Project Details
Abstract
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
External Project ID:MOST103-2321-B182-010
Status | Finished |
---|---|
Effective start/end date | 01/03/14 → 03/03/15 |
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