Abstract
Eastern equine encephalitis virus (EEEV), a mosquito-borne RNA virus, is one of the most acutely virulent viruses endemic to the Americas, causing between 30% and 70% mortality in symptomatic human cases. A major factor in the virulence of EEEV is the presence of four binding sites for the hematopoietic cell-specific microRNA, miR-142-3p, in the 3’ untranslated region (3’ UTR) of the virus. Three of the sites are “canonical” with all 7 seed sequence residues complimentary to miR-142-3p while one is “non-canonical” and has a seed sequence mismatch. Interaction of the EEEV genome with miR-142-3p limits virus replication in myeloid cells and suppresses the systemic innate immune response, greatly exacerbating EEEV neurovirulence. The presence of the miRNA binding sequences is also required for efficient EEEV replication in mosquitoes and, therefore, essential for transmission of the virus. In the current studies, we have examined the role of each binding site by point mutagenesis of the seed sequences in all combinations of sites followed by infection of mammalian myeloid cells, mosquito cells and mice. The resulting data indicate that both canonical and non-canonical sites contribute to cell infection and animal virulence, however, surprisingly, all sites are rapidly deleted from EEEV genomes shortly after infection of myeloid cells or mice. Finally, we show that the virulence of a related encephalitis virus, western equine encephalitis virus, is also dependent upon miR-142-3p binding sites.
Highlights
Eastern equine encephalitis virus (EEEV) is a mosquito-borne alphavirus that causes severe manifestations of encephalitis in humans resulting in high mortality rates and long-term neurological sequelae in symptomatic cases, [1] and is one of, if not the, most acutely virulent virus circulating in North America
A major determinant of EEEV virulence is a mammalian microRNA that is primarily expressed in hematopoietic cells, miR-142-3p
In an effort to determine if miR-142-3p bound to viral RNA and targeted it to the RNA-induced silencing complex (RISC) complex, we utilized a translation reporter that encodes the 5’ untranslated region (UTR) and the wild type (WT) EEEV 3’ untranslated region (3’ UTR) encoding the four miR-142-3p binding sites (Fig 1A) or the 11337 deletion mutant 3’ UTR that lacks all of the miR-142-3p binding sites [4], and two different experimental methodologies: 1) immunoprecipitation of the Ago2 protein to isolate RNAs interacting with the complex and 2) precipitation of biotin-labeled miR-142-3p to ascertain whether miR-142-3p interacted directly with the EEEV 3’ UTR in myeloid cells
Summary
Eastern equine encephalitis virus (EEEV) is a mosquito-borne alphavirus that causes severe manifestations of encephalitis in humans resulting in high mortality rates and long-term neurological sequelae in symptomatic cases, [1] and is one of, if not the, most acutely virulent virus circulating in North America. Even though both EEEV and the closely related Venezuelan equine encephalitis virus (VEEV) cause encephalitic disease, they exhibit drastic differences in their pathogenesis, in large part due to differential infectivity of the viruses for myeloid cells. The miR-142-3p blockade virtually eliminates EEEV translation and replication in myeloid cells [4]
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