It has become increasingly clear that influenza A viral proteins have multiple functions during infection. Our group and others have demonstrated that PB1-F2 protein from influenza A virus has multiple activities both in vitro
and in vivo
, including apoptosis induction, enhancement of secondary bacterial pneumonia, and a capacity to increase pathogenicity and induce a proinflammatory response (7
). However, the mechanism of PB1-F2's role in pathogenesis has not been fully described. There has been speculation that the apoptotic function of PB1-F2 is responsible for increased pathogenesis (34
). This possibility was investigated in the present work by performing a terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay for apoptosis on whole lung homogenates, but we observed no difference in apoptosis induction caused by the WH and WH N66S viruses (data not shown). However, these results do not eliminate the possibility that the N66S mutation may have an effect on apoptosis in a specific cell type. Increased apoptosis in the presence of PB1-F2 was shown to be specific to immune cells in a previous publication (4
). PB1-F2 N66S may contribute to this effect by increasing apoptosis even more than wt PB1-F2. However, it is likely that the significant infiltration of cells into the lungs and reduction in air space are what causes the mice to succumb to viral infection.
To determine the mechanism for the increased immunopathogenesis caused by the PB1-F2 N66S-expressing virus, the innate and adaptive immune responses were studied in detail. We have shown that elimination of key components of the adaptive immune response did not change the level of pathogenesis caused by the WH N66S virus. We expect the virulence of the WH N66S virus to decrease when an important part of the pathogenesis mechanism is eliminated. Instead, we found that regardless of what cell type (CD4−/−, CD8−/−, Rag1−/−) or cytokine (IFN-γ−/−) was knocked out, the WH N66S virus maintained its pathogenesis. While we cannot completely rule out a role for adaptive immune responses in mediating WH N66S-induced pathogenesis, our work suggests that the increase in pathogenesis results from events occurring during the acute-phase response to influenza virus infection. Inflammation and activation of proinflammatory pathways during influenza virus infection can either contribute to a protective phenotype or have a detrimental consequence to the host. Here, we report that mice infected with WH N66S virus show enhanced immunopathology, driving significant cellular accumulation in the lungs that is not observed for WH-infected mice. Infiltration of multiple leukocyte populations, including monocytes and neutrophils, is seen beginning at day 3 postinfection for WH N66S-infected mice. This increased lung cellularity is concomitant with enhanced levels of cytokines and chemokines, specifically chemoattractant molecules responsible for homing of leukocytes into lungs during infection. These observations suggest that the N66S mutation in PB1-F2 likely alters the innate immune response that may be directly or indirectly mediated by PB1-F2.
We investigated host responses to WH and WH N66S infection by using transcriptional profiling of infected mouse lungs at 12 h and 1, 3, and 5 days postinfection. This revealed differential regulation of innate immune responses, supporting a suppression of interferon-stimulated genes in WH N66S-infected mice compared to the level in WH-infected mice (Fig. ). Hence, a virus containing a single amino acid mutation in PB1-F2 found in highly pathogenic viruses allows for increased immunopathology by downregulating the early interferon response. Further studies are needed to determine whether the N66S mutation in the PB1-F2 protein of other influenza viruses also leads to a decrease in the interferon response. Based on the finding that PB1-F2 66S increases virulence in both the 1918 pandemic influenza virus and an H5N1 virus, as shown in our previous work (7
), we feel that the suppression of early innate responses may be a general feature of PB1-F2 66S proteins derived from different strains. Overall, this work suggests a novel function of PB1-F2 derived from a highly pathogenic influenza virus strain in contributing to enhanced pathogenesis through a mechanism that targets the host innate immune pathways early in infection.
To broaden our view of infection and determine how early differential regulation of interferon responses by WH and WH N66S viruses can be detected, we conducted a separate experiment that included a 12-h time point. The transcriptional profiles of the two viruses were similar at 12 h postinfection, which indicates that differences seen on day 1 postinfection are probably not due to different titers of the virus inocula. Based on the observation that the downregulation of innate immune genes by the WH N66S virus occurs at this early time point, we speculate that critical events begin by day 1 postinfection.
