Defining the proteolytic landscape during enterovirus infection

Figures

• 

  Fig 1. Analysis of protein cleavages in HeLa cells upon eGFP/CVB3 infection. (A) Subtiligase labeling workflow. HeLa cells were either infected with a virus or left uninfected, lysed in SDS buffer, and the lysates containing 3 mg total protein were subjected to subtiligase enrichment of protein N-termini. LC-MS/MS identification and bioinformatics analysis of the enriched N-termini yielded a list of virus-induced protein cleavages. (B) HeLa cells infected with eGFP/CVB3 were harvested in triplicate at the indicated times post-infection followed by subtiligase labeling. Each row indicates a unique Abu-labeled peptide, and the red and black colors are used to show the presence and absence of the peptide, respectively. The peptides are presented in no particular order. (C) Frequencies of P1 and P1´ amino acids from unique N-termini identified at the indicated times post-infection. The N-termini present in at least two of the three replicates were included in this analysis. (D) Frequencies of protein cleavages derived from the indicated P1 and P1´ amino acid pairs at various times post-infection. Only the cleavages seen in at least two of the three replicates were counted.

• 

  Fig 2. Validation of protein cleavages identified by subtiligase labeling. (A) HeLa cells were infected with eGFP/CVB3 and lysed at 0, 2, 3, 4, 5, and 6 h.p.i. followed by western blot analysis of the indicated proteins. An equal amount of total protein, as quantified by the BCA assay, was loaded for each time point. GFP expression was used to monitor the progression of infection (bottom panel). The black solid arrows indicate the full-length protein, while the cleavage products are shown with red dotted arrows. (B) The post-infection time point when the cleavage first became detectable is shown (the western blot images are shown in S4 Fig). Some proteins (CAPR1, HTSF1, MAPK3, MAVS, SP130, STAT3, and USO1) that were found to be cleaved only at 6 h.p.i by proteomic analysis were included as controls. The proteins with asterisks were found to be cleaved at 4 h.p.i. by proteomics analysis but only at 6 h.p.i. by western blot. The position and identity of P1 residues identified at either 4 or 6 h.p.i. and the biological functions of the cleaved proteins are also shown.

• 

  Fig 3. In vitro cleavage assay of the identified proteins. (A) HeLa cell lysates (200 μg protein) were incubated with CVB3 2Apro or its catalytically inactive mutant C110A (1 μg), or CVB3 3Cpro or its catalytically inactive mutant C147A (100 μg) at 37°C for 3h and analyzed by western blot. Lysates from the uninfected and eGFP/CVB3-infected HeLa cells were included as positive controls. The full-length protein is indicated with black solid arrows, while the cleavage products are indicated with red dotted arrows. (B) The proteins are grouped into three categories based on if the cleavage was mediated by 2Apro, 3Cpro or both (the western blot images are shown in S6 Fig).

• 

  Fig 4. Analysis of proteins broadly targeted by enteroviruses. (A) Venn diagram showing the numbers of cleaved proteins common or unique to different viruses. (B) STRING interaction network of proteins targeted by at least two of the five enteroviruses as rendered in Cytoscape (disconnected nodes are not shown). Coloring indicates groups of five or more proteins identified as highly interconnected by the MCODE clustering algorithm. Most significantly enriched biological processes are summarized below the network. Cleaved proteins validated by western blot are shown in diamond shaped nodes. (C) Top 15 significantly enriched biological processes targeted by at least two of the five viruses.

• 

  Fig 5. Validation of proteins targeted by multiple enteroviruses. (A) HeLa cells infected with CVB3 (eGFP-Coxsackievirus B3), PV (poliovirus type 1), HRV (human rhinovirus A16), EV70 (enterovirus D-70), EV71 (enterovirus A-71), VEEV (Venezuelan equine encephalitis virus) or VSV (vesicular stomatitis virus) were lysed at the indicated times, and equal amounts of total protein were subjected to western blot with the indicated antibodies. The black solid and red dotted arrows indicate the full-length protein and the cleavage products, respectively. (B) The western blot results are summarized to show proteins commonly targeted by all five enteroviruses and those unique to a subset of viruses (the western blot images are shown in S9 Fig).

• 

  Fig 6. Validation of LSM14A cleavage. (A) Schematics of the LSM14A constructs generated for validation of the cleavage site. The cleavage site and the flanking eight amino acids are shown in the cartoon. The P1 glycine that was mutated is shown in red. (B) HeLa cells transduced to stably express the LSM14A constructs were infected with eGFP/CVB3 or PV, and the LSM14A cleavage was analyzed by western blot. The blots were probed with mouse anti-V5 and rabbit anti-HA antibodies followed by detection with IRDye 680RD goat anti-mouse IgG (red channel) and IRDye 800CW goat anti-rabbit IgG (green channel). FL, full-length protein; CT, C-terminal cleavage product; NT, N-terminal cleavage product; M, mock; CV; eGFP/CVB3; PV, poliovirus; UBC, Ubiquitin C.

• 

  Fig 7. Functional assays of LSM14A. (A) LSM14A activated ISRE in a dose-dependent manner. The 293T cells (30,000 cells per well in 48-well plates) expressing the ISRE-FLuc and RL-RLuc reporters were transfected with the indicated amounts of GFP (vector) or LSM14A plasmids. The next day, the cells were left uninfected (left panel) or infected with SeV (right panel) for 24h followed by luciferase assay. The FLuc/RLuc ratio was calculated and plotted as fold-increase over the minimum amount of vector plasmid used (6.25 ng). (B) LSM14A cleavage fragments do not activate ISRE. The reporter assays with the indicated LSM14A mutants were performed similarly as in A. The DNA concentrations of 6.25, 12, 25, and 50 ng/well were used. (C) TEVP-mediated cleavage of LSM14A blocks its ability to activate ISRE. The 293T cells containing dox-inducible TEVP and expressing the ISRE-FLuc and RL-RLuc reporters were transfected with 12.5 ng/well, 25 ng/well, or 50 ng/well LSM14A-TEVP plasmid followed by SeV infection. The indicated amount of doxycycline was added to the culture medium 24h prior to DNA transfection and kept for the entire experiment. The reporter assay was performed as described in A. (D)…