Project 2 - Pathogenicity Factors Encoded by the Influenza Virus Polymerase Genes

Principal Investigator: Peter Palese, Ph.D.
Horace W. Goldsmith Professor & Chair, Department of Microbiology
Professor, Department of Medicine, Division of Infectious Diseases
Mount Sinai School of Medicine, New York, NY

The RNA-dependent RNA polymerase of influenza virus catalyses distinct RNA synthesis activities, which are central to the viral life cycle: replication of the viral genomic RNA (vRNA) through an antigenomic RNA (cRNA) intermediate and transcription of vRNA to produce mRNA. The mRNA is then translated by the host cell ribosomal machinery into viral proteins. Three viral proteins collaborate to perform these functions: polymerase basic 1 (PB1), polymerase basic 2 (PB2), and polymerase acidic (PA). Studies on the 1918 pandemic influenza virus and emerging highly pathogenic avian influenza viruses implicate PB1, PB2 and PA as factors contributing to viral pathogenicity and host range. Using replicon systems, we are evaluating the influence of replication and transcription efficiencies and mutations that occur in highly pathogenic strains on viral multiplication and pathogenicity. Fitness of rationally-designed mutant viruses generated by reverse genetics will be further characterized through analysis of growth properties and pathogenic effects in animal hosts and can contribute to vaccine research. Viral genome packaging signals within all eight gene segments have been mapped by this approach to better understand selective packaging and the generation of reassortants. Finally, we are identifying cellular proteins that bind the polymerase complex of influenza viruses isolated from human or avian hosts. Monoclonal antibodies raised against the PB1 proteins of human and avian influenza viruses provide reagents for these studies.

PB1-F2 and pathogenicity. The recent finding of a second protein, PB1-F2, encoded in an alternative reading frame of many influenza A virus PB1 segments, and its definition as a pro-apoptotic factor, suggests it may also contribute to viral pathogenesis. The 87 amino acid PB1-F2 peptide localizes to both the inner and outer mitochondrial membranes and leads to altered mitochondrial morphology, dissipation of mitochondrial membrane potential, and cell death. Furthermore, we have recently found that the PB1-F2 protein interacts with the mitochondrial apoptotic mediators ANT3 and VDAC1 and sensitizes cells to apoptotic stimuli through the mitochondrial pathway. Expression of PB1-F2 by influenza viruses in cell culture was shown to increase the frequency of apoptosis in immune cells, but had no effect on epithelial cell lines, implying a possible role for PB1-F2 in immunomodulation. We are approaching the study of PB1-F2 by taking advantage of the reverse genetics system available to CRIP researchers, finding that PB1-F2 contributes to pathogenesis in the mouse lung. Moreover, increased pathogenesis is observed when mice are infected with a recombinant virus containing a PB1-F2 gene from highly virulent viruses as compared to low virulence isolates. The single amino acid change in PB1-F2 that caused the increased pathogenicity is N66S. Nevertheless, the precise role of PB1-F2 in influenza A virus infection is unknown. Future studies will address the immune cell populations, cytokine and chemokine alterations, and the level of apoptosis in the infected lung.