Interplay between a New HNF3 and the HNF1 Transcriptional Factors in the Duck Hepatitis B Virus Enhancer

AbstractWe identified a new hepatocyte nuclear factor 3 (HNF3) binding site in the DHBV enhancer. This site is close to the hepatocyte nuclear factor 1 (HNF1) binding site, responsible for most of the enhancing activity. No differences in the migrating properties were found between this new site and the two other HNF3 sites recently described in this enhancer. Factor HNF1 strongly inhibits binding of the HNF3 factor in this newly characterized site. The two factors were never detected simultaneously on the DNA fragment, even when their respective concentrations were modified. Competition persisted after enlarging by 5 and 10 nucleotides the space between the two sites. On the contrary, when the HNF3 binding site was changed into the perfect consensus site, binding of the HNF3 factor was not inhibited any longer by HNF1 and a supershift, corresponding to the binding of both factors, was observed. Thus a limited mismatching appears to modulate the interaction between transcriptional proteins and DNA and allows a second transcriptional protein to interplay with the former one

Non coding extremities of the seven influenza virus type C vRNA segments: effect on transcription and replication by the type C and type A polymerase complexes

<p>Abstract</p> <p>Background</p> <p>The transcription/replication of the influenza viruses implicate the terminal nucleotide sequences of viral RNA, which comprise sequences at the extremities conserved among the genomic segments as well as variable 3' and 5' non-coding (NC) regions. The plasmid-based system for the <it>in vivo </it>reconstitution of functional ribonucleoproteins, upon expression of viral-like RNAs together with the nucleoprotein and polymerase proteins has been widely used to analyze transcription/replication of influenza viruses. It was thus shown that the type A polymerase could transcribe and replicate type A, B, or C vRNA templates whereas neither type B nor type C polymerases were able to transcribe and replicate type A templates efficiently. Here we studied the importance of the NC regions from the seven segments of type C influenza virus for efficient transcription/replication by the type A and C polymerases.</p> <p>Results</p> <p>The NC sequences of the seven genomic segments of the type C influenza virus C/Johannesburg/1/66 strain were found to be more variable in length than those of the type A and B viruses. The levels of transcription/replication of viral-like vRNAs harboring the NC sequences of the respective type C virus segments flanking the CAT reporter gene were comparable in the presence of either type C or type A polymerase complexes except for the NS and PB2-like vRNAs. For the NS-like vRNA, the transcription/replication level was higher after introduction of a U residue at position 6 in the 5' NC region as for all other segments. For the PB2-like vRNA the CAT expression level was particularly reduced with the type C polymerase. Analysis of mutants of the 5' NC sequence in the PB2-like vRNA, the shortest 5' NC sequence among the seven segments, showed that additional sequences within the PB2 ORF were essential for the efficiency of transcription but not replication by the type C polymerase complex.</p> <p>Conclusion</p> <p>In the context of a PB2-like reporter vRNA template, the sequence upstream the polyU stretch plays a role in the transcription/replication process by the type C polymerase complex.</p

Differential Effect of Nucleotide Substitutions in the 3′ Arm of the Influenza A Virus vRNA Promoter on Transcription/Replication by Avian and Human Polymerase Complexes Is Related to the Nature of PB2 Amino Acid 627

AbstractUsing a genetic system that allows the in vivo reconstitution of active ribonucleoproteins, the ability to ensure transcription/replication of a viral-like reporter RNA harboring the G3 → A3, U5 → C5, and C8 → U8 mutations (triple 3-5-8 mutations) in the 3′ arm of the promoter was examined with core proteins from human or avian strains of influenza A viruses. The efficiency of transcription/replication of the viral-like RNA with the triple 3-5-8 mutations in COS-1 cells was found to be slightly decreased as compared to the wild-type RNA when the polymerase was derived from a human virus. In contrast, it was found to be considerably increased when the polymerase was derived from an avian virus, in agreement with published observations using the avian A/FPV/Bratislava virus (G. Neumann and G. Hobom, 1995, J. Gen. Virol. 76, 1709–1717). This increase could be attributed to the compensation of the defect in transcription/replication activity in the COS-1 mammalian cell line due to the presence of a glutamic acid at PB2 residue 627, characteristic of avian strains of influenza viruses. Our results thus suggest that PB2 and/or cellular proteins interacting with PB2 could be involved in RNA conformational changes during the process of transcription/replication

