📄 Interaction of coronavirus nucleocapsid protein with the 5′- and 3′-ends of the coronavirus genome is involved in genome circularization and negative-strand RNA synthesis

© 2019 Federation of European Biochemical Societies. Synthesis of the negative-strand ((−)-strand) counterpart is the first step of coronavirus (CoV) replication; however, the detailed mechanism of the early event and the factors involved remain to be determined. Here, using bovine coronavirus (BCoV)-defective interfering (DI) RNA, we showed that (a) a poly(A) tail with a length of 15 nucleotides (nt) was sufficient to initiate efficient (−)-strand RNA synthesis and (b) substitution of the poly(A) tail with poly(U), (C) or (G) only slightly decreased the efficiency of (−)-strand synthesis. The findings indicate that in addition to the poly(A) tail, other factors acting in trans may also participate in (−)-strand synthesis. The BCoV nucleocapsid (N) protein, an RNA-binding protein, was therefore tested as a candidate. Based on dissociation constant (Kd) values, it was found that the binding affinity between N protein, but not poly(A)-binding protein, and the 3′-terminal 55 nt plus a poly(A), poly(U), poly(C) or poly(G) tail correlates with the efficiency of (−)-strand synthesis. Such an association was also evidenced by the binding affinity between the N protein and 5′- and 3′-terminal cis-acting elements important for (−)-strand synthesis. Further analysis demonstrated that N protein can act as a bridge to facilitate interaction between the 5′- and 3′-ends of the CoV genome, leading to circularization of the genome. Together, the current study extends our understanding of the mechanism of CoV (−)-strand RNA synthesis through involvement of N protein and genome circularization and thus may explain why the addition of N protein in trans is required for efficient CoV replication.

keywords 🔗 defective interfering (26) 🔗 bovine coronavirus (221) 🔗 cis-acting elements (11)

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