Cécile Baronti

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 N-terminal domain of the Arenavirus L protein is an RNA endonuclease essential in mRNA transcription

Arenaviridae synthesize viral mRNAs using short capped primers presumably acquired from cellular transcripts by a ‘cap-snatching’ mechanism. Here, we report the crystal structure and functional characterization of the N-terminal 196 residues (NL1) of the L protein from the prototypic arenavirus: lymphocytic choriomeningitis virus. The NL1 domain is able to bind and cleave RNA. The 2.13 Å resolution crystal structure of NL1 reveals a type II endonuclease α/β architecture similar to the N-terminal end of the influenza virus PA protein. Superimposition of both structures, mutagenesis and reverse genetics studies reveal a unique spatial arrangement of key active site residues related to the PD…(D/E)XK type II endonuclease signature sequence. We show that this endonuclease domain is conserved and active across the virus families Arenaviridae, Bunyaviridae and Orthomyxoviridae and propose that the arenavirus NL1 domain is the Arenaviridae cap-snatching endonuclease.

In vitro antiviral activity of arbidol against Chikungunya virus and characteristics of a selected resistant mutant.

Arbidol (ARB) is an antiviral drug originally licensed in Russia for use against influenza and other respiratory viral infections. Although a broad-spectrum antiviral activity has been reported for this drug, there is until now no data regarding its effects against alphavirus infection. Here, the in vitro antiviral effect of ARB on Chikungunya virus (CHIKV) replication was investigated and this compound was found to present potent inhibitory activity against the virus propagated onto immortalized Vero cells or primary human fibroblasts (MRC-5 lung cells) (IC(50)<10μg/ml). A CHIKV resistant mutant was then selected and adapted to growth in the presence of 30μg/ml ARB in MRC5 cells; its complete sequence analysis revealed a single amino acid substitution (G407R) localized in the E2 envelope protein. To confirm the G407R role in the molecular mechanism of ARB resistance, a CHIKV infectious clone harboring the same substitution was engineered, tested, and was found to display a similar level of resistance. Finally, our results demonstrated the effective in vitro antiviral activity of ARB against CHIKV and gave some tracks to understand the molecular basis of ARB activity.

Understanding the alphaviruses: Recent research on important emerging pathogens and progress towards their control

Abstract The alphaviruses were amongst the first arboviruses to be isolated, characterized and assigned a taxonomic status. They are globally very widespread, infecting a large variety of terrestrial animals, insects and even fish, and circulate both in the sylvatic and urban/peri-urban environment, causing considerable human morbidity and mortality. Nevertheless, despite their obvious importance as pathogens, there are currently no effective antiviral drugs with which to treat humans or animals infected by any of these viruses. The EU-supported project—VIZIER (Comparative Structural Genomics of Viral Enzymes Involved in Replication, FP6 Project: 2004-511960) was instigated with an ultimate view of contributing to the development of antiviral therapies for RNA viruses, including the alphaviruses [Coutard, B., Gorbalenya, A.E., Snijder, E.J., Leontovich, A.M., Poupon, A., De Lamballerie, X., Charrel, R., Gould, E.A., Gunther, S., Norder, H., Klempa, B., Bourhy, H., Rohayemj, J., L’hermite, E., Nordlund, P., Stuart, D.I., Owens, R.J., Grimes, J.M., Tuckerm, P.A., Bolognesi, M., Mattevi, A., Coll, M., Jones, T.A., Åqvist, J., Unger, T., Hilgenfeld, R., Bricogne, G., Neyts, J., La Colla, P., Puerstinger, G., Gonzalez, J.P., Leroy, E., Cambillau, C., Romette, J.L., Canard, B., 2008. The VIZIER project: preparedness against pathogenic RNA viruses. Antiviral Res. 78, 37–46]. This review highlights some of the major features of alphaviruses that have been investigated during recent years. After describing their classification, epidemiology and evolutionary history and the expanding geographic distribution of Chikungunya virus, we review progress in understanding the structure and function of alphavirus replicative enzymes achieved under the VIZIER programme and the development of new disease control strategies.

