The Role Of VPg Protein In Translation Initiation Of Rabbit Hemorrhagic Disease Virus

Rabbit hemorrhagic disease virus(RHDV) is the causative agent of Rabbit hemorrhagic disease(RHD), which is a highly infectious disease in rabbits. Therefore, it is necessary to strengthen the basic research of RHDV. It has been reported that the non-structural protein VPg is covalently linked to the 5′ end of RHDV genome and plays an important role in the translation initiation of Norwalk-like virus(NLV) or Feline calicivirus(FCV). However, it is not clear whether RHDV VPg has the similar functions to those of FCV VPg. In this study, we try to explore the biology functions of VP in the process of RHDV translation. Besides, to overcome the neck-bottle barrier of limiting RHDV proliferation in vitro, a RHDV mutant was constructed with revers genetics technology. A stable cell line express RHDV VPg was also constructed, which provided a good platform for studying the function of RHDV VPg. In a word, there are three parts in this study.In part I, a cell line(RK-VPg) stable expressing RHDV VPg was constructed utilizingLentivirus packaging. The stable and high express level of VPg protein in the RK-VPg cell was demonstrated by PCR, RT-PCR, IFA and western blot. This cell line is useful to study the role of VPg in the process of RHDV replication and/or translation.In part II, we constructed a recombinant plasmid pRHDV-RGD including the genomes of RHDV and two mutant sites by site-directed mutagenesis the RHDV capsid protein hyper variable region(V1) 305 th and 307 th amino acid. The pRHDV-RGD plasmids were transfected into RK-13 cells, and after blind pass three generations, the RK-13 cells have typical cytopathic effect(CPE). Then the RHDVRGD mutant strain was demonstrated by RT-PCR, IFA, western blot and transmission electron microscope(TEM). The RHDVRGD mutant strain is not only stable proliferate in RK-13 cells, but also inherite the biological characteristics of the parent strain. The experimental rabbit was dead within 48-72 h by infect the 35 generation of RHDVRGD mutant strain, and the dead rabbit has typical symptoms of rabbit hemorrhagic disease. In conclusion, The RHDVRGD mutant strain has inherited the biological characteristics of the parent and good immunogenicity, and established a good foundation to study pathogenesis and molecular biology about RHDV and research and development RHDV cells inactivated vaccines.In part III, in the research, we demonstrated that the genome-linked viral protein(VPg) was necessary for RHDV translation in RK-13 cells by constructed aVPg gene deletion replicon pRHDV-luc/?VPg and infectious clone pRHDVRGD-?VPg. Next, we confirmed that VPg interacts with the eukaryotic initiation factor 4E(eIF4E) in vitro, and silencing the expression of eIF4 E inhibited RHDV translation, which suggests that this interaction is required for RHDV translation. This work supports the hypothesis that VPg may act as a novel cap substitute during the initiation of RHDV translation.Summary, this study not only established a stable cell line expressing RHDV VPg but also constructed a RHDV mutant strain which could be proliferated in RK-13 cell line. On these basises, we first demonstrated that the genome-linked viral protein(VPg) was necessary for RHDV translation in RK-13 cells. The molecular basis of its function is that VPg interacts with the eukaryotic initiation factor 4E(eIF4E) and act as a cap analogues. The greatest innovation is that his paper has solved the problem that RHDV can’t be proliferated in cell line, which will be helpful for studying on the pathogenesis mechanism and molecular biology of RHDV, what’s more, a promising RHDV vaccine would also be developed in the future.