The title is somewhat misleading since the project focuses principally on the PB1-F2 protein product of the PB1 gene of the virus, its influence on virulence in mice and transmissibility between guinea pigs, as well as the underlying mechanisms in relation to its apoptosis-inducing and immune modulating functions. Studies of the 1918 virus identified PB1, in addition to the HA and NA, as principally responsible for its high pathogenicity. Furthermore, PB1-F2, the product of a +1 reading frame of the PB1 gene relative to that encoding the PB1 polymerase component, was shown to contribute to pathogenicity in mice and the mutation N66S was shown to increase pathogenicity. In addition the protein has been shown to increase susceptibility of mice to secondary bacterial infection. The applicant has been in the forefront of studies of the function of PB1-F2. Studies in cells have shown that PB1-F2 targets the mitochondrial membrane where it interacts with AMT3 and VDAC1 of the permeability transition pore complex causing the release of cytochrome C and consequently apoptosis. The project is to extend some of these in vivo and in vitro studies done under the current PPG and will include: 1. Investigations of the potential influence of PB1-F2 on the virulence in mice of the pandemic viruses of 1918 (H1N1), 1957 (H2N2) and 1968 (H3N2), which incorporated a PB1 avian gene in addition to those of HA (and NA), and of current H5N1 viruses, using recombinant viruses possessing a background of a recent H1N1 virus A/Texas/91, which possesses a 57 amino acid truncated version of PB1-F2 lacking the key C-terminal mitochondrial-targeting domain implicated in the immune modulation function of the protein and its effects on pathogenicity. Studies of immune responses to these recombinant viruses will include the Katze lab (Project 5). 2. More detailed studies of the molecular mechanisms underlying its apoptosis-inducing function and influence on virulence. These will focus on the effects of the N66S mutation on a) apoptosis induction, interaction of PB1-F2 with ANT3 and VDAC1 (including structural studies with Wilson, under Project 1) and stability of the protein, and b) immune modulation (cytokine and chemokine upregulation), in collaboration with the Animal Core Facility (Core B). The underlying mechanism of the in vivo pathogenic activity of a C-terminal peptide of PB1-F2 will also be examined at the cellular level. 3. Use of the guinea pig model for transmission of influenza viruses, established by the applicant, to investigate the influence on virus transmissibility of: the PB1-F2 of 1918 and H5N1 viruses and the effects of the N66S mutation, the host range determining mutation E627K in the PB2 polymerase component, and the effect of secondary bacterial infection. The overall objective of this part of the program is to better understand the mechanism of action of PB1-F2 and its role in the interplay between the virus and the immune system, and the processes underlying pathogenesis, as well as to gain additional information on the molecular correlates of influenza virus transmission. Significance: With the threat of an impending pandemic, possibly caused by highly pathogenic influenza AH5N1, a better understanding of the principal viral factors influencing transmissibility and virulence of influenza A viruses is of particular importance in assessing the potential risk of unusual viruses causing (sporadic) human infection, such as H5N1, becoming more readily transmissible (among humans) and the potential severity of a subsequent pandemic. The proposed studies focus mainly on the involvement of the pro-apoptotic PB1-F2 protein in modulating the immune response and in enhancing pathogenicity, about which relatively little is yet known. Studies of its influence on virus transmission will be complemented by studies of the effects of the host-specific mutation E627K in PB2 and important studies of potential interaction between influenza infection and secondary bacterial infection in transmission as well as in pathogenesis. The latter experiments might well be extended to studies of reduction in any synergistic transmission by anti-NA drugs, although the involvement of the NA, which was well documented in mouse studies, was not discussed. Approach: Most of the experiments are extensions of previous work using well-established techniques and are likely to yield meaningful results. Many attempt to dissect the effects of a single-mutation N66S;although this should help to identify specific effects and to integrate the results of the varied experiments, the outcome may be limited by less dramatic effects than anticipated. The experiments are described in detail and many of the potential limitations, due for example to the complexity of the systems and potential difficulties in extrapolating effects in vitro in cell culture to the situation in vivo, are discussed. Appropriate collaborative interactions have been established with Projects 1 and 5 and Core B. Innovation: The project is not particularly original, much of it being a logical extension of earlier work by the Project Leader. Innovative aspects include the collaborative structural studies of PB1-F2, the use of a relatively new Millipore cytokine capture assay and the application of the guinea pig transmission model recently developed by the applicant. The transmission experiments proposed should more thoroughly evaluate the usefulness of the model in relation to the ferret model and its applicability to the human situation. Investigators: The Project Leader's lab has made a number of very significant contributions to influenza research: in developing the reverse genetics system, used to construct the recombinant viruses;in initial identification and subsequent studies of the role of the PB1-F2 protein;and in the development of the guinea pig model for influenza virus transmission. These are key aspects of the studies proposed. The applicant has a very good publication record. The other personnel have already contributed to related work and there can be little doubt that the group encompasses the necessary expertise. Some aspects of the project rely on collaboration with other components of the program. The fact that the group has already interacted successfully in the current PPG augurs well for future success. Environment: Given the track record of the PL, including the success of the current PPG, and the expertise of the present group in utilizing the experimental systems, together with the co-localization with two of the other projects, the environment is highly appropriate to conduct this part of the program.
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