Dengue virus NS4B interacts with NS3 and dissociates it from single-stranded RNA

Abstract

Dengue virus, a member of the family Flaviviridae of positive-strand RNA viruses, has seven non-structural proteins: NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5. Except for enzymic activities contained within NS3 and NS5, the roles of the other proteins in virus replication and pathogenesis are not well defined. In this study, a physical interaction between NS4B and the helicase domain of NS3 was identified by using a yeast two-hybrid assay. This interaction was further confirmed by biochemical pull-down and immunoprecipitation assays, both with purified proteins and with dengue virus-infected cell lysates. NS4B co-localized with NS3 in the perinuclear region of infected human cells. Furthermore, NS4B dissociated NS3 from single-stranded RNA and consequently enhanced the helicase activity of NS3 in an in vitro unwinding assay. These results suggest that NS4B modulates dengue virus replication via its interaction with NS3. INTRODUCTIONDengue fever and its more severe form, dengue haemorrhagic fever, are mosquito-borne viral diseases that are caused by one of the four antigenically distinct serotypes of Dengue virus, DENV-1-DENV-4. Dengue fever affects 50-100 million people in the tropical and subtropical regions annually (Gubler, 1998(Gubler, , 2002. Contemporary demographical and lifestyle trends, such as population explosion and urbanization, have led to the spread of this disease to nonendemic regions. The pathogenesis of dengue fever remains poorly characterized and there are no antivirals or vaccines available to counter this emerging disease.Dengue virus belongs to the family Flaviviridae that consists of enveloped, positive-sense, single-stranded RNA (ssRNA) viruses, such as those that cause yellow fever, Japanese encephalitis, West Nile fever and hepatitis C. Its RNA genome is encapsulated in an icosahedral nucleocapsid (30 nm) that is enveloped in a lipid bilayer (10 nm) (Kuhn et al., 2002) consisting of the membrane and envelope proteins. The 11 kb, capped RNA genome encodes a single polyprotein that is processed co-and post-translationally by host signalases, as well as the virus-encoded serine protease, into the three structural and seven non-structural proteins (NS) in the order C (Core)-prM (pre-Membrane)-E (Envelope)-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5 (Chambers & Rice, 1987;Lindenbach & Rice, 2003).The polymerase, helicase and protease enzymic activities encoded by the dengue virus genome ensure virus replication and polyprotein processing. NS3 (618 aa) is a multifunctional protein with protease, helicase, NTPase and 59-terminal RNA triphosphatase activities (Arias et al., 1993;Benarroch et al., 2004;Falgout et al., 1991;Li et al., 1999;Zhang et al., 1992), whilst NS5 (900 aa) has RNAdependent RNA polymerase and methyltransferase activities (Ackermann & Padmanabhan, 2001;Chu & Westaway, 1987;Egloff et al., 2002;Kapoor et al., 1995;Tan et al., 1996). These two proteins form a functional complex that is vital for flavivirus replication (Brooks et al., 2002;Johansson et al., 2001;Yon et al., 2005). The role...

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

“…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.…”

Comparative Analysis of Full-Length Genomic Sequences of 10 Dengue Serotype 1 Viruses Associated with Different Genotypes, Epidemics, and Disease Severity Isolated in Thailand over 22 Years

“…Although the functions of NS2A, NS4A, and NS4B have not yet been fully elucidated, several studies suggest that they may anchor the viral replicase proteins to cellular membranes, 52 assist in virion assembly, 53,54 and lead to inhibition of the interferon a/β response. [55][56][57][58] It has been reported that NS4A may play a role in induction of membrane alterations, assumed to serve as a scaffold for the formation of the viral replicase complex, 59 and the interaction between NS1 and NS4A, which is critical for viral RNA replication. 59,60 Mutations in the NS2A protein that diminish its ability to inhibit the host interferon response could contribute to the non-predominant status of genotype III viruses.…”

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

“…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]. In addition, NS4A and NS4B, along with NS2A, as has already been referred, and NS5 proteins appear to inhibit the interferon-/ response of the host [44][45][46].…”