The ubiquitination of the influenza A virus PB1-F2 protein is crucial for its biological function.

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. IFNß 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.

A DNA vaccine expressing PB1 protein of influenza A virus protects mice against virus infection.

Although influenza DNA vaccine research has focused mainly on viral hemagglutinin and has led to promising results, other virion proteins have also shown some protective potential. In this work, we explored the potential of a DNA vaccine based on the PB1 protein to protect BALB/c mice against lethal influenza A virus infection. The DNA vaccine consisted of pTriEx4 plasmid expressing PB1. As a positive control, a pTriEx4 plasmid expressing influenza A virus HA was used. Two weeks after three subcutaneous doses of DNA vaccine, the mice were challenged intranasally with 1 LD50 of A/Puerto Rico/8/34 (H1N1) virus, and PB1- and HA-specific antibodies, survival rate, body weight change, viral mRNA load, infectious virus titer in the lungs, cytokines IL-2, IL-4 and IL-10, and granzyme-B were measured. The results showed that (i) the PB1-expressing DNA vaccine provided a fair protective immunity in the mouse model and (ii) viral structural proteins such as PB1 represent promising antigens for DNA vaccination against influenza A.