Picornavirus non-structural proteins as targets for new anti-virals with broad activity

Picornaviridae is one of the largest viral families and is composed of 14 genera, six of which include human pathogens. The best known picornaviruses are enteroviruses (including polio, PV, and rhinoviruses), foot-and-mouth disease virus (FMDV), and hepatitis A virus (HAV). Although infections often are mild, certain strains may cause pandemic outbreaks accompanied with meningitis and/or paralysis. Vaccines are available for PV, HAV and FMDV. When the oral vaccines are given to immunocompromised individuals, they may be chronically infected, and remain secretors of vaccine-derived variants of virus for years. There is no effective prophylaxis available for these or other picornaviruses. So far, only the 3C protease from viruses in three genera has been fully characterized as an anti-viral target, whereas the mode of action of compounds targeting other non-structural proteins have remained largely unaddressed. Within the EU-supported FP6 project-VIZIER (Comparative Structural Genomics of Viral Enzymes Involved in Replication), the non-structural proteins were studied to identify conserved binding sites for broadly reactive anti-virals. The putative 2C helicase from echovirus-30 was shown to form ring-shaped hexamers typical for DNA-encoded SF3 helicases, and to possess ATPase activity. Hexamer formation of 2C from enterovirus 76 was in vitro shown to be dependent on the 44 N-terminal residues. Crystal structures of three enterovirus 3C proteases were solved and shown to be similar to those of other picornaviruses. A new binding site of VPg to the bottom of the thumb domain of CV-B3 3D polymerase was identified as a potential target. Broad anti-enterovirus compounds against 2C and 3A proteins were also identified, including thiazolobenzimidazoles (active against 2C) and TTP-8307 (targeting 3A). There is a need for more potent inhibitors against PV and other picornaviruses, which are potential silent reservoirs for re-emerging PV-like disease.

Assay optimization for molecular detection of Zika virus

Abstract Objective To examine the diagnostic performance of real-time reverse transcription (RT)-polymerase chain reaction (PCR) assays for Zika virus detection. Methods We compared seven published real-time RT–PCR assays and two new assays that we have developed. To determine the analytical sensitivity of each assay, we constructed a synthetic universal control ribonucleic acid (uncRNA) containing all of the assays’ target regions on one RNA strand and spiked human blood or urine with known quantities of African or Asian Zika virus strains. Viral loads in 33 samples from Zika virus-infected patients were determined by using one of the new assays. Findings Oligonucleotides of the published real-time RT–PCR assays, showed up to 10 potential mismatches with the Asian lineage causing the current outbreak, compared with 0 to 4 mismatches for the new assays. The 95% lower detection limit of the seven most sensitive assays ranged from 2.1 to 12.1 uncRNA copies/reaction. Two assays had lower sensitivities of 17.0 and 1373.3 uncRNA copies/reaction and showed a similar sensitivity when using spiked samples. The mean viral loads in samples from Zika virus-infected patients were 5 × 104 RNA copies/mL of blood and 2 × 104 RNA copies/mL of urine. Conclusion We provide reagents and updated protocols for Zika virus detection suitable for the current outbreak strains. Some published assays might be unsuitable for Zika virus detection, due to the limited sensitivity and potential incompatibility with some strains. Viral concentrations in the clinical samples were close to the technical detection limit, suggesting that the use of insensitive assays will cause false-negative results.

Nonstructural NS1 proteins of several mosquito-borne Flavivirus do not inhibit TLR3 signaling.

