P-086 The NEP protein of influenza A virus inhibits replication and transcription of model template RNAs ROSARO BULLIDO, Paulino Gomez-Puertas, Maria Jose Saiz and Agustin Portela. Instituto de Salud Carlos III, Centro Nacional de Biologia Fundamental Madrid Spain. The Nuclear Export Protein (NEP) is a component of the influenza A virus particle. To investigate whether this protein have an effect on virus-specific RNA synthesis, we have measured the expression of an influenza A virus-like CAT RNA in cells synthesising the NEP and the four influenza A virus core proteins (NP, PB1, PB2 and PA) from recombinant plasmids. It was found that synthesis of NEP inhibited, in a dose-dependant manner, the expression of the syntetic RNA. The inhibitory effect on CAT expression mediated by the influenza A virus NEP was not observed in cells expressing the core proteins of an fluenza B virus isolate, a result which demonstrated that the inhibition was not due to a general toxic effect induced by NEP. Analysis of the CAT RNA species that accumulated in cells expressing the recombinant NEP demonstrated that there was an important reduction in the levels of vRNA, cRNA and mRNA molecules. Altogether, the results obtained suggest a regulatory role for NEP during virus-specific RNA synthesis. This finding will be discussed regarding the biological implications for the virus life cycle. P-087 Yeast 20s and 23s RNA narnaviruses exist in vivo as genomic RNA/RNA polymerase complexes in 1:1 stoichiometry ROSA ESTEBAN, Alicia Solórzano, Tsutomu Fujimura Inst. Microbiología Bioquímica CSIC/Univ. Salamanca (Spain). Plus single-stranded RNA viruses 20S RNA (ScNV-20S) and 23S RNA (ScNV-23S) belong to the family Narnaviridae and infest many Saccharomyces cerevisiae laboratory strains. 20S and 23S RNA viruses have no extracellular transmission pathway and do not render any phenotypic change in the host. These RNAs do not encode coat proteins but their RNA-dependent RNA polymerases (RDRPs), p9l and p104, respectively. Since 20S RNA and 23S RNA do not encode coat proteins they are not encapsidated into viral particles. Both RNAs, instead, form ribonucleoprotein complexes with their cognate RNA polymerases. Here we show that these complexes are localized in the cytoplasm. Cytoplasmic localization of the polymerases was demonstrated by immunofluorescence and by fluorescence emitted from green fluorescent protein (GFP)-fused polymerases. These fusion proteins were able to form ribonucleoprotein complexes as the wild type polymerases. Fluorescent observations and cell fractionation experiments suggested that the polymerases were stabilized by complex formation with their viral RNA genomes. Immunoprecipitation experiments with anti-GFP antibodies demonstrated that a single polymerase molecule binds to a single viral RNA genome in the complex. Moreover, the majority (if not all) of 20S RNA and 23S RNA molecules were found to form complexes with their cognate RNA polymerases. Since these viral RNAs were not encapsidated, ribonucleoprotein complex formation with their cognate RNA polymerases appears to be their strategy to survive in the host as persistent viruses. P-088 Reverse genetics for bunyaviridae RAMON FLICK, Johanna Veijola1, Ralf F. Pettersson2, Fredrik Elgh3 1BSL-4, Institute of Infectious Disease Control; 2LICR, Stockholm; 3BSL-4, Institute of Infectious Disease Control. To be able to manipulate the viral RNA segments of different members of Bunyaviridae, we have used an RNA polymerase I expression system. A cDNA consisting of the CAT or GFP ORF in the anti-sense orientation flanked by the 5′ and 3′ terminal noncoding sequences was inserted between an RNA polymerase I promoter and terminator. Following transfection, the cellular RNA polymerase I will generate an artificial vRNA segment without any end modification, e.g. CAP structure, poly(A)-tail. The viral proteins necessary for transcription and replication of this RNA segment were provided by either superinfection, or by plasmids transiently expressing the individual proteins. Using either approach, CAT activity/GFP fluorescence was observed. Recombinant progeny viruses were passaged several times and CAT activity could still be detected after three