Mouse hepatitis virus (MHV) isolates JHM.WU and JHM.SD promote severe central nervous system disease. However, while JHM.WU replicates robustly and induces hepatitis, JHM.SD fails to replicate or induce pathology in the liver. These two JHM variants encode homologous proteins with few polymorphisms, and little is known about which viral proteins(s) is responsible for the liver tropism of JHM.WU. We constructed reverse genetic systems for JHM.SD and JHM.WU and, utilizing these fulllength cDNA clones, constructed chimeric viruses and mapped the virulence factors involved in liver tropism. Exchanging the spike proteins of the two viruses neither increased replication of JHM.SD in the liver nor attenuated JHM.WU. By further mapping, we found that polymorphisms in JHM.WU structural protein M and nonstructural replicase proteins nsp1 and nsp13 are essential for liver pathogenesis. M protein and nsp13, the helicase, of JHM.WU are required for efficient replication in vitro and in the liver in vivo. The JHM.SD nsp1 protein contains a K194R substitution of Lys194, a residue conserved among all other MHV strains. The K194R polymorphism has no effect on in vitro replication but influences hepatotropism, and introduction of R194K into JHM.SD promotes replication in the liver. Conversely, a K194R substitution in nsp1 of JHM.WU or A59, another hepatotropic strain, significantly attenuates replication of each strain in the liver and increases IFN-␤ expression in macrophages in culture. Our data indicate that both structural and nonstructural proteins contribute to MHV liver pathogenesis and support previous reports that nsp1 is a Betacoronavirus virulence factor. IMPORTANCEThe Betacoronavirus genus includes human pathogens, some of which cause severe respiratory disease. The spread of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) into human populations demonstrates the zoonotic potential of emerging coronaviruses, and there are currently no vaccines or effective antivirals for human coronaviruses. Thus, it is important to understand the virus-host interaction that regulates coronavirus pathogenesis. Murine coronavirus infection of mice provides a useful model for the study of coronavirus-host interactions, including the determinants of tropism and virulence. We found that very small changes in coronavirus proteins can profoundly affect tropism and virulence. Furthermore, the hepatotropism of MHV-JHM depends not on the spike protein and viral entry but rather on a combination of the structural protein M and nonstructural replicase-associated proteins nsp1 and nsp13, which are conserved among betacoronaviruses. Understanding virulence determinants will aid in the design of vaccines and antiviral strategies. Mouse hepatitis virus (MHV) is an enveloped, nonsegmented, positive-strand RNA virus that belongs to the order Nidovirales, family Coronaviridae, and genus Betacoronavirus (1). Betacoronaviruses can have significant effects on human (2) and animal ...

The murine coronavirus, mouse hepatitis virus (MHV) strain A59, causes acute encephalitis and chronic demyelinating disease as well as hepatitis in mice. The JHM strain (also called MHV-4 or JHM.SD) causes fatal encephalitis and only minimal hepatitis. Previous analysis of chimeric recombinant MHVs in which the spike gene, encoding the protein that mediates viral entry and cell-to-cell fusion, was exchanged between JHM and A59 showed that the spike plays a major role in determining organ tropism and neurovirulence but that other genes also play important roles in pathogenic outcome. Here, we have investigated the role of the nucleocapsid protein in MHV-induced disease. The multifunctional nucleocapsid protein is complexed with the genomic RNA, interacts with the viral membrane protein during virion assembly, and plays an import role in enhancing the efficiency of transcription. A pair of chimeric recombinant viruses in which the nucleocapsid gene was exchanged between JHM and A59 was selected and compared to wild-type parental strains in terms of virulence. Importantly, expression of the JHM nucleocapsid in the context of the A59 genome conferred increased mortality and spread of viral antigen in the mouse central nervous system compared to the parental A59 strain, while having little effect on the induction of hepatitis. While the JHM nucleocapsid did not appear to enhance neuron-to-neuron spread in primary neuronal cultures, the increased neurovirulence it conferred may be due in part to the induction of a less robust T-cell response than that induced by strain A59.Coronaviridae are a family of large, single-stranded and positive-sense RNA viruses within the nidovirus superfamily. The murine coronavirus mouse hepatitis virus (MHV) is a collection of strains with a wide range of tropisms, inducing neurological, hepatic, enteric, and respiratory diseases, with outcomes dependent upon the viral strain and the route of infection. Infection via intracranial (i.c.) or intranasal (i.n.) routes serves as a model for studying both acute and chronic virus-induced neurological diseases; these include models of encephalitis and the demyelinating disease multiple sclerosis. Two naturally occurring neurotropic strains, A59 and JHM, have been shown to induce very different pathologies following i.c. infection. The A59 strain is a weakly neurovirulent, tissue culture-adapted strain that induces mild encephalitis and moderate hepatitis (20,40). A59 infection is cleared from the central nervous system (CNS) and liver following a robust CD8 T-cell response (24, 49) (22); however, viral RNA persists in the spinal cord, and chronic demyelination develops in animals surviving acute infection (12,19,26) In contrast, the JHM strain, which has been previously referred to as MHV-4 or JHM.SD (5, 36), is highly neurovirulent in weanling C57BL/6 (B6) mice, inducing fatal encephalitis in nearly all infected mice following inoculation with doses as low as 1 PFU. This enhanced virulence is attributed in part to its rapid spread in the CNS, w...

