Arthropod endosymbiont Wolbachia pipientis is part of a global biocontrol strategy aimed at reducing the spread of mosquito-borne RNA viruses such as alphaviruses. Our prior work examining Wolbachia-mediated pathogen blocking has demonstrated (i) the importance of a host cytosine methyltransferase, DNMT2, in Drosophila, and (ii) viral RNA as a target through which pathogen-blocking is mediated. Here we report on the role of DNMT2 in Wolbachia induced virus inhibition of alphaviruses in Aedes sp.. Mosquito DNMT2 levels were altered in the presence of both viruses and Wolbachia, albeit in opposite directions. Elevated levels of DNMT2 in mosquito salivary glands induced by virus infection were suppressed in Wolbachia colonized animals coincident with a reduction of virus replication, and decreased infectivity of progeny virus. Ectopic expression of DNMT2 in cultured Aedes cells was proviral increasing progeny virus infectivity, and this effect of DNMT2 on virus replication and infectivity was dependent on its methyltransferase activity. Finally, examination of the effects of Wolbachia on modifications of viral RNA by LC-MS showed a decrease in the amount of 5-methylcytosine modification consistent with the down-regulation of DNMT2 in Wolbachia colonized mosquito cells and animals. Collectively, our findings support the conclusion that disruption of 5-methylcytosine modification of viral RNA is an important mechanism operative in pathogen blocking. These data also emphasize the essential role of epitranscriptomic modifications in regulating fundamental processes of virus replication and transmission.

We used a fluorescently-tagged CHIKV reporter virus (CHIKV-mKate) to examine the effect of DNA MTase inhibitor DAC5 on virus replication in Wolbachia-free and Wolbachia-colonized Aedes albopictus cells using the Incucyte live-cell imaging platform. Fluorescent protein expression was used as a proxy of virus spread in cells pretreated with and without the inhibitor (DMSO), following synchronized virus adsorption at 4ºC. Post adsorption, cell monolayers were extensively washed with 1XPBS to remove any unbound viruses, followed by the addition of warm media (37ºC) to initialize virus internalization and infection. Virus spread was measured over 50 hours by quantifying mean virus-encoded red fluorescent reporter (mKate) expression observed over four distinct fields of view taken per well every 2-hours.