The Nonstructural Proteins of Nipah Virus Play a Key Role in Pathogenicity in Experimentally Infected Animals

et al. (2010) The Nonstructural Proteins of Nipah Virus Play a Key Role in Pathogenicity in Experimentally Infected Animals. PLoS ONE 5(9): e12709. doi:10.1371/journal.pone.0012709 The Nonstructural Proteins of Nipah Virus Play a Key Role in Pathogenicity in Experimentally Infected Animals Misako Yoneda 0 Vanessa Guillaume 0 Hiroki Sato 0 Kentaro Fujita 0 Marie-Claude Georges-Courbot 0 Fusako Ikeda 0 Mio Omi 0 Yuri Muto-Terao 0 T. Fabian Wild 0 Chieko Kai 0 Maria G. Masucci, Karolinska Institutet, Sweden 0 1 Laboratory Animal Research Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan, 2 International Research Center for Infectious Diseases, Institute of Medical Science, The University of Tokyo, Tokyo, Japan, 3 Institut National de la Sante et de la Recherche me dicale, U758 , Lyon, France, 4 Laboratory P4 INSERM Jean Me rieux, Lyon , France Nipah virus (NiV) P gene encodes P protein and three accessory proteins (V, C and W). It has been reported that all four P gene products have IFN antagonist activity when the proteins were transiently expressed. However, the role of those accessory proteins in natural infection with NiV remains unknown. We generated recombinant NiVs lacking V, C or W protein, rNiV(V2), rNiV(C2), and rNiV(W2), respectively, to analyze the functions of these proteins in infected cells and the implications in in vivo pathogenicity. All the recombinants grew well in cell culture, although the maximum titers of rNiV(V2) and rNiV(C2) were lower than the other recombinants. The rNiV(V2), rNiV(C2) and rNiV(W2) suppressed the IFN response as well as the parental rNiV, thereby indicating that the lack of each accessory protein does not significantly affect the inhibition of IFN signaling in infected cells. In experimentally infected golden hamsters, rNiV(V2) and rNiV(C2) but not the rNiV(W2) virus showed a significant reduction in virulence. These results suggest that V and C proteins play key roles in NiV pathogenicity, and the roles are independent of their IFN-antagonist activity. This is the first report that identifies the molecular determinants of NiV in pathogenicity in vivo. - Nipah virus (NiV) was first isolated in 1999 and was identified as the etiological agent responsible for an outbreak of fatal viral encephalitis in Malaysia and Singapore. During the first NiV outbreak, the virus infected both pigs and humans, in addition to a small number of cats, dogs and horses [1,2]. NiV causes severe encephalitis with high fatality rates in humans and the virus has been responsible for highly infectious respiratory disease with low mortality in pigs. NiV has continued to re-emerge in Bangladesh and India and person-to-person transmission appeared to be the source of the outbreak in Bangladesh [3,4,5,6]. Mortality rates recorded in outbreaks in Bangladesh reached 75%, which were significantly higher than that observed in Malaysia [7]. However, the molecular determinants of NiV in regard to severity of pathogenicity and ability to cross species barriers are not yet known, and thus remain important and urgent issues that need to be elucidated for understanding the disease and for development of effective vaccines. To address these issues, we have developed reverse genetics for NiV and a NiV recombinant expressing GFP [8]. Our study indicated that the receptor was important but was not the sole determinant of virus permissibility in cells. NiV, a member of the family Paramyxoviridae, possesses a negative-sense, non-segmented RNA genome that is 18246 nt (Malaysian isolate) or 18252 nt (Bangladesh isolate) in length [9]. It has six transcription units that encode six structural proteins, the nucleocapsid (N), phosphoprotein (P), matrix protein (M), fusion protein (F), glycoprotein (G) and polymerase (L). Similar to other paramyxoviruses, the P gene of NiV expresses four proteins, namely P, V, W and C [10,11]. V and W proteins are translated from the edited mRNA in which one or two non-templated G residues are inserted to the editing site during viral transcription by the polymerase protein [10]. C protein is encoded by an alternate open reading frame in the 59 end of the P gene, and it does not have any amino acid identity with the P protein. Viruses are subjected to various antiviral host responses upon infection, and among them, interferon (IFN) responses play important roles in early innate immunity and in the modulation of subsequent acquired immunity [12,13]. Type I IFNs mediate their biological functions by binding to the receptor on the target cells, resulting in autophosphorylation and activation of the Janus kinases (JAKs), JAK1 and tyrosine kinase 2, leading to phosphorylation of the signaling molecules STAT1 and STAT2 [14,15]. The phosphorylated STATs are associated with interferon regulatory factor 9 (IRF-9), and form an interferon-stimulated gene factor 3 (ISGF3) [16,17], which activates the transcription of a large number of genes involved in the establ (...truncated)