28Eastern equine encephalitis virus (EEEV), a mosquito-borne RNA virus, is one of the 29 most acutely virulent viruses endemic to the Americas, causing between 30% and 70% 30 mortality in symptomatic human cases. A major factor in the virulence of EEEV is the 31 presence of four binding sites for the hematopoietic cell-specific microRNA, miR-142-32 3p, in the 3' untranslated region (3' UTR) of the virus. Three of the sites are "canonical" 33 with all 8 seed sequence residues complimentary to miR-142-3p while one is "non-34 canonical" and has a seed sequence mismatch. Interaction of the EEEV genome with 35 miR-142-3p limits virus replication in myeloid cells and suppresses the systemic innate 36 immune response, greatly exacerbating EEEV neurovirulence. The presence of the 37 miRNA binding sequences is also required for efficient EEEV replication in mosquitoes 38 and, therefore, essential for transmission of the virus. In the current studies, we have 39 examined the role of each binding site by point mutagenesis of seed sequences in all 40 combinations of sites followed by infection of mammalian myeloid cells, mosquito cells 41 and mice. The resulting data indicate that both canonical and non-canonical sites 42 contribute to cell infection and animal virulence, however, surprisingly, all sites are 43 rapidly deleted from EEEV genomes shortly after infection of myeloid cells or mice. 44Finally, we show that the virulence of a related encephalitis virus, western equine 45 encephalitis virus, is also dependent upon miR-142-3p binding sites. 46 47
Author Summary 48Eastern equine encephalitis virus (EEEV) is one of the most acutely virulent mosquito-49 borne viruses in the Americas. A major determinant of EEEV virulence is a mammalian 50 microRNA (miRNA) that is primarily expressed in myeloid cells, miR-142-3p. Like 51 miRNA suppression of host mRNA, miR-142-3p binds to the 3' untranslated region 52 (UTR) of the EEEV genome only in myeloid cells suppressing virus replication and the 53 induction of the innate immune response. In this study, we used point mutations in all 54 four miR-142-3p binding sites in the EEEV 3' UTR to understand the mechanism behind 55 this miRNA suppression. We observed that decreasing the number of miR-142-3p 56 binding sites leads to virus escape and ultimately attenuation in vivo. Furthermore, 57 another virus, western equine encephalitis virus, also encodes miR-142-3p binding sites 58 that contribute to virulence in vivo. These results provide insight into the mechanism of 59 how cell-specific miRNAs can mediate suppression of virus replication. 60 61 Eastern equine encephalitis virus (EEEV) is a mosquito-borne alphavirus that 63 causes severe manifestations of encephalitis in humans resulting in high mortality rates 64 and long-term neurological sequelae in symptomatic cases [1] and is one of, if not the 65 most acutely virulent virus circulating in North America. Even though both EEEV and 66 the closely related Venezuelan equine encephalitis virus (VEEV) cause encephalitic 67 disease, th...