Fig 2: ICP0-interactome analysis. (A) Diagram of recombinant Flag-ICP0 virus. The horizontal line at the top represents HSV-1 genome and the boxes labeled with TRL, IRL, TRS, and IRS represent the repeated sequences. At the bottom are expanded ICP0 gene regions with the inserted N-terminal Flag tags indicated. (B) Virus growth curves of WT and Flag-ICP0 virus in Vero (left) and Neuro-2a (right) cells after infection at an MOI of 0.01 and 0.1, respectively. Data are presented as mean values ± SD. (C) Neuro-2a, HFF, and 293T cells were infected with Flag-ICP0 virus at an MOI of 10 and cells were lysed at the indicated time points for examination of Flag-ICP0 expression by Western blots with anti-Flag and anti-β-actin antibodies. (D) Schematic diagram of the procedures for ICP0 interactome analysis. Neuro-2a, 293T, and HFF cells were infected with WT (control) or Flag-ICP0 virus for 6 h at an MOI of 20 with three biological replicates. Proteins were immunoprecipitated by the anti-Flag antibody and examined by liquid chromatography with tandem mass spectrometry (LC-MS/MS). (E) Venn diagram showing candidate ICP0-interacting proteins identified by the interactome analysis in the three cell types. Previously reported ICP0-interacting proteins are marked in red. (F) 293T (left) or Neuro-2a (right) cells in each 100-mm plate were co-transfected with 7.5 μg of an empty vector (pcDNA) or a plasmid expressing the indicated protein linked to a Flag-tag and 7.5 μg of a plasmid expressing untagged ICP0 for 48 h. After the cells were lysed, the indicated proteins were immunoprecipitated by an anti-Flag antibody and analyzed by Western blots for ICP0 and the corresponding proteins using an ICP0 and Flag antibody, respectively. Results from immunoprecipitated samples (IP) were shown in the upper panels and those from the lysates (Input) were shown in the lower panels. Note that prolyl 4-hydroxylase subunit alpha 2 (P4HA2) and OTUD4 expression from the plasmids were not detected in the input samples due to low expression but were clearly detected in the IP samples.

Fig 3: Functions of OTUD4 and VIM in Neuro-2a cells. (A) Cells were infected with KOS or ICP0 RING finger mutant virus (RFm) at an MOI of 20 for the indicated times before the cells were harvested for Western blot analysis for the indicated proteins. (B) After infection of cells with KOS at an MOI of 20, the cells were harvested at the indicated times for qRT-PCR analysis of Otud4 mRNA levels normalized to glyceraldehyde-3 phosphate dehydrogenase (GAPDH) mRNA levels. (C) Cells were co-transfected with the indicated plasmid (400 ng/ml), pIFN-ß (80 ng/ml) (an IFN-ß promoter luciferase plasmid), and pRL-TK (40 ng/ml; a control plasmid expressing renilla luciferase) for 48 h and then infected with 7134 (left) or KOS (right) at an MOI of 2 for 16 h before measurement of luciferase activities. (D) Neuro-2a cells were co-transfected with the indicated siRNA (80 nM), pIFN-ß (80 ng/ml), and pRL-TK (40 ng/ml) for 48 h and then infected with 7134 (left) or KOS (right) at an MOI of 2 for 16 h before measurement of luciferase activities. (E) The assay was performed as in (C) except that different viruses were used. (F) Cells were transfected with the indicated plasmid (400 ng/ml) and then infected with the indicated virus at an MOI of 2 (left) or 0.5 (right) for 12 h before qRT-PCR quantification of IFN-a mRNA normalized to GAPDH mRNA. Data were analyzed by one-way ANOVA with Dunnett’s multiple comparisons tests (B–D,F) or two-tailed unpaired t-tests (E). Data are presented as mean values ± SD.