mRNA Cap Methylation Influences Pathogenesis of Vesicular Stomatitis VirusIn Vivo

Abstract

, we previously showed that a panel of recombinant VSVs carrying mutations at a predicted methyltransferase catalytic site (rVSV-K1651A, -D1762A, and -E1833Q) or S-adenosylmethionine (SAM) binding site (rVSV-G1670A, -G1672A, and -G4A) were defective in cap methylation and were also attenuated for growth in cell culture. Here, we analyzed the virulence of these recombinants in mice. We found that rVSV-K1651A, -D1762A, and -E1833Q, which are defective in both G-N-7 and 2=-O methylation, were highly attenuated in mice. All three viruses elicited a high level of neutralizing antibody and provided full protection against challenge with the virulent VSV. In contrast, mice inoculated with rVSV-G1670A and -G1672A, which are defective only in G-N-7 methylation, were attenuated in vivo yet retained a low level of virulence. rVSV-G4A, which is completely defective in both G-N-7 and 2=-O methylation, also exhibited low virulence in mice despite the fact that productive viral replication was not detected in lung and brain. Taken together, our results suggest that abrogation of viral mRNA cap methylation can serve as an approach to attenuate VSV, and perhaps other nonsegmented negative-strand RNA viruses, for potential application as vaccines and viral vectors. IMPORTANCENonsegmented negative-sense (NNS) RNA viruses include a wide range of significant human, animal, and plant pathogens. For many of these viruses, there are no vaccines or antiviral drugs available. mRNA cap methylation is essential for mRNA stability and efficient translation. Our current understanding of mRNA modifications of NNS RNA viruses comes largely from studies of vesicular stomatitis virus (VSV). In this study, we showed that recombinant VSVs (rVSVs) defective in mRNA cap methylation were attenuated in vitro and in vivo. In addition, these methyltransferase (MTase)-defective rVSVs triggered high levels of antibody responses and provided complete protection against VSV infection. Thus, this study will not only contribute to our understanding of the role of mRNA cap MTase in viral pathogenesis but also facilitate the development of new live attenuated vaccines for VSV, and perhaps other NNS RNA viruses, by inhibiting viral mRNA cap methylation.

Methyltransferase-Defective Avian Metapneumovirus Vaccines Provide Complete Protection against Challenge with the Homologous Colorado Strain and the Heterologous Minnesota Strain

“…On the other hand, inhibition of mRNA cap methylation may yield attenuated strains that can be used for the development of live vaccine candidates. In fact, the concept of using mRNA cap methylation as a target for virus attenuation has been recently proved in VSV (30). Specifically, we found that recombinant VSV (rVSV) mutants rVSV-K1651A, -D1762A, and -E1833Q, which are defective in both G-N-7 and 2=O methylation, were highly attenuated in mice.…”

“…Previously, we showed that rVSV lacking both G-N-7 and 2=-O methylations were highly attenuated, whereas rVSV lacking only G-N-7 methylation had low virulence in mouse models (30). A 2=-O MTase-defective dengue virus vaccine candidate was highly attenuated and immunogenic in mouse and macaque models (50).…”

Messenger RNA Cap Methylation in Vesicular Stomatitis Virus, a Prototype of Non‐Segmented Negative‐Sense RNA Virus

“…The virus is then disseminated to other areas in the brain through retrograde and possibly anterograde trans-neuronal transport, ultimately causing an acute brain infection. It was found that VSV mutants, rVSV-K1651A, D1762A, and E1833Q, which have mutations in the MTase catalytic site and are defective in both G-N-7 and 2'O methylation, were highly attenuated in mice [107]. Mice inoculated with these recombinant viruses did not show any clinical signs of VSV infection such as weight loss, ruffled fur, hyperexcitability, tremors, circling, and paralysis.…”

“…Furthermore, these mutant viruses were not able to enter the brain, had dramatic defects in replication in lungs, and did not cause significant histopathological changes in lungs and brain. Recombinant rVSV-G1670A and G1672A, which have mutations in the SAM binding site and are defective in G-N-7 but not 2'-O methylation, retained low virulence in mice [107]. Mice inoculated these two recombinants exhibited weight loss of approximately 2-3 g during days 3-7 post-inoculation and showed mild illnesses such as ruffled coat for 2-3 days but recovered quickly.…”

“…Recently, Ma et al, (2012) examined the pathogenicity of MTase-defective VSVs in mice [107]. VSV infects a wide range of wild and domestic animals such as cattle, horses, deer, and pigs, characterized by vesicular lesions in the mouth, tongue, lips, gums, teats, and feet.…”