Zika Virus Disrupts the Nonsense-mediated mRNA Decay Pathway feat. A Clinical Application of Next Generation Sequencing

Zika virus (ZIKV) is a mosquito-borne illness responsible for an outbreak of microcephaly in Brazil in 2015. To better understand the molecular mechanisms behind the neurodevelopmental delay seen, we explored the pathways perturbed by ZIKV to establish an infection and cause the neurological sequelae seen in patients. We focused particularly on the Nonsense-mediated mRNA Decay (NMD) pathway: a cellular RNA quality control pathway responsible for degrading mRNAs with premature termination codons that also regulates a significant subset of normal mRNAs. To best study the impacts of ZIKV, we used neural progenitor cells (NPCs) derived from induced pluripotent stem cells. We found that during ZIKV infection, the viral Capsid protein interacted with and degraded UPF1, the master regulator of the NMD pathway, in the nucleus. UPF1 was a viral restriction factor, and removal of UPF1 from the cell leads to increased permissivity to ZIKV infection. We next found that ZIKV-mediated UPF1 degradation led to a loss of UPF1 occupancy on host transcripts, which caused a shift in cellular localization and trapped specific transcripts in the nucleus. An extracellular matrix protein involved in fetal development, FREM2, had mRNA retained in the nucleus, leading to decreased protein levels. Depletion of FREM2 in NPCs led to premature neuronal differentiation. Overall, it leads to a model system where ZIKV, using its capsid protein, causes destruction of UPF1 to promote a productive infection As we find UPF1 linked to many neurodevelopment pathways, we propose that the disruption of the NMD and the lack of host mRNA export contributes to virally induced neurodevelopmental disorders in ZIKV infection. Lastly, we show an example of how laboratory techniques used in the study of viral infections in vitro can be used in a clinical application. An outbreak of EV-A71 in Catalonia, Spain caused widespread brainstem encephalitis in a pediatric population. To better understand the outbreak, we compare metagenomic next generation sequencing (mNGS) to quantitative PCR (qPCR) as a diagnostic tool in the CSF of patients. We show that mNGS is capable of identifying virus in samples that qPCR can’t. Using mNGS data, we obtained several full-length genomes, allowing for phylogenetic analyses and the identification of a single mutation potentially responsible for the enhanced neuroinvasive characteristics. We then used VirScan, a phage display library expressing a wide variety of human viral protein peptides, to identify patients exposed to virus and characterize which viral epitopes were the most immunoreactive, complementing the mNGS data. Overall, we show that this standard laboratory technique can play an important role in patient care, especially for viral detection in typically hard-to-diagnose body sites. This also applies to surveillance of current and future viral outbreaks. Given the circumstances—writing a dissertation regarding the molecular pathogenesis of a virus responsible for an epidemic concurrent with the COVID-19 pandemic highlights the need for translation of cutting-edge laboratory techniques to improve virus detection and comprehension for more personalized therapeutics