passages. Different optimization strategies for production of recombinant virus will be discussed. The result suggest that this RNA polymerase I system can be used for the manipulation of the genomes of Bunyaviridae members with a high efficiency, despite the fact that the replication cycle of these viruses takes place exclusively in the cytoplasm. P-089 Opposite translational efficiencies of hepatitis C virus quasispecies 5′ untranslated regions depending on cell type JULIEN LAPORTE, I. Malet, T. Andrieu, H. Agut and A. Cahour CERVI (Virologie), Hôpital Pitié Salpêtrière, Paris, France. Hepatitis C virus (HCV) is the major cause of chronic liver disease worldwide. Infection evolves to chronicity in 80% of cases, and can conduce to cirrhosis and hepatocellular carcinoma. Like others RNA viruses, genetic heterogeneity leads to evolution of quasispecies, closely related but distinct virions, allowing virus to escape immune system. It is now clear that initiation of translation of HCV RNA occurs by a capindependent mechanism mediated by an internal ribosome entry site (IRES) that comprises most of the 5′ untranslated region (UTR) and extends at least to the first 12–30 nucleotides of the coding sequence. Our aim was to establish how 5′ UTR diversity displayed in a viral population could influence IRES functions in vitro and in vivo. We took advantage of studying authentic, biologically derived, HCV 5′ LITRs isolated from a HCV-infected patient serum, using a bicistronic reporter system designed to quantify translational efficiency. Following RTPCR, we were able to isolate 15 variants, with one to four mutations, in a 43 clones population. These quasispecies were tested for translational efficiencies in vitro (reticulocyte lysate) and in vivo, by transfection of three cell types: Hep G2, Jurkat, and Vero. Dual luciferase assays to quantify translational efficiency showed dramatic changes in IRES activity (up to 95%). Furthermore, we observed opposite patterns of IRES activity depending on the cell type transfected. Variants exhibiting a high efficiency in HepG2 and Vero cells were much less active in Jurkat cells and vice versa. Our results highlight the influence of variability in a well-conserved area of HCV genome on the initiation of translation. Opposite effects depending on cell type have conducted us to distinguish between lymphoid (Jurkat) and non-lymphoid (Hep G2) tropism. This could be of interest to study viral compartimentation, interactions of cellular factors on IRES translational activity, and their respective part in viral persistence. P-090 Rotavirus gene expression in monkey kidney cells treated with interferon alpha GARAN MACINUSSON, Alexis Fuentes Uppsala University, Department of Medical Biochemistry and Microbiology. Cells infected with many types of RNA viruses respond to interferon by a general inhibition of protein synthesis. In rotavirus-infected cells, the formation of most cellular polypeptides is inhibited by an, as yet, incompletely characterised mechanism which favours the translation of viral mRNA. The effect of interferon on the expression of the rotavirus strain SA1 1 genes was examined in monkey kidney MA1 04 and Cos7 cells. The cells were treated with interferon alpha (IFN-alpha) 12 h prior to infection, and maintained with interferon throughout the infection period. Virus production, measured by titration, was not significantly inhibited within the range of interferon doses we tested. The synthesis of viral RNA and polypeptides was monitored during an infection period of 8 h. These processes showed similar kinetics in cells treated with IFN-alpha and in control cells. Moreover, the rotavirus induced shut-off of cellular protein synthesis was preserved. The insensitivity of rotavirus infection to IFN-alpha was not caused by a failure of this substance to induce the antiviral state. Treatment of cells with IFN-alpha and double-stranded RNA induced the synthesis of the double-strand RNA activated protein kinase (PKR) which was active, since analysis of eIF2a showed a strongly increased phosphorylation. Thus, rotavirus appears to have a mechanism to evade the effect of PKR, both in the absence and presence of IFN-alpha. P-091 Does influenza C virus CM2 