Unraveling the Packaging Mechanism of Coronavirus Ribonucleocapsid

Abstract

Short communicationJ 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 bindingcoupled 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 do...