The Provisional Unit “Antiviral Strategies” (UPSA) has a long lasting involvement in basic and translational research on rabies and its etiologic agents, the rabies virus (RABV) and the other members of the Lyssavirus genus. More recently UPSA has extended its interest towards emerging members of the Bunyaviridae family, particularly in the genera Hantavirus (up to BSL3 level) and Nairovirus (up to BSL4 level).
Antiviral strategies against NSRV:
In the recent years, we focused on antiviral approaches, mostly targeting the transcription/replication machinery (RNP), which is similar among Negative Strand RNA Viruses (NSRV). The RNP is composed of an encapsidated template (RNA genome / nucleoprotein N) and an enzymatic complex (RNA-dependent RNA polymerase L and, in the case of lyssaviruses, its cofactor the phosphoprotein P). We study in parallel NSRV provoking severe (sometimes neglected) human pathologies to favour the identification of wide-spectrum antiviral drugs without (or with low) cellular toxicity. We also look for antivirals blocking other steps of the viral cycle, such as cell entry. Our strategy combines: (1) a “cognitive” approach by investigation of the structure/function relationships within the RNP and its interactions with host factors in order to identify targets for specific inhibitors; (2) a “random” approach using high throughput (HTP) screening methods (2-hybrid, phage display) to design interfering peptides (or molecules) that inhibit the polymerase activity.
The “cognitive” approach: identifying and destabilizing functional interactions in the transcription/replication complex of NSRV:
The polymerase L of NSRV is multifunctional (RNA synthesis, capping, polyadenylation…) and composed of autonomous active modules interacting each other and with cellular partners. In collaboration with P.O. Vidalain (Unit Viral Genomic and Vaccination) we look for these partners using 2-hybrid screening of cDNA libraries. The functional role of the identified partners during viral cycle is further explored using over-expression or inhibition with siRNA in cells hosting full infectious virus or pseudotypes/minireplicon to address specific steps of viral infection. The phosphoprotein P has two key functions during replication: it mediates the physical link between the L polymerase and the N-RNA template and interacts with monomeric unbound N (N°) to keep it available for encapsidation of the nascent viral RNA genome. In collaboration with R. Ruigrok (EMBL, Inst. Virologie, Grenoble) we showed that N-residues 4-40 and 40-70 of the RABV-P are binding to N° (Mavrakis et al, 2004) and L (Castel et al, 2009), respectively. Thus, we designed competitive peptides mimicking different lengths of this N-extremity and showed their rapid, strong and long lasting inhibitory effect on cells hosting RABV minireplicon (expressing a reporter gene) or infected with RABV or other Lyssaviruses (Castel et al, 2009; see Figure). The delineation of the inhibitory domain(s) and their co-crystallisation with N° may lead to drug design of smaller molecules.
The “random” approach: screening combinatorial libraries to identify antiviral drugs:
We follow several strategies for HTP screening of random libraries for antiviral discovery. From classical libraries of chemical compounds tested against RABV minireplicons or RABV infected cells to more sophisticated systems. For example, phage-display libraries presenting a large diversity (107 to 109 ) of random peptides (12 to 20 aa) were screened for binding to the N-RNA template of RABV and other NRSV such as respiratory syncytial virus (Castel et al, 2011). The purpose was to select wide-spectrum inhibitory peptides with similar affinity for different NRSV N-RNA complexes. In addition, combinatorial libraries mimicking natural bioactive peptides (conotoxins, apidaecins, lebocins) were screened by 2-hybrid for their affinity to RABV P protein. Validation of the binding candidates using minireplicon or infection assays identified 4 peptides showing >80% RABV inhibition. The destabilized protein/protein interaction was further identified by SELDI-Tof (Réal et al., 2004). Finally, dermaseptines are 28-34 aa peptides secreted by the skin of amphibians, that destabilize membranes and have a broad antimicrobial activity against yeast, bacteria, fungi, parasites and enveloped viruses like HSV and HIV. In collaboration with K. Hani (Sousse Medical Faculty, Tunisia) we showed a strong in vitro anti-RABV effect of dermaseptines S4 from Pyllomedusa sauvagei. Most of the inhibitory potential concentrates in the 5 NH2-aa. We also demonstrated an in vivo anti-RABV effect of several mutants against intra-muscular infection of mice. The dissection of S4 is currently performed to reduce the dermaseptine to its smallest effective peptide. Recently, UPSA has extended its interest towards Hantaviruses and Nairoviruses. In this context, UPSA is leading the WHO Collaborative Centre for “Arboviruses and Viral Haemorrhagic Fevers”, a consortium including two other Pasteurian structures: (1) the Unit “Molecular Interactions Flavivirus-Hosts” (Dr. P. Desprès); (2) the Laboratory “Urgent Response to Biological Threats” (Dr. J.-C. Manuguerra). In addition, UPSA participates in the OIE Reference Laboratory (Head: M. Bouloy) for “Rift Valley Fever Virus (RVFV) and Crimean Congo Haemorrhagic Fever Virus” (CCHFV).
