Heterogeneous Nuclear Ribonucleoprotein A2 Participates in the Replication of Japanese Encephalitis Virus through an Interaction with Viral Proteins and RNA

Abstract

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that is kept in a zoonotic transmission cycle between pigs and mosquitoes. JEV causes infection of the central nervous system with a high mortality rate in dead-end hosts, including humans. Many studies have suggested that the flavivirus core protein is not only a component of nucleocapsids but also an important pathogenic determinant. In this study, we identified heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2) as a binding partner of the JEV core protein by pulldown purification and mass spectrometry. Reciprocal coimmunoprecipitation analyses in transfected and infected cells confirmed a specific interaction between the JEV core protein and hnRNP A2. Expression of the JEV core protein induced cytoplasmic retention of hnRNP A2 in JEV subgenomic replicon cells. Small interfering RNA (siRNA)-mediated knockdown of hnRNP A2 resulted in a 90% reduction of viral RNA replication in cells infected with JEV, and the reduction was cancelled by the expression of an siRNA-resistant hnRNP A2 mutant. In addition to the core protein, hnRNP A2 also associated with JEV nonstructural protein 5, which has both methyltransferase and RNA-dependent RNA polymerase activities, and with the 5-untranslated region of the negative-sense JEV RNA. During one-step growth, synthesis of both positive-and negativestrand JEV RNAs was delayed by the knockdown of hnRNP A2. These results suggest that hnRNP A2 plays an important role in the replication of JEV RNA through the interaction with viral proteins and RNA.Japanese encephalitis virus (JEV) belongs to the genus Flavivirus within the family Flaviviridae. Members of the genus Flavivirus are predominantly arthropod-borne viruses, such as dengue virus (DEN), West Nile virus (WNV), yellow fever virus (YFV), and tick-borne encephalitis virus, and frequently cause significant morbidity and mortality in mammals and birds (46). JEV is distributed in the south and southeast regions of Asia and is kept in a zoonotic transmission cycle between pigs or birds and mosquitoes (46,69). JEV spreads to dead-end hosts, including humans, through the bite of JEVinfected mosquitoes and causes infection of the central nervous system, with a high mortality rate (46). JEV has a singlestranded positive-strand RNA genome of approximately 11 kb, which is capped at the 5Ј end but lacks modification of the 3Ј terminus by polyadenylation (38). The genomic RNA carries a single large open reading frame, and a polyprotein translated from the genome is cleaved co-and posttranslationally by host and viral proteases to yield three structural proteins-the core, precursor membrane, and envelope protein-and seven nonstructural (NS) proteins-NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 (61).The core protein of flaviviruses has RNA-binding activity through basic amino acid clusters located in both the amino and carboxyl termini, indicating that the core protein forms a nucleocapsid interacting with viral RNA (23). In spite of the replication of flaviviruses in the cyto...

Downregulation of Nipah Virus N mRNA Occurs through Interaction between Its 3′ Untranslated Region and hnRNP D

“…Similar relocalizations of hnRNP A1, C1/C2, and K were also reported (24). In addition to VSV, other RNA viruses, such as enterovirus (25,26), rhinovirus (27), Japanese encephalitis virus (28), and Junin virus (29), have also been shown to relocalize hnRNP proteins. To the best of our knowledge, relocalization of hnRNP proteins by NiV infection has not been reported previously.…”

Hepatocyte Factor JMJD5 Regulates Hepatitis B Virus Replication through Interaction with HBx

“…Infectious titers of HCV and JEV were determined by focus-forming assay. Mouse monoclonal antibodies against HCV NS5A (5A27) (44) and JEV NS3 (1791) (45) were used to visualize their foci. The titers of HSV-1 were determined by plaque assay.…”

A Japanese Encephalitis Virus Genotype 5 Molecular Clone Is Highly Neuropathogenic in a Mouse Model: Impact of the Structural Protein Region on Virulence

“…HB-112), and a highly purified antibody preparation was produced by RD Biotech (Besançon, France). Mouse monoclonal antibody anti-JEV NS5 and rabbit polyclonal antibody anti-JEV C were kindly provided by Yoshiharu Matsuura (19,20). The antibody against calnexin was purchased from Enzo Life Sciences (catalog no.…”