Influenza A viruses control expression of proviral human p53 isoforms p53ß and Delta133p53α.

Previous studies have described the role of p53 isoforms, including p53ß and Δ133p53α, in the modulation of the activity of full-length p53, which regulates cell fate. In the context of influenza virus infection, an interplay between influenza viruses and p53 has been described, with p53 being involved in the antiviral response. However, the role of physiological p53 isoforms has never been explored in this context. Here, we demonstrate that p53 isoforms play a role in influenza A virus infection by using silencing and transient expression strategies in human lung epithelial cells. In addition, with the help of a panel of different influenza viruses from different subtypes, we also show that infection differentially regulates the expressions of p53ß and Δ133p53α. Altogether, our results highlight the role of p53 isoforms in the viral cycle of influenza A viruses, with p53ß and Δ133p53α acting as regulators of viral production in a p53-dependent manner.

Characterization of a key residue for hyperfusogenic phenotype in human parainfluenza virus type 2 (hPIV-2) fusion glycoprotein.

Human parainfluenza viruses (hPIV) are pathogens responsible for upper and lower respiratory tract infections. We previously described clinical variant strains of hPIV-2 that display unusual large syncytial cytopathic effects. Their molecular characterization revealed a recurrent conserved specific amino acid substitution: A96T in the F2 subunit of the fusion glycoprotein F. The objective of this study was to investigate the contribution of this A96T substitution to the specific hyperfusogenic properties of the hPIV-2 variant strains. Based on a transient expression strategy, quantification of cell-cell fusion assays, and flow cytometry, we have shown that the A96T mutation strongly alters the fusogenic properties of F hPIV-2, highlighting this key residue in the F2 subunit and its possible role in fusion regulation. This work highlights the benefits of monitoring genetic and phenotypic changes of circulating strains to complete our understanding of Paramyxovirus fusion and related pathogenesis.

Cellular transcriptional profiling in human lung epithelial cells infected by different subtypes of influenza A viruses reveals an overall down-regulation of the host p53 pathway.

BACKGROUND: Influenza viruses can modulate and hijack several cellular signalling pathways to efficiently support their replication. We recently investigated and compared the cellular gene expression profiles of human lung A549 cells infected by five different subtypes of human and avian influenza viruses (Josset et al. Plos One 2010). Using these transcriptomic data, we have focused our analysis on the modulation of the p53 pathway in response to influenza infection. RESULTS: Our results were supported by both RT-qPCR and western blot analyses and reveal multiple alterations of the p53 pathway during infection. A down-regulation of mRNA expression was observed for the main regulators of p53 protein stability during infection by the complete set of viruses tested, and a significant decrease in p53 mRNA expression was also observed in H5N1 infected cells. In addition, several p53 target genes were also down-regulated by these influenza viruses and the expression of their product reduced. CONCLUSIONS: Our data reveal that influenza viruses cause an overall down-regulation of the host p53 pathway and highlight this pathway and p53 protein itself as important viral targets in the altering of apoptotic processes and in cell-cycle regulation.

Influenza A viruses alter the stability and antiviral contribution of host E3-ubiquitin ligase Mdm2 during the time-course of infection.

The interplay between influenza A viruses (IAV) and the p53 pathway has been reported in several studies, highlighting the antiviral contribution of p53. Here, we investigated the impact of IAV on the E3-ubiquitin ligase Mdm2, a major regulator of p53, and observed that IAV targets Mdm2, notably via its non-structural protein (NS1), therefore altering Mdm2 stability, p53/Mdm2 interaction and regulatory loop during the time-course of infection. This study also highlights a new antiviral facet of Mdm2 possibly increasing the list of its many p53-independent functions. Altogether, our work contributes to better understand the mechanisms underlining the complex interactions between IAV and the p53 pathway, for which both NS1 and Mdm2 arise as key players.

Echovirus 6 strains derived from a clinical isolate show differences in haemagglutination ability and cell entry pathway.

