The aim of the present study was to identify what influences the short half-life of the influenza A virus PB1-F2 protein and whether a prolonged half-life affects the properties of this molecule. We hypothesized that the short half-life of PB1-F2 could conceal the phenotype of the protein. Because proteasome degradation might be involved in PB1-F2 degradation, we focused on ubiquitination, a common label for proteasome targeting. A cluster of lysine residues was demonstrated as an ubiquitination acceptor site in evolutionary and functionally distinct proteins. The PB1-F2 sequence alignment revealed a cluster of lysines on the carboxy terminal end of PB1-F2 in almost all of the GenBank sequences available to date. Using a proximity ligation assay, we identified ubiquitination as a novel posttranslational modification of PB1-F2. Changing the lysines at positions 73, 78, and 85 to arginines suppressed the ubiquitination of A/Puerto Rico/8/1934 (H1N1)-derived PB1-F2. The mutation of the C-terminal lysine residue cluster positively affected the overall expression levels of avian A/Honk Kong/156/1997 (H5N1)- and mammalian A/Puerto Rico/8/1934 (H1N1)-derived PB1-F2. Moreover, increased PB1-F2 copy numbers strengthened the functions of this virus in the infected cells. The results of a minigenome luciferase reporter assay revealed an enhancement of viral RNA-dependent RNA polymerase activity in the presence of stabilized PB1-F2, regardless of viral origin. IFNβ antagonism was enhanced in 293T cells transfected with a plasmid expressing stabilized K→R mutant variants of PB1-F2. Compared with PB1-F2 wt, the loss of ubiquitination enhanced the antibody response after DNA vaccination. In summary, we revealed that PB1-F2 is an ubiquitinated IAV protein, and this posttranslational modification plays a central role in the regulation of the biological functions of this protein.

Summary. -In this work we simulated in a mouse model a naturally occurring situation of humans, who overcame an infection with epidemic strains of influenza A, and were subsequently exposed to avian influenza A viruses (IAV). The antibody response to avian IAV in mice previously infected with human IAV was analyzed. We used two avian IAV (A/Duck/Czechoslovakia/1956 (H4N6) and the attenuated virus rA/Viet Nam/1203-2004 (H5N1)) as well as two human IAV isolates (virus A/Mississippi/1/1985 (H3N2) of medium virulence and A/Puerto Rico/8/1934 (H1N1) of high virulence). Two repeated doses of IAV of H4 or of H5 virus elicited virus-specific neutralizing antibodies in mice. Exposure of animals previously infected with human IAV (of H3 or H1 subtype) to IAV of H4 subtype led to the production of antibodies neutralizing H4 virus in a level comparable with the level of antibodies against the human IAV used for primary infection. In contrast, no measurable levels of virus-neutralizing (VN) antibodies specific to H5 virus were detected in mice infected with H5 virus following a previous infection with human IAV. In both cases the secondary infection with avian IAV led to a significant increase of the titer of VN antibodies specific to the corresponding human virus used for primary infection. Moreover, cross-reactive HA2-specific antibodies were also induced by sequential infection. By virtue of these results we suggest that the differences in the ability of avian IAV to induce specific antibodies inhibiting virus replication after previous infection of mice with human viruses can have an impact on the interspecies transmission and spread of avian IAV in the human population.Keywords: avian influenza A viruses; sequential influenza A infection; antibody response; virus-neutralizing antibodies; HA2-specific antibodies; original antigenic sin * Corresponding author. E-mail: viruevar@savba.sk; phone: +421-2-59302427. Abbreviations: HA = hemagglutinin; IAV = influenza A virus; MAb = monoclonal antibody; OAS = original antigenic sin; VN = virus-neutralizing

Summary. -Human infections with avian infl uenza A viruses (IAVs) without or with clinical symptoms of disease were recently reported from several continents, mainly in high risk groups of people, who came into the contact with infected domestic birds or poultry. It was shown that avian IAVs are able to infect humans directly without previous adaptation, however, their ability to replicate and to cause a disease in this new host can diff er. No spread of these avian IAVs among humans has been documented until now, except for one case described in Netherlands in the February of 2003 in people directly involved in handling IAV (H7N7)-infected poultry. Th e aim of our work was to examine whether a low pathogenic avian IAV can induce a virus-specifi c immune response of biological relevancy, in spite of its restricted replication in mammals. As a model we used a low pathogenic virus A/Duck/Czechoslovakia/1956 (H4N6) (A/Duck), which replicated well in MDCK cells and produced plaques on cell monolayers, but was unable to replicate productively in mouse lungs. We examined how the immune system of mice responds to the intranasal application of this non-adapted avian virus. Th ough we did not prove the infectious virus in lungs of mice following A/Duck application even aft er its multiple passaging in mice, we detected virus-specifi c vRNA till day 8 post infection. Moreover, we detected virus-specifi c mRNA and de novo synthesized viral nucleoprotein (NP) and membrane protein (M1) in lungs of mice on day 2 and 4 aft er exposure to A/Duck. Virus-specifi c antibodies in sera of these mice were detectable by ELISA already aft er a single intranasal dose of A/Duck virus. Not only antibodies specifi c to the surface glycoprotein hemagglutinin (HA) were induced, but also antibodies specifi c to the NP and M1 of IAV were detected by Western blot and their titers increased aft er the second exposure of mice to this virus. Importantly, antibodies neutralizing virus A/Duck were proved in mouse immune sera aft er the second dose of virus and a slight increase of mRNA expression of immune mediators tumor necrosis factor alpha (TNF-α) and IP10 has been observed in lungs of these mice 48 hr aft er the infection. Th ese observations correspond to the limited replication ability of the virus in mice and provided an important information about its ability to induce virusspecifi c antibodies, including those neutralizing virus, even without the previous virus adaptation to the new mammalian host. Such antibodies could consequently infl uence the immune potential of exposed individuals and their defensive capability against the newly emerged, even more virulent IAV.