No abstract

For positive-strand RNA viruses, genomic RNA (gRNA) functions both as a message for translation and as a template for the viral RNA-dependent RNA polymerase. As part of their infectious cycle, these viruses often extensively remodel host cellular membranous compartments to facilitate replication and transcription of gRNA. These expropriated membranes are thought to provide surfaces for the concentration of enzymatic and substrate components of RNA synthesis, to furnish general protection from cellular ribonucleases, and to allow evasion of host innate immunity through the sequestration of double-stranded viral RNA intermediates. Among all known RNA viruses, coronaviruses have the largest genomes, ranging from 27 to 32 kb, and the most complex machinery for RNA synthesis (21,35,74). The coronavirus replicase-transcriptase complex (here called the replicase) is assembled from 16 nonstructural proteins (nsp), which collectively total some 803 kDa for mouse hepatitis virus (MHV). The nsp are autoproteolytically processed from two overlapping polyprotein precursors, which are produced by translation of gRNA. Almost all of the replicase components are associated with or embedded in convoluted membranes and double-membrane vesicles that are generated during coronavirus infection (29).In addition to the replicase, the nucleocapsid protein (N) is known to play an important role in coronavirus RNA synthesis, although that role is, as yet, poorly defined (1,51,60,76). Multiple investigations have shown intracellular colocalization of N with replicase components at early stages of infection by MHV or by severe acute respiratory syndrome coronavirus (SARS-CoV) (14,55,58,61). The participation of N protein in an early event in RNA synthesis is also implied by the poor adherence of coronaviruses to the nearly universal rule that the gRNA of positive-strand RNA viruses is completely infectious when introduced into susceptible host cells. Some time ago, we noted that the infectivity of purified MHV gRNA was markedly enhanced by cotransfection of synthetic N mRNA (36); this phenomenon was rediscovered when full-length reversegenetic systems were devised for coronaviruses. For systems that are launched by transfection of gRNA, the provision of cotransfected N mRNA or N-encoding plasmid either is greatly stimulatory (10,(69)(70)(71) or is absolutely required (6) for the recovery of infectious virus.In the present study, we analyzed the consequences of replacing the MHV N open reading frame (ORF) with its counterpart from the closely related bovine coronavirus (BCoV). This substitution resulted in the discovery of a previously unsuspected genetic interaction between the serine-and argininerich (SR) region of the N protein and a region of the nsp3 subunit of the viral replicase. We confirmed this interaction through biochemical pulldown experiments. Moreover, we found that there is a correlation between the N-nsp3 interaction and the ability of N protein to stimulate the infectivity of MHV gRNA. Our results offer an explanation for t...

The coronavirus nucleocapsid protein (N), together with the large, positive-strand RNA viral genome, forms a helically symmetric nucleocapsid. This ribonucleoprotein structure becomes packaged into virions through association with the carboxy-terminal endodomain of the membrane protein (M), which is the principal constituent of the virion envelope. Previous work with the prototype coronavirus mouse hepatitis virus ( The assembly of coronaviruses is driven principally by homotypic and heterotypic interactions between the two most abundant virion proteins, the membrane protein (M) and the nucleocapsid protein (N) (14, 32). The M protein is a triplespanning transmembrane protein residing in the virion envelope, which is derived from the cellular budding site, the endoplasmic reticulum-Golgi intermediate compartment. More than half of the M molecule, its carboxy-terminal endodomain, is situated in the interior of the virion, where it contacts the nucleocapsid (46, 50). Also found in the virion envelope is the spike protein (S), which, although crucial for viral infectivity, is not an essential participant in assembly. The other canonical component of the coronavirus envelope is the small envelope protein (E), the function of which is enigmatic. Some evidence suggests that the E protein does not make sequence-specific contacts with other viral proteins (27) but instead functions by modifying the budding compartment, perhaps as an ion channel (56, 57). Alternatively, or additionally, E could act in a chaperone-like fashion to facilitate homotypic interactions between M protein monomers or oligomers (4).The N protein is the only protein constituent of the helically symmetric nucleocapsid, which is located in the interior of the virion. Coronavirus N proteins are largely basic phosphoproteins that share a moderate degree of homology across all three of the phylogenetic groups within the family (29). Some time ago, we proposed a model that pictured the N protein as comprising three domains separated by two spacers (A and B) (40). This arrangement was originally inferred from a sequence comparison of the N genes of multiple strains of the prototypical group 2 coronavirus, mouse hepatitis virus (MHV), and its validity seemed to be reinforced by numerous sequences that later became available. Part of this model, the delineation of spacer B and the acidic, carboxy-terminal domain 3, has been well supported by subsequent work (22,25,41,42). However, a wealth of recent, detailed structural studies of bacterially expressed domains of the N proteins of the severe acute respiratory syndrome coronavirus (SARS-CoV) and of infectious bronchitis virus (IBV) has much more precisely mapped boundaries within the remainder of the N molecule (8,16,21,23,47,51,60). The latter studies have shown that the N protein contains two independently folding domains, designated the N-terminal domain (NTD) and the C-terminal domain (CTD). It should be pointed out that this nomenclature can be misleading: the NTD does not contain the amino terminus of ...