Factor-Dependent Internal Ribosome Entry Site and -1 Programmed Frameshifting Signal in the Bemisia-Associated Dicistrovirus 2.

The dicistrovirus intergenic (IGR) IRES uses the most streamlined translation initiation mechanism: the IRES recruits ribosomes directly without using protein factors and initiates translation from a non-AUG codon. Several subtypes of dicistroviruses IRES have been identified; typically, the IRESs adopt two -to three overlapping pseudoknots with key stem-loop and unpaired regions that interact with specific domains of the ribosomal 40S and 60S subunits to direct translation. We previously predicted an atypical IGR IRES structure and a potential -1 programmed frameshift (-1 FS) signal within the genome of the whitefly Bemisia-associated dicistrovirus 2 (BaDV-2). Here, using bicistronic reporters, we demonstrate that the predicted BaDV-2 -1 FS signal can drive -1 frameshifting in vitro via a slippery sequence and a downstream stem-loop structure that would direct the translation of the viral RNA-dependent RNA polymerase. Moreover, the predicted BaDV-2 IGR can support IRES translation in vitro but does so through a mechanism that is not typical of known factorless dicistrovirus IGR IRES mechanisms. Using deletion and mutational analyses, the BaDV-2 IGR IRES is mapped within a 140-nucleotide element and initiates translation from an AUG codon. Moreover, the IRES does not bind directly to purified ribosomes and is sensitive to eIF2 and eIF4A inhibitors NSC1198983 and hippuristanol, respectively, indicating an IRES-mediated factor-dependent mechanism. Biophysical characterization suggests the BaDV-2 IGR IRES contains several stem-loops; however, mutational analysis suggests a model whereby the IRES is unstructured or adopts distinct conformations for translation initiation. In summary, we have provided evidence of the first -1 FS frameshifting signal and a novel factor-dependent IRES mechanism in this dicistrovirus family, thus highlighting the diversity of viral RNA-structure strategies to direct viral protein synthesis.

Japanese encephalitis virus persists in the human reproductive epithelium and porcine reproductive tissues.

Japanese encephalitis virus (JEV) is the emerging and geographically expanding flavivirus and the major causative agent of encephalitis in humans in Asia. There are risks of JEV introduction into the Americas given a large population of amplifying hosts-pigs and wild boars, and insect vectors-Culex mosquitoes. There are emerging concerns about vector-free ways of flavivirus transmission, for example sexual and transplacental Zika virus transmissions, which may change flavivirus epidemiology and expand the geographical range to territories with no insect vectors. It is unknown whether JEV has tropism in the female lower reproductive tract and the potential for sexual transmission in humans. While clinical outcomes of transplacental JEV infection are described in humans and pigs, cellular targets and tissue tropism in the upper reproductive tract are also unknown. Here, we studied JEV infection phenotypes and host transcriptional responses in human reproductive epithelial cells. We found that JEV caused persistent infection and cytopathology in the vaginal epithelium, endometrial epithelium, and trophoblast. Human vaginal epithelial cells infected with JEV had altered transcriptional responses associated with inflammation and disruption of epithelial barrier function. Also, using pigs-the native amplifying host for JEV, we confirmed JEV tropism in the female lower and upper reproductive tracts. We discovered that JEV persists in the vaginal mucosa for at least 28 days and pigs shed the virus in vaginal secretions. We also found JEV persistence in the endometrium and placenta with transplacental and fetal infections. Altogether, we discovered that JEV targets the vaginal epithelium and has the potential for sexual transmission in humans. We also contributed to a better understanding of JEV pathogenesis during transplacental infection. Further studies are needed to better understand the interactions of JEV with reproductive tissues, how persistent infection affects female reproductive functions, and the risks for non-vector transmission.