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Link to original content: https://pubmed.ncbi.nlm.nih.gov/32995777
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[Preprint]. 2020 Sep 18:2020.09.18.302901.
doi: 10.1101/2020.09.18.302901.

SARS-CoV-2 Nsp1 suppresses host but not viral translation through a bipartite mechanism

Ming Shi  1   2   3   4 , Longfei Wang  1   2   4 , Pietro Fontana  1   2   4 , Setu Vora  1   2   4 , Ying Zhang  2   5 , Tian-Min Fu  1   2   6   7 , Judy Lieberman  2   5 , Hao Wu  1   2   8
Affiliations

SARS-CoV-2 Nsp1 suppresses host but not viral translation through a bipartite mechanism

Ming Shi et al. bioRxiv. .

Update in

Abstract

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a highly contagious virus that underlies the current COVID-19 pandemic. SARS-CoV-2 is thought to disable various features of host immunity and cellular defense. The SARS-CoV-2 nonstructural protein 1 (Nsp1) is known to inhibit host protein translation and could be a target for antiviral therapy against COVID-19. However, how SARS-CoV-2 circumvents this translational blockage for the production of its own proteins is an open question. Here, we report a bipartite mechanism of SARS-CoV-2 Nsp1 which operates by: (1) hijacking the host ribosome via direct interaction of its C-terminal domain (CT) with the 40S ribosomal subunit and (2) specifically lifting this inhibition for SARS-CoV-2 via a direct interaction of its N-terminal domain (NT) with the 5' untranslated region (5' UTR) of SARS-CoV-2 mRNA. We show that while Nsp1-CT is sufficient for binding to 40S and inhibition of host protein translation, the 5' UTR of SARS-CoV-2 mRNA removes this inhibition by binding to Nsp1-NT, suggesting that the Nsp1-NT-UTR interaction is incompatible with the Nsp1-CT-40S interaction. Indeed, lengthening the linker between Nsp1-NT and Nsp1-CT of Nsp1 progressively reduced the ability of SARS-CoV-2 5' UTR to escape the translational inhibition, supporting that the incompatibility is likely steric in nature. The short SL1 region of the 5' UTR is required for viral mRNA translation in the presence of Nsp1. Thus, our data provide a comprehensive view on how Nsp1 switches infected cells from host mRNA translation to SARS-CoV-2 mRNA translation, and that Nsp1 and 5' UTR may be targeted for anti-COVID-19 therapeutics.

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Conflict of interest statement

