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The 5’ end of the flavivirus genome contains a type 1 cap structure formed by sequential N-7 and 2’-O methylations by viral methyltransferase (MTase). Cap methylation of flavivirus genome is an essential structural modification to ensure the normal proliferation of the virus. Tembusu virus (TMUV) (genus Flavivirus) is a causative agent of duck egg drop syndrome and has zoonotic potential. Here, we identified the in vitro activity of TMUV MTase and determined the effect of K61-D146-K182-E218 enzymatic tetrad on N-7 and 2’-O methylation. The entire K61-D146-K182-E218 motif is essential for 2’-O MTase activity, whereas N-7 MTase activity requires only D146. To investigate its phenotype, the single point mutation (K61A, D146A, K182A or E218A) was introduced into TMUV replicon (pCMV-Rep-NanoLuc) and TMUV infectious cDNA clone (pACYC-TMUV). K-D-K-E mutations reduced the replication ability of replicon. K61A, K182A and E218A viruses were genetically stable, whereas D146A virus was unstable and reverted to WT virus. Mutant viruses were replication and virulence impaired, showing reduced growth and attenuated cytopathic effects and reduced mortality of duck embryos. Molecular mechanism studies showed that the translation efficiency of mutant viruses was inhibited and a higher host innate immunity was induced. Furthermore, we found that the translation inhibition of MTase-deficient viruses was caused by a defect in N-7 methylation, whereas the absence of 2’-O methylation did not affect viral translation. Taken together, our data validate the debilitating mechanism of MTase-deficient avian flavivirus and reveal an important role for cap-methylation in viral translation, proliferation, and escape from innate immunity.

The 5' end of the flavivirus genome contains a type 1 cap structure formed by sequential N-7 and 2'-O methylations by viral methyltransferase (MTase). Cap methylation of flavivirus genome is an essential structural modification to ensure the normal proliferation of the virus. Tembusu virus (TMUV) (genus Flavivirus) is a causative agent of duck egg drop syndrome and has zoonotic potential. Here, we identified the in vitro activity of TMUV MTase and determined the effect of K61-D146-K182-E218 enzymatic tetrad on N-7 and 2'-O methylation. The entire K61-D146-K182-E218 motif is essential for 2'-O MTase activity, whereas N-7 MTase activity requires only D146. To investigate its phenotype, the single point mutation (K61A, D146A, K182A or E218A) was introduced into TMUV replicon (pCMV-Rep-NanoLuc) and TMUV infectious cDNA clone (pACYC-TMUV). K-D-K-E mutations reduced the replication ability of replicon. K61A, K182A and E218A viruses were genetically stable, whereas D146A virus was unstable and reverted to WT virus. Mutant viruses were replication and virulence impaired, showing reduced growth and attenuated cytopathic effects and reduced mortality of duck embryos. Molecular mechanism studies showed that the translation efficiency of mutant viruses was inhibited and a higher host innate immunity was induced. Furthermore, we found that the translation inhibition of MTase-deficient viruses was caused by a defect in N-7 methylation, whereas the absence of 2'-O methylation did not affect viral translation. Taken together, our data validate the debilitating mechanism of MTase-deficient avian flavivirus and reveal an important role for cap-methylation in viral translation, proliferation, and escape from innate immunity. [ABSTRACT FROM AUTHOR]

According to the American Centers for Disease Control and Prevention, people in all age groups catch two or more "colds" per year, at least half of which are caused by human rhinoviruses. Despite decades of effort, there are no vaccines or drugs against rhinovirus infections and even social distancing measures that were effective in reducing the spread of the pandemic coronavirus, SARS-CoV-2, did not reduce the rate of rhinovirus detection. Fortunately, most rhinovirus strains are naturally attenuated in that they are not associated with serious illness, hospitalization or mortality. Instead, rhinoviruses are one of the most frequent viruses found in nasal swabs of asymptomatic, healthy people. Since rhinovirus infections cannot be avoided, a rational approach would be to engineer them for the benefit of their human hosts. Rhinovirus infections naturally induce robust mucosal and serum immune responses to all virus-expressed proteins. Several replication-competent, human rhinovirus vaccine vectors able to express protective antigens for other pathogens have already been designed and tested in animal models. With this strategy, the inevitable common cold would be able to induce immunity not just to a specific rhinovirus serotype but to other more pathogenic respiratory viruses as well. This article reviews existing rhinovirus vaccine vector technology and describes the characteristics that make live-attenuated rhinoviruses attractive vaccine candidates for SARS-CoV-2 and other pathogenic respiratory viruses in the future., Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kasman.)