The Studies Of Protein-Protein Interactions Between RNA Viruses And Host Cells

RNA viruses are a large group of viruses containing RNAs as their genomes,which include around half of all the viruses,and can cause a variety of diseases in all kinds of organisms ranging from bacteria to mammals.Thus,the studies of RNA viruses are very valuable for better understanding of these important pathogens,as well as providing basis for developing antiviral drugs and diagnostic tools.During viral infections,viruses need to constantly adapt and interact with intracellular environment within host cells,which will help them complete viral life cycles or keep latency.On the other hand,in order to defend against viral infection,host cells utilize a variety of cellular responses,including antiviral immune pathways,apoptosis,and etc.And most of these processes require the participation of protein-protein interactions.Therefore,it is very important to uncover the protein interaction network of viral and cellular proteins during viral infection,which can help us to deepen the understanding of the mechanism of viral infection and corresponding host responses.In the study of the interaction between RNA virus and the host cell histones,we used human enterovirus 71(EV71)to infect human embryonic malignant rhabdomyoma cells(RD)to study the functions of histone modifications and epigenetic regulation mechanisms in viral infection.We found that cellular histone H3 undergoes a N-terminal cleavage when being infected with EV71.By using mass spectrometry,we identified the site of histone H3 cleavage and simultaneously identified changes in various modifications of histones.Furthermore,we found that the N-terminal cleavage of histone H3 is mediated by the protease 3C encoded by EV71,which can enter the nucleus and complete the cleavage by directly interact with histone H3.In addition,we generated and purified recombinant wild-type and protease catalytic site mutant 3C proteins(3C wt and 3C-C147S)in vitro.Using the in vitro cleavage assay of histone H3,we found that 3C can induce the cleavage of histone H3 while the mutant 3C-C147 S cannot.Our studies uncovered a novel epigenetic regulation of histone H3 by EV71-encoded protease,which may have global impact to the transcription and regulation of host and even viral genes.In the proteomics studies of dengue virus infection,we used a stable isotope dimethylation labeling and a TiO2 phospho-enrichment strategy,and total proteins and phosphoproteins were fractionated by HILIC.Through the LC-ESI-MS/MS,we investigated the proteomic and phosphoproteomic changes of the human erythroleukemia K562 cell line infected with Dengue virus type 2(DENV-2).During our study,a total of 2263 proteins,799 phosphoproteins and 2440 phosphosites were quantified.Among them,there are 321 significantly down-or up-regulated proteins,189 significantly down-or up-regulated phosphoproteins,and 501 different phosphosites.Mass spectrometry results were analyzed by bioinformatic analyses,including protein functional classification analysis,GO analysis and protein-protein interaction analysis.We have uncovered b iological processes that are significantly enriched during DENV-2 infection,including RNA splicing,Cellular macromolecule biosynthetic process,mRNA processing,and Regulation of transcription.In further protein interaction analysis,we found that several proteins such as HDAC1,BID,EIF3 E,GRB2 and SAP18 are at the center of the protein interaction network in the enriched biological processes.And these target proteins were further verified by Western blots.