| The vesicular stomatitis virus(VSV)is RNA virus that infects mammalian cells and strongly induces the production of interferon,thereby initiating innate and adapted immune responses.RNA viruses induce interferon production by activating signaling pathways through RNA receptors such as RIG-I and MAD5.When the pathogen RNA is recognized by RNA receptors,the sensors are activated and interact with the downstream adaptor protein MAVS.MAVS forms large polymers and recruits multiple kinases and ubiquitin ligases,ultimately leading to the activation of transcription factors such as IRF3 and interferon secretion.Although RNA virus innate immune signaling pathways have been studied for many years,there remains some elusive questions.Are there any new mechanisms for regulation of RIG-I and MAVS?Are there other signaling molecules downstream of RIG-I or upstream of MAVS?In this study,quantitative proteomics is used to offer some hints on these questions.We systematically analyzed the proteome dynamics of THP1 cells during VSV infection using high-sensitivity data-independent acquisition(DIA)based quantitative mass spectrometry.After optimaztiion of DIA-MS data analysis,we quantified more than 8000 proteins in VSV-stimulated 20 samples,and found 6 human proteins which are regulated in abudance under VSV stimulation.Among them,we experimentally verified that HMOX1 can inhibit VSV replication.In addition,we compared phosphoproteome in VSV-treated wild-type,RIG-I knockout,and MAVS knockout THP1 cells.We quantified more than 18,000 phosphorylopeptides in 18 samples.Bioinformatic analysis revealed the RIG-I and MAVS-dependent or independent phosphorylation events both occurred in THP1 cells.Furthermore,we quantified more than 38,000 phosphopeptides in VSV-stimulated wild-type,RIG-I knockout and MAVS knockout L929 cells.The quantification results showed that,similar to the phosphorylation results in THP1 cells,there are RIG-I and MAVS-dependent orindependent phosphorylation events in L929 cells,such as S811 phosphorylation on Tns2 that is RIG-I-dependent and MAVS-independent.In summary,these results provide a panoramic view of the protein and phosphorylation dynamics during VSV infection and provide a valuable resource for later functional experiments. |
