SARS-CoV-2 Nsp1 binds the ribosomal mRNA channel to inhibit translation

Abstract

ARS-CoV-2 is the causing agent of the COVID-19 pandemic and belongs to the genus of beta-coronaviruses, with enveloped, positive sense and single-stranded genomic RNA 1. On entering host cells, the viral genomic RNA is translated by the cellular protein synthesis machinery to produce a set of non-structural proteins (NSPs) 2. NSPs render the cellular conditions favorable for viral infection and viral mRNA synthesis 3. Coronaviruses have evolved specialized mechanisms to hijack the host gene expression machinery and employ cellular resources to regulate viral protein production. Such mechanisms are common for many viruses and include inhibition of host protein synthesis and endonucleolytic cleavage of host messenger RNAs (mRNAs) 4,5. In cells infected with the closely related SARS-CoV, one of the most enigmatic viral proteins is the host shutoff factor, Nsp1. Nsp1 is encoded by the gene closest to the 5′ end of the viral genome and is among the first proteins to be expressed after cell entry and infection to repress multiple steps of host protein expression 6-9. Initial structural characterization of the isolated SARS-CoV Nsp1 protein revealed the structure of its N-terminal domain, whereas its C-terminal region was flexibly disordered 10. Furthermore, it was shown that SARS-CoV Nsp1 suppresses host innate immune functions, mainly by targeting type I interferon expression and antiviral signaling pathways 11. Taken together, Nsp1 serves as a potential virulence factor for coronaviruses and represents an attractive target for live attenuated vaccine development 12,13. To provide molecular insights into the mechanism of SARS-CoV-2 Nsp1-mediated translation inhibition, we solved the structures of ribosomal complexes isolated from HEK293 lysates supplemented with recombinant purified Nsp1 as well as of an in vitro reconstituted 40S-Nsp1 complex using cryo-EM. We complement our findings by reporting in vitro and in vivo translation inhibition in the presence of Nsp1 that is relieved after mutating key interacting residues. Furthermore, we show that the translation output of reporters containing full-length viral 5′ untranslated regions (UTRs) is significantly enhanced, which could explain how Nsp1 inhibits global translation while still translating sufficient amounts of viral mRNAs. Results C-terminal domain of SARS-CoV-2 Nsp1 binds to the mRNA entry channel. To elucidate the mechanism of how Nsp1 inhibits translation, we aimed to identify the structures of potential ribosomal complexes as binding targets (Fig. 1). Previously, it has been suggested that Nsp1 mainly targets the ribosome at the translation initiation step 9. We thus treated lysed HEK293E cells with bacterially expressed and purified Nsp1 and loaded the cleared lysate on a sucrose gradient. Fractions containing ribosomal particles were then analyzed for the presence of Nsp1. Interestingly, Nsp1 not only co-migrated with 40S particles, but also with 80S ribosomal complexes (Fig. 1c), suggesting that it interacts with a range of different ribosomal states. W...

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“…In all these distinct intermediates, Nsp1 was found to bind in the position and conformation to that observed in the in vitro complex, confirming its role as inhibitor of translation initiation (Thoms et al, 2020). These findings were confirmed in an independently resolved structure of Nsp1 in complex with several ribosomal components (Schubert et al, 2020).…”

“…Recent studies have confirmed that Nsp1 from SARS-CoV-2 plays the same roles as its counterpart in SARS-CoV during virus infection (Schubert et al, 2020;Thoms et al, 2020). In the study by Thoms et al (2020) a complex between purified Nsp1 and 40S ribosomal subunits was reconstituted in vitro by cryo-EM.…”

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“…Papain-like protease 137 PLpro (ORF 1a, Nsp3 138 ), cleaves 139 proteinaceous post-translational (ref 131) modifications on host proteins to evade host anti-viral immune responses. Nsp1 140 suppresses host translation by cleaving cell mRNAs 141 and competes 142 with mRNAs for binding to human 40S ribosomal mRNA channel 143 (as well as 43S, 80S subunits). Type 1 144 interferon 145 (IFN-1) response 146 is modulated by Nsp1, Nsp 6 and Nsp13, which interferes indirectly with IFN-1 by suppressing the phosphorylation and/or nuclear translocation of other cellular molecules 147 involved in catalyzing the IFN-1 response.…”

Aptamers for Detection and Diagnostics (ADD) is a proposed mobile app acquiring optical data from conjugated quantum nanodots to identify molecules indicating presence of SARS-CoV-2 virus: Why public health and healthcare need smartphone sensors as a platform for early detection and prevention

“…Papain-like protease 137 PLpro (ORF 1a, Nsp3 138 ), cleaves 139 proteinaceous post-translational (ref 131) modifications on host proteins to evade host anti-viral immune responses. Nsp1 140 suppresses host translation by cleaving cell mRNAs 141 and competes 142 with mRNAs for binding to human 40S ribosomal mRNA channel 143 (as well as 43S, 80S subunits). Type 1 144 interferon 145 (IFN-1) response 146 is modulated by Nsp1, Nsp 6 and Nsp13, which interferes indirectly with IFN-1 by suppressing the phosphorylation and/or nuclear translocation of other cellular molecules 147 involved in catalyzing the IFN-1 response.…”