Torres, F. J. | bioRxiv Science

bioRxiv2021

Structural analysis of 3'UTRs in insect flaviviruses reveals novel determinant of sfRNA biogenesis and provides new insights into flavivirus evolution

Insect-specific flaviviruses (ISFs) circulate in nature due to vertical transmission in mosquitoes and do not infect vertebrates. ISFs include two distinct lineages - classical ISFs (cISFs) that evolved independently and dual host associated ISFs (dISFs) that are proposed to diverge from mosquito-borne flaviviruses (MBFs). Compared to pathogenic flaviviruses, ISFs are relatively poorly studied, and their molecular biology remains largely unexplored. In this study we focused on the characterisation of ISF 3UTRs and their ability to produce subgenomic flaviviral RNAs - noncoding viral RNAs that are known as important determinants of transmission and replication of pathogenetic flaviviruses. We demonstrated that cISFs and dISFs produce sfRNAs by employing a highly conserved mechanism of resistance to degradation by the cellular 5-3 exoribonuclease XRN1. We determined the secondary structures of complete 3UTRs and experimentally identified structured RNA elements that resist degradation by XRN1 (xrRNAs) in divergent representatives of cISF and dISF clades. We discovered a novel class of xrRNAs in dISFs and identified structurally divergent xrRNA in Anopheles-associated cISFs. Phylogenetic analyses based on sequences and secondary structures of xrRNAs and complete 3UTRs reveal that xrRNAs of cISFs and MBFs/dISFs evolved from a common xrRNA ancestor similar to the xrRNA of Anopheles-associated cISFs. Additionally, we found that duplications of xrRNAs occurred independently in ISF and MBF clades. Using ISF mutants deficient in the production of sfRNAs, we found that individual sfRNAs of ISFs have redundant functions. We conclude that duplicated xrRNAs were selected in the evolution of flaviviruses to ensure that sfRNA is produced if one of the xrRNAs lose XRN1 resistance due to mutations or misfolding.

microbiology↗

bioRxiv2021

Zika virus noncoding RNA cooperates with the viral protein NS5 to inhibit STAT1 phosphorylation and facilitate viral pathogenesis

Zika virus (ZIKV) is a re-emerging pathogenic flavivirus, which causes microcephaly in infants and poses a continuing threat to public health. ZIKV, like all other flaviviruses, produces highly abundant noncoding RNA known as subgenomic flaviviral RNA (sfRNA). Herein we utilized wild-type and mutant ZIKV defective in production of sfRNA to elucidate for the first time how production of sfRNA affects all aspects of ZIKV pathogenesis. We found that in mouse pregnancy model of infection sfRNA is required for trans-placental dissemination of ZIKV and subsequent infection of fetal brain. Using human brain organoids, we showed that sfRNA promotes apoptosis of neural progenitor cells leading to profound cytopathicity and disintegration of organoids. We also found by transcriptome profiling and gene network analysis that in infected human placental cells sfRNA inhibits multiple antiviral pathways and promotes apoptosis with STAT1 identified as a key shared factor linking these two interconnected sfRNA activities. We further showed for the first time that sfRNA inhibits phosphorylation and nuclear translocation of STAT1 by a novel mechanism which involves binding to and stabilizing viral protein NS5. This allows accumulation of NS5 at the levels required for efficient inhibition of STAT1 phosphorylation. Thus, we elucidated the molecular mechanism by which ZIKV sfRNA exerts its functions in vertebrate hosts and discovered a co-operation between viral noncoding RNA and a viral protein as a novel strategy employed by viruses to counteract antiviral responses.

microbiology↗