Nucleotides at the extremities of the viral RNA of influenza C virus are involved in type-specific interactions with the polymerase complex

International audienceInfluenza A and C viruses share common sequences in the terminal noncoding regions of the viral RNA segments. Differences at the 5h-and 3h-ends exist, however, that could contribute to the specificity with which the transcription/replication signals are recognized by the cognate polymerase complexes. Previously, by making use of a transient expression system for the transcription and replication of a reporter RNA template bearing either type A or type C extremities, it was shown that a type C RNA template is transcribed and replicated with equal efficiency by either the type A or the type C polymerase complex, whereas a type A RNA template is less efficiently transcribed and replicated by the type C polymerase complex than by the type A complex. To explore the contribution of the nucleotides at the extremities of the RNAs to this type-specificity, the effect of mutations introduced either alone or in combination at nucleotide 5 at the 3h-end and at nucleotides 3h, 6h or 8h at the 5h-end of type A or C RNA templates were studied in the presence of either the type A or the type C polymerase complex. The results indicate that the nature of nucleotides 5 and 6h contribute to type-specificity. Moreover, these results underline the importance of the base pairing between nucleotide 3h and 8h at the 5h-end of the RNA. Thus, it could be suggested that the nature of the nucleotides as well as the stability of the secondary structure at the extremities of the viral RNA are important determinants of type-specificity

Rescue of Influenza C Virus from Recombinant DNA▿

The rescue of influenza viruses by reverse genetics has been described only for the influenza A and B viruses. Based on a similar approach, we developed a reverse-genetics system that allows the production of influenza C viruses entirely from cloned cDNA. The complete sequences of the 3′ and 5′ noncoding regions of type C influenza virus C/Johannesburg/1/66 necessary for the cloning of the cDNA were determined for the seven genomic segments. Human embryonic kidney cells (293T) were transfected simultaneously with seven plasmids that direct the synthesis of each of the seven viral RNA segments of the C/JHB/1/66 virus under the control of the human RNA polymerase I promoter and with four plasmids encoding the viral nucleoprotein and the PB2, PB1, and P3 proteins of the viral polymerase complex. This strategy yielded between 103 and 104 PFU of virus per ml of supernatant at 8 to 10 days posttransfection. Additional viruses with substitutions introduced in the hemagglutinin-esterase-fusion protein were successfully produced by this method, and their growth phenotype was evaluated. This efficient system, which does not require helper virus infection, should be useful in viral mutagenesis studies and for generation of expression vectors from type C influenza virus

Comparative Analysis of the Ability of the Polymerase Complexes of Influenza Viruses Type A, B and C to Assemble into Functional RNPs that Allow Expression and Replication of Heterotypic Model RNA Templates In Vivo

International audienceInfluenza viruses type A, B, and C are human pathogens that share common structural and functional features, yet they do not form natural reassortants. To determine to what extent type-specific interactions of the polymerase complex with template RNA contribute to this lack of genotypic mixing, we investigated whether homotypic or heterotypic polymerase complexes support the expression and replication of model type A, B, or C RNA templates in vivo. A plasmid-based expression system, as initially described by Pleschka et al. [(1996) J. Virol. 70, 4188–4192] for influenza A virus, was developed for influenza viruses B/Harbin/7/94 and C/Johannesburg/1/66. The type A core proteins expressed heterotypic model RNAs with similar efficiencies as the homotypic RNA. The influenza B virus model RNA was efficiently expressed by all three types of polymerase complexes. Although no functional polymerase complex could be reconstituted with heterotypic P protein subunits, when the influenza A virus P proteins were expressed together with heterotypic nucleoproteins, significant, albeit limited, expression of RNA templates of all influenza virus types was detected. Taken together, our results suggest that less strict type-specific interactions are involved for the polymerase complex of influenza A compared with influenza B or C viruses

