Defining Particle-Associated Rotavirus NSP4

About This Grant

PROJECT SUMMARY Our long-term goal is to understand how rotaviruses (RVs) cause life-threatening diarrhea and to develop vaccines to combat this pathogen. Despite the global introduction of vaccines for RV over a decade ago, RV infections still cause >200,000 deaths annually, mostly in low-income countries, creating an urgent need for better vaccines to overcome this mortality. The structure of this large icosahedral virus is complex, consisting of three concentric capsid layers (triple-layered particles, TLPs) that encapsidate 11 genomic dsRNA segments. TLP assembly is unique and requires the viral nonstructural protein 4 (NSP4). NSP4, initially synthesized as an endoplasmic reticulum transmembrane 175 amino acid glycoprotein, serves as an intracellular receptor for nascent immature double layered particles (DLPs). DLPs bind to the cytoplasmic C-terminus of NSP4 and bud through NSP4-containing membranes and acquire a transient membrane. Through a poorly understood mechanism, the transient membrane is lost and the outer capsid proteins, the glycoprotein VP7 and the spike protein VP4, are assembled forming the infectious TLP. We previously characterized a domain of NSP4 that interacts with the stalk domain of VP4, which may be instrumental in outer capsid protein assembly onto TLPs. Unexpectedly, more recently, we discovered a domain of NSP4 is part of infectious animal and human RV TLPs but not DLPs. Immuno-electron microscopy clearly shows NSP4 associates with TLPs as detected by rabbit polyclonal anti-NSP4 antibody. Rabbits parenterally immunized with CsCl and sucrose gradient purified, psoralen-inactivated RV TLPs develop antibodies against NSP4 but not against any other known RV nonstructural proteins. Importantly, we previously demonstrated NSP4 interacts with integrins that have been implicated as RV receptors. Integrins are primarily located on the basolateral surface of intestinal epithelial cells where human RVs infect. Preliminary data shows NSP4 antibodies neutralize RV infectivity. Together, these data indicate a domain of NSP4 is a previously unrecognized component of virus particles. Our central hypotheses are a domain of NSP4 is retained on TLPs, NSP4 mediates the basolateral infection of human epithelial cells and NSP4 antibodies will neutralize RV infectivity. We propose experiments to answer: (1) Which domain of NSP4 is retained on RV, and where is NSP4 located on the TLP? (2) Do NSP4 antibodies neutralize human RV basolateral infection of human intestinal enteroids? While a correlate of protection against RV has not been determined, NSP4 antibodies ameliorate NSP4 induced diarrhea, and studies indicate NSP4 antibodies acquired by natural RV infection are associated with reduced RV diarrhea and seizures. NSP4 may become a new component of non-replicating injectable vaccines, either inactivated RV or subunit vaccines. Such vaccines that bypass the gut could be more effective than oral vaccines in low-income settings by avoiding factors that may limit immune responses in the intestine, such as gut inflammation, malnutrition, or co-infections.