Hantaviruses and related human diseases:
Hantaviruses are hosted and transmitted by mammals (rodents, insectivores, bats). They have co-evolved with their reservoirs for which they appear non pathogenic. Pathogenicity is exacerbated when the virus is jumping across the species barrier to infect humans. Hantaviruses transmitted by Old World rodents provoke Haemorrhagic Fever with Renal Syndrome (HFRS), from mild (Puumala virus – PUUV; Viel et al, 2011) to more severe (Hantaan virus – HTNV) forms. Hantaviruses carried by New World rodents provoke Haemorrhagic Fever with Cardio-Pulmonary Syndrome (HCPS), which is very severe (Sin Nombre Virus – SNV). No human case has been associated so far with hantaviruses carried by insectivores or bats. Our objectives are (1) to elucidate the molecular basis explaining why hantaviruses become pathogenic when jumping to human and (2) to explore hantavirus diversity in nature and understand the dynamics of their circulation in reservoirs and transmission to human. This research is notably supported by EU-FP7 programs: (1) “Antigone” (2011-16) is devoted at elucidating the fundamental mechanisms governing the jump across the species barrier; (2) “Empirie” (2008-13) and “Edenext” (2011-14) are intended to explore the diversity and dynamics of viruses in their natural reservoirs, to evaluate the risk and to be better prepared in case of human transmission.
Jumping across the species barrier:
To understand the molecular basis involved in crossing the species barrier and inducing pathogenicity we are exploring in two directions: (1) comparing the same virus in two species where it is differently pathogenic (ex: PUUV in rodents and humans); (2) comparing two viruses showing different pathogenesis in humans (ex : PUUV pathogenic and Tula virus – TULV non pathogenic). Using 2-hybrid technology, we are screening comparatively human and rodent cDNA libraries, looking for cell partners of hantavirus proteins that could explain their differential pathogenicity. Libraries from genuine reservoirs (Myodes, Apodemus, etc) can be generated whenever necessary. In parallel, we study the infection of various cell types from blood donors or reservoirs to explore if pathogenicity might result from the inhibition/activation of innate/adapted immune responses. The molecular mechanisms underneath infection are further dissected in primary cells (coll. C. Drosten, R. Ulrich, Germany) or in cell lines relevant for hantavirus infection: endothelial, respiratory, derived from reservoirs…. Reagents necessary for cell infection (MAbs, isolates) are developed or acquired in collaboration with Scandinavian teams (A. Plyusnin, O. Vapalahti, Haartman Inst. Finland; A. Lundkvist, Karolinska Inst., Sweden). Animal models up to wild reservoirs are handled in collaboration with the Virology Unit in the Anses Laboratory, Lyon (P. Marianneau), which enjoys a sophisticated BSL3 animal facility.
Surveillance and dynamics of Hantaviruses and Nairoviruses in their reservoir hosts:
In the fields, we maintain active surveillance programs of animal reservoirs in France and abroad both to discover new hantaviruses or nairoviruses (like CCHFV) and to understand the relationships between the virus dynamics in animal populations and the occurrence of human cases. Recombinant proteins expressed in drosophila S2 cells (coll. P. Desprès) are produced for large serology screening by ELISA. For molecular study, the re-sequencing microarray PathogenID-v3.0 (coll. Institut Pasteur), largely enriched in bunyavirus sequences, is used to screen pool of animals from different countries (Filippone et al, 2013).