Iminosugars as Antivirals Against Cytopathic and non Cytopathic Bovine Viral Diarrhea Virus Mark Hussey Dr Zitzmann Prof Dwek

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

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

Antivirals A successful antiviral agent may target cellular or viral processes involved in… Viral binding Entry Uncoating RNA replication Packaging Secretion

Iminosugars as antivirals Iminosugars are monosaccharide analogs in which the ring oxygen has been replaced by an imino group Glucose analogues (DNJ compounds) inhibit ER  -glucosidase enzymes causing viral proteins to misfold and reduce virus secretion Galactose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

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.

Plaque formation co-infection Non cytopathic infection Cytopathic infection Mock infected plaques

Cytopathic plaque reduction assay of 231B drug concentration  M % plaques IC50 IC untreated neg 231B concentration  M plaque number Plaque assay Yield assay n = 4 n > 50 untreated 1mM treated untreated 1mM treated WB heterodimer concentrated cp virus purified on sucrose gradient and pelleted at 22,000g homo hetero

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

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

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

cp BVDV envelope and endosomal membrane fusion R18 dequenching assay at room temperature

R18 fusion assay cont… R18 dequenching assay room temperature time / mins fluorescence at 590nm untreated1mM 231B treatednon infected cells supernatant

Neat untreated 2.5  M5M5M10  M50  M 100  M 1mM 5mM 10mMNegative control Immunofluorescent MDBK cells with 231B treated and untreated cp BVDV

RNA is secreted from cells infected with non plaque forming virions

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

MDBK growth curves in 231B Dr Steve Woodhouse Not possible for cytopathic BVDV

G1 synchronised cells 2.5% DMSO G1 SG2M Normal cell cycle

Cell cycle analysis of infected cells 20hpi Infected + 50  M 231B infected Mock-infected Infected  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

BVDV does not arrest cell cycle G1 SG2M Normal cell cycle Non infected v’s infected cell cycle

cp BVDV induced apoptosis Moi 0.1 Moi 0.01

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

ncp BVDV

Non cytopathic BVDV

Ncp BVDV infected MBDK cells with 231B drug is added 1 hour post infection

Establishment of persistent infection in absence / presence of 231B Model based on results 1 2 ncp infected moi 0.01 ncp infected moi uM 231B ncp infected moi 0.1 ncp infected moi uM 231B  -Interferon PCR product 500bp non infected drug treated 10uM

Drug treatment of persistently infected MDBK cells (p34) P = % RNA copies N=10

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

DNJ compounds

NB-DNJ Tested for activity against ncp BVDV in persistently infected cells

NN-DNJ Tested for activity against ncp BVDV in persistently infected cells

Summary Inhibition of viral RNA secretion DRUGIC 50IC90 NB DNJ ~2.5  M Not reached NN-DNJ ~2.5  M15-30  M 231BNone

NB-DNJ and NN-DNJ treatment of MDBK cells Work carried out with Dom Alonzi [G3M5N] relative to [M5N]

RNA copies per reaction ncp RNA secretion

IF reduction assay for NB-DNJ and NN-DNJ cytopathic infection Drug concentration  M  cells + for NS2/3

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

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

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

Cytopathic v’s 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