A Primary Study Of Infection Mechanism Of Hepatitis E Virus On Hepatocytes

Hepatitis E Virus [HEV] has emerged to be a dominant cause of acute hepatitis, replacing hepatitis A virus in some areas and comparable in others. Research into pathogenesis and epidemiology of infection caused by this virus has been severely hampered, because the virus could not be efficiently propagated in cell culture.Previous studies suggest that p239, a recombinant protein specified by ORF2 of the HEV genome, models the HEV neutralization sites identified by the HEV neutralizing antibodies, 8C11 and 8H3, as well as other structural features of the protrusion projecting from the basal shell of the virus capsid identified by other monoclonal antibodies. Taking advantage of this structural resemblance, the objective of my thesis is to use this peptide as a probe to study interaction of HEV with its host cells. To this end, I have established a transient infection model for HEV, using the HepG2 and a variety of other cell line and analyzed the interactions between these cells and p239.The results suggest that binding of p239 to HepG2 cells may occur via at least 2 separate sites locating to aa423-438 and aa459-606, respectively, of the HEV structural protein. These sites were distinguished by using a panel of monoclonal antibodies and also by mutagenesis.Further analysis identified at least 7 host cell proteins which bind with p239, and suggest that these p239 binding host cell proteins include HSP90,GRP78/Bip,alpha tubulin,P43,ATPase beta subunit and an unknown cellular protein. Moreover, it was shown that binding of p239 to one of these cellular proteins, GRP78/Bip, is ATP dependent.The most critical step of infection is for virus to gain entry into its host cells. The present study took advantage of the structural resemblance of a recombinant protein, p239, to neutralization sites and other structural features of the HEV capsid, and use it as a probe to analyze the mechanism by which the virus gain entry to its host cells. The findings that p239 could effectively block HEV infection of a variety of cell lines susceptible to infection by this virus suggests that this novel approach is valid for studying virus-cell interaction. The findings described in this thesis suggest that this is a complex process, brought about by concerted actions involving direct and indirect binding of the virus structural protein with multiple of host cell proteins locating to spatially distinct sites on the virus capsid, and the process may require energy expenditure. Further studies will be required to identify the major cellular proteins involved in direct and indirect binding with the virus and how the interactions act in concert to bring about HEV entry to host cells.