Two echovirus 6 (EV6) strains were isolated from a clinical sample after successive sub-cultures in PLC (human hepatocellular carcinoma) and HeLa (human cervical adenocarcinoma) cells. The first strain retained its haemagglutinating capacity (HAEV6) while the second became non-haemagglutinating (NHAEV6). Virus binding assay showed that HAEV6 was capable of binding to DAF-expressing cells but not NHAEV6 confirming the role of DAF in EV6 haemagglutination. The lack of competition between the two viral strains during coinfections suggested that each strain used a different cell entry pathway. We provide evidence showing that HAEV6 used preferentially the lipid raft-dependent caveolae pathway, whereas NHAEV6 followed the clathrin-mediated pathway. Comparison of the sequences of HAEV6 and NHAEV6 revealed five amino acid changes in the VP1, VP2 and VP3 capsid proteins distributed in domains which are known to be highly immunogenic or suggested to be involved in receptor binding, virion stability and pathogenicity.

Nucleolin interacts with influenza A nucleoprotein and contributes to viral ribonucleoprotein complexes nuclear trafficking and efficient influenza viral replication.

Influenza viruses replicate their single-stranded RNA genomes in the nucleus of infected cells and these replicated genomes (vRNPs) are then exported from the nucleus to the cytoplasm and plasma membrane before budding. To achieve this export, influenza viruses hijack the host cell export machinery. However, the complete mechanisms underlying this hijacking remain not fully understood. We have previously shown that influenza viruses induce a marked alteration of the nucleus during the time-course of infection and notably in the nucleolar compartment. In this study, we discovered that a major nucleolar component, called nucleolin, is required for an efficient export of vRNPs and viral replication. We have notably shown that nucleolin interacts with the viral nucleoprotein (NP) that mainly constitutes vRNPs. Our results suggest that this interaction could allow vRNPs to "catch" the host cell export machinery, a necessary step for viral replication.

Divergent genetic evolution of hemagglutinin in influenza A H1N1 and A H1N2 subtypes isolated in the south-France since the winter of 2001-2002.

BACKGROUND: Influenza A viruses are divided into subtypes based on their hemagglutinin (H1 to H15) and neuraminidase (N1 to N9) glycoproteins. Of these, three A subtypes H1N1, H3N2 and H1N2 circulate in the human population. Influenza A viruses display a high antigenic variability called "antigenic drift" which allows the virus to escape antibody neutralization. OBJECTIVES: Evaluate the mutations apparition that might predict a divergent antigenic evolution of hemagglutinin in influenza A H1N1 and A H1N2 viruses. STUDY DESIGN: During the three winters of 2001-2002 to 2003-2004, 58 A H1N1 and 23 A H1N2 subtypes have been isolated from patients with influenza-like illness in the south of France. The HA1 region was analyzed by RT-PCR and subsequently sequenced to compare the HA1 genetic evolution of influenza A H1N1 and A H1N2 subtypes. RESULTS: Our results showed that 28 amino acid substitutions have accumulated in the HA1 region since the circulation of A/New Caledonia/20/99-like viruses in France. Of these, fifteen were located in four antigenic sites (B, C, D and E). Six of them were observed only in the A H1N2 isolates, six only in the A H1N1 isolates and three in both subtypes. Furthermore, nine of twenty two A H1N2 isolates from the winter of 2002-2003 shared a T90A amino acid change which has not been observed in any A H1N1 isolate; resulting in the introduction of a new glycosylation site close to the antigenic site E. This might mask some antigenic E determinants and therefore, modify the A H1N2 antigenicity. CONCLUSIONS: The divergent genetic evolution of hemagglutinin may ultimately lead to a significant different antigenicity between A H1N1 and A H1N2 subtypes that would require the introduction of a new subtype in the vaccine batches.

Ultrastructural fingerprints of avian influenza A (H7N9) virus in infected human lung cells.

In this study, we investigated the ultrastructural modifications induced by influenza A (H7N9) virus in human lung epithelial cells. One particular characteristic of H7N9 viral infection is the formation of numerous M1-associated striated tubular structures within the nucleus and the cytoplasm, which have only previously been observed for a limited number of influenza A viruses, notably the 2009 pandemic (H1N1) virus.

Influenza NS1 interacts with p53 and alters its binding to p53-responsive genes, in a promoter-dependent manner.