DECLARATION OF INTERESTS The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. Suppression of Protein Synthesis by SARS-CoV-2 Nsp1
(A) Schematic representation of CMV 5’ UTR mScarlet reporter (upper panel) and immunofluorescence images of HeLa cells transfected with mScarlet (red) and MBP or MBP-Nsp1 (green), and DNA (blue) (lower panel). (B) Quantification of relative fluorescence intensity of mScarlet in MBP- or MBP-Nsp1- expressing HeLa cells. (C) Gel filtration profiles of MBP-Nsp1 and the MBP-Nsp1–40S complex. Schematic representation of MBP-Nsp1 is shown. Peak positions are labeled. (D) SDS PAGE of MBP-Nsp1 and the MBP-Nsp1–40S complex. (E) A cryo-EM micrograph of the MBP-Nsp1–40S complex.
Figure 2.
Figure 2.. Blockage of the mRNA Channel in the 40S by the Nsp1 C-terminal Helical Hairpin
(A) Cryo-EM density of the Nsp1–40S complex. Nsp1 is in cyan. Subunits of 40S that interact with Nsp1 are color-coded. (B) Ribbon diagram of the Nsp1–40S complex. Nsp1 (cyan) binds at the mRNA channel in the cleft between the head and body of the 40S ribosomal subunit. Subunits of 40S that interact with Nsp1 are color-coded. (C) Ribbon diagram and cryo-EM density of SARS-CoV-2 Nsp1 with schematics highlighting its interfaces to 40S. (D) Electrostatic surface representations of Nsp1 binding surfaces to the 40S subunits and rRNA. Buried surface areas are marked. (E) Detailed interactions between Nsp1 and the 40S ribosomal subunit. See also Supplemental Figure 1
Figure 3.
Figure 3.. Evasion of Nsp1-Mediated Translation Inhibition by SARS-CoV-2 5′ UTR
(A) Summary of Nsp1 constructs and SARS-CoV-2 5’ UTR mScarlet reporter levels. The various Nsp1 constructs are: Nsp1-FL, Nsp1-NT, Nsp1-CT, both Nsp1-NT and Nsp1-CT (Nsp1-NT+CT), Nsp1-linker1, and Nsp1-linker2. (B) Immunofluorescence images of HeLa cells transfected with SARS-CoV-2 5’ UTR mScarlet reporter (red) and MBP, MBP-Nsp1-FL, MBP-Nsp1-NT, MBP-Nsp1-CT, or MBP-Nsp1-N+C (green). (C) Quantification of relative fluorescence intensity of mScarlet in indicated groups from (B) and (E). (D) Quantification of the relative luciferase activity in HEK293T cells transfected with SARS-CoV-2 5’ UTR luciferase reporter and various Nsp1 constructs. Values are means ± S.E.M. obtained from three independent experiments. (E) Immunofluorescence images of HeLa cells transfected with SARS-CoV-2 5’ UTR mScarlet reporter (red) and Nsp1-FL, Nsp1-linker1, or Nsp1-linker2 (green).
Figure 4.
Figure 4.. Direct interaction between SARS-CoV-2 5′ UTR and Nsp1-NT, and Requirement of the SL1 of the 5’ UTR in Viral Evasion
(A) Gel filtration profile of the complex between Nsp1-NT (Strep-tagged) and SARS-CoV-2 5’ UTR purified by the Strep-Tactin® affinity resin from the HeLa cell lysate transfected with the SARS-CoV-2 5’ UTR Nsp1-NT-Strep construct. (B) Anti-Strep tag Western blot (upper panel) and RT-PCR of SARS-CoV-2 5’ UTR (lower panel) of the gel filtration peak fractions in (A). Nsp1-NT co-migrated with SARS-CoV-2 5’ UTR. (C) Construct design of SARS-CoV-2 SL1 5’ UTR and SARS-CoV-2 ΔSL1 5’ UTR-mScarlet reporters (upper panel) and immunofluorescence images of HeLa cells transfected with a mScarlet reporter (red) and MBP or MBP-Nsp1-FL (green) (lower panel) (D) Quantification of data in (C) showing the relative fluorescence intensity of mScarlet in HeLa cells transfected with MBP or MBP-Nsp1-FL. (E) High throughput quantification of mean cellular mScarlet fluorescence intensity when placed downstream of either SARS-CoV-2 5’ UTR, SARS-CoV-2 ΔSL1 5’ UTR, or control 5’UTR, with or without co-expression of MBP-Nsp1. (F) Relative luciferase activity in HEK293T cells after co-transfection of a luciferase reporter and MBP or MBP-Nsp1. The luciferase reporter was placed downstream of SARS-CoV-2 5’ UTR, SARS-CoV-2 SL1 5’ UTR or SARS-CoV-2 ΔSL1 5’ UTR. NC: negative control without adding the luciferase substrate in cells co-transfected with luciferase reporter and MBP. Values are means ± S.E.M. obtained from three independent experiments.
Figure 5.
Figure 5.. Model of a Bipartite Mechanism for Nsp1-Mediated Translation Inhibition and Evasion by SARS-CoV-2 5’ UTR
A schematic model depicting the bipartite roles of SARS-CoV-2 Nsp1 during infection. First, Nsp1 blocks host mRNA from binding to the 40S ribosomal subunit due to physical occlusion by the bound Nsp1-CT. Second, Nsp1 supports viral mRNA translation by interacting with SARS-CoV-2 5’ UTR using Nsp1-NT, which results in dissociation of the Nsp1-CT-40S complex to overcome inhibition. This mechanism of evasion of Nsp1-mediated translation inhibition is illustrated by the failure of linker-lengthened Nsp1 to support viral mRNA translation. With the longer linker, the Nsp1-NT-5’ UTR complex can co-exist with the Nsp1-CT-40S complex.
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