protein form an ion channel? YASUSHI MURAKI, M. Murthy, D. Ogden, A. Hay National Instiute for Medical Research. The CM2 protein (115 amino acids) is an integral membrane protein of the influenza C virus, of unknown function. Certain biochemical characteristics of CM2 are similar to those of influenza A virus M2 and influenza B virus NB proteins, suggesting that CM2 may have a similar function to those of M2 and NB and act as an ion channel during virus uncoating. The M2 protein of influenza A virus (97 amino acids) expressed in mouse erythroleukaemia (MEL) cells forms an H+ selective channel which can selectively transfer protons across membranes with the H+ electrochemical gradient. The NB protein of influenza B virus (100 amino acids), the counterpart of M2, expressed in MEL cells forms channels permeable to H+ and Cl−, indicating that NB also acts as a proton channel but has properties different from those of M2, e.g. H+ permeation requires Na+ for activation and cotransport of Cl- promotes the electroneutral transfer of H+.
In the present study the CM2 protein has been expressed in MEL cells and the channel activity due to expressed CM2 has been studied by whole cell patch clamp measurements under conditions used to identify the M2 and NB channel activities. Preliminary results have shown H+ current in response to transmembrane pH gradients, indicating that CM2 forms an ion channel. The characteristics of the CM2 channel activity are being investigated and comparisons of channel activities between CM2, M2 and NB will be described. P-092 The lyssavirus RNP complex analyzed by two-hybrid and functional reverse genetic approaches: interchanging N and P proteins from different genotypes ELEONORE REAL, Y. Jacob and N. Tordo Lyssavirus Laboratory, Institut Pasteur, 75724, Paris, France. Rabies virus and Mokola virus (Rhabdoviridae family), belong to the Lyssavirus genus comprising 7 genotypes. Mokola virus (genotype 3) is phylogenetically one of the most distant from rabies virus (genotype 1). The Lyssavirus phosphoprotein (P) and nucleoprotein (N), together with RNA polymerase (L) and genomic RNA form a ribonucleoproteic complex (RNP) essential for RNA genome transcription/replication. To localize N and P oligornerization domains, a dual approach combining the yeast two-hybrid system and a reverse-genetic assay based on the reconstitution of a transcriptionaly functional RNP complex, was realised. For each genotype, the yeast two-hybrid system was used with P and N deletion mutants to localize multimerization and N-P interaction domains. We extended this approach by interchanging genotypes and identify interacting regions shared by both genotypes: P self-association required the N-terminal half. Two Nbinding domains were determined on P: a main one (C-terminal halo and a weaker one (N-terminal half). Moreover a yeast reverse two-hybrid screen was used to isolate P N-interaction domain mutants that lost the property to interact. Three lysine (213–216), highly conserved among all Lyssaviruses, are implicated in this strong interaction. The reverse genetic assay demonstrates that heterologous RNP containing combinations of proteins from divergent lyssaviruses can be functional. Interestingly, recognition of heterologous RNPs by the viral polymerase requires that N but not P is homologous. Futhermore, replacement of the conserved three lysines by three glutamic acids results in a loss of function mutant, highlighting the central role of these residues in a functional N–P interaction. P-093 Spontaneous mutant of RSV long strain deficient in soluble g protein showed higher infectivity in cell culture and an animal model CHRISTIAN RODER, Juergen Schwarze, Markus Vossmann, Kerstin ReimersMedical Microbiology and Virology, Ruhr-University Bochum, Bochum. We characterise a spontaneous mutant (RSVmut) of human RSV strain long. The genes encoding F and SH are conserved, while the sequence for the attachment protein G is marked by several hypermutations. Most interestingly, the second start codon responsible for initiation of soluble G is changed to GTG. RSVmut displays growth kinetics different from the wild type strain and it reaches 10 fold higher titres in cell culture. In a murine model