Genetic analysis of the compatibility between polymerase proteins from human and avian strains of influenza A viruses

International audienceIn order to determine how efficiently the polymerase proteins derived from human and avian influenza A viruses can interact with each other in the context of a mammalian cell, a genetic system that allows the in vivo reconstitution of active ribonucleoproteins was used. The ability to achieve replication of a viral-like reporter RNA in COS-1 cells was examined with heterospecific mixtures of the core proteins (PB1, PB2, PA and NP) from two strains of human viruses (A/Puerto Rico/8/34 and A/Victoria/3/75), two strains of avian viruses (A/Mallard/NY/6750/78 and A/FPV/-Rostock/34), and a strain of avian origin (A/Hong Kong/156/97) that was isolated from the first human case of H5N1 influenza in Hong Kong in 1997. In accordance with published observations on reassortant viruses, PB2 amino acid 627 was identified as a major determinant of the replication efficiency of heterospecific complexes in COS-1 cells. Moreover, the results showed that replication of the viral-like reporter RNA was more efficient when PB2 and NP were both derived from the same avian or human virus or when PB1 was derived from an avian virus, whatever the origin of the other proteins. Furthermore, the PB1 and PB2 proteins from the A/Hong-Kong/156/97 virus exhibited intermediate properties with respect to the corresponding proteins from avian or human influenza viruses, suggesting that some molecular characteristics of PB1 and PB2 proteins might at least partially account for the ability of the A/Hong Kong/156/97 virus to replicate in humans

Growth kinetics of rescued influenza viruses.

<p>(A) Type C rescued viruses. Infections were performed in SK cells infected at a low m.o.i. (0.001 PFU/cell) in the presence of 0.25 µg/ml of TPCK-trypsin. (B) Type A rescued viruses. Infections were performed in MDCK cells infected at a low m.o.i. (0.001 PFU/cell) in the presence of 1 µg/ml of TPCK-trypsin. Titers were determined by standard plaque assays in duplicate. Results are representative of two independent experiments. </p

Evaluation of mRNA/vRNA and NS1/NP ratios during single-cycle infection with rescued influenza A viruses.

<p>(A) Quantification of vRNA and mRNA in infected cells. Six hours after MDCK infection with rescued influenza A viruses at high m.o.i. (2 PFU/cell), viral vRNA and mRNA levels for each segment were evaluated by specific two-step RT-qPCRs previously described [23]. Results were expressed as the mean of mRNA/vRNA Cp (crossing-point) ratios calculated on data obtained from 12 independent qPCRs (corresponding to two infections, two independent cDNA syntheses per infection and three independent qPCRs for each cDNA). Statistics were performed using the ANOVA test. Black bars: wild-type 5’A/3’A; hatched bars: 5’A/3’C(C5U); grey bars: 5’C/3’C-proxA. (B) Levels of NP and NS1 proteins after infection with the rescued influenza A viruses. Four hours after MDCK infection at a high m.o.i. (2 PFU/cell), cell lysates were analyzed by Western-blot for viral NS1 and NP proteins and for β-actin as cellular control. Following chemiluminescence acquisition with a G. Box (SYNGENE, Cambridge, UK), band densities for viral proteins were determined using GeneTools software (SYNGENE, Cambridge, UK), and were used to calculate NS1/NP ratios for each virus. Due to the high levels of viral protein expression during infection, protein extracts were diluted in uninfected cellular extracts prior to electrophoresis. Results are from one representative assay out of three independent infections.</p