Mechanism Of Targeted Attenuation For Japanese Encephalitis Virus Based On Viral 2’-O Methyltransferase

The Japanese encephalitis virus(JEV), an arthropod-born Flavivirus, is one of the major causes of viral encephalitis in Asia. JE is induced by infection with Japanese encephalitis virus(JEV) and is prevalent in southern and Southeast Asia. In recent years, the JEV distribution area has been large and continuously extending toward new Asian and Australasian regions. Approximately 35,000~50,000 people suffer from this disease every year, causing approximately 10,000~15,000 deaths. Of the JE survivors, 30%~50% have permanent sequelae for the remainder of their lives. Despite large effective immunization campaigns, Japanese encephalitis remains a disease of global health concern due to its high fatality rate and epidemic expansion.JEV is a single-stranded, positive-sense RNA virus with a genome size of approximately 11 kb. The genome contains a single open reading frame encoding three structural proteins(capsid [C], premembrane [pr M/M], and envelope [E]) and seven nonstructural proteins(NS1, NS2 A, NS2 B, NS3, NS4 A, NS4 B, and NS5), and two nontranslating regions flank the 5’ and 3’ ends. The 5’ of JEV genome contains a type I cap structure(m7Gppp Am G) but lack the ploy(A) tail in its 3’ terminal. The cap structure is important for the stability and transportation of viral RNA, as well as the translation process.Usually, the RNA capping needs four enzymes, which are RNA triphosphatase, RNA guanylyltransferase, N-7 and 2’-O methyltransferase. Flavivirus replicate in the cytoplasm and cannot use the cellular nuclear capping machinery and thus have evolved their own viral methyltransferase(MTase). The N-7 and 2’-O MTases are located on the N-terminal domain of NS5, which can facilitate cap N-7 methylations and cap 2’-O methylations in a sequential manner. Currently, the knowledge about flavivirus MTase and cap methylation are mainly based on the studies of WNV and DENV. However, limited information is available for the JEV MTase and N7 & 2’-O methylation, and the methylation assay method is not established yet. In this research, we firstly established the methylation assay method for JEV, and screened the key amino acid in K61-D146-K182-E218 tetrad which would affect the MTase activities. Further, we evaluated the effection of 2’-O MTase deficiency on the biological properties of JEV. Lastly, we explored the possibility of using the 2’-O MTase deficient JEV as a live attenunated vaccine candidate. 1. Establishment of N-7 & 2’-O methylation assays for JEV and screening of key amino acid in MTase.To establish the methylation assay method for JEV, recombinant MTase representing the N-terminal 300 amino acids of NS5 derived from JEV was expressed and purified from E.coli. cells. SDS-PAGE analysis showed that the purified proteins were over 95% pure with the expected molecular mass of 35 k Da. Then, Substrate Gppp A-RNA representing the first 190 nucleotides of the 5’ end of the JEV genome was obtained by PCR and in-vitro transcription, next, the capping reaction uses a vaccinia virus capping enzyme, the P33-labeled G*ppp A-RNA and m7G*ppp A-RNA(the asterisk indicates that the following phosphate is labeled) were used for N-7 and 2’-O methylations, respectively. The N-7 and 2’-O methylation assays for JEV were established based on that of DENV with some optimization on salt concentration, tempreture and time. Then we tested the effect of K61 A, D146 A, K182 A, and E218 A mutations(in conserved K61-D146-K182-E218 tetrad) on JEV N-7 and 2’-O MTase activities. As a result, the K61 A, D146 A, K182 A and E218 A mutations completely abolished the 2’-O methylation activity. And for N-7 MTase activity, except for the D146 A mutation which completely knocked out the N-7 MTase activity, the K61 A, K182 A and E218 A mutation retained part of N-7 MTase activity. These results demonstrated that the K61, D146, K182 and E218 were all essential for 2’-O methylation, while only the D146 was essential for the N-7 methylation. 2. The effect of 2’-O methylation deficiency on the biological properties of JEvirusTo investigate the effect of 2’-O methylation deficiency on the biological properties of JE virus, we introduced K61 A,D146A, K182 A or E218 A mutations into the full-length infectious clone of JEV, after linearization and in-vitro transcription, the transcript RNAs were transfected into BHK-21 cells, as a result, two recombinant viruses, K61 A and E218 A, were obtained, while the D146 A and K182 A mutants were lethal. Then, we tested the biological properties of the 2’-O methylation defective JE viruses, including the expression of viral protein, plaque morphology, growth kinetics on cells, genetic stability, the neuroinvasiveness and neurovirulence in mice, as well as the sensitivities to the IFN and IFIT. As a result, transfection of BHK-21 cells with genome-length RNAs generated equivalent numbers of viral E protein-positive cells between the WT and mutant; the 2’-O methylation defective JE virus exhibited smaller plaque morphology than the WT virus did; One-step growth curves in mammalian(BHK-21,Vero and A549) and mosquito(C6/36) cells showed that the mutant viruses replicated slightly slower than the WT virus; Continuous passaging of the mutant virus on Vero cells for 10 rounds did not change its small-plaque morphology, meanwhile, sequencing of the passage 10 virus showed that the engineered E218 A change was retained without any extra mutation; The mutant viruses were highly attenuated in mice, the neuroinvasiveness and neurovirulence of mutant viruses were weaker than WT; o viremia was observed in the mutant virus-infected mice; The mutant viruses replicated slower than the WT in mice brain. Accordingly, the mutant viruses replicated slower than WT in nerve cells. Most importantly, the 2’-O methylation defective JE virus was more sensitive to the anti-viral effect of IFN and IFIT than WT. Collectively, the 2’-O methylation defective JE virus were highly attenuated in vitro and in vivo, which was attributed to its enhanced sensitivity to the antiviral effects of interferon and IFIT proteins. 3. The immunogenicity and protective immunity of 2’-O methylation defective virus.To further explore the possibility of using the 2’-O MTase deficient JEV as a live attenunated vaccine candidate, we tested the immunogenicity and protective immunity of 2’-O methylation defective virus. 104 PFU mutant virus was s.c. injected into BALB/c mice, at 2 weeks and 4 weeks post immunization, the sera were collected and the titers of Ig G and JEV-specfic neutralizing antibodies were measured. Meanwhile, the celluar immunity was also tested. As a result, a single immunization of mutant viruses could induce robust JEV-specific Ig G and neutralizing antibodies, notably, neutralizing antibody against JEV reached 97.4 at 4 weeks post-immunization. Meanwhile, splenocyte proliferative response and IFN-γ production in splenocyte revealed a robust cellular immunity in mice which were induced by single dose immunization of mutant virus. Furthter, the protection experiment shown that a single dose of 2’-O MTase deficient JEV immunization provides full protection against lethal challenges with different JEV genotypes in mice.Overall, in this research, we established the JEV N-7 & 2’-O methylation assay methods, based on this method, we further checked the effect of K-D-K-E mutations on N-7 & 2’-O MTase activities. Further, we investigate the effect of 2’-O methylation deficiency on the biological properties of JE virus. These findings not only enriched our knowledge on flavivirus RNA cap methylation, but also deepened the understanding on the interaction between host and virus. On the other hand, the 2’-O methylation defective virus was genetically stable, attenuated in mice, elicited robust humoral and cellular immune responses. Mechanistically, the attenuation phenotype was attributed to the enhanced sensitivity of the mutant virus to the antiviral effects of interferon and IFIT proteins. So this 2’-O methylation defective JE virus could be seen as a live attenuated vaccine candidate. Moreover, this rational design approach could be readily applicable to other viruses with defined 2’-O MTase activity for potential vaccine development.