAM111B in U-2 OS cells expressing LT or mock (M). Levels of FAM111A, FAM111B, LT, and vinculin (VIN) were determined by western blot. (D) Immunoprecipitations of FAM111A on U-2OS, BSC40, and CV-1P cells 48 hours post-infection with wild type SV40 (SV) or mock infected (M). (E) Immunoprecipitation of FAM111A in U-2 OS cells transfected with viral DNA encoding wild type SV40 (WT), K697R acetylation mutant (KR), T701A phosphorylation mutant (TA) and host range mutants HR684 (HR) and dl1066 (dl).

Figure 6. 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.

Figure 7. 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.

Figure 8. 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.

Figure 9. LT interactome perturbs host gene expression, overcomes host range restriction and enables viral gene expression.

SV40 LT interaction with p130 (RBL2) disrupts the DREAM complex leading to cell cycle entry and increased FAM111A expression. LT reduces the expression of p53-responsive genes. LT binding to FAM111A overcomes SV40 host range restriction and enables the adenovirus helper effect.