Mechanisms of VP4 Promoting Rotavirus Cell-to-Cell Transmission and Viral Pathogenesis

Project Summary Viruses spread from infected cells to uninfected “bystander” cells by two fundamentally distinct modes, by releasing into extracellular environment or by direct cell-cell contact. For enteric viruses, which are mostly non- enveloped viruses, the current paradigm is that they predominantly egress host cells via an extracellular manner, either as naked cell-free viruses or in membrane-associated forms. Whether enteric viruses can efficiently spread through cell-to-cell transmission has not been studied. Rotaviruses (RVs) are highly pathogenic human viruses that infect intestinal epithelial cells (IECs) and cause more than 200,000 children deaths every year. RV infections are also associated with severe diarrheal diseases in the elderly and immunocompromised. The current RV vaccines are significantly less effective in low-to-middle-income countries. There is also a lack of RV-specific and broad-spectrum anti-enteric virus therapeutics. In a recent proteomics study from our laboratory, we found that the RV outer capsid protein VP4 co-precipitates with multiple subunits of the actin-related protein 2/3 (Arp2/3) complex. IEC-intrinsic Arp2/3 mediates the migration of IECs from crypts towards the top of the intestinal villi. However, the role of Arp2/3 in enteric virus infections is not known. Our new preliminary data suggests that RV cell-to-cell transmission was defective in CRISPR/Cas9 knockout cells that lack Arp2/3 subunits. In addition, chemical inhibition of Arp2/3 activity significantly reduced RV fecal shedding and diarrhea in a neonatal mouse model of RV infection. RV replication was also attenuated by Arp2/3 inhibition in fully differentiated human intestinal organoids. We hypothesize that VP4 protein binds and co-opts the host Arp2/3 complex in the infected IECs to enable RV cell-to-cell spread, which contributes to viral replication and pathogenesis. To test this hypothesis, we will leverage our expertise in RV reverse genetics systems, a highly tractable mouse model of RV diseases, and state-of-the-art biochemical and imaging techniques. In Aim 1, we will use hydrogen-deuterium exchange mass spectrometry and proximity ligation assays to determine the biochemical properties, subcellular localization, and timing of VP4-Arp2/3 interactions during RV infection. In Aim 2, we will investigate the physiological relevance of the Arp2/3 complex in RV infection in vivo using conditional knockout mice, 5-ethynyl-2-deoxyuridine labeling, and infectious RVs expressing an mScarlet reporter and chimeric VP4s. Collectively, we expect that this study will establish a new model of a successful enteric pathogen taking advantage of cell-to-cell transmission in the milieu of the mucus layer in the intestine to complement the traditional extracellular way of egress. We may also reveal novel mechanisms by which RV alters the migration patterns of infected and bystander IECs to its benefit. Our findings will be instrumental for designing new antiviral inhibitors and next-generation live-attenuated RV vaccine candidates based on targeting VP4-Arp2/3 interactions to alleviate diarrhea and mortality.