Adenovirus E1B-55K controls SUMO-dependent degradation of antiviral cellular restriction factors

Abstract

Human adenoviruses (HAdVs) generally cause mild and self-limiting diseases of the upper respiratory and gastrointestinal tracts but pose a serious risk to immunocompromised patients and children. Moreover, they are widely used as vectors for vaccines and vector-based gene therapy approaches. It is therefore vital to thoroughly characterize HAdV gene products and especially HAdV virulence factors. Early region 1B 55 kDa protein (E1B-55K) is a multifunctional HAdV-encoded oncoprotein involved in various viral and cellular pathways that promote viral replication and cell transformation. We analyzed the E1B-55K dependency of SUMOylation, a post-translational protein modification, in infected cells using quantitative proteomics. We found that HAdV increases overall cellular SUMOylation and that this increased SUMOylation can target antiviral cellular pathways that impact HAdV replication. Moreover, we showed that E1B-55K orchestrates the SUMO-dependent degradation of certain cellular antiviral factors. These results once more emphasize the key role of E1B-55K in the regulation of viral and cellular proteins in productive HAdV infections.

Keywords: DNA damage response; E1B-55K; SUMO E3-ligase; SUMO proteomics; cell cycle regulation; chromatin assembly; post-translational modification (PTM); protein network analysis; small ubiquitin-like modifier (SUMO); viral transcription.

Fig 1

Forty-eight-hour adenovirus infection triggers a net increase in SUMO2 conjugation in cultured human cells. (A) Western blots of indicated proteins from crude lysates (crude) and Ni²⁺-NTA affinity purified (pure) samples from HAdV-C5 WT- and ΔE1B-55K-infected HeLa SUMO2 cells (antibodies are listed in Table 1). (B) Stable isotope labeling by amino acids in cell culture (SILAC)-based proteomic experiment workflow. HeLa cells overexpressing 6His-SUMO2 were grown in light (L; isotopically normal, K0R0) or heavy (H; K8R10) SILAC medium prior to infection and 6His-SUMO2 enrichment. Proteins were separated by SDS-PAGE and the gel was cut into 12 slices for subsequent processing as indicated.

Fig 2

Changes in cellular protein levels and protein SUMOylation during HAdV-C5 WT and ΔE1B-55K infections. (A and B) Scatter plots comparing infected/uninfected ratios from crude cell extracts (x-axes) with 6His-SUMO2 purifications (y-axes) for HAdV-C5 WT (A) and HAdV-C5 ΔE1B-55K (B) infections. Pink areas show putative SUMO substrates that respond to infection with increased conjugation, blue areas are those showing an apparent reduction in SUMOylation. Dotted circles indicate the positions of proteins for which changes in abundance in crude extracts and Ni²⁺-NTA purifications are similar. In all charts, y = x is indicated with a broken line and fold changes less than a factor of 2 are bound by broken boxes.

Fig 3

Networks of proteins with regulated SUMOylation during WT adenovirus infection. (A to I) Data for 4,231 proteins identified from 6His-SUMO2 purifications were uploaded to STRING using the log₂ infected/uninfected ratio data for HAdV-C5 WT as quantification. “Proteins with values/ranks” analysis was conducted to identify any functional enrichments in proteins at either end of the ratio scale (see Supplementary Data File S2). STRING interaction networks were made for representative functional groups and uploaded to Cytoscape for visualization. Nodes are colored by ratio (see key). Gray nodes had no data for this set. Nodes with a thick border have already had a SUMO2 conjugation site identified through published high-throughput proteomic analyses. FDR, false discovery rate for the STRING analysis.

Fig 4

Influence of E1B-55K on the cellular SUMO proteome during infection. (A) Relationship between abundance difference and total intensity for proteins identified in both 6His-SUMO2 purifications from HAdV-C5 WT- and ΔE1B-55K-infected cells. Dotted lines indicate double or half abundance thresholds. Red markers are viral proteins, blue are SigB < 0.01 and gray SigB > 0.01. Selected outliers are indicated. (B) Scatter plot showing the relationship between infected/uninfected ratios for HAdV-C5 WT infection (x-axis) and HAdV-C5 ΔE1B-55K infection (y-axis) in Ni²⁺-NTA purified samples. Yellow and orange regions in (B) indicate areas of the greatest differences between virus types. (C) Log₂-fold changes of SUMOylated protein abundances of FAM111B and MRN complex components in crude and pure lysates from HAdV-C5 WT and HAdV-C5 ΔE1B-55K infections. (D–F) Western blot confirmation for FAM111B (D), Nibrin (E), and Mre11 (F) of the data summarized in part (C). Here, the cells were harvested 48 h p.i. and 10% of each sample was separated for input preparation. Ninety percent of the cells were lysed in GuHCl and subjected to Ni²⁺-NTA purification. Input and Ni²⁺-NTA purified samples were analyzed by SDS-PAGE and western blotting (antibodies are listed in Table 1). Asterisks in A and B indicate proteins previously described as a SUMO substrate.

Fig 5

Hierarchical clustering analysis of significantly affected SUMO substrates during adenovirus infection. Hierarchical clustering analysis of the proteomic data from HAdV-C5 WT- and ΔE1B-55K-infected cells relating to both SUMOylation changes and total protein abundance changes. Data derived from 329 proteins previously identified as SUMO substrates with data in both SILAC experiments and significantly differing according to at least one ratio comparison. Clustering only by rows. The full data set is shown as an insert, and the region expanded in the figure is boxed in red. Individual clusters are indicated with a brief summary of the group.

Fig 6

SUMO polymerization and SUMO substrate ubiquitination increase during infection. (A) Left: ratio data for peptides diagnostic of SUMO2-SUMO2 and SUMO2-SUMO3 polymerization via lysine 11 (K11). (B) Left: ratio data for ubiquitin-ubiquitin polymerization via lysines 11, 48, and 63. Protein abundance changes are shown to the right of (A) and (B) using ratio data for all peptides from SUMO2, SUMO3, and ubiquitin, respectively. Bars show the median normalized ratio and MaxQuant-derived coefficient of variability and values indicate SigB for infected/uninfected ratios.