Unraveling the Packaging Mechanism of Coronavirus Ribonucleocapsid

Abstract

Short communication J Hum Virol Retrovirol 2017, 5(2): 00148 the RNA-dependent RNA polymerase complex. EM studies of coronaviruses have shown that coronavirus RNPs are helical, consisting of coils of 9-16 nm in diameter and a hollow interior of approximately 3-4 nm (For a review see Chang and Huang, 2014) [2] Accommodation of the ~30 kb CoV genome into the newly formed virion spherules <100 nm in size necessitates an extremely well-packed, largely helical, supercoiling of the nucleic acid within the RNP core. The pleomorphic nature of the coronavirus particle has hampered the effort to obtain high-resolution virion image at atomic resolution. The inability to observe a well-structured RNP layer inside the SARS-CoV particle and only short coiled fragments of RNP in MHV in the cryo-EM reconstructions strongly suggests that the helical nucleocapsid is a very flexible structure that extensively twists and folds upon itself [3]. Extensive biophysical studies suggest that all CoV N proteins share the same modular organization containing two structured domains, the N-terminal domain (NTD) and the C-terminal domain (CTD), surrounded by three intrinsically disordered regions (IDR) called the N-terminal arm (N-arm), the central linker region (LKR), and the C-terminal tail (C-tail), respectively [4]. N protein forms a dimer, which constitutes the basic building block of the nucleocapsid, through its CTD [4, 5]. Studies from several laboratories have established that the NTD is involved in RNA binding, whereas the CTD is involved in RNA binding and oligomerization. All three IDRs of coronaviral N protein can modulate the RNA-binding and oligomerization properties of NTD and CTD, respectively [6]. In addition, the LKR of the N protein with Ser-Arg-rich sequences has also been shown to contain an RNA-binding region and putative phosphorylation sites that might regulate N protein functions [7, 8] and N-M interaction. No existing data supports the presence of a long-lived SARS-CoV N oligomer or intermediate in solution and the SARS-CoV genomic ssRNA by itself is unlikely to exist as a helix of the length observed in cryo-EM. Thus, packaging of SARS-CoV RNP proceeds most likely through a RNA binding-coupled packaging mechanism. This suggests that coronavirus RNA synthesis is coupled to the encapsidation of nascent RNA, The modular organization with three long IDRs provides the N protein with considerable flexibility, as also the RNA molecule. Based on available detailed 3D structural information of the N protein modules and our understanding of N-RNA interaction we proposed a probable CoV RNP packaging model derived from the crystal structure of the CTD [9], which was shown to exist transiently in solution by disulfide trap experiment [10]. A putative scenario of the molecular events leading to the formation of RNP may involve the following steps: Initial binding: I. RNA first binds at either NTD or CTD which facilitates binding of other modules to RNA in a coupled-allostery manner. RNA molecule threads in between the two structural domains. This initial N-RNA binary complex (RNP0) may have several N protein bound at a particular time. II. Growth: The RNP0 could grow by either recruiting more N to the adjacent RNA sites, or it could slide or hop along the linear RNA molecule and combine with other smaller N-RNA oligomers to form a larger oligomer (RNPN) of various sizes. RNPN would pack in a structure with CTD forming the helical core and RNA wraps and twists around the helical groove through mostly electrostatic interaction between the positively charge residues in the groove and the phosphate backbone of the RNA molecule. III. Packaging of NTD: The NTD module will cap on the outside of the helical CTD-RNA complex with the positively charged surface covering the free phosphate groups of the RNA molecule. Furthermore, RNA bases sticking out of the CTD groove could intercalate in between the aromatic rings at the bottom of the β-sheet on the NTD core. The presence of IDRs permits the two structural domains considerable freedom to adapt a wide range of orientations and positions for optimal packing of the RNP complex. Likewise, the RNA molecule can adjust to local conformation by an induced-fit process.