DISSECTION OF HOST-VIRUS INTERACTIONS IN THE HUMAN CENTRAL NERVOUS SYSTEM

Viral encephalitis is an inflammatory brain that can lead to severe neurological deficits, despite antiviral treatment. Neurotropic viruses cause viral encephalitis due to their ability to invade the central nervous system (CNS), and the most common cause is HSV-1. Inborn errors of immunity (IEIs) have demonstrated that neuron, cell type-specific, antiviral immunity is essential to prevent viral infection and encephalitis. However, despite these key advances in human genetics, relatively little is known about what regulates neuron-specific antiviral immunity and how this may differ against specific neurotropic viruses. Human pluripotent stem cells (hPSCs) can, theoretically, be differentiated into any given cell type of the human body. Here, we leverage hPSC technology by differentiating cortical neurons at scale to study the neurotropism of Herpes simplex virus-1 (HSV-1), Monkeypox virus (MPXV), Varicella zoster virus (VZV) and Influenza A viruses (IAV). Our comparative virology approach demonstrated a susceptibility and permissiveness to herpesvirus infection, while in contrast neurons did not show any productive infection to either IAV or MPXV. By developing a genome-wide CRISPR/Cas9 screening platform in human neurons, we were able to identify several candidate host factors that regulate neuronal anti-HSV-1 immunity, such as MTBP, SIRT7, GNAI3, NPPA, POLR3E and RNGTT. In a complementary approach, we performed high temporal resolution transcriptional profiling of human cortical neurons upon infection with herpesviruses. This allowed us to dissect neuronal responses from 30 minutes to 48 hours post infection (hpi) to HSV-1 and VZV infections, identifying a delayed antiviral response in neurons compared to fibroblasts. Interestingly, both human cortical neurons and fibroblasts showed the upregulation of cilium-related genes starting from 24hpi onwards. We validated several cilia-related genes, such as ARL13b and DNAH5 by immunostaining, demonstrating both an increased expression and mislocalisation of these proteins upon HSV-1 infection. siRNA- mediated knockdown experiments of ARL13b show reduced intracellular replication in fibroblasts, identifying a potential proviral role for cilia-related proteins during herpesvirus infection. To conclude, through our genome-wide screens and transcriptional profiling we identified candidate neuronal anti- and proviral host factors of HSV-1 infection, providing a better understanding of intrinsic neuronal antiviral immunity. Our datasets yield a unique resource to the virology and neuroscience fields and, especially, in the context of viral encephalitis. Moreover, our candidate anti- and proviral factors may provide therapeutic targets for viral encephalitis.