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Virol Res J 2017 Volume 1 Issue 3
International Virology Conference
October 30-31, 2017 | Toronto, Canada
Enterovirus subverts autophagy through cleavage of fusion adaptor proteins and selective autophagy receptors
Yasir Mohamud, Junyan Shi, Junyan Qu, Yuan Chao Xue, Haoyu Deng, Jingchun Zhang
and
Honglin Luo
University of British Columbia, Canada
Background:
Myocarditisisaninflammatorydiseaseoftheheart
often caused by viral infection, particularly the enteroviruses,
such as coxsackievirus B3 (CVB3). Autophagy, an evolutionarily-
conserved intracellular degradation pathway, targets misfolded
proteins, damaged organelles, and invading pathogens for
lysosomal clearance. Although traditionally considered a non-
selective degradative process, it’s now clear that autophagy can
mediate targeted clearance of protein aggregates/damaged
organelles via selective autophagy receptors, which harbor
highly conserved ubiquitin-associated and LC3 interacting
domains. Contrary to previous understanding of autophagy as
ananti-viral pathway,weandothershaveshownthat thecellular
autophagic machinery can be hijacked by enterovirus to disrupt
its degradative capacity (or autophagic flux) and promote the
accumulation of autophagosomes that serve as membrane
scaffolds for viral replication. Moreover, we discovered that two
selective autophagic receptors, namely p62/sequestosome 1
and neighbor of BRCA1 gene 1 (NBR1), are cleaved upon CVB3
infection, resulting in not only loss-of-function, but also the
generation of dominant-negative fragments that further impair
selective clearance of ubiquitinated protein aggregates. Despite
these intriguing findings, the exact mechanism by which CVB3
inhibits autophagic flux and disrupts protein/organelle quality
control is not fully understood. We hypothesize that CVB3
infection impairs the autophagic pathway through virus-
encodedproteinases that specifically target autophagic proteins
required for autophagosome-lysosome fusion and/or selective
cargo recruitment, ultimately leading to cardiac dysfunction by
facilitating viral replication and via preventing the clearance of
toxic protein aggregates/damaged organelles.
Methods & Results:
Our previous
in vivo
findings that CVB3-
infected mouse hearts display an abnormal accumulation
of autophagosomes and misfolded proteins/damaged
mitochondria, and the in vitro evidence that CVB3 infection
inhibits autophagic flux, suggest that the fusion process
of autophagy is disrupted during infection. To delineate
the possible mechanism involved, we focused on proteins
previously reported to be involved in autophagosome fusion.
Notably, we found that the autophagosomal SNARE (soluble
N-ethylmaleimide-sensitive factor activating protein receptor)
protein SNAP29 (synaptoxomal-associated protein 29) and
the tethering protein PLEKHM1 (pleckstrin homology domain
containing protein family member 1), two critical proteins
known to regulate autophagosome-lysosome fusion, were
cleaved upon CVB3 infection. Further
in vivo
(in cells transfected
with protease constructs) and in vitro (using recombinant
proteases) cleavage assays demonstrated that CVB3-encoded
proteinase 3Cpro, not 2Apro or caspases, is responsible for
these cleavages. Combining a bioinformatics approach with
site-directed mutagenesis, we identified the cleavage sites on
SNAP29 (Q161) and PLEKHM1 (Q668), respectively, leading to
impaired SNARE complex formation.Moreover, we showed that
gene-silencing of SNAP29 and PLEKHM1 inhibited autophagic
flux, resulting in a significant increase in viral growth, likely
due to enhanced accumulation of autophagosomes that
provide sites for viral RNA replication and assembly. Finally,
we also identified the autophagic receptor protein, NDP52
(nuclear domain 10 protein 52), as a bona fide substrate of
viral proteinase 3Cpro. The cleavage of NDP52 takes place at
Q139, separating the N-terminal LC3-interacting region from
the C-terminal ubiquitin-binding domain. The functional
significance of NDP52 cleavage is currently under investigation.
Conclusion:
We identified a novel underlying mechanism by
which enterovirus, through viral encoded proteinases, subverts
the host autophagic pathway to promote viral propagation and
cause cardiac damage. Our findings in this study provide strong
evidence of a potential therapeutic benefit by targeting the
autophagy-virus interface.
Speaker Biography
Yasir Mohamud, is a PhD student from University of British Columbia, Canada,
Centre for Heart Lung Innovation, St. Paul’s Hospital 2 Department of Pathology and
Laboratory Medicine, University of British Columbia, Canada.
e:
Yasir.Mohamud@hli.ubc.ca