Michela Bollati

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

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.

The SARS-Unique Domain (SUD) of SARS Coronavirus Contains Two Macrodomains That Bind G-Quadruplexes

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 cells response to the viral infection. Possible interference with poly(ADP-ribose) polymerase-like domains is also discussed.

Flaviviral methyltransferase/RNA interaction: Structural basis for enzyme inhibition

Abstract 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.

Structural bases for substrate recognition and activity in Meaban virus nucleoside-2′-O-methyltransferase

Viral methyltransferases are involved in the mRNA capping process, resulting in the transfer of a methyl group from S‐adenosyl‐L‐methionine to capped RNA. Two groups of methyltransferases (MTases) are known: (guanine‐N7)‐methyltransferases (N7MTases), adding a methyl group onto the N7 atom of guanine, and (nucleoside‐2′‐O‐)‐methyltransferases (2′OMTases), adding a methyl group to a ribose hydroxyl. We have expressed and purified two constructs of Meaban virus (MV; genus Flavivirus) NS5 protein MTase domain (residues 1–265 and 1–293, respectively). We report here the three‐dimensional structure of the shorter MTase construct in complex with the cofactor S‐adenosyl‐L‐methionine, at 2.9 Å resolution. Inspection of the refined crystal structure, which highlights structural conservation of specific active site residues, together with sequence analysis and structural comparison with Dengue virus 2′OMTase, suggests that the crystallized enzyme belongs to the 2′OMTase subgroup. Enzymatic assays show that the short MV MTase construct is inactive, but the longer construct expressed can transfer a methyl group to the ribose 2′O atom of a short GpppAC5 substrate. West Nile virus MTase domain has been recently shown to display both N7 and 2′O MTase activity on a capped RNA substrate comprising the 5′‐terminal 190 nt of the West Nile virus genome. The lack of N7 MTase activity here reported for MV MTase may be related either to the small size of the capped RNA substrate, to its sequence, or to different structural properties of the C‐terminal regions of West Nile virus and MV MTase‐domains.

Crystal structure of LptH, the periplasmic component of the lipopolysaccharide transport machinery from Pseudomonas aeruginosa

Lipopolysaccharide (LPS) is the main glycolipid present in the outer leaflet of the outer membrane (OM) of Gram‐negative bacteria, where it modulates OM permeability, therefore preventing many toxic compounds from entering the cell. LPS biogenesis is an essential process in Gram‐negative bacteria and thus is an ideal target pathway for the development of novel specific antimicrobials. The lipopolysaccharide transport (Lpt) system is responsible for transporting LPS from the periplasmic surface of the inner membrane, where it is assembled, to the cell surface where it is then inserted in the OM. The Lpt system has been widely studied in Escherichia coli, where it consists of seven essential proteins located in the inner membrane (LptBCFG), in the periplasm (LptA) and in the OM (LptDE). In the present study, we focus our attention on the Pseudomonas aeruginosa PAO1 Lpt system. We identified an LptA orthologue, named LptH, and solved its crystal structure at a resolution of 2.75 Å. Using interspecies complementation and site‐directed mutagenesis of a conserved glycine residue, we demonstrate that P. aeruginosa LptH is the genetic and functional homologue of E. coli LptA, with whom it shares the β‐jellyroll fold identified also in other members of the canonical E. coli Lpt model system. Furthermore, we modeled the N‐terminal β‐jellyroll domain of P. aeruginosa LptD, based on the crystal structure of its homologue from Shigella flexneri, aiming to provide more general insight into the mechanism of LPS binding and transport in P. aeruginosa. Both LptH and LptD may represent new targets for the discovery of next generation antibacterial drugs, targeting specific opportunistic pathogens such as P. aeruginosa.

Structures of the lamin A/C R335W and E347K mutants: Implications for dilated cardiolaminopathies.

