title: Roles of the Rabies Virus Phosphoprotein Isoforms in Pathogenesis.
abstract: Rabies virus (RABV) P gene mRNA encodes five in-frame start codons, resulting in expression of full-length P protein (P1) and N-terminally truncated P proteins (tPs), designated P2, P3, P4, and P5. Despite the fact that some tPs are known as interferon (IFN) antagonists, the importance of tPs in the pathogenesis of RABV is still unclear. In this study, to examine whether tPs contribute to pathogenesis, we exploited a reverse genetics approach to generate CE(NiP)ΔP2-5, a mutant of pathogenic CE(NiP) in which the P gene was mutated by replacing all of the start codons (AUG) for tPs with AUA. We confirmed that while CE(NiP) expresses detectable levels of P2 and P3, CE(NiP)ΔP2-5 has an impaired ability to express these tPs. After intramuscular inoculation, CE(NiP)ΔP2-5 caused significantly lower morbidity and mortality rates in mice than did CE(NiP), indicating that tPs play a critical role in RABV neuroinvasiveness. Further examinations revealed that this less neuroinvasive phenotype of CE(NiP)ΔP2-5 correlates with its impaired ability to replicate in muscle cells, indicative of the importance of tPs in viral replication in muscle cells. We also demonstrated that CE(NiP)ΔP2-5 infection induced a higher level of Ifn-β gene expression in muscle cells than did CE(NiP) infection, consistent with the results of an IFN-β promoter reporter assay suggesting that all tPs function to antagonize IFN induction in muscle cells. Taken together, our findings strongly suggest that tPs promote viral replication in muscle cells through their IFN antagonist activities and thereby support infection of peripheral nerves.
  
  
  
  Despite the fact that previous studies have demonstrated that P2 and P3 of RABV have IFN antagonist activities, the actual importance of tPs in pathogenesis has remained unclear. Here, we provide the first evidence that tPs contribute to the pathogenesis of RABV, especially its neuroinvasiveness. Our results also show the mechanism underlying the neuroinvasiveness driven by tPs, highlighting the importance of their IFN antagonist activities, which support viral replication in muscle cells.
authors.affiliation: The United Graduate School of Veterinary Sciences, Gifu University, Gifu, Japan.
authors.forename: Kazuma
authors.initials: K
authors.lastname: Okada
authors.affiliation: The United Graduate School of Veterinary Sciences, Gifu University, Gifu, Japan Laboratory of Zoonotic Diseases, Faculty of Applied Biological Sciences, Gifu University, Gifu, Japan.
authors.forename: Naoto
authors.initials: N
authors.lastname: Ito
authors.affiliation: The United Graduate School of Veterinary Sciences, Gifu University, Gifu, Japan.
authors.forename: Satoko
authors.initials: S
authors.lastname: Yamaoka
authors.affiliation: The United Graduate School of Veterinary Sciences, Gifu University, Gifu, Japan.
authors.forename: Tatsunori
authors.initials: T
authors.lastname: Masatani
authors.affiliation: Molecular Virology and Host-Pathogen Interaction Unit, Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rocky Mountain Laboratories, Hamilton, Montana, USA.
authors.forename: Hideki
authors.initials: H
authors.lastname: Ebihara
authors.affiliation: Laboratory of Zoonotic Diseases, Faculty of Applied Biological Sciences, Gifu University, Gifu, Japan.
authors.forename: Hideo
authors.initials: H
authors.lastname: Goto
authors.affiliation: The United Graduate School of Veterinary Sciences, Gifu University, Gifu, Japan.
authors.forename: Kento
authors.initials: K
authors.lastname: Nakagawa
authors.affiliation: The United Graduate School of Veterinary Sciences, Gifu University, Gifu, Japan.
authors.forename: Hiromichi
authors.initials: H
authors.lastname: Mitake
authors.affiliation: The United Graduate School of Veterinary Sciences, Gifu University, Gifu, Japan.
authors.forename: Kota
authors.initials: K
authors.lastname: Okadera
authors.affiliation: The United Graduate School of Veterinary Sciences, Gifu University, Gifu, Japan Laboratory of Zoonotic Diseases, Faculty of Applied Biological Sciences, Gifu University, Gifu, Japan sugiyama@gifu-u.ac.jp.
authors.forename: Makoto
authors.initials: M
authors.lastname: Sugiyama
mesh_terms: D000818:Animals; D001921:Brain / Q000821:virology; D002460:Cell Line; D006224:Cricetinae; D007155:Immunologic Factors / Q000235:genetics / Q000378:metabolism*; D007273:Injections, Intramuscular; D016899:Interferon-beta / Q000037:antagonists & inhibitors*; D051379:Mice; D018832:Molecular Chaperones; D009132:Muscles / Q000821:virology; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D011818:Rabies / Q000473:pathology* / Q000821:virology; D011820:Rabies virus / Q000235:genetics / Q000472:pathogenicity*; D059386:Reverse Genetics; D016019:Survival Analysis; D015678:Viral Structural Proteins / Q000235:genetics / Q000378:metabolism*; D014774:Virulence; D014779:Virus Replication
chemical_list: D007155:Immunologic Factors; D018832:Molecular Chaperones; C103961:P phosphoprotein, Rabies virus; D010750:Phosphoproteins; D020033:Protein Isoforms; D015678:Viral Structural Proteins; D016899:Interferon-beta