Flaviviruses are single-stranded positive RNA viruses that replicate through double stranded RNA (dsRNA) intermediates. These dsRNA may be recognized as pathogen-associated molecular patterns by cellular receptors including membrane-bound Toll-like receptor 3 (TLR3) and cytosolic helicases RIG-I and MDA5. dsRNA stimulation results in signaling cascades converging to activation of interferon (IFN) regulatory factor 3 (IRF3) and to transcriptional activation of several interferon stimulated genes, including IFNss and inflammatory cytokines. There are conflicting reports concerning the ability of West Nile virus to counteract TLR3 signaling. In our analyses, transiently or stably expressed NS1 proteins from two West Nile viruses, two dengue 2 viruses and a yellow fever virus failed to inhibit TLR3 signaling in two different mammalian cell lines. Moreover, using siRNA inhibiting the helicase signalization pathway, we show that viral infection did not impede TLR3 responses to poly(I:C). We conclude that NS1 proteins from distinct mosquito-borne flaviviruses do not inhibit TLR3 signaling.

Simple reverse genetics systems for Asian and African Zika viruses

Zika virus (ZIKV), a typical example of a re‐emerging pathogen, recently caused large outbreaks in Pacific islands and the Americas, associated with congenital diseases and neurological complications. Deciphering the natural history, ecology and pathophysiology of this mosquito-borne pathogen requires effective reverse genetics tools. In the current study, using the bacterium-free ‘Infectious Subgenomic Amplicons’ (ISA) method, we generated and made available to the scientific community via the non-profit European Virus Archive collection, two simple and performing reverse genetics systems for ZIKV. One is based on an Asian ZIKV strain belonging to the outbreak lineage (French Polynesia 2013). The second was designed from the sequence of a low-passaged ZIKV African strain (Dakar 1984). Using the ISA procedure, we derived wild-type and a variety of specifically engineered ZIKVs in days (intra- and inter-lineage chimeras). Since they are based on low-passaged ZIKV strains, these engineered viruses provide ideal tools to study the effect of genetic changes observed in different evolutionary time-scales of ZIKV as well as pathophysiology of ZIKV infections.

Molecular Epidemiological Analysis of Dengue Fever in Bolivia from 1998 to 2008

Dengue fever was first recognized in Bolivia in 1931. However, very limited information was available to date regarding the genetic characterization and epidemiology of Bolivian dengue virus strains. Here, we performed genetic characterization of the full-length envelope gene of 64 Bolivian isolates from 1998 to 2008 and investigated their origin and evolution to determine whether strains circulated simultaneously or alternatively, and whether or not multiple introductions of distinct viral variants had occurred during the period studied. We determined that, during the last decade, closely related viruses circulated during several consecutive years (5, 6, and 6 years for DENV-1, DENV-2, and DENV-3, respectively) and the co-circulation of two or even three serotypes was observed. Emergence of new variants (distinct from those identified during the previous episodes) was identified in the case of DENV-1 (2007 outbreak) and DENV-2 (2001 outbreak). In all cases, it is likely that the viruses originated from neighboring countries.

Comparative Production Analysis of Three Phlebovirus Nucleoproteins under Denaturing or Non-Denaturing Conditions for Crystallographic Studies

Nucleoproteins (NPs) encapsidate the Phlebovirus genomic (-)RNA. Upon recombinant expression, NPs tend to form heterogeneous oligomers impeding characterization of the encapsidation process through crystallographic studies. To overcome this problem, we set up a standard protocol in which production under both non-denaturing and denaturing/refolding conditions can be investigated and compared. The protocol was applied for three phlebovirus NPs, allowing an optimized production strategy for each of them. Remarkably, the Rift Valley fever virus NP was purified as a trimer under native conditions and yielded protein crystals whereas the refolded version could be purified as a dimer. Yields of trimeric Toscana virus NP were higher from denaturing than from native condition and lead to crystals. The production of Sandfly Fever Sicilian virus NP failed in both protocols. The comparative protocols described here should help in rationally choosing between denaturing or non-denaturing conditions, which would finally result in the most appropriate and relevant oligomerized protein species. The structure of the Rift Valley fever virus NP has been recently published using a refolded monomeric protein and we believe that the process we devised will contribute to shed light in the genome encapsidation process, a key stage in the viral life cycle.