Tracking SARS-CoV-2 one molecule at a time: Spatiotemporal investigation of coronavirus replication dynamics and host response in single cells in vitro and in vivo

Project Summary The current pandemic has highlighted fundamental gaps in our knowledge about the replication strategies of coronaviruses, and how these are affected by the host at both the organismal and cellular level. There is a pressing need to understand how SARS-CoV-2 infection and host-cell responses trigger such a diverse set of pathologies, and the roles played by viral variation, host genetics and underlying preconditions. As studies of SARS-CoV-2 frequently utilize population-based assays that look hours to days post infection, information on cellular and spatial variability are lost. Furthermore, host responses are communicative spatial processes subject to signaling gradients that vary between cells. Thus, averages over populations obscure heterogeneity and spatial separations, and miss the earliest viral and host behaviors due to lack of sensitivity. To fill this gap, we developed experimental and computational approaches to quantify individual virion entrance, establishment of the first replicative events, and production of viral RNAs and host responses in single cells, all while maintaining sample spatial integrity. This project's long-term objective is to apply this novel approach to gain insights into SARS-CoV-2 biology distinct from those gleaned using traditional strategies. This knowledge will provide new insight into the spectrum of COVID-19 disease outcomes and help guide future therapeutic strategies. To this end, single-molecule in situ analyses, including single molecule fluorescence in situ hybridization (smFISH) and multiplexed error-robust FISH (MERFISH) will be applied to the study of SARS-CoV-2. Aim 1 will quantify SARS-CoV-2 entry, replication and spread, and host transcriptional responses in cells of varying tissue origin. These data will be used to develop a stochastic computational model to address the determinants of early viral replication and the resulting cellular response. Aim 2 will examine the effect of host mutations or pre-existing conditions that affect the type I interferon (IFN) response and have been associated with severe COVID-19, as well as emerging viral Variants of Concern. Aim 3 will model patient comorbidities in vivo using mouse models of SARS-CoV-2 infection, identifying the functional and spatial consequences of host responses, including IFN and other cytokine expression, in the respiratory tract and lung. We will further utilize these in vivo models to understand why pathogenesis and disease outcome differ depending on the inoculum dose and the age of the animal. Together, our multidisciplinary approach utilizing techniques and information from systems-level virology, spatial transcriptomics, host genetics, computational biology, and innate immunity provides a powerful means of probing questions central to understanding clinical outcome and informing life-saving interventions.