Abstract
Coronaviruses are enveloped, single-stranded RNA viruses that are distributed worldwide. They include transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), and the human coronaviruses severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), many of which seriously endanger human health and well-being. Only alphacoronaviruses and betacoronaviruses harbor nonstructural protein 1 (nsp1), which performs multiple functions in inhibiting antiviral host responses. The role of the C terminus of betacoronavirus nsp1 in virulence has been characterized, but the location of the alphacoronavirus nsp1 region that is important for virulence remains unclear. Here, using TGEV nsp1 as a model to explore the function of this protein in alphacoronaviruses, we demonstrate that alphacoronavirus nsp1 inhibits host gene expression. Solving the crystal structure of full-length TGEV at 1.85-Å resolution and conducting several biochemical analyses, we observed that a specific motif (amino acids 91–95) of alphacoronavirus nsp1 is a conserved region that inhibits host protein synthesis. Using a reverse-genetics system based on CRISPR/Cas9 technology to construct a recombinant TGEV in which this specific nsp1 motif was altered, we found that this mutation does not affect virus replication in cell culture but significantly reduces TGEV pathogenicity in pigs. Taken together, our findings suggest that alphacoronavirus nsp1 is an essential virulence determinant, providing a potential paradigm for the development of a new attenuated vaccine based on modified nsp1.
Highlights
Coronaviruses are enveloped, single-stranded RNA viruses that are distributed worldwide
Using Renilla luciferase (Rluc) assays, we assessed several representative ␣-CoV nsp1 proteins for their ability to interfere with host-cell gene expression
The results showed that transmissible gastroenteritis virus (TGEV), feline infectious peritonitis virus (FIPV), HCoV-229E, HCoV-NL63, and porcine epidemic diarrhea virus (PEDV) nsp1 significantly reduced luciferase reporter gene expression
Summary
CoV, coronavirus; HCoV, human coronavirus; FIPV, feline infectious peritonitis virus; MHV, murine hepatitis virus; TGEV, transmissible gastroenteritis virus; PEDV, porcine epidemic diarrhea virus; SARS-CoV, severe acute respiratory syndrome coronavirus; MERS-CoV, Middle East respiratory syndrome coronavirus; nsp, nonstructural protein 1; IFN, interferon; Rluc, Renilla luciferase; PDB, Protein Data Bank; sg, SerGly-Ser-Gly; BAC, bacterial artificial chromosome; ST, swine testicle; TCID50, 50% tissue culture infective dose; DMEM, Dulbecco’s modified Eagle’s medium; r.m.s.d., root mean square deviation; H&E, hematoxylin and eosin; NS1, nonstructural protein 1; vhs, virion host shutoff; HA, hemagglutinin; sgRNA, single guide RNA; Ab, antibody; GAPDH, glyceraldehyde-3phosphate dehydrogenase. SARS-CoV nsp prevents mRNA translation and promotes its degradation by binding to the 40S ribosomal subunit [13]. MERS-CoV nsp selectively targets mRNA synthesized in the host cell nucleus for degradation and inhibits translation in host cells [14]. The specific pathway by which ␣-CoV nsp inhibits host gene expression is unknown. SARS-CoV nsp suppresses interferon (IFN) expression and host antiviral signaling pathways in infected cells [10, 17]. Further structural and biochemical analyses indicated that a motif (amino acids 91–95) is important for the inhibition of host gene expression by ␣-CoV nsp. We demonstrate that the loss of nsp1-induced inhibition of host protein synthesis does not affect the replication of TGEV but can significantly reduce its virulence in piglets. This research improves our understanding of why ␣-CoV nsp is necessary for virulence and may aid in the development of a new attenuated vaccine
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