Structural and functional studies of alphavirus receptors

ABSTRACT Alphaviruses are mosquito-transmitted, positive-strand enveloped RNA viruses that cause severe diseases in humans, including lethal encephalitis. Despite their epidemic potential, there are no FDA-approved antivirals or vaccines against any alphavirus infection. Infections by certain alphaviruses have increased in recent years, likely secondary to climate change. Eastern equine encephalitis virus, Venezuelan equine encephalitis virus, and Western equine encephalitis virus cause outbreaks in horses and humans in the Americas. While all three viruses pose threats to public health, among them, eastern equine encephalitis virus carries the highest case fatality rate (30-80%). Of those who survive eastern equine encephalitis virus infection, most are left with permanent neurological damage. The cellular receptors that most alphaviruses bind to when infecting cells have remained elusive, and this knowledge gap has limited our understanding of key determinants of alphavirus pathogenesis and of pathways for potential therapeutic intervention through molecules that block virus receptor binding. Our lab recently discovered that very low-density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2, two LDLR-related proteins that are also expressed in the brain, are cellular receptors for eastern equine encephalitis virus. Western equine encephalitis virus had unknown receptors. We identified related and unrelated membrane proteins that are highly enriched in the brain as receptors for western equine encephalitis virus. We hypothesize that binding to VLDLR, ApoER2, or the newly identified receptors explains mechanisms for differential cellular entry, tissue distribution, and viral pathogenesis of different encephalitic alphaviruses. In Aim 1, we will study whether eastern equine encephalitis virus entry into physiologically relevant brain cell types (neurons, astrocytes, and microglial cells) critically depends on VLDLR and ApoER2 and determine if blockade of these receptors can protect mice from viral encephalitis. In Aim 2, we will evaluate whether western equine encephalitis virus binding to its receptors is also a critical determinant of infection of human brain cell types in vitro and of neuropathogenesis in mice. In Aim 3, we will determine high-resolution cryo-electron microscopy structures of virus-like particles bound to different alphavirus receptors. Our studies may pave the way for the design of inhibitors that help combat alphavirus encephalitis by preventing attachment to cellular receptors and help address an urgent need for antivirals against these important human pathogens.