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.

Since the outbreak of severe acute respiratory syndrome (SARS) in 2003, the three-dimensional structures of several of the replicase/transcriptase components of SARS coronavirus (SARS-CoV), the non-structural proteins (Nsps), have been determined. However, within the large Nsp3 (1922 amino-acid residues), the structure and function of the so-called SARS-unique domain (SUD) have remained elusive. SUD occurs only in SARS-CoV and the highly related viruses found in certain bats, but is absent from all other coronaviruses. Therefore, it has been speculated that it may be involved in the extreme pathogenicity of SARS-CoV, compared to other coronaviruses, most of which cause only mild infections in humans. In order to help elucidate the function of the SUD, we have determined crystal structures of fragment 389–652 (“SUDcore”) of Nsp3, which comprises 264 of the 338 residues of the domain. Both the monoclinic and triclinic crystal forms (2.2 and 2.8 Å resolution, respectively) revealed that SUDcore forms a homodimer. Each monomer consists of two subdomains, SUD-N and SUD-M, with a macrodomain fold similar to the SARS-CoV X-domain. However, in contrast to the latter, SUD fails to bind ADP-ribose, as determined by zone-interference gel electrophoresis. Instead, the entire SUDcore as well as its individual subdomains interact with oligonucleotides known to form G-quadruplexes. This includes oligodeoxy- as well as oligoribonucleotides. Mutations of selected lysine residues on the surface of the SUD-N subdomain lead to reduction of G-quadruplex binding, whereas mutations in the SUD-M subdomain abolish it. As there is no evidence for Nsp3 entering the nucleus of the host cell, the SARS-CoV genomic RNA or host-cell mRNA containing long G-stretches may be targets of SUD. The SARS-CoV genome is devoid of G-stretches longer than 5–6 nucleotides, but more extended G-stretches are found in the 3′-nontranslated regions of mRNAs coding for certain host-cell proteins involved in apoptosis or signal transduction, and have been shown to bind to SUD in vitro. Therefore, SUD may be involved in controlling the host cell's response to the viral infection. Possible interference with poly(ADP-ribose) polymerase-like domains is also discussed.

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Flaviviral NS3 is a multifunctional protein displaying N-terminal protease activity in addition to C-terminal helicase, nucleoside 5'-transferase (NTPase), and 5'-terminal RNA triphosphatase (RTPase) activities. NS3 is held to support the separation of RNA daughter and template strands during viral replication.We solved the three-dimensional structure (at 3.1 Å resolution) of the NS3 helicase domain (residues 186-619; NS3:186-619) from Kunjin virus, an Australian variant of the West Nile virus (1). We showed that NS3:186-619 displays both ATPase and RTPase activity, that it can unwind a double-stranded RNA substrate, being however inactive on a double-stranded DNA substrate. Analysis of different constructs shows that full length NS3 displays increased helicase activity, suggesting that the protease domain plays an assisting role in the RNA unwinding process. The structural interaction between the helicase and protease domain has been assessed using small angle X-ray scattering on full length NS3, disclosing that the protease and helicase domains build a rather elongated molecular assembly differing from that observed in the NS3 protein from hepatitis C virus. S.A.X.S. data were collected on the X33 camera of the EMBL on the storage ring DORIS III (DESY, Hamburg, Germany). The data were recorded using a MAR345 two-dimensional image plate detector at a sample-detector distance of 2.7 m and a wavelength of λ = 1.5 Å, covering the range of momentum transfer 0.012 < s < 0.45 Å -1 (s = 4π sinθ/λ, where 2θ is the scattering angle). The solutions containing NS3FL at 3.0, 1.5 and 0.75 mg/ml in Hepes 10 mM, NaCl 300 mM and protease inhibitors cocktail (Roche), with the addition of 2 mM DTT to reduce radiation damage, were placed in a 70 µl cell with polystyrene windows and exposed to X-ray beam. To check for radiation damage, the data were collected in two successive 2-minute exposures, and no changes were observed in the scattering patterns with time, i.e., there was no measurable radiation damage. All measurements were performed at 15°C. The data were averaged after normalization to the intensity of the incident beam and the scattering of the buffer was subtracted. The difference curves were scaled for the solute concentration and extrapolated to infinite dilution. All data manipulations were performed using the program package PRIMUS (2).The forward scattering I(0) and the radius of gyration (R g ) were evaluated using the Guinier approximation (3); the maximum diameter D max of the particle were computed from the entire scattering patterns using program GNOM (4), and the excluded volume V p of the particle was computed from the Porod invariant (5). The molecular masses of the solutes were evaluated by comparison of the forward scattering with that from reference solutions of bovine serum albumin (66 kDa). The ab initio shape reconstruction was performed using the program GASBOR (6), using the total number of the residues in NS3FL (619).The estimated molecular mass of the NS3FL sample, 65±5 kDa, is compatible with mon...

Flaviviruses are the causative agents of severe diseases such as Dengue or Yellow fever. The replicative machinery used by the virus is based on few enzymes including a methyltransferase, located in the N-terminal domain of the NS5 protein. Flaviviral methyltransferases are involved in the last two steps of the mRNA capping process, transferring a methyl group from S-adenosyl-L-methionine onto the N7 position of the cap guanine (guanine-N7 methyltransferase) and the ribose 2'O position of the first nucleotide following the cap guanine (nucleoside-2'O methyltransferase). The RNA capping process is crucial for mRNA stability, protein synthesis and virus replication. Such an essential function makes methyltransferases attractive targets for the design of antiviral drugs. In this context, starting from the crystal structure of Wesselsbron flavivirus methyltransferase, we elaborated a mechanistic model describing protein/RNA interaction during N7 methyl transfer. Next we used an in silico docking procedure to identify commercially available compounds that would display high affinity for the methyltransferase active site. The best candidates selected were tested in vitro to assay their effective inhibition on 2'O and N7 methyltransferase activities on Wesselsbron and Dengue virus (Dv) methyltransferases. The results of such combined computational and experimental screening approach led to the identification of a high-potency inhibitor.