Integrative Interactome and Ubiquitinome Analyses Reveal Multiple Regulatory Pathways Targeted by ICP0 in HSV-1 Infected Neuronal Cells

Herpes simplex virus 1 (HSV-1) is a neurotropic virus that can undergo both productive and latent infection in neurons. ICP0 is an HSV-1 E3 ubiquitin ligase crucial for productive infection and reactivation from latency. However, its targets have not been systematically investigated in neuronal cells. After confirming the importance of ICP0 in HSV-1 neuronal replication using an ICP0-null virus, we identified many ICP0-interacting proteins in infected neuronal and non-neuronal cells by mass-spectrometry-based interactome analysis. Co-immunoprecipitation assays validated ICP0 interactions with ACOT8, C1QBP, OTUD4, SNX9 and VIM in both Neuro-2a and 293T cells. Overexpression and knockdown experiments showed that SNX9 restricted replication of the ICP0-null but not wild-type virus in Neuro-2a cells. Ubiquitinome analysis by immunoprecipitating the trypsin digested ubiquitin reminant followed by mass spectrometry identified numerous candidate ubiquitination substrates of ICP0 in infected Neuro-2a cells, among which OTUD4 and VIM were novel substrates confirmed to be ubiquitinated by transfected ICP0 in Neuro-2a cells despite no evidence of their degradation by ICP0. Expression of OTUD4 was induced independently of ICP0 during HSV-1 infection. Overexpressed OTUD4 enhanced type I interferon expression during infection with the ICP0-null but not wild-type virus. In summary, by combining two proteomic approaches followed by confirmatory and functional experiments, we identified and validated multiple novel targets of ICP0 in neuronal cells, and revealed potential restrictive activities of SNX9 and OTUD4 as well as ICP0-dependent antagonism of these activities. Author Summary Herpes simplex virus 1 (HSV-1) establishes latent infection in neurons. ICP0 is known for its critical role in antagonizing cellular restrictive functions thereby initiating productive infection. It has been demonstrated to be important for both acute infection and reactivation from latency in neurons. However, little is known about its targets in neuronal cells. Here we combined two proteomic approaches, interactome and ubiquitinome analyses, to integratively identify interaction partners and substrates of ICP0 in HSV-1 infected neuronal cells. The results identified many novel targets as well as confirming previously reported ones. We also further validated some of the binding interactions and ubiquitin modifications. Functional studies revealed that the ICP0-interacting protein SNX9 restricted HSV-1 replication and the ICP0 substrate OTUD4 was induced to enhance type I interferon expression during HSV-1 neuronal infection. Moreover, the activities of these proteins appeared to be antagonized by ICP0-dependent mechanisms. This study provided comprehensive insight into ICP0 targets in neuronal cells and might prompt further investigation into the newly identified targets of ICP0.


ICP0 interactome analysis in HSV-1 infected cells
To analyze the ICP0 interactome, we infected Neuro-2a, HFF and 293T cells with Flag-156 ICP0 or WT virus for 6 h before co-immunoprecipitation (Co-IP) with a Flag antibody To validate the proteomics data, we selected some top novel host candidates according 174 to Flag-ICP0/WT ratios. Priorities were given to the candidates identified in Neuro-2a cells. However, considering that results from the mouse cells might miss interactions 176 occurring in human cells, we also included some top candidates identified in both 293T 177 and HFF cells. Thus, we selected a total of 17 top candidates and cloned their human 178 genes into expressing plasmids with Flag tags. After co-transfection of plasmids with 179 an ICP0 expressing plasmid into Neuro-2a and 293T cells, we performed IP in a reverse 180 way relative to the interactome analysis. We confirmed that untagged ICP0 could be 181 pulled down by Flag-tagged C1QBP, VIM, SNX9, ACOT8 and OTUD4 in both Neuro-182 2a and 293T cells (Fig. 2F). We note that although SNX9, ACOT8 and OTUD4 were 183 not identified in the interactome analysis in Neuro-2a cells possibly due to limited 184 sensitivity of mass spectrometry or the human-mouse differences, the reverse Co-IP 185 showed that their human forms were all able to interact with ICP0 in Neuro-2a cells. 186 Therefore, these proteins were included in the following functional studies. Hist1H2BB   replication was caused by specific knockdown of SNX9. Growth curve analysis showed 211 that even the less effective SNX9 siRNA (SNX9-si2) significantly increased replication 212 kinetics of 7134 virus at an MOI of 0.2 but it had no effect at an MOI of 5 (Fig. 3D). 213 Therefore endogenous SNX9 restricted HSV-1 replication at a low MOI. 215 To identify ubiquitination substrates of ICP0, we compared the ubiquitinome during 216 HSV-1 infection in the presence versus absence of ICP0. Absence of ICP0 was 217 achieved using the ICP0-null virus 7134, which was compared with 7134R. To increase 218 the stringency of the experiment, we also considered rescuing the absence of ICP0 by 219 transfection. Therefore we designed the following three groups: group A, control 220 transfection + 7134 virus infection; group B, ICP0 transfection + 7134 virus infection; group C, control transfection + 7134R virus infection. Only ubiquitination events that 222 were induced both by transfected ICP0 (comparing B and A) and by ICP0 expressed 223 from the virus (comparing C and A) were considered, which should greatly reduce 224 frequencies of false-positive discoveries. Neuro-2a cells were transfected for 24 h and 225 then infected for 6 h at an MOI of 20 before total protein was digested by trypsin and 226 immunoprecipitated with ubiquitin remnant K-ε-GG antibody followed by TMT 227 labeling and LC-MS/MS analysis (Fig. 4A). Overall, ratios of protein quantities in 228 group B versus A (B/A) and group C versus A (C/A) comparisons correlated well with 229 each other (Fig. 4B). Ubiquitinated sites potentially modified by ICP0 were selected by 230 applying criteria of either false discover rate (FDR) < 0.05 or fold change > 1.25 in both 231 comparisons. After mapping these sites to proteins, we identified 1022 proteins in the 232 B/A comparison and 522 proteins in the C/A comparison. The 351 proteins identified 233 by both B/A and C/A comparisons were considered as potential ICP0 substrates (Fig. 234 4C and Table S1). Functional enrichment analysis showed that the potential host 235 substrates are enriched in multiple pathways, including metabolism of RNA/proteins, 236 rRNA processing, cell cycle and deubiquitination etc. (Fig. 4C (Table S3). Regarding viral proteins, out of 55 viral proteins with detected 244 ubiquitination, 7 proteins, UL12, UL21, UL5, UL51, UL54, UL9 and US3 showed 245 increased ubiquitination in the presence of ICP0 (Fig. S3), indicating that they might 246 be potential viral substrates of ICP0.

