Structural Basis for the Inhibition of Host Gene Expression by Porcine Epidemic Diarrhea Virus nsp1

Abstract

Porcine epidemic diarrhea virus (PEDV), an enteropathogenic , has caused enormous economic losses in the pork industry. Nonstructural protein 1 (nsp1) is a characteristic feature of alpha- and betacoronaviruses, which exhibits both functional conservation and mechanistic diversity in inhibiting host gene expression and antiviral responses. However, the detailed structure and molecular mechanisms underlying the nsp1 inhibition of host gene expression remain unclear. Here, we report the first full-length crystal structure of nsp1 from PEDV. The structure displays a six-stranded β-barrel fold in the middle of two α-helices. The core structure of PEDV nsp1 shows high similarity to those of severe acute respiratory syndrome coronavirus (SARS-CoV) nsp1 and transmissible gastroenteritis virus (TGEV) nsp1, despite its low degree of sequence homology. Using ribopuromycylation and luciferase reporter assays, we showed that PEDV nsp1 can dramatically inhibit general host gene expression. Furthermore, three motifs (amino acids [aa] 67 to 71, 78 to 85, and 103 to 110) of PEDV nsp1 create a stable functional region for inhibiting protein synthesis, differing considerably from nsp1. These results elucidate the detailed structural basis through which PEDV nsp1 inhibits host gene expression, providing insight into the development of a new attenuated vaccine with nsp1 modifications. Porcine epidemic diarrhea virus (PEDV) has led to tremendous economic losses in the global swine industry. PEDV nsp1 plays a crucial role in inhibiting host gene expression, but its functional mechanism remains unclear. Here, we report the full-length structure of PEDV nsp1, the first among coronaviruses to be reported. The 1.25-Å resolution crystal structure of PEDV nsp1 shows high similarity to severe acute respiratory syndrome coronavirus (SARS-CoV) nsp1 and transmissible gastroenteritis virus (TGEV) nsp1, despite a lack of sequence homology. Structural and biochemical characterization demonstrated that PEDV nsp1 possesses a stable functional region for inhibition of host protein synthesis, which is formed by loops at residues 67 to 71, 78 to 85, and 103 to 110. The different functional regions in PEDV nsp1 and SARS-CoV nsp1 may explain their distinct mechanisms. Importantly, our structural data are conducive to understanding the mechanism of PEDV nsp1 inhibition of the expression of host genes and may aid in the development of a new attenuated vaccine.

A conserved region of nonstructural protein 1 from alphacoronaviruses inhibits host gene expression and is critical for viral virulence

“…Subsequently, Western blot analysis indicated that nsp1(91-95sg) expression was significantly higher than nsp1 expression ( Fig. 2C), again demonstrating that nsp1 could inhibit its own expression (9,15). We examined Rluc mRNA expression in HEK-293T cells by real-time quantitative PCR and found that TGEV nsp1(91-95sg) and TGEV nsp1(37-40sg) did not inhibit Rluc mRNA synthesis at different time points ( Fig.…”

“…To identify which region in TGEV nsp1 was essential for its inhibitory activity, we used methods that have been previously used to study important functional regions in PEDV nsp1 (9). Based on the crystal structure of TGEV nsp1, we designed the following series of plasmids in which random coils were replaced with flexible Ser-Gly-Ser-Gly (sg) linkers: nsp1(1-3sg), nsp1(11-15sg), nsp1(19 -22sg), nsp1(37-40sg), nsp1(46 -48sg), nsp1(52-60sg), nsp1(66 -69sg), nsp1(76 -84sg), nsp1(91-95sg), and nsp1(105-109sg) (Fig.…”

“…MHV nsp1 interferes efficiently with the type I IFN system to enhance virulence (11). Nonetheless, structural analysis indicates that CoV nsp1s have a common origin (7)(8)(9) and that the nsp1 proteins of ␣-CoVs and ␤-CoVs share a biological function to inhibit host gene expression. Furthermore, previous studies have revealed that the C terminus of MHV and SARS-CoV nsp1 is an important functional region for inhibiting host protein synthesis (10,11).…”

“…Furthermore, MERS-CoV nsp1 selectively targets mRNA synthesized in the host cell nucleus for degradation and thus inhibits translation in host cells (14). TGEV and PEDV nsp1 proteins cannot bind the 40S ribosomal subunit to inhibit the translation of host mRNA; however, HCoV-229E and HCoV-NL63 nsp1 proteins might bind the 40S ribosomal subunit to affect host mRNA stability (9,15,16).…”

Porcine Epidemic Diarrhea Virus and the Host Innate Immune Response

“…The TGEV nsp1 considerably suppresses host protein expression during viral infection [77]. Structural studies show that the core structure of PEDV nsp1 is highly similar to those of SARS-CoV nsp1 and TGEV nsp1 [183]. PEDV nsp1 inhibits host gene expression and three motifs (amino acids 67 to 71, 78 to 85, and 103 to 110) form a stable functional region for inhibition of host protein synthesis, differing considerably from SARS-CoV nsp1 [183].…”