Iminosugars as Antivirals Against Cytopathic and non Cytopathic
Bovine Viral Diarrhea Virus Mark Hussey Dr Zitzmann Prof Dwek
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Bovine Viral Diarrhea Virus (BVDV) Pestivirus of the
Flaviviridae family Enveloped Positive sense RNA genome 12.7kb
Exists as two biotypes ( cytopathic (cp) and non-cytopathic (ncp) )
Infected animals shed virus in nasal and oral secretions and faeces
Symptoms include diarrhea and fatal thrombocytopenia NS2/3
Non-cytopathic cytopathic
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Virus life cycle Virus binding And internalisation via
endosomal pathway Viral and endosomal membranes fuse Capsid
breakdown RNA translation Virus Secretion ER Golgi RNA + + + -
Template RNA Apoptosis cp BVDV Persistent infection ncp BVDV
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Antivirals A successful antiviral agent may target cellular or
viral processes involved in Viral binding Entry Uncoating RNA
replication Packaging Secretion
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Iminosugars as antivirals Iminosugars are monosaccharide
analogs in which the ring oxygen has been replaced by an imino
group Glucose analogues (DNJ compounds) inhibit ER -glucosidase
enzymes causing viral proteins to misfold and reduce virus
secretion Galactose analogues (DGJ compounds) do not inhibit ER
glucosidase enzymes but those with long alkyl chains show antiviral
activity with an unknown mechanism
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Aims of my project To investigate the antiviral mechanism of
the iminosugar N7-oxanonyl-6deoxy-DGJ (231B) as an example of a
long chain DGJ compound against cp and ncp BVDV Binding
Internalisation Membrane fusion Capsid breakdown Expression of
viral proteins Look for a decrease in the efficacy of drug treated
virions to undergo N OH O H OH CH 3 O Re-visit DNJ compounds as
antivirals.
Cytopathic plaque reduction assay of 231B drug concentration M
% plaques IC50 IC90 0 20 40 60 80 100 120 140 160 untreated 2.5 5
10 100 10005000 10000 neg 231B concentration M plaque number Plaque
assay Yield assay n = 4 n > 50 untreated 1mM treated untreated
1mM treated WB166214 heterodimer 62 83 concentrated cp virus
purified on sucrose gradient and pelleted at 22,000g 62 83 homo
hetero
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Secretion of cp BVDV is not affected by 231B 0, 2.5, 5, 10, 50,
100, 1000 M 5mM 10mM Standard curve from which RNA is
quantified
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BVDV life cycle Nucleus Virus binding and internalisation via
endosomal pathway Viral and endosomal membranes fuse Capsid
breakdown RNA translation Virus secretion ER Golgi RNA
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cp BVDV internalisation assay Conclusion Virus grown in the
presence of 1mM 231B is capable of binding and entering MDBK cells
as efficiently as untreated virus, ie the antiviral effect observed
is not a function of binding or entry via receptor-mediated
endocytosis. + control untreated treated
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cp BVDV envelope and endosomal membrane fusion R18 dequenching
assay at room temperature
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R18 fusion assay cont R18 dequenching assay room temperature
-20 30 80 130 180 1611162126 time / mins fluorescence at 590nm
untreated1mM 231B treatednon infected cells supernatant
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Neat untreated 2.5 M5M5M10 M50 M 100 M 1mM 5mM 10mMNegative
control Immunofluorescent MDBK cells with 231B treated and
untreated cp BVDV
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RNA is secreted from cells infected with non plaque forming
virions
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Plaque formation Clear plaque formation is dependent on the
rate of cell proliferation and spread of virus infection. If cell
density is too high or low prior to infection plaques are not
representative of titre If the multiplicity of infection is too
high, plaques do not form or are too many to be counted Check cell
proliferation in drug Check infected cell proliferation in
drug
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MDBK growth curves in 231B Dr Steve Woodhouse Not possible for
cytopathic BVDV
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G1 synchronised cells 2.5% DMSO G1 SG2M Normal cell cycle
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Cell cycle analysis of infected cells 20hpi Infected + 50 M
231B infected Mock-infected Infected + 500 M 231B Cells have
completed one cell cycle passing through Mitosis and back to G1
(38%) Only 18% of cells have entered G1, 20% fewer than in non
infected cells. These cells can be found in G2M Cells now move
