NS4A regulates the ATPase activity of the NS3 helicase: a novel cofactor role of the non-structural protein NS4A from West Nile virus

Abstract

Using constructs that encode the individual West Nile virus (WNV) NS3helicase (NS3hel) and NS3hel linked to the hydrophilic, N-terminal 1-50 sequence of NS4A, we demonstrated that the presence of NS4A allows NS3hel to conserve energy in the course of oligonucleotide substrate unwinding. Using NS4A mutants, we also determined that the C-terminal acidic EELPD/E motif of NS4A, which appears to be functionally similar to the acidic EFDEMEE motif of hepatitis C virus (HCV) NS4A, is essential for regulating the ATPase activity of NS3hel. We concluded that, similar to HCV NS4A, NS4A of WNV acts as a cofactor for NS3hel and allows helicase to sustain the unwinding rate of the viral RNA under conditions of ATP deficiency.After flavivirus entry into the host cell, its approximately 11 kb genome is uncoated and serves as a template for the translation of a single C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5 polyprotein precursor (Chambers et al., 1990;Sampath & Padmanabhan, 2009). The polyprotein is inserted into the endoplasmic reticulum membrane and processed by host and viral proteinases into three structural proteins (C, prM and E) and seven non-structural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) (Padmanabhan et al., 2006). The structural proteins are components of mature virus particles. The NS proteins are expressed only in the infected host cell and are not packaged into mature particles. Similar to other flaviviruses, the fulllength NS3 peptide sequence of West Nile virus (WNV) represents a multifunctional protein in which the Nterminal 184 residues encode serine proteinase (NS3pro) and the C-terminal 440 residues code for an RNA triphosphatase, an NTPase and an RNA helicase (NS3hel) (Benarroch et al., 2004;Borowski et al., 2001). NS3hel is a member of the DEAH/D-box family within the helicase superfamily 2 (Gorbalenya et al., 1989;Li et al., 1999;Luking et al., 1998;Luo et al., 2008). NS3hel is required for unwinding of RNA during replication. The ATPase activity of NS3hel does not require the binding of the enzyme to its nucleotide substrate (Chernov et al., 2008). As a result, high concentrations of ATP (in the mM range) are necessary to support the unwinding activity of NS3hel in vitro.The presence of a cofactor, NS2B, is necessary for NS3pro to exhibit its proteolytic activity (Aleshin et al., 2007;Erbel et al., 2006). Although evidence suggests that the small, hydrophobic NS4A protein (approx. 16 kDa) is required for viral replication, and that NS4A may serve as a central 'organizer' of the replication complex of flaviviruses (Lindenbach & Rice, 1999;Paredes & Blight, 2008;Umareddy et al., 2006), its precise functional role is not characterized sufficiently. NS4A associates with membranes via four internal hydrophobic regions. Its C-terminal region (frequently designated 2K fragment) serves as a signal sequence for the translocation of the adjacent NS4B into the endoplasmic reticulum lumen. In the lumen, the 2K fragment is removed from the N terminus of NS4B by the host signallase. Proteolytic remo...

Genetic interaction between NS4A and NS4B for replication of Japanese encephalitis virus

“…NS4A is regarded as a central 'organizer' of the replication complex of flaviviruses (Lindenbach & Rice, 1999;Nemésio et al, 2012;Shiryaev et al, 2009), and is composed of a hydrophilic N-terminal portion residing in the cytoplasm, three internal hydrophobic regions associated with endoplasmic reticulum (ER) membranes (pTMS1-pTMS3) and a C-terminal fragment called 2K that acts as the signal sequence for translocation of the NS4B protein into the ER lumen (Miller et al, 2007). After translocation, the 2K fragment is cleaved off the N terminus of NS4B by the host signallase in the ER lumen.…”

“…A recent study identified that NS4B existed as dimers either when the protein was expressed alone in cells or in cells infected with DENV (Zou et al, 2014). Additionally, both NS4A and NS4B proteins have been demonstrated to be able to interact genetically or physically with other viral proteins, including NS3 localized in the cytoplasm (Shiryaev et al, 2009;Umareddy et al, 2006) and NS1 positioned in the ER lumen (Lindenbach & Rice, 1999;Youn et al, 2012) to modulate viral replication. For example, the hydrophilic NS4A sequence functions as a cofactor of NS3 helicase and regulates the ATPase activity of NS3 helicase (Shiryaev et al, 2009).…”

Characterization of Dengue Virus NS4A and NS4B Protein Interaction

“…(iii) Flavivirus NS4A protein induces autophagy to prevent cell death and facilitate viral replication (24). (iv) WNV NS4A regulates the ATPase activity of NS3 helicase (25), while DENV NS4B interacts with the helicase domain of NS3 and dissociates it from singlestrand RNA (26). As noted in the accompanying article by Zou et al (27), the DENV NS3-NS4B interaction was mapped to the NS3 helicase (subdomains 2 and 3) and the NS4B cytoplasmic region (amino acids 129 to 165).…”

West Nile Virus: Basic Principles, Replication Mechanism, Immune Response and Important Genetic Determinants of Virulence

“…Proteolytic removal of the 2K peptide also induces membrane alterations [40]. Recently NS4A was proven to act as a cofactor for NS3 helicase allowing the helicase to sustain the unwinding rate of the viral RNA under conditions of ATP deficiency [41]. NS4B colocalizes with viral replication complexes and proved to dissociate NS3 from single-stranded RNA, thereby enabling it to bind to a new dsRNA duplex, consequently enhancing the helicase activity and modulating viral replication [42,43].…”