Crystal Structure of a Novel Conformational State of the Flavivirus NS3 Protein: Implications for Polyprotein Processing and Viral Replication

Abstract

The flavivirus genome comprises a single strand of positive-sense RNA, which is translated into a polyprotein and cleaved by a combination of viral and host proteases to yield functional proteins. One of these, nonstructural protein 3 (NS3), is an enzyme with both serine protease and NTPase/helicase activities. NS3 plays a central role in the flavivirus life cycle: the NS3 N-terminal serine protease together with its essential cofactor NS2B is involved in the processing of the polyprotein, whereas the NS3 C-terminal NTPase/helicase is responsible for ATP-dependent RNA strand separation during replication. An unresolved question remains regarding why NS3 appears to encode two apparently disconnected functionalities within one protein. Here we report the 2.75-Å-resolution crystal structure of full-length Murray Valley encephalitis virus NS3 fused with the protease activation peptide of NS2B. The biochemical characterization of this construct suggests that the protease has little influence on the helicase activity and vice versa. This finding is in agreement with the structural data, revealing a single protein with two essentially segregated globular domains. Comparison of the structure with that of dengue virus type 4 NS2B-NS3 reveals a relative orientation of the two domains that is radically different between the two structures. Our analysis suggests that the relative domain-domain orientation in NS3 is highly variable and dictated by a flexible interdomain linker. The possible implications of this conformational flexibility for the function of NS3 are discussed.Flaviviruses such as dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV), and Japanese encephalitis virus (JEV) belong to the family Flaviviridae and are the causative agents of a range of serious human diseases including hemorrhagic fever, meningitis, and encephalitis (37). They remain a global health priority, as many viruses are endemic in large parts of the Americas, Africa, Australia, and Asia, and vaccines remain unavailable for most members (31,46,57).Flaviviruses have a positive-sense single-stranded RNA (ssRNA) genome (approximately 11 kb) that encodes one large open reading frame containing a 5Ј type 1 cap and conserved RNA structures at both the 5Ј and 3Ј untranslated regions that are important for viral genome translation and replication. The genomic RNA is translated into a single polyprotein precursor (11) consisting of three structural (C [capsid], prM [membrane], and E [envelope]) and seven nonstructural (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5) proteins arranged in the order C-prM-E-NS1-NS2a-NS2b-NS3-NS4a-NS4b-NS5 (reviewed in reference 33) (Fig. 1). Only the structural proteins become part of the mature, infectious virion, whereas the nonstructural proteins are involved in polyprotein processing, viral RNA synthesis, and virus morphogenesis (33, 43). The precursor protein is directed by signal sequences into the host endoplasmic reticulum (ER), where NS1 and the exogenous domains of prM and E face the lumen, while C, ...

A Crystal Structure of the Dengue Virus Non-structural Protein 5 (NS5) Polymerase Delineates Interdomain Amino Acid Residues That Enhance Its Thermostability and de Novo Initiation Activities

“…Interestingly, as was observed for the NS3 protease-helicase linker (25,26), most residues from the interdomain linker of NS5 have been poorly conserved during flavivirus evolution (Fig. 1a), suggesting limited functional constraints on the precise amino acid sequence of this region.…”

Flexibility between the Protease and Helicase Domains of the Dengue Virus NS3 Protein Conferred by the Linker Region and Its Functional Implications

“…Interestingly, the Gly insertion mutant also crystallizes in conformation I, which lends further support to the notion that at least two significantly populated conformations with different orientations between the protease and helicase co-exist in solution. Indeed, recently Assenberg et al (27) reported a crystal structure for the Murray Valley encephalitis virus NS3 protein linked to 40 amino acids of NS2B. This conformation resembles conformation II of NS2B 18 NS3 from DENV4 as it also leaves the RNA-binding site fully accessible.…”

A macrocyclic HCV NS3/4A protease inhibitor interacts with protease and helicase residues in the complex with its full-length target

“…Two alternative conformations related by a ca. 160°r otation of the protease domain with respect to the helicase in the interdomain linker region have been reported (19)(20)(21). This interdomain flexibility may have mechanistic implications for flaviviral NS3 proteins.…”

“…Several crystal structures of full-length NS3 proteins from another genus of the flaviviridae family, the flaviviruses, have been reported, those of Dengue virus and Murray Valley encephalitis virus (19)(20)(21). Flaviviral NS3 proteins employ NS2B rather than NS4A as protease cofactor (see ref.…”