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OTUD4 and VIM were ubiquitinated by ICP0 in Neuro-2a cells 248 Comparison of the interactome and ubiquitinome data identified 3 viral and 8 host 249 proteins which both interacted with ICP0 and exhibited elevated ubiquitination in 250 ICP0's presence indicating that they were highly likely to be substrates of ICP0 (Fig. 251 5A). All of these are novel potential substrates of ICP0 except for the deubiquitinase 252 USP7, whose ubiquitination by ICP0 has been reported elsewhere [53,59]. Because host 253 proteins VIM and OTUD4 were confirmed to robustly interact with ICP0 ( Fig. 2F), we 254 focused on these two proteins for ubiquitination analysis. VIM was previously 255 documented to be decorated with 11 SUMO2 modifications by another large-scale MS 256 study [60]. Our MS data detected 17 potential ubiquitination sites in VIM (Fig. 4B). 257 We note that the trypsin digestion resulted in different reminants in ubiquitinated or showing increased ubiquitination due to ICP0 presence has not been reported to be 264 SUMOylated. The MS data also detected 4 potential ubiquitination sites in OTUD4, all 265 of which are near the OTU domain and not far from the previously annotated 266 phosphorylation sites. One of the sites (K264) showed increased ubiquitination due to 267 ICP0 presence and therefore could be an ICP0 target site. To examine whether VIM 268 and OTUD4 were ubiquitinated by ICP0, we co-transfected ubiquitin, VIM or OTUD4 and ICP0 or its control (empty vector or ring finger mutant) into Neuro-2a cells before 270 immunoprecipitation of VIM and OTUD4 and analysis of ubiquitination levels by 271 western blots. Both VIM and OTUD4 exhibited substantial enhancement of 272 ubiqutination in the presence of ICP0 relative to the respective controls suggesting that 273 they could be ubiquitinated by ICP0 (Fig. 5C). However transfected ICP0 did not cause 274 decreases in VIM and OTUD4 levels indicating that ubiquitination of these proteins 275 may not lead to degradation.  To learn more about the roles OTUD4 and VIM, although they showed no effect on 289 HSV-1 replication (Fig. 3A, B), we wondered whether they could regulate innate

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After confirming that ICP0 is important for HSV-1 lytic infection in neuronal cells, we 313 combined quantitative interactome and ubiquitinome approaches to identify targets of ICP0 in neuronal cells followed by validation of the interaction and ubiquitination 315 events. Functional studies on the validated targets established SNX9 as a restriction 316 factor and OTUD4 as a regulator of type I IFN production and provided evidence that 317 the functions of these proteins might be modulated in an ICP0-dependent manner.

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The use of two proteomic approaches allowed us to dissect binding and 319 ubiquitination events, which may not always be coupled. Binding interactions may 320 function in ways independent of ubiquitination, and some ubiquitin modifications  Table S3. HSV-1 infection of cells and plaque 386 assays for viral titer measurements were performed as previously described [45].  Table S3.

Sample preparation for quantitative ubiquitinome investigation
The following three groups were compared to determine the effects of ICP0 on Ubiquitin-F cgGGATCCgATGCAGATCTTCGTGAAAACCCT T Ubiquitin-R CCaagcttTTAACAGCCACCCCTCAGGC