Taken together, our findings indicate that PB1-F2 plays an important role in promoting lung pathology in both primary viral infection and secondary bacterial infection. The numbers of IAV-associated deaths vary from season to season, and secondary bacterial pneumonia is a significant contributor to this yearly toll (Thompson et al., 2003
). Differences in viral virulence factors likely contribute to fluctuations in secondary bacterial pneumonia and annual mortality (Peltola et al., 2005
; McCullers, 2006
). IAVs are subtyped by differences in their hemagglutinin (HA) and neuraminidase (NA) glycoproteins. Over the last 2 decades infections with H1N1 strains have caused less morbidity and mortality than H3N2 strains (Thompson et al., 2003
; Thompson et al., 2004
; Simonsen et al., 2000
). Post-1956 H1N1 viruses possess PB1 gene segments that encode truncated PB1-F2 proteins of 67 amino acids due to the introduction of a stop codon. Such PB1-F2s lack the MTS required for induction of apoptosis, which is in the region important for induction of inflammation in our experiments with synthesized peptides (Gibbs et al., 2003
). This defect might contribute to the decreased pathogenicity of contemporary H1N1 strains and their reduced capacity to promote secondary bacterial infections (Peltola et al., 2006
). The predicted amino acid sequence of the PB1-F2 from the 1918 strain differs at eight positions from the sequence of PR8, five of them in the C-terminal region. It is of obvious interest and importance to correlate genetic variation in this and other PB1-F2 proteins with alterations in PB1-F2 function. The enhancing effect of these substitutions on viral replication, completely unexpected from previous findings that PB1-F2 knockdown does not effect replication in vitro
(Chen et al., 2001
; Zamarin et al., 2006
), suggests that PB1-F2 can modify the function of one or more IAV gene products that are required for viral replication.
The mechanism by which PB1-F2 facilitates inflammation and secondary bacterial pneumonia is of prime interest. A synthetic version of PB1-F2 has been shown to be a potent inducer of cell death (Chen et al., 2001
) and a synthetic peptide from the C-terminal region provided a strong pro-inflammatory stimulus in mice (). In vivo
, the protein may be released from moribund or dead cells and promote cell death in areas surrounding foci of infection or in first responder infected mononuclear cells. Release of cell wall components from pneumococci, such as lipoteichoic acids and peptidoglycan, along with the cytotoxin pneumolysin, activates the innate immune system through Toll-like receptors 2 and 4 leading to production of pro-inflammatory cytokines (Yoshimura et al., 1999
; Malley et al., 2003
; McCullers and Tuomanen, 2001
). PB1-F2 mediated cell death may trigger a positive feedback cytokine loop, amplified by bacterial superinfection, that enhances the pulmonary inflammatory response to IAV leading to the immunopathological death of the host. This hypothesis builds on earlier work demonstrating that influenza infection prior to bacterial superinfection causes a synergistic increase in the cytokine response with a resultant adverse outcome (Smith et al., 2007
), and treatment of secondary pneumonia by antibiotic-mediated lysis of pneumococcus does not reduce mortality (McCullers, 2004
). Recent work from Kash et al.
supports this view, as expression of inflammatory and death receptor genes linked to mitochondrial apoptosis is increased by infection with the reconstructed 1918 virus (Kash et al., 2006
). Although these effects could not be tied directly to a specific gene product through analyses using the full virus, our data suggest that the PB1-F2 is a leading candidate. However, differences in lung cytokines were not seen in our experiments until after bacterial challenge, suggesting that expression of PB1-F2 in the context of the entire 1918 genome may be necessary for the full impact on inflammation.
While this model is compelling, the pro-inflammatory effect of PB1-F2 may be completely unrelated to its pro-apoptotic functions. PB1-F2 may be directly recognized by pattern recognition receptors of the innate immune system, or serve as a chemoattractant similar to human beta-defensins released from dead cells (Yang et al., 1999
). The predicted homology of the C-terminal cationic helical domain of PB1-F2 to similar regions of the human beta-defensins lends some credence to this theory. The immune effector cells recruited through this mechanism may contribute to immunopathology while accomplishing their primary function of clearing the infection. Alternatively, expression of PB1-F2 may alter the expression or function of other viral proteins that impact viral pathogenesis. Further studies are necessary to investigate these hypotheses in detail.
One intriguing result was the shortened time to peak viral titer observed both in vitro () and in vivo in the virus expressing the 1918-like PB1-F2 (). This appears to be a unique feature of the 1918-like PB1-F2, since deletion of PB1-F2 from wt
PR8 (data not shown) or WSN (Zamarin et al., 2006
) did not alter viral replication in vitro. In mice, virulence is not diminished by deletion of PB1-F2 from wt
PR8 (data not shown) or WSN (Zamarin et al., 2006
), and the kinetics of viral infection do not differ in a PR8 background (). Transfer of the PR8 PB1 gene to a WSN background did reveal differences in virulence and clearance of virus from the lungs of mice, although viral titers did not differ at an early timepoint (day 3) and in vitro replication was again not affected (Zamarin et al., 2006
). Thus, it could be postulated that the accelerated tempo of early lung infection of the virus expressing the 1918-like PB1-F2 allows the virus to outpace innate immune mechanisms of control. This could contribute to virulence as has been suggested for PR8 (Grimm et al., 2007
), highly pathogenic H5N1 viruses (Gao et al., 1999
) and the 1957 H2N2 pandemic strain (Legge and Braciale, 2003
). While this is an interesting hypothesis to explain the increased virulence of the 1918 strain, it fails to explain the enhancement of bacterial superinfections because this enhancement is seen in both the wt
PR8 knockout model (), where there are no differences in viral kinetics (), as well as in the 1918 PB1-F2 model (). Several factors suggest that other mechanisms must be considered for the exacerbation of secondary bacterial infections with the 1918 PB1-F2, or at the least that there are both direct and indirect contributions of this protein to the pathogenesis. First, the differences in cellularity of the BALF () were initially detected on d7, a time when viral titers () and lung pathology (data not shown) did not significantly differ. Second, the enhanced secondary bacterial pneumonia and elevated cytokines () also occurred after viral lung loads had equalized. Had the increased tempo of early infection been responsible these differences would have been expected to be detectable earlier as was seen in experiments with H5N1 viruses (Gao et al., 1999
). Third, even when wt
PR8 was given in much higher doses, or with much higher doses of bacteria, it never induced pathological alterations of similar character and magnitude as PB8-PB1-F21918 (data not shown). And finally, topical administration of a peptide derived from the C-terminal end of the PB1-F2 protein recapitulates the effects on bacterial superinfection independent of viral infection ().
Clearly, much remains to be learned about PB1-F2 and its contribution to viral virulence. We have demonstrated here that the protein is pro-inflammatory, can contribute to virulence, and facilitates secondary bacterial infections. The ability of 1918 PB1-F2 to enhance the pathogenicity of PR8 may only represent the tip of the iceberg of the full pathogenic potential of this protein when expressed in its natural context of the complete 1918 strain genome. Given the importance of IAV as a leading cause of virus-induced morbidity and mortality year in and year out, and its potential to kill tens of millions in the inevitable pandemic that may have its genesis in the viruses currently circulating in southeast Asia, it is imperative to understand the role of PB1-F2 in IAV pathogenicity and transmission in humans and animals. Our data help explain why the 1918 pandemic strain was so efficient at supporting bacterial pneumonia and reinforce the recent suggestion of the American Society of Microbiology that nations should stockpile antibiotics in anticipation for the next pandemic (American Society for Microbiology, 2006