The Study On The Mechanism Of Anti-Ifn-α Effects Of Hepatitis B Virus Polymerase TP-257

Infection of Hepatitis B virus (HBV) is a serious public health problem. Infection of HBV causes hepatic failure, cirrhosis and hepatocellular carcinoma (HCC). HBV belongs to Hepadnaviridae with a partially double stranded and circular DNA genome. HBV replicates via a unique reverse transcription process. The structure and function of the DNA polymerase/reverse transcriptase (DNA pol/RT) play an important role in HBV replication. IFN-αhad been used for the therapy of chronic hepatitis B since 1976. It can inhibit and ultimately clear the HBV by immunomodulation and establishing intracellular antiviral state. However, it was found that approximately two thirds of IFN-αtreated patients showed IFN-αnon-responseness. It was considered that the IFN-αnon-responseness of HBV was associated with HBV DNA polymerase. Foster et al demonstrated that terminal protein (TP) of HBV DNA polymerase could dramatically inhibit the cellular response to IFN-αby blocking the IFN-αpathway. We had found that terminal protein of polymerase with additional spacer region (partial or whole) showed anti-IFN-αeffect, especially of terminal protein with part of Spacer Region as a total of 257 amino acids (TP257) residues. But the mechanism is still unclear. The aim of this study was to preliminary illustrated the mechanism of HBV DNA polymerase anti-IFN-αeffect by applying AdEasy XL Adenoviral Vector System to express TP257 protein and immunoprecipitation to screen for anti-IFN-αeffect of TP257-related hepatocellular proteins.For packaging adenovirus AD-TP257 and expressing TP257 protein in Huh7 cells, we first constructed the shuttle vector pShuttle-IRES-hrGFP-TP257, and recombined the vector into adenovirus backbone vector pADEasy-1.The adenovirus particles were packaged in 293A cells and infected Huh7 cells, then the expression of TP257 protein was verified by Western-blot. For screening for anti-IFN-αeffect of TP257-related cellular proteins, Huh7 cells were infected with recombinant adenoviral particles before or after adding IFN-α, then cell protein was harvested for immunoprecipitation and the different proteins were identified by mass spectrometry. The result showed that recombinant adenovirus particles AD-TP257 can efficiency infect Huh7 cells and highly express TP257 protein. Adding IFN-αafter infected Huh7 cells with recombinant adenovirus particles AD-TP257, we found three different candidate proteins by immunoprecipitation. The mass spectrometry identified that they were 60 kDa heat shock protein, HIST1H2BC protein and myosin regulatory light chain 12A, respectively. Therefore, it was concluded that TP257 may affect viral anti-IFN-αeffect through interacting with above mentioned cellular proteins.