Dahai-Lab‎ > ‎Research‎ > ‎

Flavivirus Replication

1. Introduction

  • 2. Our work

    2.1 NonStructural protein 3 - NS3 - NTPase, RTPase, RNA helicase, and beyond ... 

            2.1.1 Structure of the scNS2B18NS3 protein from Dengue 4 : the first full length NS3 Proease-Helicase from flaviviruses.
            Atomic structures are available for the individual protease and helicase domains of NS3 separately but a molecular view of the full length flavivirus NS3 polypeptide is still lacking. In order to study its overall architecture and the implications on the enzymatic activities of the NS2B-NS3 protease-helicase, a complete NS3 molecule fused with 18 residues of the NS2B cofactor through a flexible linker (scNS2B18NS3) was engineered. The recombinant protein was successfully expressed, purified and crystallized. The first crystal structure of scNS2B18NS3 was determined as apo enzyme to 3.1 Å. The relative orientation between the protease and helicase domains is drastically different compared to the scNS3-NS4A molecule from hepatitis C virus (HCV), which was caught in the act of cis cleavage at the NS3-NS4A junction. For dengue NS3, the protease domain sits beneath the ATP binding site, giving the molecule an elongated shape. The domain arrangement found in the crystal structure fits nicely into an envelope determined ab-initio using small angle X-ray scattering experiments in solution. 

            2.1.2 Alternative Conformation of the NS2B18NS3 protein: the naughty protease domain and the flexible domain linker. 
            The dengue virus (DENV) NS3 protein is essential for viral polyprotein processing and RNA replication. It contains an N-terminal serine protease region (residues 1 to 168) joined to a superfamily-2 RNA-helicase (residues 180 to 618) by an 11-amino-acids linker (16 to 179). The structure at 3.15 A of the soluble NS3 protein from DENV4 covalently attached to 18 residues of the NS2B cofactor region (NS2B18NS3) revealed an elongated molecule with the protease domain abutting sub-domains I and II of the helicase (Luo et al., J. Virol. 2008, 82, 173-83). Unexpectedly, using similar crystal growth conditions, we observed an alternative conformation where the protease domain has rotated by ~161 degrees with respect to the helicase domain. We report this new crystal structure bound to ADP-Mn2+ refined to a resolution of 2.2 A. The biological significance for interdomain flexibility conferred by the linker region was probed by either inserting a Gly residue between Glu-173 and Pro-174 or replacing Pro-174 with a Gly residue. Both mutations resulted in significantly lower ATPase and helicase activities. We next increased flexibility in the linker by introducing a Pro-176 to Gly mutation in a DENV2 replicon system. A 70% reduction in luciferase reporter signal as well as a similar reduction in the level of viral RNA synthesis was observed. Our results indicate that the linker region has evolved to an optimum length to confer flexibility to the NS3 protein that is required both for polyprotein processing and RNA replication.

            2.1.3 Structures of NS3 helicase domain : ssRNA recognition and ATP hydrolysis by NS3 helicase.
            Together with the NS5 polymerase, the NS3 helicase has a pivotal function in flavivirus RNA replication and constitutes an important drug target. We captured the dengue virus NS3 helicase at several stages along the catalytic pathway including bound to single-stranded (ss) RNA, to an ATP analogue, to a transition-state analogue and to ATP hydrolysis products. RNA recognition appears largely sequence independent in a way remarkably similar to eukaryotic DEAD box proteins Vasa and eIF4AIII. On ssRNA binding, the NS3 enzyme switches to a catalyticcompetent state imparted by an inward movement of the P-loop, interdomain closure and a change in the divalent metal coordination shell, providing a structural basis for RNA-stimulated ATP hydrolysis. These structures demonstrate for the first time large quaternary changes in the flaviviridae helicase, identify the catalytic water molecule and point to a b-hairpin that protrudes from subdomain 2, as a critical element for dsRNA unwinding. They also suggest how NS3 could exert an effect as an RNA anchoring device and thus participate both in flavivirus RNA replication and assembly. 


    2.1.4 The flavivirus NS2B–NS3 protease–helicase as a target for antiviral drug development
    • Structural and functional studies on flavivirus NS2B–NS3 protease–helicase/NTPase/RTPase.
    • Recent advances in drug discovery targeting NS2B–NS3.
    • NS2B–NS3 and the assembly of the flavivirus replication complex.

    2.2 NonStructural protein 5 - NS5 - MTase, RdRP, and beyond... 

            The N-terminal domain of the flavivirus NS5 protein functions as a methyltransferase (MTase). It sequentially methylates the N7 and 2′-O positions of the viral RNA cap structure (GpppA→7meGpppA→7meGpppA2′-O-me). The same NS5 domain could also have a guanylyltransferase activity (GTP+ppA-RNA→GpppA). The mechanism by which this protein domain catalyzes these three distinct functions is currently unknown. Here we report the crystallographic structure of DENV-3 MTase in complex with a 5′-capped RNA octamer (GpppAGAACCUG) at a resolution of 2.9 Å. Two RNA octamers arranged as kissing loops are encircled by four MTase monomers around a 2-fold non-crystallography symmetry axis. Only two of the four monomers make direct contact with the 5′ end of RNA. The RNA structure is stabilised by the formation of several intra and intermolecular base stacking and non-canonical base pairs. The structure may represent the product of guanylylation of the viral genome prior to the subsequent methylation events that require repositioning of the RNA substrate to reach to the methyl-donor sites. The crystal structure provides a structural explanation for the observed trans-complementation of MTases with different methylation defects.

               DENV causes widespread mosquito-borne viral infections worldwide and nearly 40% of the world’s population is at risk of being infected. Currently, no licensed vaccines or specific drugs are available to treat severe infections by DENV. NS5 is a large protein of 900 amino acids composed of two domains with several key enzymatic activities for viral RNA replication in the host cell and constitutes a prime target for the design of antiviral inhibitors. We succeeded in trapping a stable conformation of the full-length NS5 protein and report its crystal structure at a resolution of 2.3 Å. This conformation reveals the entire inter-domain region and clarifies the determinants of NS5 flexibility. The inter-domain interface is stabilized by several polar contacts between residues projecting from the MTase and RdRp domains of NS5. Several evolutionarily conserved residues at the interface play a crucial role for virus replication as shown by reverse genetics, although the analogous mutations mostly do not abolish the in vitro enzymatic activities of the recombinant proteins.

                We examined the function of the conserved Val/Ile residue within the dengue virus NS5 interdomain linker (residues 263-272) by site-directed mutagenesis. Gly substitution or Gly/Pro insertion after the conserved residue increased the linker flexibility and created slightly attenuated viruses. In contrast, Pro substitution abolished virus replication by imposing rigidity in the linker and restricting NS5's conformational plasticity. Our biochemical and reverse genetics experiments demonstrate that NS5 utilizes conformational regulation to achieve optimum viral replication.

    Dengue is the most prevalent mosquito-borne viral disease, endemic in more than a hundred tropical and subtropical countries. NS5, the largest viral protein, is a key replication enzyme with both methyltransferase and RNA polymerase activities. We present to our knowledge the first crystal structure of the full-length NS5 protein from dengue virus bound to the authentic 5′-end viral RNA fragment. This structure captures the viral enzyme in the act of transferring a methyl group to the 2′-O-ribose of the first nucleotide of the viral genome, providing an atomic-level understanding of specific 2′-O-methylation and cap formation by the flavivirus methyltransferase. The structure also suggests an evolutionary origin for the methyltransferase domain of NS5 and strategies for designing novel antiviral inhibitors.

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