The recent emergence of several new coronaviruses, including the etiological cause of severe acute respiratory syndrome, has significantly increased the importance of understanding virus-host cell interactions of this virus family. We used mouse hepatitis virus (MHV) A59 as a model to gain insight into how coronaviruses affect the type I alpha/beta interferon (IFN) system. We demonstrate that MHV is resistant to type I IFN. Protein kinase R (PKR) and the alpha subunit of eukaryotic translation initiation factor are not phosphorylated in infected cells. The RNase L activity associated with 2,5-oligoadenylate synthetase is not activated or is blocked, since cellular RNA is not degraded. These results are consistent with lack of protein translation shutoff early following infection. We used a well-established recombinant vaccinia virus (VV)-based expression system that lacks the viral IFN antagonist E3L to screen viral genes for their ability to rescue the IFN sensitivity of the mutant. The nucleocapsid (N) gene rescued VV⌬E3L from IFN sensitivity. N gene expression prevents cellular RNA degradation and partially rescues the dramatic translation shutoff characteristic of the VV⌬E3L virus. However, it does not prevent PKR phosphorylation. The results indicate that the MHV N protein is a type I IFN antagonist that likely plays a role in circumventing the innate immune response.

Coronavirus envelope (E) proteins are small (ϳ75-to 110-amino-acid) membrane proteins that have a short hydrophilic amino terminus, a relatively long hydrophobic membrane domain, and a long hydrophilic carboxyterminal domain. The protein is a minor virion structural component that plays an important, not fully understood role in virus production. It was recently demonstrated that the protein forms ion channels. We investigated the importance of the hydrophobic domain of the mouse hepatitis coronavirus (MHV) A59 E protein. Alanine scanning insertion mutagenesis was used to examine the effect of disruption of the domain on virus production in the context of the virus genome by using a MHV A59 infectious clone. Mutant viruses exhibited smaller plaque phenotypes, and virus production was significantly crippled. Analysis of recovered viruses suggested that the structure of the presumed ␣-helical structure and positioning of polar hydrophilic residues within the predicted transmembrane domain are important for virus production. Generation of viruses with restored wild-type helical pitch resulted in increased virus production, but some exhibited decreased virus release. Viruses with the restored helical pitch were more sensitive to treatment with the ion channel inhibitor hexamethylene amiloride than were the more crippled parental viruses with the single alanine insertions, suggesting that disruption of the transmembrane domain affects the functional activity of the protein. Overall the results indicate that the transmembrane domain plays a crucial role during biogenesis of virions.

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Mutations in the Fused in sarcoma/Translated in liposarcoma gene (FUS/TLS, FUS) have been identified among patients with amyotrophic lateral sclerosis (ALS). FUS protein aggregation is a major pathological hallmark of FUS proteinopathy, a group of neurodegenerative diseases characterized by FUS-immunoreactive inclusion bodies. We prepared transgenic Drosophila expressing either the wild type (Wt) or ALS-mutant human FUS protein (hFUS) using the UAS-Gal4 system. When expressing Wt, R524S or P525L mutant FUS in photoreceptors, mushroom bodies (MBs) or motor neurons (MNs), transgenic flies show age-dependent progressive neural damages, including axonal loss in MB neurons, morphological changes and functional impairment in MNs. The transgenic flies expressing the hFUS gene recapitulate key features of FUS proteinopathy, representing the first stable animal model for this group of devastating diseases.