Structure and functionality in flavivirus NS-proteins: perspectives for drug design

Abstract

Flaviviridae are small enveloped viruses hosting a positive-sense single-stranded RNA genome. Besides yellow fever virus, a landmark case in the history of virology, members of the Flavivirus genus, such as West Nile virus and dengue virus, are increasingly gaining attention due to their re-emergence and incidence in different areas of the world. Additional environmental and demographic considerations suggest that novel or known flaviviruses will continue to emerge in the future. Nevertheless, up to few years ago flaviviruses were considered low interest candidates for drug design. At the start of the European Union VIZIER Project, in 2004, just two crystal structures of protein domains from the flaviviral replication machinery were known. Such pioneering studies, however, indicated the flaviviral replication complex as a promising target for the development of antiviral compounds. Here we review structural and functional aspects emerging from the characterization of two main components (NS3 and NS5 proteins) of the flavivirus replication complex. Most of the reviewed results were achieved within the European Union VIZIER Project, and cover topics that span from viral genomics to structural biology and inhibition mechanisms. The ultimate aim of the reported approaches is to shed light on the design and development of antiviral drug leads.

Fig. 1

Model representation of NS3 (full-length) anchoring via NS2B to the ER membrane. The N-terminal NS3Pro domain is shown in blue, the NS3Hel domain in green. The crystal structure of DENV4 NS2B/NS3 (PDB entry 2VBC, Luo et al., 2008a) was used for model preparation. The NS2B protein is shown in yellow, modeled regions are shown as dashed lines and helices anchoring the complex to the membrane.

Fig. 2

Crystal structures of NS3Pro from DENV2 and WNV viruses. (a) Overall fold of NS2B/NS3Pro from DENV2 (PDB entry 2FOM, Erbel et al., 2006). NS3Pro is shown in blue, the NS2B region, ordered in the crystal structure, is shown in yellow. (b) Superposition of DENV2 NS2B/NS3Pro as depicted in (a) and the crystal structure of DENV2 NS3Pro without the stabilizing cofactor shown in orange (PDB entry 1BEF, Murthy et al., 1999). Remarkably, substantial differences with respect to secondary structure elements are observed. (c) Superposition of the WNV NS2B/NS3Pro in ligand-bound and uncomplexed state. The NS3Pro covalently linked to the inhibitor (PDB entry 2FP7, Erbel et al., 2006) is shown in blue with the cofactor and ligand colored in orange and light blue, respectively. In the uncomplexed state (H51A mutant, PDB entry 2GGV, Aleshin et al., 2007) shown in green, the NS2B colored in yellow exhibits remarkable plasticity compared to the ligand-bound conformer.

Fig. 3

DENV2 NS3Pro complexed with a Bowman-Birk inhibitor from Mung Bean (PDB entry 1DF9, Murthy et al., 2000). The representation shows a superimposition of the two protein molecules present in the asymmetric unit and the relevant peptide region of the inhibitor (lysine head, depicted in orange). The crystal structure suggests a pronounced mobility for the region 126–136 lining the specificity pockets of the NS3Pro. Particularly Asp129 (equivalent to Asp189 in trypsin) is capable of pointing either towards the solvent or contributing to the S1 pocket.

Fig. 4

Induction of the oxyanion hole in WNV NS3B/NS3Pro by the polypeptide-type inhibitor aprotinin (for clarity, only Pro13-Lys15 are shown in yellow). Residues of the uncomplexed NS3Pro (H51A mutant, PDB entry 2GGV, Aleshin et al., 2007) are shown as green sticks, residues of the aprotinin-bound enzyme are depicted in blue (PDB entry 2IJO, Aleshin et al., 2007). The peptide bond Thr132-Gly133 flips and contributes via its backbone nitrogen atom to the formation of the oxyanion hole. H-bonding interactions between the ligand carbonyl oxygen and the backbone nitrogens of Gly133, Thr134 and Ser135 are shown as orange dashes.

Fig. 5

The structure of DENV NS3Hel with its three domains (I red, II blue and III green) bound to AMPPNP (left, molecule in green) and RNA (7 bases are visible: AGACUAA in yellow), adapted from PDB entry 2JLV (Luo et al., 2008a).

Fig. 6

Comparison of the NS2B-NS3 structures of MVEV (upper panel) and DENV4 (lower panel); in both panels the NS3Hel domain is in the upper part of the figure, the NS3Pro domain is in blue–cyan colors, hosting the NS2B segment (red color).

Fig. 7

Models for membrane association of MVEV and DENV4 NS2B-NS3. (a) Schematic diagram of the flavivirus polyprotein organization and processing. The upper figure shows the linear organization of the structural and non-structural proteins within the polyprotein. The lower figure shows the putative membrane topology of the polyprotein, as predicted from biochemical and cellular analyses, which is then processed by cellular and viral proteases (denoted by arrows). (b) Predicted structural organization of MVEV NS2B₄₅NS3 and DENV4 NS2B₁₈NS3 at the cellular membrane. A model for the membrane is shown as van der Waals balls, atomic structures are shown in a surface representation and color coded according to the following convention: NS3 protein (pale yellow) and NS2B stretches (blue). The NS4A (shown schematically in pink) was positioned at the NS3 C-terminus (domain3) and the RNA (shown schematically in grey) is positioned in the ssRNA binding groove.

Fig. 8

Crystal structure of DENV NS5MTase in complex with AdoHcy. A ball-and-stick representation is used for AdoHcy, whereas DENV NS5MTase is drawn as a ribbon (Egloff et al., 2002). The loops differing between NS5MTases representative of the three Flaviviral branches are highlighted with a star and an identification number referring to what has been described in the text.

Fig. 9

Stereo view of the complex formed by MVEV NS5MTase with AdoHcy and two molecules of GpppA: the first cap analogue binds in the HBS, the second is adjacent and interacting with the positively charged residues near the AdoMet-binding cleft in the LBS (Assenberg et al., 2007).

Fig. 10

Overview of the flavivirus RdRp structure based on WNV NS5Pol (Malet et al., 2007) as an example; a “Front” view is presented here in ribbon representation. Fingers, palm and thumb subdomains are colored in blue, green and red, respectively. The ssRNA template entry and the dsRNA exit are shown by black arrows. A dotted arrow points to the NTP entry tunnel at the back of the RdRp. Motifs A, C, E, F, the G-loop and the priming loop are colored in orange, yellow, grey, magenta, cyan and purple, respectively. The Asp residues of catalytic motifs A and C (Asp-533, Asp-663 and Asp-664) are represented as stick models. N-ter and C-ter indicate the termini of the RdRp domain.

Fig. 11

Structural formulae of (a) ribavirin and (b) EICAR.

Fig. 12

Mechanism of ribavirin action. Target enzyme: IMP dehydrogenase. Ribavirin 5′ monophosphate inhibits the conversion of IMP to XMP resulting in a reduced supply of GTP, and, indirectly, also a reduced supply of ATP.

Fig. 13

Structural formulae of (a) 2′-c-methylcytidine, (b) 4′-azidocytidine and (c) T-705.