N Protein Nexus: Rewiring Host Translation Machinery for SARS-CoV-2's Early Replicative Advantage

ABSTRACT: Dependence on host-cell machinery for protein synthesis poses a significant challenge to coronavirus replication, particularly at the onset of infection when viral genomic RNA must compete with an abundance of host mRNAs for translation. To overcome this hurdle, viruses have evolved sophisticated strategies to commandeer the host translation machinery. While multiple mechanisms by which SARS-CoV-2 hijacks host translation have been elucidated, almost all involve non-structural viral proteins. This raises a fundamental question: how does SARS- CoV-2 establish a translational foothold during the early stages of infection, before non-structural proteins are synthesized? The viral nucleocapsid (N) protein is the primary viral factor present at this early stage of infection and has been shown to manipulate cell machinery to facilitate infection. We have found that N protein physically and functionally interacts with the human translation machinery, facilitating preferential viral translation. Moreover, our results suggest that the viral genome's 5ʹ untranslated region exploits high-affinity N protein binding to potentiate selective viral RNA recognition for translation. We hypothesize that N protein is a key mediator of viral translational hijacking in early SARS-CoV-2 infection, establishing a new paradigm within the N- protein functional repertoire. This proposal now seeks to elucidate the molecular mechanisms of host protein- synthesis modulation by N protein for viral benefit during early infection. Through two specific aims, we will (1) identify the viral determinants responsible for the impact of N protein on viral RNA translation and (2) delineate the roles of host factors in N protein viral-translation enhancement. By combining biochemical, biophysical, and genetic approaches, we will establish a comprehensive understanding of unanticipated host-virus interactions that govern SARS-CoV-2 pathogenesis, uncovering novel viral vulnerabilities that can be exploited to develop targeted antiviral therapy. Ultimately, this study will provide new insights for innovative therapeutic strategies that can be extended to other viruses with RNA-binding proteins, offering a promising avenue for smothering infection at its onset. 3