RNA-protein interactions: involvement of NS3, NS5, and 3' noncoding regions of Japanese encephalitis virus genomic RNA

Abstract

The mechanism of replication of the flavivirus Japanese encephalitis virus (JEV) is not well known. The structures at the 3 end of the viral genome are highly conserved among divergent flaviviruses, suggesting that they may function as cis-acting signals for RNA replication and, as such, might specifically bind to cellular or viral proteins. UV cross-linking experiments were performed to identify the proteins that bind with the JEV plus-strand 3 noncoding region (NCR). Two proteins, p71 and p110, from JEV-infected but not from uninfected cell extracts were shown to bind specifically to the plus-strand 3 NCR. The quantities of these binding proteins increased during the course of JEV infection and correlated with the levels of JEV RNA synthesis in cell extracts. UV cross-linking coupled with Western blot and immunoprecipitation analysis showed that the p110 and p71 proteins were JEV NS5 and NS3, respectively, which are proposed as components of the RNA replicase. The putative stem-loop structure present within the plus-strand 3 NCR was required for the binding of these proteins. Furthermore, both proteins could interact with each other and form a protein-protein complex in vivo. These findings suggest that the 3 NCR of JEV genomic RNA may form a replication complex together with NS3 and NS5; this complex may be involved in JEV minus-strand RNA synthesis.

The DEAD-box RNA helicase DDX5 acts as a positive regulator of Japanese encephalitis virus replication by binding to viral 3′ UTR

“…By using GST-pulldown and coimmunoprecipitation, we demonstrated that DDX5 can interact with JEV core, NS3, NS5-MTase and NS5-RdRp, suggesting that DDX5 might act as a cofactor for JEV proteins in virus replication. It has been suggested that cellular proteins can bind to viral 5 0 and/or 3 0 UTR to modulate viral translation and/or replication (Chen et al, 1997). Our studies showed that DDX5 can only interact with viral 3 0 UTR and colocalize with viral RNA.…”

“…It has been shown JEV core, NS3 and NS5 proteins play important roles in viral genome replication (Chen et al, 1997;Uchil and Satchidanandam, 2003a). By using GST-pulldown and coimmunoprecipitation, we demonstrated that DDX5 can interact with JEV core, NS3, NS5-MTase and NS5-RdRp, suggesting that DDX5 might act as a cofactor for JEV proteins in virus replication.…”

“…3.6. DDX5 can bind to JEV 3 0 UTR and colocalize with the viral RNA It has been shown that NS3 and NS5 of JEV can bind to 3 0 -UTR of viral RNA and engage in virus replication (Chen et al, 1997). We also determined whether DDX5 can bind to JEV 5 0 or/and 3 0 -UTR.…”

“…We next determined whether DDX5 is involved in JEV replication by interacting with JEV proteins related to viral RNA replication. It has been shown that JEV core, NS3 and NS5 are involved in the JEV replication (Chen et al, 1997;Uchil and Satchidanandam, 2003a). In flavivirus, NS5 can be separated into two domains: methyltransferase (MTase) domain and RNA-dependent RNA polymerase (RdRp) domain (Ackermann and Padmanabhan, 2001;Egloff et al, 2002;Guyatt et al, 2001;Koonin, 1993).…”

Cellular DDX3 regulates Japanese encephalitis virus replication by interacting with viral un-translated regions

“…The viral replication is initiated by the replication complex through a process of RNA-dependent RNA polymerization in the perinuclear endoplasmic reticulum membranes (Westaway et al, 2003). Nonstructural proteins 3 and 5 are components of the replication complex, which associates with the 3′ noncoding region of genomic RNA to initiate viral replication (Chen et al, 1997;Edward and Takegami, 1993). NS5, the largest and most conserved viral protein, contains methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) domain.…”