Proteomics Analysis Of EV71 And HBV Virus Interactions With Host Cells

Quantitative proteomics has now becoming an indispensable tool in life science research. In this dissertation, we utilized biochemical methods such as stable isotope labeling and subcellular fractionation, coupled with high performance mass spectrometry techniques to systematically analyze the host cellular proteome changes upon the infection of EV71 and HBV.As a major threat to public health in the Asian-Pacific region, enterovirus 71 (EV71) can cause severe neurological and systemic illness in children under the age of five. Since very little information is available regarding EV71 and host cell interactions, our understanding about EV71 virus and its pathogenesis have been limited. In Chapter two of this dissertation, we investigated the host cell global proteome alterations at two different EV71 infection stages. We used triple-SILAC labeled host cells and infected with EV71 virus. At 8 h.p.i. (hours post infection) and 20 h.p.i., cellular proteins were extracted and the proteome changes were analyzed and compared. A total of 4114 proteins were quantitated, and～17% of the proteins were found as significantly changed (p< 0.01) at either 8 or 20 hp.i. Bio informatics analysis showed that five biological processes and seven protein classes underwent significant changes, with histones significantly decreased at both time points. Proteins in mitochondrion were mostly changed either in the 8 hp.i group or in the 20 h.p.i. group, but rarely in both. These results were also validated by hierarchical clustering analysis. Moreover, protein classes related to virus infection, such as ubiquitination and SUMOylation, vesicular transporters and tumor necrosis factors, were also changed significantly. Functional screening and validation studies of selected proteins revealed that a host mitochondrial protein, CHCH2, could influence the replication of EV71 by means of regulating host cell innate immune responses.With estimated 93 million HBV-infected patients in China, studies on HBV infection and replication are important and with practical significance. In Chapter Three of this dissertation, we systematically analyzed the global and subcellular proteomes of Huh7.37 cells, which was derived from Huh7 cells with stably expressed HBV incorporated. We enriched and labeled the nucleus and mitochondrion of Huh7.37 and Huh7. Quantitative proteomics analysis results revealed that multiple signaling pathways and protein classes were significantly affected by HBV replication, and these effects were different among whole cell, nuclear and mitochondrial perspetive. Nineteen peroxisomal proteins were found significantly changed in mitochondrial fraction which impled that there is a close connection between mitochondrion and peroxisome in Huh7.37 cells. By comparing the nuclear and mitochondrial proteomes, we found that 9.6% proteins entered nucleus from cytoplasma and 29.8% proteins translocated to mitochondrion from cytoplasma, which suggested that HBV replication altered the subcellular localization of host proteins. The STRING analyses of 30 proteins revealed that their expression in nucleus decreased while in the whole cell and mitochondrion, they did not change, or even increased. Based on this, we speculated that these proteins may shuttle among subcellular structures. In addition, we studied the influence of 30 host proteins on HBV replication. About 10 proteins could affect the expression of HBeAg, but only DDX1 could affect the expression of HBsAg at the same time.