The role of cell surface heparan sulfate in enterovirus A71 infections and the development of antiviral agents targeting viral attachment and RNA translation initiation / Tan Chee Wah
Enterovirus A71 (EV-A71) is the main causative agent of hand, foot and mouth disease (HFMD). Recent EV-A71 outbreaks in Asia-Pacific were not limited to mild HFMD, but were associated with neurological complications including aseptic meningitis, brainstem encephalitis and deaths. The absence of l...
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Format: | Thesis |
Published: |
2014
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Online Access: | http://studentsrepo.um.edu.my/4619/1/Tan_Chee_Wah_PhD_Thesis.pdf http://studentsrepo.um.edu.my/4619/ |
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Summary: | Enterovirus A71 (EV-A71) is the main causative agent of hand, foot and mouth disease
(HFMD). Recent EV-A71 outbreaks in Asia-Pacific were not limited to mild HFMD,
but were associated with neurological complications including aseptic meningitis,
brainstem encephalitis and deaths. The absence of licensed therapeutics for clinical use
has intensified research into anti-EV-A71 development. Since virus-host receptor
interaction is the first essential event during virus infection, inhibitors that block this
event could act as potential therapeutics. EV-A71 VP1 capsid protein is involved in
viral-host receptor interactions and carries multiple receptor binding sites. Screening of
95 overlapping peptides covering the entire EV-A71 VP1 capsid protein was
hypothesized to identify potential viral attachment inhibitors, as well as unknown
receptors. Out of 95 overlapping peptides, a peptide designated as SP40 peptide
significantly inhibited EV-A71-induced cytopathic effect, plaque formation, RNA
synthesis and viral protein synthesis. Mechanism of action analysis revealed that SP40
peptide is not virucidal, but blocked EV-A71 attachment to the cell surface. Alanine
scanning analysis showed that positively charged amino acids were critical for the
antiviral activities. Sequence analysis revealed that SP40 peptide carried a heparan
sulfate-specific binding domain (-RRKV-), which led to the hypothesis that EV-A71
could use cell surface heparan sulfate as an attachment receptor. Highly sulfated heparin,
dextran sulfate and suramin significantly inhibited EV-A71 infections in a dosedependent
manner. Interference with heparan sulfate biosynthesis either by sodium
chlorate treatment or through transient knockdown of N-deacetylase/N-sulfotransferase-
1 and exostosin-1 expression reduced EV-A71 infection. Enzymatic removal of cell
surface heparan sulfate by heparinase I/II/III inhibited EV-A71 infection. Biochemistry
analysis revealed that EV-A71 interacts with heparan sulfate through electrostatic
interactions. These findings support the hypothesis and confirmed that EV-A71 uses
iv
cell surface heparan sulfate as an attachment receptor. Other than the attachment
inhibitor, this study also tested DNA-like antisense-mediated morpholino oligomers as
anti-EV-A71 agents. Two octaguanidinium-conjugated morpholino oligomers (vivo-
MOs) targeting EV-A71 internal ribosome entry site (IRES) significantly inhibited EVA71
infections at multiple time points, in a dose-dependent manner. EV-A71 resistance
to vivo-MO-1 arose after 8 blind passages in the presence of increased concentrations of
vivo-MO-1, but not vivo-MO-2. A single nucleotide mutation at the extreme 3’ end (T
to C substitution at position 533) was sufficient to confer vivo-MO-1 resistance. In
mismatch tolerance analysis, results demonstrated that the positions and the number of
mismatches affect vivo-MO-1 efficacy. A single mismatch at the center of the targeted
region was more tolerable compared to a mismatch at the end of the targeted region. In
conclusion, this study has identified an antiviral peptide that potentially blocks viral
attachment to heparan sulfate, and led to the discovery of a novel EV-A71 attachment
receptor. This study also identified two antisense-mediated vivo-MOs targeted sites for
antiviral intervention. This study suggests that blocking of viral attachment and viral
RNA translation are good strategies for antiviral intervention. |
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