Investigation of Host Adaptation/Transmissibility of the Acidic Polymerase (PA) of the Avian Influenza a Virus (H7N9) (I)

Project: National Science and Technology CouncilNational Science and Technology Council Academic Grants

Project Details


An epidemic of an avian-origin H7N9 influenza virus has recently emerged in China and still poses a threat of pandemic outbreak as a result of cross-transmission from birds to humans. As November 15, 2013, the WHO has documented a total of 139 laboratory-confirmed human cases of avian influenza A (H7N9) virus infection, including 45 deaths. The human H7N9 virus has a preference to replicate in the lower respiratory tract and is difficult to detect. The heterotrimeric influenza virus polymerase comprises the acidic polymerase (PA), basic polymerase 1 (PB1), and basic polymerase 2 (PB2) subunits, which catalyze viral transcription and replication. In contrast to most RNA viruses, cap-snatching is a unique mechanism used by influenza viruses to initiate genome transcription. During this process, PA (via its endonuclease activity) cleaves capped RNA leader sequences from host cellular mRNAs and uses these to prime transcription of the vRNA genome. Ninety-one human and avian H7N9 PA sequences downloaded from the NCBI revealed that several amino acids have changed between low-pathogenic and high-pathogenic strains. Thus, we speculate that PA may play a role in host adaptation or virulence. First, we will substitute the H7N9 PA subunit in the background of the seasonal influenza virus and measure RNP activity, assess the growth kinetics of the recombinant virus in ex vivo lung epithelial cells, and study its virulence in mice and ferrets. Subsequently, we will pinpoint which altered amino acids contribute to host adaptation in humans by engineering the particular amino acids in the H7N9 PA subunit. Alternatively, we will culture H7N9 viruses in ex vivo upper respiratory tract cells, which are not usually infected by the H7N9 virus, to obtain adapted viruses that might have a higher chance to spread. We will then sequence the viral genome, in an attempt to identify the adapted amino acids that may contribute to enhanced infection. Based on the observation that PA possesses endonuclease activity and has both a nuclear and cytoplasmic distribution, analogous to the subcellular localization of precursor (primary) and mature microRNAs, we propose to investigate whether PA’s intrinsic endonuclease activity plays an additional role in cleaving host microRNAs to modulate viral pathogenicity. We will validate this proof-of-concept using wild-type and endonuclease-inactive mutants of the H7N9 PA subunit in A549 lung cells, and compare their miRNA expression profile using deep-sequencing approaches. The results of this experiment will then be confirmed by viral infection in cells. The target genes that are regulated by the miRNA of interest will be identified by proteomics approaches, such as stable isotope labeling by/with amino acids in cell culture (SILAC). These proposed specific aims will provide insight into the role of PA in host adaptation and viral pathogenesis.

Project IDs

Project ID:PC10304-0016
External Project ID:MOST103-2321-B182-010
Effective start/end date01/03/1403/03/15


Explore the research topics touched on by this project. These labels are generated based on the underlying awards/grants. Together they form a unique fingerprint.