RSVmut showed higher infectivity as determined by in-situ hybridisations and viral subcultivating of lung homogenisates. In addition, infection of mice with the mutant strain resulted in increased numbers of lung eosinophils compared to wild type. Airway function at baseline and following methacholine provocation was assessed by barometric body plethysmography, monitoring “enhanced pause” (Penh), a calculated value which correlates closely with lung resistance. Airway function was decreased in RSVmut infected mice, while baseline Penh was increased as well as airway responsiveness to methacholine. RSV deficient of soluble G therefore seems to be more infectious than wild type RSV. RSV G protein beside its role in attachment to the cell surface may have an important function in regulating infectivity and the host response to the viral infection. P-094 Limited durability of anti-E2 protective antibodies in haemodialyzed patients infected with hepatitis G virus MANUEL RODRIGUEZ-IGLESIAS, M.T. Pérez-Gracia, F. Galán, A. Lozano, B.O. Benavides, J.A. Girón-Gonzalez, E. Fernandez Laboratory of Microbiology and Haemodialysis Unit, Puerto Real Univ. Hosp. and Service of Internal Medicine, Puerta del Mar Univ. Hosp., Cádiz, Spain. In order to analyze the evolution of hepatitis G virus (HGV) infection in a cohort of 58 haemodialyzed patients (mean follow-up time: 4.88 + 0.42 years) a prospective observational study was designed. Study subjects were analyzed every twelve months for HGV RNA (by reverse transcriptase-polymerase chain reaction) and antibodies against the envelope protein E2 region of the virus (by ELISA). Twenty nine patients (50%) presented, at the beginning or during evolution, markers of hepatitis G infection. Duration of haernodialysis and coinfection by hepatitis C virus were significantly higher in patients with past infection by HGV, in comparison with patients not presenting HGV markers. Several different patterns of evolution of the HGV infection in haernodialyzed patients were detected: 1) Persistent positive viremia (1/29 cases, 3.4%). 2) Persistent positive anti-E2 antibodies (9/29 cases, 31,0%). 3) Negativization of anti-E2 antibodies with (4/29 cases, 13.8%) or without (9/29 cases, 31,0%) the appearance of viremia. In those cases in which viremia was detected, a subsequent negativization of HGV RNA, without reappearance of anti-E2 antibodies was observed. 4) Infection during follow-up in patients without previous markers of hepatitis G infection, detected either by appearance of viremia (4/29 cases, 13.8%) or by appearance of anti-E2 antibodies (2/29 cases, 6.9%). The existence of patients who lose antibodies during the follow-up requires a reconsideration of the natural history of HGV infection in haernodialyzed patients. This finding raises the possibility of underestimating HGV prevalence in transversal studies, and implies a temporally limited protection in previously infected patients. P-095 Cis - and trans -complementation of classical swine fever virus E2 deletion mutants NICOLAS RUCIALI, J.D. Tratschin, P. Stettler, and M.A. Hofmann Institute of Virology and Immunoprophylaxis, CH-3147 Mittelhäusern, Switzerland. A set of classical swine fever virus (CSFV) cDNA clones with different in-frame deletions in the gene encoding the major structural protein E2 was constructed. After transfection of the respective in vitro transcribed defective RNAs into SK-6 porcine kidney cells all RNAs replicated, although with varying efficiency. Packaging of the defective genomes was mediated by expressing E2 in cis from a second reading frame introduced in the 3′nontranslated region of the respective mutant genomes downstream of an EMCV IRES. Alternatively, E2 was provided in trans by transfecting the defective RNAs into SK-6 cells constitutively expressing E2 from a CMV promoter (SK-6/E2 cells). Defective CSFV RNA partially or completely lacking the E2 gene could be rescued either as virus by ciscomplementation or as defective particles by trans-complementation in SK-6/E2 cells. No recombination between the truncated E2 gene and the complete copy of E2 was observed so far. The bicistronic virus showed similar growth kinetics as the parent CSFV vA187–1 but maximal virus titers