Previous studies by our lab have shown that PB1-F2 contributes to the pathogenesis of the influenza A virus [20
]. When expression of PB1-F2 was knocked out of a moderately virulent virus in mice, there was a significant loss in pathogenicity, indicating that PB1-F2 plays an important role in virulence [20
]. In the present study, we show that a single aa change in PB1-F2 from highly virulent viruses increases pathogenicity in mice and modulates the immune response. It has been proposed that PB1-F2 causes apoptosis of immune cells, which may lead to decreased antigen presentation and a decrease in the adaptive immune response [17
]. Humans infected with highly pathogenic viruses consistently have decreased lymphocytes and impaired immune response to influenza virus infection [4
]. We wondered if these effects could be caused in part by PB1-F2. In this study, we provide evidence that PB1-F2 does contribute to the high pathogenicity phenotype and that the N66S mutation, also found in the 1918 H1N1 virus, contributes to virulence in highly pathogenic viruses.
After aligning the PB1-F2 sequences from H5N1 viruses that exhibited high- and low-pathogenicity phenotypes, a single aa change was found to correlate with high pathogenicity. The location of the N66S mutation also made it an excellent candidate for affecting the proapoptotic function of PB1-F2. Position 66 is in the α-helical structure of PB1-F2, in the mitochondrial targeting sequence. The location of aa 66 in the C-terminal mitochondrial targeting sequence of the protein could affect PB1-F2 interactions with ANT3 and VDAC1, potentially increasing the induction of apoptosis by PB1-F2 [19
Recombinant A/WSN/33 viruses were created to specifically examine the effects of the N66S mutation during viral infection. The recombinant virus WH has decreased pathogenesis in mice compared with that of A/WSN/33 (unpublished data), likely due to the mismatched polymerase genes, resulting in less efficient replication in the host. The N66S mutation within the PB1-F2 protein partially reversed this attenuating effect.
Within a natural setting, the presence of a “virulent” PB1-F2 may be important when influenza viruses cross species barriers or when new pandemic strains are generated by reassortment. In fact, the PB1
gene has been one of the segments found to reassort to create the pandemic strains of 1957 and 1968, potentially giving these viruses a more pathogenic PB1-F2 and thus a higher virulence [13
]. It is possible that the PB1-F2 protein could allow a newly reassorted virus to replicate in a new host efficiently enough to spread, and develop mutations to create a more efficient polymerase complex. In addition, influenza surveillance data shows that in recent history (1970 onward), H3N2 infections cause almost 14 times the number of influenza related deaths than H1N1 infections and are associated with a higher epidemic severity index (as measured by the rate of increase in pneumonia and influenza mortality) [31
]. Interestingly, recent H1N1 isolates contain a truncated PB1-F2, which possibly plays a role in their decreased virulence [19
]. The mutation we investigate here is not currently found in recent H5N1 isolates; however, it is possible for those viruses to acquire the mutation either through the error-prone RNA polymerase or through reassortment with a virus that contains the N66S mutation.
The observation that the WH N66S virus grew to higher titers in the lung and persisted at high titers for a longer time than the WH virus supports the role of PB1-F2 in allowing for increased replication. This may also explain the impairment of viral clearance in the mice infected with WH N66S. In addition, the 1918 wt virus showed higher lung titers and slower viral clearance when compared with the 1918 S66N virus. We suspect that the delay in viral clearance due to expression of PB1-F2 protein may allow for prolonged viral replication and development of irreversible pulmonary immunopathology, the findings observed with highly pathogenic influenza strains. CD8+
T cells are mainly responsible for viral clearance in the host, and it is possible that their function could be impaired by PB1-F2 [35
]. In support of this, we observed that the WH N66S and wt 1918 viruses caused a significant increase in IFN-γ and TNF-α cytokine production over the WH and 1918 S66N viruses, respectively. Whether this change in cytokine levels is through the direct action of PB1-F2 or through its impact on viral replication in the lung is difficult to determine. However, the cytokine dysregulation is of special interest because it has been associated with both H5N1 and 1918 H1N1 virus infections. In previous studies, cytokine dysregulation was associated with high virulence and death in animal models [29
]. Our study supports these findings and suggests that PB1-F2 could be one of the factors contributing to the cytokine dysregulation seen in H5N1 virus–infected patients and 1918 H1N1 virus–infected animals [4