Murine coronavirus, mouse hepatitis virus (MHV), causes various diseases depending on the strain and route of inoculation. Both the JHM and A59 strains, when inoculated intracranially or intranasally, are neurovirulent. Comparison of the highly virulent JHM isolate, JHM.SD, with less virulent JHM isolates and with A59 has been used to determine the mechanisms and genes responsible for high neuropathogenicity of MHV. The focus of this review is on the contributions of viral spread, replication and innate and adaptive immunity to MHV neuropathogenesis. JHM.SD spreads more quickly among neurons than less neurovirulent MHVs, and is able to spread in the absence of the canonical MHV receptor, CEACAM1a. The observation that JHM.SD infects more cells and expresses more antigen, but produces less infectious virus per cell than A59 implies that efficient replication is not always a correlate of high neurovirulence. This is likely due to the unstable nature of the JHM.SD spike protein (S). JHM.SD induces a generally protective innate immune response; however, the strong neutrophil response may be more pathogenic than protective. In addition JHM.SD induces only a minimal T cell response, while the strong T cell response and the concomitant IFNγ induced by the less neurovirulent A59 is protective. Differences in the S and nucleocapsid (N) proteins between A59 and JHM.SD contribute to JHM.SD neuropathogenicity. The hemmagglutinin-esterase (HE) protein may enhance neuropathogenicity of some MHV isolates, but is unlikely a major contributor to the high neuroviruence of JHM.SD. Further data suggests that neither the internal (I) protein, nor nonstructural proteins ns4, and ns2 are significant contributors to neurovirulence.

Murine coronavirus, mouse hepatitis virus (MHV), causes various diseases depending on the strain and route of inoculation. Both the JHM and A59 strains, when inoculated intracranially or intranasally, are neurovirulent. Comparison of the highly virulent JHM isolate, JHM.SD, with less virulent JHM isolates and with A59 has been used to determine the mechanisms and genes responsible for high neuropathogenicity of MHV. The focus of this review is on the contributions of viral spread, replication and innate and adaptive immunity to MHV neuropathogenesis. JHM.SD spreads more quickly among neurons than less neurovirulent MHVs, and is able to spread in the absence of the canonical MHV receptor, CEACAM1a. The observation that JHM.SD infects more cells and expresses more antigen, but produces less infectious virus per cell than A59 implies that efficient replication is not always a correlate of high neurovirulence. This is likely due to the unstable nature of the JHM.SD spike protein (S). JHM.SD induces a generally protective innate immune response; however, the strong neutrophil response may be more pathogenic than protective. In addition JHM.SD induces only a minimal T cell response, while the strong T cell response and the concomitant IFNγ induced by the less neurovirulent A59 is protective. Differences in the S and nucleocapsid (N) proteins between A59 and JHM.SD contribute to JHM.SD neuropathogenicity. The hemmagglutinin-esterase (HE) protein may enhance neuropathogenicity of some MHV isolates, but is unlikely a major contributor to the high neuroviruence of JHM.SD. Further data suggests that neither the internal (I) protein, nor nonstructural proteins ns4, and ns2 are significant contributors to neurovirulence.

The transcriptional co-regulator host cell factor-1 (HCF-1) plays critical roles in promoting cell cycle progression in diverse cell types, and in maintaining self-renewal of embryonic stem cells, but its role in pancreatic β-cell function has not been investigated. Immunhistochemistry of mouse pancreas revealed nuclear expression of HCF-1 in pancreatic islets. Reducing HCF-1 expression in the INS-1 pancreatic β-cell line resulted in reduced cell proliferation, reduced glucose-stimulated insulin secretion, and reduced expression of the critical β-cell transcription factor Pdx1. HCF-1 is a known co-activator of the E2F1 transcription factor, and loss of E2F1 results in pancreatic β-cell dysfunction and reduced expression of Pdx1. Therefore we wondered whether HCF-1 might be required for E2F1 regulation of Pdx1. Chromatin immunoprecipitation experiments revealed that HCF-1 and E2F1 co-localize to the Pdx1 promoter. These results indicate that HCF-1 represents a novel transcriptional regulator required for maintaining pancreatic β-cell function.