Abstract
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.
Significance Statement Presence of the endosymbiont Wolbachia pipientis in the arthropod host reduces establishment and dissemination of several emerging arboviruses within the insect and prevents virus transmission to a vertebrate host. However, the precise mechanisms mediating this inhibition are unknown. In this study, we demonstrate that the host RNA cytosine methyltransferase DNMT2 is an important regulator of this process. Our findings establish DNMT2 as a host factor targeting the viral RNA and as a conserved determinant of Wolbachia-mediated pathogen blocking. Importantly, we reveal a previously understudied role of virion encapsidated RNA methylation in regulating alphavirus particle infectivity in naïve cells.
Competing Interest Statement
The authors have declared no competing interest.
