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SARS-CoV-2 viral proteins NSP1 and NSP13 inhibit interferon activation through distinct mechanisms.

ABSTRACT: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a devastating global pandemic, infecting over 43 million people and claiming over 1 million lives, with these numbers increasing daily. Therefore, there is urgent need to understand the molecular mechanisms governing SARS-CoV-2 pathogenesis, immune evasion, and disease progression. Here, we show that SARS-CoV-2 can block IRF3 and NF-κB activation early during virus infection. We also identify that the SARS-CoV-2 viral proteins NSP1 and NSP13 can block interferon activation via distinct mechanisms. NSP1 antagonizes interferon signaling by suppressing host mRNA translation, while NSP13 downregulates interferon and NF-κB promoter signaling by limiting TBK1 and IRF3 activation, as phospho-TBK1 and phospho-IRF3 protein levels are reduced with increasing levels of NSP13 protein expression. NSP13 can also reduce NF-κB activation by both limiting NF-κB phosphorylation and nuclear translocation. Last, we also show that NSP13 binds to TBK1 and downregulates IFIT1 protein expression. Collectively, these data illustrate that SARS-CoV-2 bypasses multiple innate immune activation pathways through distinct mechanisms.

SUBMITTER: Vazquez C

PROVIDER: S-EPMC8224853 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe new Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), which was first detected in Wuhan (China) in December of 2019 is responsible for the current global pandemic. Phylogenetic analysis revealed that it is similar to other betacoronaviruses, such as SARS-CoV and Middle-Eastern Respiratory Syndrome, MERS-CoV. Its genome is ∼ 30 kb in length and contains two large overlapping polyproteins, ORF1a and ORF1ab that encode for several structural and non-structural proteins. The non-structural protein 1 (nsp1) is arguably the most important pathogenic determinant, and previous studies on SARS-CoV indicate that it is both involved in viral replication and hampering the innate immune system response. Detailed experiments of site-specific mutagenesis and in vitro reconstitution studies determined that the mechanisms of action are mediated by (a) the presence of specific amino acid residues of nsp1 and (b) the interaction between the protein and the host's small ribosomal unit. In fact, substitution of certain amino acids resulted in reduction of its negative effects.MethodsA total of 17,928 genome sequences were obtained from the GISAID database (December 2019 to July 2020) from patients infected by SARS-CoV-2 from different areas around the world. Genomes alignment was performed using MAFFT (REFF) and the nsp1 genomic regions were identified using BioEdit and verified using BLAST. Nsp1 protein of SARS-CoV-2 with and without deletion have been subsequently modelled using I-TASSER.ResultsWe identified SARS-CoV-2 genome sequences, from several Countries, carrying a previously unknown deletion of 9 nucleotides in position 686-694, corresponding to the AA position 241-243 (KSF). This deletion was found in different geographical areas. Structural prediction modelling suggests an effect on the C-terminal tail structure.ConclusionsModelling analysis of a newly identified deletion of 3 amino acids (KSF) of SARS-CoV-2 nsp1 suggests that this deletion could affect the structure of the C-terminal region of the protein, important for regulation of viral replication and negative effect on host's gene expression. In addition, substitution of the two amino acids (KS) from nsp1 of SARS-CoV was previously reported to revert loss of interferon-alpha expression. The deletion that we describe indicates that SARS-CoV-2 is undergoing profound genomic changes. It is important to: (i) confirm the spreading of this particular viral strain, and potentially of strains with other deletions in the nsp1 protein, both in the population of asymptomatic and pauci-symptomatic subjects, and (ii) correlate these changes in nsp1 with potential decreased viral pathogenicity.

Project description:UnlabelledMouse hepatitis virus (MHV) isolates JHM.WU and JHM.SD promote severe central nervous system disease. However, while JHM.WU replicates robustly and induces hepatitis, JHM.SD fails to replicate or induce pathology in the liver. These two JHM variants encode homologous proteins with few polymorphisms, and little is known about which viral proteins(s) is responsible for the liver tropism of JHM.WU. We constructed reverse genetic systems for JHM.SD and JHM.WU and, utilizing these full-length cDNA clones, constructed chimeric viruses and mapped the virulence factors involved in liver tropism. Exchanging the spike proteins of the two viruses neither increased replication of JHM.SD in the liver nor attenuated JHM.WU. By further mapping, we found that polymorphisms in JHM.WU structural protein M and nonstructural replicase proteins nsp1 and nsp13 are essential for liver pathogenesis. M protein and nsp13, the helicase, of JHM.WU are required for efficient replication in vitro and in the liver in vivo. The JHM.SD nsp1 protein contains a K194R substitution of Lys194, a residue conserved among all other MHV strains. The K194R polymorphism has no effect on in vitro replication but influences hepatotropism, and introduction of R194K into JHM.SD promotes replication in the liver. Conversely, a K194R substitution in nsp1 of JHM.WU or A59, another hepatotropic strain, significantly attenuates replication of each strain in the liver and increases IFN-β expression in macrophages in culture. Our data indicate that both structural and nonstructural proteins contribute to MHV liver pathogenesis and support previous reports that nsp1 is a Betacoronavirus virulence factor.ImportanceThe Betacoronavirus genus includes human pathogens, some of which cause severe respiratory disease. The spread of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) into human populations demonstrates the zoonotic potential of emerging coronaviruses, and there are currently no vaccines or effective antivirals for human coronaviruses. Thus, it is important to understand the virus-host interaction that regulates coronavirus pathogenesis. Murine coronavirus infection of mice provides a useful model for the study of coronavirus-host interactions, including the determinants of tropism and virulence. We found that very small changes in coronavirus proteins can profoundly affect tropism and virulence. Furthermore, the hepatotropism of MHV-JHM depends not on the spike protein and viral entry but rather on a combination of the structural protein M and nonstructural replicase-associated proteins nsp1 and nsp13, which are conserved among betacoronaviruses. Understanding virulence determinants will aid in the design of vaccines and antiviral strategies.

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SARS-CoV-2 viral proteins NSP1 and NSP13 inhibit interferon activation through distinct mechanisms.

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