The interplay between influenza A viruses (IAV) and p53 has only been reported in a limited number of studies, mainly focusing on the antiviral role of p53. We investigated the impact of IAV infection on p53 stability and transcriptional activity. Our results indicate that IAV-induced stabilization of p53 only partially correlates with modulation of p53 transcriptional activity measured during infection. Moreover, we show that the viral non-structural protein 1 (NS1) is able to inhibit p53 transcriptional activity, in a promoter-dependent manner. Based on these data, we propose that NS1 may contribute to p53-mediated cell fate decision during IAV infection.

The influenza fingerprints: NS1 and M1 proteins contribute to specific host cell ultrastructure signatures upon infection by different influenza A viruses.

Influenza A are nuclear replicating viruses which hijack host machineries in order to achieve optimal infection. Numerous functional virus-host interactions have now been characterized, but little information has been gathered concerning their link to the virally induced remodeling of the host cellular architecture. In this study, we infected cells with several human and avian influenza viruses and we have analyzed their ultrastructural modifications by using electron and confocal microscopy. We discovered that infections lead to a major and systematic disruption of nucleoli and the formation of a large number of diverse viral structures showing specificity that depended on the subtype origin and genomic composition of viruses. We identified NS1 and M1 proteins as the main actors in the remodeling of the host ultra-structure and our results suggest that each influenza A virus strain could be associated with a specific cellular fingerprint, possibly correlated to the functional properties of their viral components.

New enteroviruses, EV-93 and EV-94, associated with acute flaccid paralysis in the Democratic Republic of the Congo.

Surveillance of acute flaccid paralysis often identifies enteroviruses not typeable by virus neutralization in cell culture. During 2000 and 2001, 186 isolates from 138 children with acute flaccid paralysis in the Democratic Republic of the Congo were sent for typing to the National Reference Centre for Enteroviruses in Lyon, France. The 5' UTR of the viral genome could be amplified by PCR for 158 isolates from 114 patients. Isolates from 89 patients were neutralizable, and contained non-polio enterovirus types. Seventeen children were infected with more than one entero- or adenovirus; another three were co-infected with both these viruses. Serological typing failed with 19 isolates from 13 (9%) patients. The VP1 region of these strains could be amplified by PCR and sequenced, which revealed that five children were infected with CV-A17, EV-70, EV-76, EV-77, or CV-A13. Two patients were doubly infected, one with CV-A24 and E-9, and another with E-27 and EV-81. Isolates from six children contained strains with divergent VP1 region. The amino acid sequences of these complete VP1 regions diverged >or=28% from published types indicating that they represented two new enterovirus types, tentatively designated EV-93 belonging to HEV-B and EV-94 within HEV-D. The latter enterovirus has in parallel been isolated from sewage in Egypt. In conclusion, there was a high frequency of "untypable" enterovirus isolates from cases with acute flaccid paralysis in the Democratic Republic of the Congo. Six of these were shown to represent two enteroviruses not previously described.

Outbreak of acute hemorrhagic conjunctivitis in French Guiana and West Indies caused by coxsackievirus A24 variant: phylogenetic analysis reveals Asian import.

An outbreak of acute hemorrhagic conjunctivitis occurred in French Guiana between April and July 2003, with approximately 6,000 cases in the two major cities Kourou and Cayenne. Since acute hemorrhagic conjunctivitis is not a notifiable disease in France, there was no registration of the number of cases. Therefore, these were estimated by comparing the consumption of antibiotic eye drops and ophthalmic ointments during 2002 and 2003. The outbreak rapidly spread into the Caribbean Islands, causing an outbreak in Guadeloupe in October. Viral isolates from conjunctival swabs of 16 patients were confirmed to be enterovirus by PCR directed to the 5' UTR of the genome. The isolates could not be neutralized by the Melnick intersecting pools, but were shown to be CV-A24 variant by limited sequencing within the VP1 and 3C regions of 12 strains. Phylogenetic analysis revealed that they were similar to the genotype III strains causing outbreaks in Korea 2002 and Malaysia 2003. The previous outbreak of conjunctivitis caused by CV-A24 in the Caribbean in the 1980s was also introduced from Asia, and disappeared after 3 years. This new introduction from Asia and its rapid spread into the Caribbean, where the infection disappeared after a few months, indicates that the CV-A24 variant has a different epidemiological pattern in this region compared to South East Asia, since it has not established an endemic infection. It had to be reintroduced from Asia, where it has been circulating since the 1970s.