Parsing the role of NSP1 in SARS-CoV-2 infection

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 19 (COVID-19) pandemic. Despite its urgency, we still do not fully understand the molecular basis of SARS-CoV-2 pathogenesis and its ability to antagonize innate immune responses. SARS-CoV-2 leads to shutoff of cellular protein synthesis and over-expression of nsp1, a central shutoff factor in coronaviruses, inhibits cellular gene translation. However, the diverse molecular mechanisms nsp1 employs as well as its functional importance in infection are still unresolved. By overexpressing various nsp1 mutants and generating a SARS-CoV-2 mutant in which nsp1 does not bind ribosomes, we untangle the effects of nsp1. We uncover that nsp1, through inhibition of translation and induction of mRNA degradation, is the main driver of host shutoff during SARS-CoV-2 infection. Furthermore, we find the propagation of nsp1 mutant virus is inhibited specifically in cells with intact interferon (IFN) response as well as in-vivo, in infected hamsters, and this attenuation is associated with stronger induction of type I IFN response. This illustrates that nsp1 shutoff activity has an essential role mainly in counteracting the IFN response. Overall, our results reveal the multifaceted approach nsp1 uses to shut off cellular protein synthesis and uncover the central role it plays in SARS-CoV-2 pathogenesis, explicitly through blockage of the IFN response.

Protocol for assessing translational regulation in mammalian cell lines by OP-Puro labeling

“…Minor optimizations might be required for other cell lines, which will be highlighted in the text. Although we have not applied the protocol to non-mammalian and suspension cell lines, similar protocols based on OP-Puro incorporation have been widely used to study protein synthesis in mouse small intestine ( Liu et al., 2012 ), Chinese hamster ovary (CHO) cells ( Nagelreiter et al., 2018 ), hematopoietic stem cells ( Signer et al., 2014 ), muscle stem cells ( Zismanov et al., 2016 ), skin stem cells ( Blanco et al., 2016 ), colorectal cancer organoids ( Morral et al., 2020a , 2020b ), and Calu-6 cells upon SARS-CoV-2 infection ( Fisher et al., 2022 ). Thaw cells of interest.…”