Unit: Molecular Genetics of Respiratory Tract Viruses - URA 1966 CNRS
Director: van der WERF Sylvie
The activities of the unit are focused on molecular genetics of positive and negative strand RNA viruses (influenza viruses, SARS coronavirus, hepaciviruses). They include analysis of the molecular mechanisms of expression and replication of the viral genomes and of their use as expression vectors, study of virus host interactions and of the evolution and genetic variability of the viral genomes, as well as the development of vaccines and diagnostic tools, for instance for SARS.
I. "MOLECULAR GENETICS OF RESPIRATORY VIRUSES" directed by Sylvie van der WERF
a) Molecular genetics of influenza virus transcription/réplication complexes ( Nadia NAFFAKH, Bernadette CRESCENZO-CHAIGNE, Emmanuel DOS-SANTOS, Pierre DUARTE-RODRIGUES, Monika MARASESCU)
Les séquences des extrémités des segments génomiques sont partiellement conservées pour les 3 types de virus grippaux A, B et C et constituent le promoteur pour l'initiation de la transcription et de la réplication par le complexe polymérase du virus. Nous avions montré précédemment à l'aide d'un système de transcription/réplication transitoire que la nature de certains nucléotides et la stabilité de la structure secondaire des extrémités des segments
The sequences at the extremities of the viral gnome segments are partially conserved for influenza viruses type A, B and C. They constitute the promoter for intiation of transcription and replication. By making use of a transient transcription/replication system we had previously shown that the nature of the nucleotides and the stability of the secondary structure at the extremities of the viral RNA are important determinants of type specificity. Analysis of the effect of mutations of these determinants when introduced by reverse genetics in the context of an infectious virus allowed us to confirm the role of these determinants for viral multiplication and to underline the importance of the 5' extremity for recognition by the polymerase complex.
To complete our analysis of the determinants of host specificity harbored by the polymerase complexes derived from avian or human viruses in the context of avian versus mammalian cells, we performed the molecular cloning of the avian polI polymerase and developped a reverse genetics system for influenza viruses in avian cells. This system should allow us to study the functional interactions of the polymerase complex with host proteins in avian cells. With the aim to identify cellular interactors of the polymerase complex, we pursued the analysis of influenza viruses expressing a tagged PB2 (one of the subunits of the polymerase complex).
b) Packaging of the viral RNA segments (Nicolas ESCRIOU, Nadia NAFFAKH, India LECLERCQ, Sylvie GERBAUD, Emmanuel DOS-SANTOS, Monika MARASESCU)
Using various approaches we confirmed the importance for viral multiplication of sequences spanning the extremities of the coding sequences in the case of the NA and PB2 segments. By making use of pseudo-viral RNAs harboring reporter genes we further established the role of these sequences for packaging of the RNA segments during the morphogenesis of the viral particles and mapped the signals involved more precisely.
c) Transfectant influenza viruses with a dicistronic segment as vectors
(Nicolas ESCRIOU, Sylvie GERBAUD)
Transfectant influenza viruses harboring a dicistronic segment by means of an internal duplication of the 3' non-coding sequences were produced by reverse genetics. These were shown to be able to induce a humoral or T-cytotoxic immune response towards the heterologous antigens. We pursued our studies in order to generalize this approach to the other viral segments as well as to the expression of other heterologous sequences (fluorescent markers, HBs antigen, LCMV NP).
Expression of structural proteins of SARS-CoV (Nicolas ESCRIOU, Benoît CALLENDRET, Valérie LORIN)
After molecular cloning of the sequences corresponding to the various structural proteins of the SARS coronavirus their expression by means of different prokaryotic and eukaryotic vectors was achieved with the aim to develop vaccine and/or diagnostic tools based on the recombinant proteins. We also contributed to the evaluation of animal models of infection by the SARS-CoV.
c) National Influenza Center (Northern-France) and WHO Collaborating Center for Reference and Research on influenza viruses and other respiratory viruses (Sylvie van der WERF, Jean-Claude MANUGUERRA, Jean-Thierry AUBIN, Ana-Maria BURGUIERE, Maryse TARDY-PANIT, Saliha AZEBI, Frédérique CUVELIER, Patricia JEANNIN, Valérie LORIN, Claudine ROUSSEAUX)
As a Reference Center, the unit contributes to the surveillance of influenza and other respiratory viruses at the national level through the RENAL network of hospital laboratories and the GROG network of sentinel general practitioners and pediatricians, as well as on the international, particularly european, level by electronic exchange of data within the European Influenza Surveillance Scheme (EISS) and by piloting the EUROGROG network. Isolation, identification and antigenic characterization of respiratory viruses from specimens from cases of influenza-like illness are carried out. The unit also performs the antigenic and genetic characterization of viruses isolated from swine, horses or from wild birds. Large-scale sequencing should provide a spatio-temporal analysis of the evolution of human and animal (porcine, equine, avian) influenza viruses as well as information about the frequency of reassortments between various lineages.
In relation with the recent occurrence of human cases of infection by avian H5N1 viruses that circulate in Asia the unit has been part of the WHO reference laboratories for H5 infleunza viruses. In this context the unit isolated and characterized H5N1 viruses from different bird species specimens that were provided by the Pasteur Institute in Pnomh Penh, Cambodia. Differnet formats of RT-PCR for specific detection of these H5 viruses were developped and implemented for the evaluation of suspect cases in France.
II. "VIRUS des HÉPATITES", directed by Annette MARTIN
Study of in vitro and in vivo replication of hepatitis C and GB-B viruses. (Annette MARTIN, Lisette COHEN, David GHIBAUDO, Christophe CHEVALIER, Lucile WARTER)
Studies of hepatitis C virus (HCV) have been hindered by the lack of in vitro cell culture systems able to support efficient replication of HCV and the increasing unavailability of chimpanzees, the only experimental animal model susceptible to HCV. GB virus B (GBV-B), which is phylogenetically the most closely related virus to HCV, is an interesting surrogate model, in that it causes hepatitis in small New World primates such as tamarins and marmosets and replicates efficiently in primary hepatocytes of these species.
We are interested in structural proteins of these viruses and have shown that GBV-B capsid protein can assemble with HCV envelope glycoproteins to form virus-like particles, opening the way to the creation of chimeric GBV-B viruses bearing HCV envelope proteins, hence HCV antigenicity. Following the identification of a new p13 protein in GBV-B that shares partial homology with HCV p7 protein, we undertook to study its potential function as an ion channel. The role of this type of proteins in the infectious cycle of these viruses is being explored by a reverse genetics approach.
We are developing and using HCV and GBV-B subgenomic RNAs (replicons), that are capable of autonomous replication in cell culture, to investigate molecular requirements for polyprotein processing and RNA replication. Using HCV replicons, we identified small interfering RNAs (siRNAs) that target a functional domain of the HCV internal ribosome entry segment (IRES) and inhibit HCV RNA replication in cell culture (coll. Lab. "Enterovirus et Stratégies Antivirales", I.P.). The engineered, chimeric GBV-B which genome contains the corresponding HCV IRES domain allows us to study the antiviral potential of these siRNAs in vitro and in vivo in small primates.
Keywords: influenza, hepatitis C virus, SARS coronavirus, replication, packaging, vaccine, vector, virology, molecular epidemiology, animal model