The involvement of host proteins in the replication and transcription of viral RNA is a poorly understood area for many RNA viruses. For coronaviruses, it was long speculated that replication of the giant RNA genome and transcription of multiple subgenomic mRNA species by a unique discontinuous transcription mechanism may require host cofactors. To search for such cellular proteins, yeast two-hybrid screening was carried out by using the nonstructural protein 14 (nsp14) from the coronavirus infectious bronchitis virus (IBV) as a bait protein, leading to the identification of DDX1, a cellular RNA helicase in the DExD/H helicase family, as a potential interacting partner. This interaction was subsequently confirmed by coimmunoprecipitation assays with cells coexpressing the two proteins and with IBV-infected cells. Furthermore, the endogenous DDX1 protein was found to be relocated from the nucleus to the cytoplasm in IBV-infected cells. In addition to its interaction with IBV nsp14, DDX1 could also interact with the nsp14 protein from severe acute respiratory syndrome coronavirus (SARS-CoV), suggesting that interaction with DDX1 may be a general feature of coronavirus nsp14. The interacting domains were mapped to the C-terminal region of DDX1 containing motifs V and VI and to the N-terminal portion of nsp14. Manipulation of DDX1 expression, either by small interfering RNA-induced knockdown or by overexpression of a mutant DDX1 protein, confirmed that this interaction may enhance IBV replication. This study reveals that DDX1 contributes to efficient coronavirus replication in cell culture.Coronaviruses cause severe diseases in humans and many other animal species. Severe acute respiratory syndrome coronavirus (SARS-CoV) is the causative agent of SARS (34, 45). Viruses in this family contain a single-stranded, positive-sense RNA genome of 27 to 31 kb. In cells infected with coronaviruses, six to nine mRNA species, including the genome-length mRNA1 and five to eight subgenomic mRNAs (mRNAs 2 to 9), are produced by a discontinuous RNA transcription mechanism (40,46,47). Avian infectious bronchitis virus (IBV), a prototype group 3 coronavirus, causes an acute and highly contagious disease in chickens, with a significant impact on the poultry industry worldwide. In IBV-infected cells, six mRNA species are produced (5). Subgenomic mRNAs 2, 3, 4, and 6 encode the four structural proteins, i.e., spike glycoprotein (S), envelope protein (E), membrane protein (M), and nucleocapsid protein (N). The 5Ј two-thirds of mRNA1 comprises two large open reading frames (ORFs), 1a and 1b, and encodes polyproteins 1a and 1ab. The two polyproteins are proteolytically cleaved by virus-encoded proteinases into 15 functional nonstructural proteins (nsp2 to nsp16) (18, 25-27, 30-32, 37-39, 54, 59).The functional roles of coronavirus nonstructural proteins in replication and transcription of viral RNAs are beginning to emerge. For example, nsp14, nsp15, and nsp16 are predicted to possess exonuclease (ExoN), uridylate-specific endoribonuclease...

Genetic manipulation of the RNA genomes by reverse genetics is a powerful tool to study the molecular biology and pathogenesis of RNA viruses. During construction of an infectious clone from a Vero cell-adapted coronavirus infectious bronchitis virus (IBV), we found that a G-C point mutation at nucleotide position 15526, causing Arg-to-Pro mutation at amino acid position 132 of the helicase protein, is lethal to the infectivity of IBV on Vero cells. When the in vitro-synthesized full-length transcripts containing this mutation were introduced into Vero cells, no infectious virus was rescued. Upon correction of the mutation, infectious virus was recovered. Further characterization of the in vitro-synthesized full-length transcripts containing the G15526C mutation demonstrated that this mutation may block the transcription of subgenomic RNAs. Substitution mutation of the Arg132 residue to a positively charged amino acid Lys affected neither the infectivity of the in vitro-synthesized transcripts nor the growth properties of the rescued virus. However, mutation of the Arg132 residue to Leu, a conserved residue in other coronaviruses at the same position, reduced the recovery rate of the in vitro-synthesized transcripts. The recovered mutant virus showed much smaller-sized plaques. On the contrary, a G-C and a G-A point mutations at nucleotide positions 4330 and 9230, respectively, causing Glu-Gln and Gly-Glu mutations in or near the catalytic centers of the papain-like (Nsp3) and 3C-like (Nsp5) proteinases, did not show detectable detrimental effect on the rescue of infectious viruses and the infectivity of the rescued viruses.