We have shown that the WH N66S virus, possessing only a single amino acid substitution in the PB1-F2 protein compared to its isogenic counterpart, causes a delayed expression of innate immune pathway genes. This suggests that there may be a blockade in the sensing capabilities for membrane-bound or cytosolic pathogen recognition receptors responsible for detecting viral RNA and transmitting downstream signals to initiate induction of interferon and proinflammatory responses. The downregulation of interferon-related genes by the PB1-F2 N66S protein may occur via two mechanisms. PB1-F2 N66S could be a traditional interferon antagonist, binding to a molecule in the type I interferon pathway and reducing the production of interferon. With the impairment of interferon expression, there would then be a reduction of interferon-stimulated genes compared to that found with wt infection. Alternatively, PB1-F2 N66S could inhibit a transcription factor that is common among interferon-stimulated genes. This inhibition by PB1-F2 N66S would either prevent the transcription factor from binding the promoter complex or block the action of the promoter complex.
A recent study by Le Goffic et al. reports that PB1-F2 proteins of certain influenza virus strains increase the expression of IFN during viral infection (19
). The authors suggest that the PB1-F2-mediated IFN induction is dependent on the mitochondrial antiviral signaling protein (MAVS, also known as IPS-1, Cardif, or VISA) and the NF-κB transcription factor. This interferon-inducing effect of PB1-F2 was found in epithelial cells only. Future studies are necessary to sort out whether this pro/anti-interferon effect is tissue specific or whether these effects are strain or backbone specific.
We believe that PB1-F2 proteins of highly pathogenic influenza viruses, such as the WH N66S virus, may have evolved to reduce the property of IFN induction and thus contribute to increased virulence. The N66S mutation in the PB1-F2 protein may specifically mediate inhibition of innate immune responses through a newly gained direct physical interaction with key modulators of IFN signaling responses. The interaction with IFN signaling mediators may not occur with PB1-F2 proteins derived from low-pathogenic strains or may have lower affinity. Alternatively, the N66S mutation may result in PB1-F2 adopting an entirely different function. It is probable that the N66S mutation could alter the function of PB1-F2, because the N66S mutation is located in an exposed portion of the α-helix in the C-terminal region of PB1-F2. The C-terminal region is important for other functions of PB1-F2, such as apoptosis, and the α-helix is known to be the only secondary structure of the molecule (2
). However, a molecular mechanism for the decreased IFN induction by PB1-F2 N66S has yet to be uncovered.
The transcriptional profiles observed for WH N66S at days 3 and 5 postinfection are representative of an infection involving significant cell infiltration and extreme upregulation of cytokines and chemokines. The cytokines and chemokines with increased protein levels at day 3 were also shown to have increased gene expression on the same day. This not only validates the microarray data but also indicates that the upregulation is due to increased gene expression and not just increased translation or stored protein release.
Previous publications indicate that the PB1-F2 proteins vary in ability to contribute to pathogenesis. Here, we suggest that this may be due to point mutations that can enhance or decrease the function of PB1-F2. The microarray data compiled show an important suppression of interferon-stimulated genes early during infection, allowing for higher viral titers and increased pathogenesis later during infection. What this demonstrates is that initially the PB1-F2 N66S protein is able to suppress the early antiviral response, which allows for unchecked viral growth. This increased viral growth then triggers an overwhelming inflammatory response that overloads the lung environment with cells. Our data support this mechanism through the timeline of events occurring; on day 1, there was a suppression of interferon signaling that would allow for increased viral replication, and on day 3, there were increased levels of proinflammatory genes and high expression of proinflammatory molecules. The excessive infiltration of cells then makes it difficult for the mice to maintain lung function, and they eventually succumb to infection. Alternatively, not only the increase in viral titer but also the increase in cytokine and chemokine levels may be responsible for WH N66S-mediated virulence.
Recent studies have identified additional viral proteins besides the well-characterized IFN antagonist NS1 that limit the host IFN response (13
). Graef et al. show that the polymerase protein PB2 interacts with the MAVS protein at the mitochondria and thus inhibits the induction of IFN (13
). These studies highlight that the IFN antagonism strategy of influenza viruses is more complex than previously thought. Our study supports the hypothesis that the PB1-F2 protein has anti-interferon activity in addition to previously published functions, and future studies should unravel the molecular mechanism of PB1-F2 N66S-mediated interferon suppression. The importance of studying this protein from influenza A viruses is essential to understanding virulence in different influenza A virus strains.