reached were lower. Defective particles rescued by trans-complementation were obtained at titers of up to 5.7 log10 TCID50/ml. Such particles are unable to spread and are potential candidates for future vaccine development. P-096 Dissection and functional analysis of the subgenomic promoter of turnip yellow mosaic tymovirus JAN SCHIRAWSKI, Ariane Voyatzakis1 and Anne-Lise Haenni1 1Institut Jacques Monod; 2 Place Jussieu, 75251 Paris, France. Turnip yellow mosaic virus (TYMV) is the type member of the Tymoviridae and infects Cruciferae. It has one single-stranded, positive sense genomic (g) RNA of 6318 nucleotides, that is capped at its 5′ end, folds into a tRNA-like structure at its 3′ end, and carries three open reading frames (ORFs). The third ORF codes for the viral coat protein (CP) and is expressed via the production of a subgenomic (sg) RNA. The sg RNA is a 5′ truncated version of the g RNA containing only the CP ORF. It is presumably produced by the action of the viral RNA dependent RNA polymerase (RdRp), using the g minus strand as template. We have investigated which elements characterize the sg promoter of TYMV and are thus required for the in vivo production of the sg RNA by mutational modification of the RNA genome and subsequent northern blot analysis of RNA production in Arabidopsis thaliana protoplasts. We show that the sg promoter of TYMV contains highly conserved sequence elements, in which single nt exchanges abolish promoter function. A stretch of 500 nt surrounding the conserved sequences contains all the elements necessary for sg RNA production. The sg promoter sequence can be displaced from its natural environment without loss of function. The potential use of the sg promoter of TYMV for the construction of a plant viral vector for A. thaliana is proposed. P-097 A reverse genetic system for coronaviruses S. SIDELL, V. Thiel1, R. Casais2, D. Cavanagh2, P. Britton2 1Institute of Virology, University of Wuerzburg, Germany; 2Division of Molecular Biology, Institute for Animal Health, United Kingdom. The coronavirus genome is a positive strand RNA of extraordinary size and complexity. Here, we describe a reverse genetic system for coronaviruses based upon the generation of synthetic RNA from cloned cDNA copies of coronavirus genomes. First, we constructed a 22.3 kbp cDNA encoding a human coronavirus (HCoV-229E) based autonomously replicating RNA and a single reporter gene, GFP. This cDNA was cloned as a recombinant vaccinia virus genome, wHCoV-rep-1, and used for the in vitro transcription of HCoV-rep1 RNA. When this RNA was transfected into baby hamster kidney fibroblast (BHK-21) cells, a coronavirus specific transcript, which functions as a GFP mRNA was detected. Second, we constructed a cDNA encoding a complete copy of the HCoV-229E genome (27.3 kb), and cloned this cDNA as a recombinant vaccinia virus genome, wHCoV-inf-1. This recombinant vaccinia virus genome was used for the in vitro transcription of HCoV-inf-1 genomic RNA. When HCoV-inf-1 RNA was transfected into human lung fibroblast (MRC-5) cells, a recombinant human coronavirus was recovered. To test whether the described vaccinia virus cloning system is applicable to other coronaviruses we constructed a cDNA encoding a complete copy of the infectious bronchitis virus (IBV) genome and inserted this 27.6 kb cDNA into the vaccinia virus genome. A recombinant vaccinia virus with the full-length IBV-cDNA has been isolated. The system we describe here should find wide application in the analysis of the molecular biology and pathogenesis of RNA viruses in general and coronaviruses in particular. P-098 The sensitivity of polymerase chain reaction (RT-PCR) versus other immunological methods for the detection of hepatitis C virus in human sera MOKHTAR TAHA Assiut University Dept of Pathology Fac of Vet Medicine Assiut 71526 Egypt. A total of 118 hepatitis patients were referred to the Molecular Biology Research Center fo Assiut University, Egypt for confirming the diagnosis of hepatitis C infection. All the patients were screened for the presence of specific antibodies to HCV in their sera using commercially available enzyme immunoassays methods (CAL-TECH Diagnostics, INC). Antibody response was detected in 101 