Dilated cardiomyopathy (DCM) is a condition whereby the normal muscular function of the myocardium is altered by specific or multiple aetiologies. About 25-35% of DCM patients show familial forms of the disease, with most mutations affecting genes encoding cytoskeletal proteins. Most of the DCM-related mutations fall in the Lamin AC gene, in particular in the Coil2B domain of the encoded protein. In this context, we focussed our studies on the crystal structures of two lamin Coil2B domain mutants (R335W and E347K). Both R335 and E347 are higly conserved residues whose substitution has little effects on the Coil2B domain three-dimensional structure; we can thus hypothesize that the mutations may interfere with the binding of components within the nuclear lamina, or of nuclear factors, that have been proposed to interact/associate with lamin A/C.

Crystal structure of a methyltransferase from a no-known-vector Flavivirus

Presently known flaviviruses belong to three major evolutionary branches: tick-borne viruses, mosquito-borne viruses and viruses with no known vector. Here we present the crystal structure of the Yokose virus methyltransferase at 1.7A resolution, the first structure of a methyltransferase of a Flavivirus with no known vector. Structural comparison of three methyltransferases representative of each of the Flavivirus branches shows that fold and structures are closely conserved, most differences being related to surface loops flexibility. Analysis of the conserved residues throughout all the sequenced flaviviral methyltransferases reveals that, besides the central cleft hosting the substrate and cofactor binding sites, a second, almost continuous, patch is conserved and points away from active site towards the back of the protein. The high level of structural conservation in this region could be functional for the methyltransferase/RNA interaction and stabilization of the ensuing complex.

Structure and activity of Kunjin virus NS3 helicase domain

Page s290 Acta Cryst. (2007). A63, s290 A prerequisite for efficient high throughput protein crystallisation screening is the accurate pipetting and positioning of the low volume drops used in hanging and sitting drop setups. Screening the many different conditions under which a protein crystal may form lends itself to automation, since it requires hundreds of similar experiments to be set up to find the few ‘hits’. Automated solutions exist for low volume pipetting, however, the variable viscosities of protein and reservoir/screen solutions present significant challenges for many liquid handling systems. Another challenge is that of drop positioning. The mosquito® (TTP LabTech) offers fast positive displacement pipetting for accurate and reproducible aspiration and dispensing throughout the 50 nL 1.2 μL range, producing CVs of <8% at 50 nL irrespective of viscosity. This, plus its columnar arrangement of pipettes, allows it to automate hanging drop as well as sitting drop set-ups. Mosquito’s micropipettes are also disposable, thus guaranteeing zero cross-contamination where required.

MTases and helicases: a medium-throughput approach to viral protein structures

In the context of the European VIZIER project, our lab is involved in the study of flaviviral methyltransferases (MTase) and helicases (Hel). Flaviviruses are enveloped positive-strand RNA viruses, which code for 3 structural and 7 non-structural (NS) proteins. Among the NS, particularly important are the multifunctional proteins NS3 (protease/helicase) and NS5 (Mtase/RNA-polymerase). Flaviviral Hel participate in RNA replication separating the RNA template and daughter strands. We report here the three-dimensional structure (at 3.1 A resolution) of the NS3 helicase domain (residues 186-619; NS3:186-619) from Kunjin virus, an Australian variant of the West Nile virus. As for homologous helicases, NS3:186-619 is composed of three domains, two of which are structurally related and held to host the NTPase and RTPase active sites. The third domain (C-terminal) is involved in RNA binding/recognition. In addition, we analyzed the activity of the full-length protein and its structure in solution using small angle X-ray scattering (SAXS). Our results show a strong influence of the NS3 protease domain on the helicase activity that can scarcely be explained in term of domains organization and requires further investigations. MTases are involved in the mRNA capping process, resulting in the transfer of methyl groups from the cofactor S-adenosyl-L-methionine (AdoMet) to a capped RNA substrate. We solved the crystal structures of the Wesselsbron virus methyltransferase (MTase) in complex with AdoMet and with both the cofactor and the capped substrate GpppG, at 2.0 A and 1.9 A resolution, respectively. Wesselsbron is an African mosquito-borne Flavivirus belonging to the Yellow Fever virus group that affects animals and human beings. Comparison of the two structures shows that the presence of GpppG stabilizes the N-terminal subdomain, as indicated by the higher B-factor values relative to the other MTases. In order to further characterize the function and catalytic activity of MTase, assays with different substrates are in progress.