The fixed rabies virus (RV) strain Nishigahara kills adult mice after intracerebral inoculation, whereas the chicken embryo fibroblast cell-adapted strain Ni-CE causes nonlethal infection in adult mice. We previously reported that the chimeric CE(NiP) strain, which has the phosphoprotein (P protein) gene from the Nishigahara strain in the genetic background of the Ni-CE strain, causes lethal infection in adult mice, indicating that the P gene is responsible for the different pathogenicities of the Nishigahara and Ni-CE strains. Previous studies demonstrated that RV P protein binds to the interferon (IFN)-activated transcription factor STAT1 and blocks IFN signaling by preventing its translocation to the nucleus. In this study, we examine the molecular mechanism by which RV P protein determines viral pathogenicity by comparing the IFN antagonist activities of the Nishigahara and Ni-CE P proteins. The results, obtained from both RV-infected cells and cells transfected to express P protein only, show that Ni-CE P protein is significantly impaired for its capacity to block IFN-activated STAT1 nuclear translocation and, consequently, inhibits IFN signaling less efficiently than Nishigahara P protein. Further, it was demonstrated that a defect in the nuclear export of Ni-CE P protein correlates with a defect in its ability to cause the mislocalization of STAT1. These data provide the first evidence that the capacity of the RV P protein to inhibit STAT1 nuclear translocation and IFN signaling correlates with the viral pathogenicity.

Rabies is a zoonotic disease caused by the Lyssavirus rabies virus (RABV) that can infect most mammals, including humans, where it has a case-fatality rate of almost 100%. Although preventable by vaccination, rabies causes c. 59,000 human fatalities every year worldwide. Thus, there exists an urgent need to establish an effective therapy and/or improve dissemination of vaccines for humans and animals. These outcomes require greater understanding of the mechanisms of RABV pathogenesis to identify new molecular targets for the development of therapeutics and/or live vaccines with high levels of safety. Importantly, a number of studies in recent years have indicated that RABV specifically suppresses host immunity through diverse mechanisms and that this is a key process in pathogenicity. Here, we review current understanding of immune modulation by RABV, with an emphasis on its significance to pathogenicity and the potential exploitation of this knowledge to develop new vaccines and antivirals.

The large (L) protein of rabies virus (RABV) plays multiple enzymatic roles in viral RNA synthesis and processing. However, none of its putative enzymatic activities have been directly demonstrated in vitro. In this study, we expressed and purified a recombinant form of the RABV L protein and verified its guanosine 5′-triphosphatase and GDP polyribonucleotidyltransferase (PRNTase) activities, which are essential for viral mRNA cap formation by the unconventional mechanism. The RABV L protein capped 5′-triphosphorylated but not 5′-diphosphorylated RABV mRNA-start sequences, 5′-AACA(C/U), with GDP to generate the 5′-terminal cap structure G(5′)ppp(5′)A. The 5′-AAC sequence in the substrate RNAs was found to be strictly essential for RNA capping with the RABV L protein. Furthermore, site-directed mutagenesis showed that some conserved amino acid residues (G1112, T1170, W1201, H1241, R1242, F1285, and Q1286) in the PRNTase motifs A to E of the RABV L protein are required for cap formation. These findings suggest that the putative PRNTase domain in the RABV L protein catalyzes the rhabdovirus-specific capping reaction involving covalent catalysis of the pRNA transfer to GDP, thus offering this domain as a target for developing anti-viral agents.

By using a cultured neuroblastoma cell line, the present authors recently showed that the N protein of virulent rabies virus fixed strain Nishigahara (Ni), but not that of the attenuated derivative Ni‐CE, mediates evasion of induction of type I interferon (IFN). In this study, to determine whether Ni N protein indeed fulfills this function in vivo, the abilities to suppress IFN responses in the mouse brain of Ni‐CE and the virulent chimeric virus CE(NiN), which has the N gene from Ni in the genetic background of Ni‐CE, were compared. It was demonstrated that CE(NiN) propagates and spreads more efficiently than does Ni‐CE in the brain and that IFN response in brains infected with CE(NiN) is weaker than in those infected with Ni‐CE. It was also shown that amino acids at positions 273 and 394 in the N protein, which are known as pathogenic determinants, affect the ability of the viruses to suppress IFN response in the brain. These findings strongly suggest that, in the brain, rabies virus N protein plays important roles in evasion of innate immune responses and thereby in efficient propagation and spread of virus leading to lethal outcomes of infection.

Rotavirus non-structural protein (NSP) 4 can induce aqueous secretion in the gastrointestinal tract of neonatal mice through activation of an age- and Ca²⁺-dependent plasma membrane anion permeability. Accumulating evidence suggests that nitric oxide (NO) plays a role in the modulation of aqueous secretion and the barrier function of intestinal cells. This study investigated transcriptional changes in inducible NO synthase (iNOS), an enzyme responsible for NO production, after rotavirus infection in mice and after treatment of intestinal cells with NSP4. Diarrhoea was observed in 5-day-old CD-1 mice from days 1 to 3 after inoculation with 10⁷focus-forming units of different rotavirus strains. Ileal iNOS mRNA expression was induced as early as 6 h post-inoculation, before the onset of clinical diarrhoea in infected mice, and was upregulated during the course of rotavirus-induced diarrhoea.Ex vivotreatment of ilea excised from CD-1 suckling mice with NSP4 resulted in upregulation of ileal iNOS mRNA expression within 4 h. Furthermore, NSP4 was able to induce iNOS expression and NO production in murine peritoneal macrophages and RAW264.7 cells. The specificity of NSP4 inducibility was confirmed by the inhibitory effect of anti-NSP4 serum. Using a series of truncated NSP4s, the domain responsible for iNOS induction in macrophages was mapped to the reported enterotoxin domain, aa 109–135. Thus, rotavirus infection induces ileal iNOS expressionin vivoand rotavirus NSP4 also induces iNOS expression in the ileum and macrophages. Together, these findings suggest that NO plays a role in rotavirus-induced diarrhoea.