faster through the cell cycle. Nearly all cells have completed one
cycle Cells now even further through the progression of the second
cycle
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BVDV does not arrest cell cycle G1 SG2M Normal cell cycle Non
infected vs infected cell cycle
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cp BVDV induced apoptosis Moi 0.1 Moi 0.01
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Conclusion Viral RNA secretion is unaffected by 231B 231B
treated particles bind, enter and fuse with host cells to translate
their genomes as efficiently as their plaque forming counterparts
Monolayers without plaques still secreted viral RNA at comparable
levels to monolayers with plaques Cp BVDV infection causes cell
cycle progression to slow but not arrest at any checkpoint
Apoptosis may be delayed in drug treated cells infected with BVDV
accounting in part for plaque reduction
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ncp BVDV
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Non cytopathic BVDV
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Ncp BVDV infected MBDK cells with 231B drug is added 1 hour
post infection
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Establishment of persistent infection in absence / presence of
231B Model based on results 1 2 ncp infected moi 0.01 ncp infected
moi 0.01 + 10uM 231B ncp infected moi 0.1 ncp infected moi0.1 +
10uM 231B -Interferon PCR product 500bp non infected drug treated
10uM
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Drug treatment of persistently infected MDBK cells (p34) P =
0.633 % RNA copies N=10
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Conclusion Ncp BVDV viral RNA tires are naturally reduced with
each passage to a steady state level Reduction in viral titre is
enhanced by 231B during the first few passages but does not affect
viral RNA load for subsequent passages 231B does not alter ncp BVDV
RNA secretion at 100 M in persistently infected cells
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DNJ compounds
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NB-DNJ Tested for activity against ncp BVDV in persistently
infected cells
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NN-DNJ Tested for activity against ncp BVDV in persistently
infected cells
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Summary Inhibition of viral RNA secretion DRUGIC 50IC90 NB DNJ
~2.5 M Not reached NN-DNJ ~2.5 M15-30 M 231BNone
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NB-DNJ and NN-DNJ treatment of MDBK cells Work carried out with
Dom Alonzi [G3M5N] relative to [M5N]
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RNA copies per reaction ncp RNA secretion
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IF reduction assay for NB-DNJ and NN-DNJ cytopathic infection
Drug concentration M cells + for NS2/3
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Conclusions NB-DNJ and NN-DNJ reduce viral secretion
effectively in persistently infected cells whereas 231B does not
NN-DNJ access to the ER is about 3-4 times greater than that of
NB-DNJ, as validated by real- time RT-PCR and IF reduction
assay
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Summary Plaque assays may not be a reliable method of screening
antiviral compounds Plaque reduction assays may be replaced by
immunofluorescence (IF) assays and real time RT-PCR IF is much
quicker, easier and negative controls can eliminate any chance of
ncp co-infection (possible in persistently infected cells) ncp BVDV
and cp BVDV should be considered as completely different viruses
for drug screening despite almost identical genomes Only cells
after established persistently infection should be used for drug
screening 231B is not antiviral against BVDV NB-DNJ and NN-DNJ
reduce infectivity by reducing viral RNA secretion
Slide 39
Acknowledgements Supervisors Dr Zitzmann Prof Dwek Dr Patil Dr
Argaud Dr Woodhouse Dr Smith Dom Alonzi Dr Butters Dr Neville Aruna
Jeans Dr Maria Pardo-perez Dr Narayan I Popescu T Whitfield Dr S.
Etti All first floor Glycobiology BBSRC 9am, 10:30am, 2pm, 5:05pm
Daily visits and discussion Technical help support and discussions
FOS work on MDBK cells FACS and cell cycle analysis For being great
people
Slide 40
Cytopathic vs non-cytopathic Nucleus RNA + - Template RNA IFR-3
translocated dsRNA-activated protein kinase (PKR) 2-5
oligoadenylate synthase (OAS)/RNase L system eIF-2 dsRNA APOPTOSIS
cytopathic Non-cytopathic Interferonalpha/beta response IFNs
Phosphorylation cascade ISRE activated MHC class proteins
upregulated mitochondria Bax translocation Cyt c released
Expression of NS2/3 Inhibition of IFR-3 IFR-3 translocated
Interferon beta response Bcl2 upregulated Cyt c not released PTP
pore Caspase 3 and 9 activated