Hunter Olsen

Introduction: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent for coronavirus disease 2019 (COVID-19)¹. Disease presentation can range from asymptomatic to severe, with serious cases resulting in acute respiratory distress syndrome or multiorgan failure². SARS-CoV-2 has a positive sense, single-stranded, RNA genome³. Upon infection, the genome is translated into 16 nonstructural proteins (nsps)³. Nsp1 functions to inhibit host mRNA translation³. While the mechanism of translation suppression is not fully understood, recent studies have increased our understanding of the process. Methods: Various in vitro and cell biologic assays were used to study the effect of nsp1 on translation of host and viral mRNAs. The structural basis for the inhibition of host cell translation was examined by cryo-electron microscopy (Cryo-EM)¹. Affinity chromatography, used in conjunction with luciferase-capped FLAG-tag antibodies, was utilized to monitor translation in human embryonic kidney (HEK) 293T cells¹. Glutathione S-transferase (GST) pull-down assays identified the intracellular interactions of nsp1^1,4. The significance of segments of the viral genome were probed using mScarlet reporters cotransfected with nsp1⁵. Host transcript degradation rates were measured in Calu 3 cells using Thiol (SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq)^6,7. mScarlet reporters with SARS-CoV-2 5’ UTRs were used to evaluate antisense oligonucleotide (ASO) effectiveness⁵. Clinical efficacy of ASOs was studied using intranasal administration in K18-hACE2 mice⁸. Results: Cryo-EM showed two alpha-helices belonging to the C-terminus of nsp1 in the mRNA entry tunnel of the 40S ribosomal subunit¹. Affinity chromatography, using luciferase-capped FLAG-tag antibodies, revealed that the K165 and H165 residues of nsp1 are crucial for ribosomal interaction¹. GST pull-down assays showed that nsp1 interacts with the NXF1 protein⁴. mScarlet reporters revealed the importance of the Stem-loop 1 (SL1) motif found in the 5’UTR of viral mRNA transcripts⁵. Furthermore, they showed that the entirety of the nsp1 protein is needed for more efficacious binding to the host ribosome⁵. RNA SLAM-seq revealed that the presence of nsp1 is correlated with a decrease in host mRNA levels^6,7. ASO use, in vitro, resulted in a decrease in viral mRNA translation⁵. In vivo use led to a better prognosis in mice upon viral infection⁵. Conclusions: The KH motif of nsp1 enables it to interact with the 40S ribosome¹. Nsp1 reduces host gene expression, nuclear export, and is associated with host transcript degradation^3,4,6,7. The SL1 segment of the viral 5’ UTR allows viral transcripts to avoid translation inhibition⁵.

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2. Hu B, Guo H, Zhou P, Shi ZL. Characteristics of SARS-CoV-2 and COVID-19. Nat Rev Microbiol. Mar 2021;19(3):141-154. doi:10.1038/s41579-020-00459-7
3. Malone B, Urakova N, Snijder EJ, Campbell EA. Structures and functions of coronavirus replication-transcription complexes and their relevance for SARS-CoV-2 drug design. Nat Rev Mol Cell Biol. Jan 2022;23(1):21-39. doi:10.1038/s41580-021-00432-z
4. Zhang K, Miorin L, Makio T, et al. Nsp1 protein of SARS-CoV-2 disrupts the mRNA export machinery to inhibit host gene expression. Sci Adv. Feb 2021;7(6)doi:10.1126/sciadv.abe7386
5. Vora SM, Fontana P, Mao T, et al. Targeting stem-loop 1 of the SARS-CoV-2 5′ UTR to suppress viral translation and Nsp1 evasion. Proceedings of the National Academy of Sciences. 2022;119(9):e2117198119. doi:10.1073/pnas.2117198119
6. Finkel Y, Gluck A, Nachshon A, et al. SARS-CoV-2 uses a multipronged strategy to impede host protein synthesis. Nature. Jun 2021;594(7862):240-245. doi:10.1038/s41586-021-03610-3
7. Mendez AS, Ly M, González-Sánchez AM, et al. The N-terminal domain of SARS-CoV-2 nsp1 plays key roles in suppression of cellular gene expression and preservation of viral gene expression. Cell Rep. Oct 19 2021;37(3):109841. doi:10.1016/j.celrep.2021.109841
8. Nakagawa K, Makino S. Mechanisms of Coronavirus Nsp1-Mediated Control of Host and Viral Gene Expression. Cells. Feb 2 2021;10(2)doi:10.3390/cells10020300