cases (85.5%) while 17 cases (14.4%) were negative. This may suggest that some persons do not have an antibody response to infection with HCV or are infected with a variant of HCV that elicited antibodies unrecognized by the assays used. Meanwhile, sera of all patients were tested for the presence of viral nucleic acids (HCV RNA) using reverse amp Viral RNA Mini Kit (QIAGEN). c-DNA synthesis and nested DNA amplification were done by Omniscript RT Kit and taq PCR Master Mix Kit (Germany). Confirmed positive and negative controls were run with all PCRs to ensure specific amplication. Seventy cases (59.3%) were PCR positive while the rest 48 cases (40.67%) were negative. Interesting, all the Ab seronegative patients proved positive by PCR which may indicate the specificity and sensitivity of PCR for confirming the diagnosis. In addition, the incidence of PCR positivity related to age and sex was recorded. Ninety one patients were 40-55 years old and the percentage positivity was 62.6%. Twenty seven patients were 30–40 years old and the percentage of positivity was 48%. Male patients were 113 and the percentage positivity was 61.06% while female patients were 5 and the percentage positivity was 20%. These data were discussed and interpreted. P-099 Biochemical characterisation of RNA-dependent RNA polymerase (RdRs) activities of hepatitis C virus (HCV), bovine viral diarrhoea virus (BVDV) and GB virus B (GBV-B) JOANNE WAYNE, Malcolm Ellis, Janette Dillon, Umesh Kumar, Pia Thommes Virology Unit, GlaxoWellcome Medicines Research Centre. In order to assess the suitability of BVDV and GBV-B as surrogate viruses for HCV, the biochemical characteristics of their RNA dependent RNA polymerases (RdRp) were compared. The RdRps of HCV, BVDV and GBV-B are encoded by the non-structural gene 5B (NS5B) located at the 3′ end of the open reading frame of each of the viruses. The NS5B genes of HCV, BVDV and GBV-B were expressed as histidine tagged proteins using recombinant baculoviruses and purified by nickel affinity chromatography. In vitro activity was compared on homopolymeric and heteropolymeric templates. All three polymerases demonstrated activity on both types of template. The effects of magnesium and manganese on the polymerases were tested, with manganese having the greater stimulatory effect. The Km values for the nucleotide substrates were similar for all three enzymes. GTP has a stimulatory effect on all three polymerases, with high concentrations becoming inhibitory. In conclusion, the biochemical properties of the three NS5B RdRps are similar enough to expect inhibitors of one to be active against the others, and also demonstrate activity in the BVDV cell culture system and GBV-B small animal model. P-100 Identification of a domain in the respiratory syncytial virus nucleocapsid protein that interacts with the phosphoprotein PAUL YEO, J. Murray, L. B. Rowlands and C. Loney MRC Virology Unit The interaction between the Nucleocapsid (N) protein and the Phosphoprotein (P) of Human Respiratory Syncytial Virus (RSV) was investigated using monoclonal antibodies that prevent the N and P proteins from associating. Seven monoclonal antibodies were partially epitope mapped to sites within the amino and carboxy domains of the N protein. Blocking of P protein binding to N protein was investigated with two monoclonal antibodies, one from each of the domains. The observed blocking of the N-P interaction by the monoclonal antibodies was probably due to steric interference between the competing ligands. This suggests that widely separated regions of the N protein are folded at, or close to, the P protein-binding site giving an indication of the conformational nature of N protein. A panel of overlapping 20-mer peptides was utilized to map a site involved in P protein binding on the N protein. Four peptides that bound to the P protein were identified. One peptide was subsequently demonstrated to block binding in vitro between recombinant RSV N and P proteins. We suggest that contrary to previous reports the amino and carboxy domains of the N protein perform a structural role rather than binding directly to P protein, changes in the termini resulting in conformations that are unfavourable for N-P binding.