An interesting question posed by the current evidence that severe acute respiratory syndrome coronavirus may be originated from an animal coronavirus is how such an animal coronavirus breaks the host species barrier and becomes zoonotic. In this report, we study the chronological order of genotypic changes in the spike protein of avian coronavirus infectious bronchitis virus (IBV) during its adaptation to a primate cell line. Adaptation of the Beaudette strain of IBV from chicken embryo to Vero cells showed the accumulation of 49 amino acid mutations. Among them, 26 (53.06%) substitutions were located in the S protein. Sequencing analysis and comparison of the S gene demonstrated that the majority of the mutations were accumulated and fixed at passage 7 on Vero cells and minor variants were isolated in several passages. Evidence present suggests that the dominant Vero cell-adapted IBV strain may be derived from the chicken embryo passages by selection of and potential recombination between the minor variants. This may explain why adaptation is a rapid process and the dominant strain, once adapted to a new host cell, becomes relatively stable.

The most striking difference between the subgenomic mRNA8 of severe acute respiratory syndrome coronavirus isolated from human and some animal species is the deletion of 29 nucleotides, resulting in splitting of a single ORF (ORF8) into two ORFs (ORF8a and ORF8b). ORF8a and ORF8b are predicted to encode two small proteins, 8a and 8b, and ORF8 a single protein, 8ab (a fusion form of 8a and 8b). To understand the functions of these proteins, we cloned cDNA fragments covering these ORFs into expression plasmids, and expressed the constructs in both in vitro and in vivo systems. Expression of a construct containing ORF8a and ORF8b generated only a single protein, 8a; no 8b protein expression was obtained. Expression of a construct containing ORF8 generated the 8ab fusion protein. Site‐directed mutagenesis and enzymatic treatment revealed that protein 8ab is modified by N‐linked glycosylation on the N81 residue and by ubiquitination. In the absence of the 8a region, protein 8b undergoes rapid degradation by proteasomes, and addition of proteasome inhibitors inhibits the degradation of protein 8b as well as the protein 8b‐induced rapid degradation of the severe acute respiratory syndrome coronavirus E protein. Glycosylation could also stabilize protein 8ab. More interestingly, the two proteins could bind to monoubiquitin and polyubiquitin, suggesting the potential involvement of these proteins in the pathogenesis of severe acute respiratory syndrome coronavirus.

Coronaviruses are RNA viruses with a large zoonotic reservoir and propensity for host switching, representing a real threat for public health, as evidenced by severe acute respiratory syndrome (SARS) and the emerging Middle East respiratory syndrome (MERS). Cellular factors required for their replication are poorly understood. Using genome-wide small interfering RNA (siRNA) screening, we identified 83 novel genes supporting infectious bronchitis virus (IBV) replication in human cells. Thirty of these hits can be placed in a network of interactions with viral proteins and are involved in RNA splicing, membrane trafficking, and ubiquitin conjugation. In addition, our screen reveals an unexpected role for valosin-containing protein (VCP/p97) in early steps of infection. Loss of VCP inhibits a previously uncharacterized degradation of the nucleocapsid N protein. This inhibition derives from virus accumulation in early endosomes, suggesting a role for VCP in the maturation of virus-loaded endosomes. The several host factors identified in this study may provide avenues for targeted therapeutics. The other human coronaviruses (HCoV)-HCoV-229E, HCoV-OC43, NL63, and HKU1-are collectively responsible for about 10 to 30% of common colds. Generally harmless and selflimiting, these HCoV are also implicated in severe clinical outcomes, particularly in immunocompromised individuals, infants, and the elderly (3). Other coronaviruses cause considerable economic concern to the livestock industry as they readily infect farmed animals such as cows (4), pigs (5), and chickens (6).In addition to the diverse range of species that they infect, coronaviruses have a propensity for host switching. For instance, HCoV-OC43 bears a strong resemblance to a bovine coronavirus, from which it probably originated (7). SARS-CoV is postulated to have originated from bats and then transferred to palm civets and finally humans (8). The MERS coronavirus probably also has its origin in bats and is responsible for severe respiratory and renal failure in humans (9). Although human-to-human transmission is low at present (10), this new beta-coronavirus has raised global health concerns because its mortality rate is more than 30% (1, 11). This "interspecies jumping" continuously threatens to initiate a novel epidemic and presents a challenge for vaccine-based containment.It is thus critical to have a better understanding of the infectious cycle of CoV. This multistep process includes attachment of the spike protein (S) to cell surface receptors, endocytosis, and then fusion of the viral and endocytic membranes (12). The viral capsid then undergoes an uncoating process to deliver the viral genome into the cytosol.Host ribosomes then translate the viral genome, yielding nonstructural proteins that modulate virus pathogenesis (13) and form with host membranes the viral transcription/replication complex (RTC). The RTC is responsible for transcription of full-length genomic RNA as well as subgenomic RNA species via a nidovirusspecific discontinuous tran...