A Major Determinant for Membrane Protein Interaction Localizes to the Carboxy-Terminal Domain of the Mouse Coronavirus Nucleocapsid Protein

Abstract

Coronavirus assembly results from an accumulation of interactions among four structural proteins, the positive-sense RNA genome, and a host membrane envelope obtained from the site of budding, which is the endoplasmic reticulum-Golgi intermediate compartment. Three of the four structural proteins are embedded in the virion envelope: the spike protein (S), the membrane protein (M), and the small envelope protein (E). The fourth, the nucleocapsid protein (N), resides in the virion interior, wrapping the positive-strand RNA genome into a helical nucleocapsid.The most abundant viral constituent is M, a 25-kDa protein containing three transmembrane segments flanked by a short amino-terminal ectodomain and a large carboxy-terminal endodomain. The M protein is the central organizer of assembly, in that it self-associates (8, 10, 21), captures S for virion incorporation (36, 37), and selectively packages the fraction of N protein that is bound to genomic RNA (32-34). The S protein is responsible for viral attachment to host cell receptors and for the membrane fusion event that initiates infection. This type I membrane protein, shortly after its synthesis, folding, and oligomerization, forms complexes with M protein in the endoplasmic reticulum (36, 37). The M-interacting domain of the S protein was localized to the transmembrane-endodomain region of the molecule, based on the assembly of chimeric S proteins into virus-like particles (VLPs) (16) or virions (18, 23). More recently, this determinant was further localized to the endodomain of S, which could confer virion assembly competence if transferred to a heterologous transmembrane protein (48).The general (1, 3, 4, 31, 45), but not universal (20), consensus from studies of VLPs is that formation of coronaviruses is mediated by just the M and the E proteins, and that neither S protein nor the nucleocapsid plays an obligate role in virion morphogenesis. The precise role of E protein in this process is enigmatic, with some evidence pointing to a direct interaction between E and M (1, 5) and other observations suggesting that E acts independently of M in the budding compartment (4,26,41). A very recent finding that may shed light on the workings of the E protein is the demonstration that the E protein of severe acute respiratory syndrome coronavirus has the properties of a cation-selective ion channel (47). The construction of E protein mutants of mouse hepatitis virus (MHV) has confirmed the critical role of E in viral assembly (14). Surprisingly, however, MHV remains viable, although severely impaired, following deletion of the E gene (25), whereas disruption of the E gene of porcine transmissible gastroenteritis virus (TGEV) is lethal (6,38).The interaction between M protein and N protein has been previously explored by biochemical and molecular biological methods for both MHV (33,34, 44) and TGEV (12). In a genetic approach, we constructed and analyzed a highly defective MHV mutant with a carboxy-terminal truncated M protein, M⌬2, and we identified suppressors of this...

Mouse Hepatitis Coronavirus Nucleocapsid Phosphorylation

“…Through its interactions with the viralRNA, the M protein and itself, N plays important roles in virus assembly (Escors et al, 2001;Hurst et al, 2005;Narayanan et al, 2003;Narayanan and Makino, 2001;Verma et al, 2006). Through its interactions with the viralRNA, the M protein and itself, N plays important roles in virus assembly (Escors et al, 2001;Hurst et al, 2005;Narayanan et al, 2003;Narayanan and Makino, 2001;Verma et al, 2006).…”

Characterization of an Immunodominant Epitope in the Endodomain of the Coronavirus Membrane Protein

“…M protein self-interactions occur among the transmembrane domains [49,50]. The ectodomain of the CoV M protein plays an important role in interactions with other viral proteins, such as the N protein of MHV [51,52,53], SARS-CoV [54,55,56], and TGEV [28] and the S protein of MHV [25] and SARS-CoV [13]. TGEV M aa 233–257 (AYYVKSKAAGD YSTEARTDNLSEQEK ), which contains the epitopes recognized by the mAbs 1C3 and 4C7 (underlined), is involved in M-N binding to allow virion morphogenesis [28].…”

The assembly of virus-like particles of porcine transmissible gastroenteritisvirus in vitro by baculovirus expression system

“…Previous studies on coronaviruses, including TGEV, have demonstrated that the sM protein is essential for coronavirus particle assembly (6). Coronaviruses are assembled intracellularly at membranes of the intermediate compartment, between the endoplasmic reticulum (ER) and the Golgi complex (7)(8)(9)(10)(11). The highly basic N interacts with genomic RNA to form helical nucleocapsids (12), and M interacts with nucleocapsids on cell membranes at the ER or Golgi complex (13,14).…”