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

Abstract

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.

Effects of Coronavirus Persistence on the Genome Structure and Subsequent Gene Expression, Pathogenicity and Adaptation Capability

“…It has been well characterized that the 5′ and 3′ termini of the coronavirus genome harbor cis-acting RNA elements that are required for efficient gene expression [ 2 ]. To determine whether the alterations in the 5′ and 3′ termini of the genome and subgenomes ( Figure 1 ) were connected to the attenuation of gene expression during persistence, BCoV defective interfering (DI) RNA ( Figure 3 A), which is a surrogate for the BCoV genome and has been extensively used to study cis-acting RNA elements required for coronavirus gene expression, was employed for this aim [ 7 , 8 , 9 , 31 , 34 , 35 , 36 ]. To determine whether the replication efficiency was affected by the alterations of 5′-terminal sequences, DI RNA was engineered to contain the 5′-modified sequences ( Figure 3 B) identified during persistence ( Figure 1 C–G).…”

Multiple recombination events between field and vaccine strains resulted in the emergence of a novel infectious bronchitis virus with decreased pathogenicity and altered replication capacity

“…Interestingly, LJL/110302 and LLN/111169 showed different replication patterns in the tissues of SPF chickens, with the more virulent LJL/110302 isolate showing a greater replication capacity than the LLN/111169 isolate in the earlier stages of infection and lower replication capacity at 8 dpi. This difference in replication patterns might be associated with the recombination events in the N gene and 3 0 UTR of isolate LLN/111169 (Tsai et al, 2018;Lo et al, 2019).…”

Using nucleocapsid proteins to investigate the relationship between SARS-CoV-2 and closely related bat and pangolin coronaviruses

“…4,19 The N protein is multifunctional and involved in a variety of processes such as genome encapsidation and circularization, transcription, RNA packaging, genome transport to viral budding sites, repressing interferon production, cell cycle inhibition, and blocking RNA interference. [19][20][21][22] Furthermore, the N protein is also highly immunogenic and largely overexpressed during replication, thereby capable of inducing large-scale immune responses against CoVs. 23,24 Overall, the N protein possesses two highly conserved domains: the N-Terminal Domain (NTD) and C-Terminal Domain (CTD).…”