Rational Design of a Flavivirus Vaccine by Abolishing Viral RNA 2′- O Methylation

Abstract

Viruses that replicate in the cytoplasm cannot access the host nuclear capping machinery. These viruses have evolved viral methyltransferase(s) to methylate N-7 and 2=-O cap of their RNA; alternatively, they "snatch" host mRNA cap to form the 5= end of viral RNA. The function of 2=-O methylation of viral RNA cap is to mimic cellular mRNA and to evade host innate immune restriction. A cytoplasmic virus defective in 2=-O methylation is replicative, but its viral RNA lacks 2=-O methylation and is recognized and eliminated by the host immune response. Such a mutant virus could be rationally designed as a live attenuated vaccine. Here, we use Japanese encephalitis virus (JEV), an important mosquito-borne flavivirus, to prove this novel vaccine concept. We show that JEV methyltransferase is responsible for both N-7 and 2=-O cap methylations as well as evasion of host innate immune response. Recombinant virus completely defective in 2=-O methylation was stable in cell culture after being passaged for >30 days. The mutant virus was attenuated in mice, elicited robust humoral and cellular immune responses, and retained the engineered mutation in vivo. A single dose of immunization induced full protection against lethal challenge with JEV strains in mice. Mechanistically, the attenuation phenotype was attributed to the enhanced sensitivity of the mutant virus to the antiviral effects of interferon and IFIT proteins. Collectively, the results demonstrate the feasibility of using 2=-O methylationdefective virus as a vaccine approach; this vaccine approach should be applicable to other flaviviruses and nonflaviviruses that encode their own viral 2=-O methyltransferases. Live attenuated vaccine represents the best medical intervention to prevent many viral diseases, such as those caused by vaccinia virus, poliovirus (Sabin), yellow fever virus (YFV; YF-17D), Japanese encephalitis virus (JEV SA14-14-2), and MMR (measles, mumps, and rubella viruses). The attenuated vaccine replicates to a low level but induces immune response and memory that are sufficient to prevent virulent virus infection. The traditional method of developing an attenuated vaccine is by passaging the virus through a foreign host (e.g., tissue culture or live animals). The attenuation of a vaccine strain is empirically achieved through accumulation of random mutations during passaging while maintaining immunogenicity. The function of each accumulated mutation in the vaccine strain needs to be analyzed to understand the mechanism of attenuation. As an alternative approach for vaccine development, viral attenuation could be rationally designed by altering the ability of virus to antagonize innate immunity (1). Such rationally designed virus is replicative and induces protective immunity; however, the virus is quickly eliminated due to its enhanced sensitivity to the antiviral effect of the host innate immune response.RNA and DNA viruses that replicate in the cytoplasm cannot use the cellular nuclear capping machinery and thus have evolved viral methyltransferas...

Zika Virus Methyltransferase: Structure and Functions for Drug Design Perspectives

“…In this work, we focus on the structure/function analysis of ZIKV NS5-MTase, closely related to that of DENV, and involved in the cap structure methylation. The latter is essential for viral RNA translation into proteins and the hiding of viral genomes from detection by cellular antiviral pathways (9,15). The ZIKV MTase presented here crystallizes as a homodimer.…”

“…DENV mutant viruses lacking N-7-methyltransferase (MTase) activity show strong replication defects in infected cells, whereas those altering the 2=-O-MTase activity show only attenuated phenotypes (7,14), as most cell lines used in the laboratory for virus replication are deprived of the RIG-I/MDA5 antiviral pathway. In contrast, it was demonstrated that a 2=-O-MTase knockout virus barely replicates in infected mice and elicits a strong humoral and cellular antiviral response (15,16). Thus, viral RNA capping represents an attractive antiviral strategy (17,18) since it should inhibit viral replication and/or accelerate virus clearance upon stimulation of the innate antiviral response.…”

Genotype-specific neutralization determinants in envelope protein: implications for the improvement of Japanese encephalitis vaccine

“…Virus stocks were stored in aliquots at 280 uC until use. Virus titres were determined by a standard plaque-forming assay in BHK-21 cells (Li et al, 2013a).…”

“…Xho I-linearized plasmids were purified and used as DNA templates for in vitro transcription as described previously (Li et al, 2013a) in the presence of an m 7 GpppA cap analogue with a RiboMax Large Scale RNA Production system (Promega). RNA transcripts were transfected into BHK-21 cells with Lipofectamine 2000 (Invitrogen).…”

Dendritic Cell Targets for Self-Replicating RNA Vaccines

“…By inference to the role of capping with mRNA vaccines, the capping of Alphavirus replicons should enhance their applicability without the need for a delivery vehicle. Flaviviruses also require 5'-capping to promote both polyprotein translation and protection of the RNA [42], although an uncapped flavivirus vaccine has been developed [43]. In contrast, application of Pestivirus replicon RNA circumvents the necessity to provide a capping step in the construction and production of the vaccine [40,44], due to the existence of the ribosomal entry site in the 5'-UTR (Figure 2).…”