Search for cellularattachment receptors usedby a GII.4 Norovirus DijonGaurav Dutta DwivediSupervisors: Niklas Arnberg and Carin Årdahl
IntroductionNorovirus are a group of viruses that cause non bacterial gastroenteritis inhumans.The first outbreak of Norovirus infection was reported in Norwalk, Ohio in1968.Human Norovirus infection is popularly known as winter vomiting disease.Norovirus outbreaks frequently occur in community surroundings.Noroviruses are predominantly infectious and highly stable in the environmentand immunity following infection generally is short-lived.
Symptoms of Norovirus infection Nausea and Vomiting Dehydration Fever Abdominal cramping DiarrheaIncubation period is 24 to 48 hours, with the symptoms lasting 12 to 60 hours.Infected hosts can shed virus in stool for up to 2 weeks.
Human Noroviruses recognize histo-blood group antigens (HBGAs)that are expressed on the surface of mucosal epithelial cells.Noroviruses infect individuals with a functional alpha-1, 2-fucosyltransferase(FUT2) gene and are designated as secretor-positive and those with defectedFUT2 gene are termed as secretor-negative ,they are resistant to the commonGII.4 strain, however they can still get infected with other Norovirus strains.
Norovirus ClassificationThe genogroup II, genotype 4 Noroviruses, designated GII.4, are currentlyresponsible for 70–80% of Norovirus outbreaks worldwide.
Viral Morphology Family Caliciviridae. Single-stranded, positive-sense RNA genome of ~7.7 kb. Non-enveloped and icosahedral. 27-40 nm. The viral genome encodes one major structural protein of 60 kDa which forms viral capsid. Capsid 180 molecules, folds into 90 dimers.
Norovirus genome comprises of three open reading frames (ORFs).ORF1 encodes the non-structural proteins that are crucial for virus replication, ORF2 and ORF3encode a major capsid protein VP1 and a minor structural protein VP2, respectively.VP1 consists of shell domain (S) and the protruding domain (P).P domain is further divided into two subdomains known as P1 and P2.Unpublished observations indicate that the presence of specific integrin-binding motifs plays arole in interactions for binding to integrins and allows virus particles attachment to the hostcells.
AimTo explore cellular receptors used by the GII.4 Human Norovirus(NoV) Dijon Virus Like Particles (VLPs) and analyse its bindingcharacteristics across various integrin expressing cells.
Why we used Norovirus like particles (NoV-LPs) in thepresent study ?The study of NoV has been hamperedby the lack of an efficient cell culturesystem, which is still not available forHuman NoV.
MethodsThe Dijon NoV-LPs used were produced in Sf9 insect cells.The VLPs were clarified using ultracentrifugation and sucrosedensity gradient methods , finally the purity was confirmed byusing SDS-PAGE and Western blot.Flow cytometry was used for evaluation of binding characteristicsof NoV-LPs across various integrin expressing cells.
Production of VLPsSf9 cell culture Incubate at 130 rpm,28 ˚C, 5 days Western blot SDS-PAGE Centrifuge at 3000 g,30 min Ultracentrifuge supernatant at 30000 rpm,2hrs,4˚C Loading on gel Ultracentrifuge at 60% 50% 320000 rpm,4hrs,4˚C. 40% 30% Collect the fractions 20% 1 2 3 4 5 Collected fractions Sucrose density gradient
Binding Assay Cell culture Detach cells and Transfer 200 000 cells/well. Incubate with added VLPs NoV-LPs FACS BD LSRII96 well plate Wash 3 X with 100 µl of PFN and centrifuge every time. Wash 3X with 100 µl of PFN and transfer Centrifuge at 1300 rpm,4˚C for 5 mins Into FACS tubes and analyse the binding. goat-anti-rabbit Alexa Flour 647 rabbit-anti-NoV Wash with PFN buffer and incubate with the antibodies Incubate with primary and secondary antibody Centrifuge
Results Pooled 3-5 Pooled 6-7SDS-PAGE showing Fractions1-9 of the SDS-PAGE of cell lysate from the sf9 cell cultureproduced VLPs
Protein gel of pooled concentrated GII.4 Dijon (100K, Amicon filter)
Binding characteristics of NoV-LPs to integrin expressing cellsFigure 1 Figure 2Figure 1 and Figure 2 represents the binding percentage of various integrin expressing cells toNoV-LPs .
Western blot for the produced VLPs Figure 3Detection of produced NoV-LPs. Figure 3 showing the Western blot results ofDijon NoV-LPs in various concentrations probed with primary antibody(rabbit-anti-NoV, serum) and secondary antibody (HRP-conjugated-swine-anti-rabbit).
ConclusionIn conclusion, the production and characterization of GII.4 NoV-LPsin insect cells was validated in this study.Western blot detected NoV-LPs using NoVs raised antibodies fromrabbit sera.This receptor binding study indicates approximately 2-6 timesincreased binding of NoV-LPs to various integrin expressing cellssignifying the importance of integrins as candidate receptors forNoV.
Future directions Repetition of binding experiments with more integrin expressing cell lines. Immortalization and replication studies using human cells. Blocking studies for analyzing Norovirus binding.
Acknowledgement Carin Årdahl thesis supervisor. Professor Niklas Arnberg Division of Virology, Umeå university. Division of Medical Microbiology, Department of Clinical and Experimental Medicine, LiU(Linköping University), Linköping, Sweden.