Norovirus Dijon

1. Search for cellular attachment receptors used by a GII.4 Norovirus Dijon (30 hp)
Gaurav Dutta Dwivedi
Supervisors: Niklas Arnberg and Carin Årdahl
Division of Virology, Department of Clinical Microbiology, Umeå University, Umeå -90187
Introduction
Noroviruses (NoVs) belongs to the family Caliciviridae and NoV infection is now considered as the supreme cause of acute gastroenteritis in people of all ages (1). NoVs are approximately 38 nm icosahedral
viruses and contain a 7.5 kb single-stranded, positive-sense RNA genome that encodes three open reading frames (ORFs), including the ORF1 replicase polyprotein and the major and minor structural ORFs 2
and 3, respectively. For the last 20 years, genogroup II genotype 4 (GII.4) accounts for the majority of human cases (2). Recent studies demonstrate that human histo-blood group antigens (HBGAs) function as
receptors for NoVs infection (3). The development of vaccines or antivirals against human NoVs has been hampered by the nonexistence of an in vitro cell culture system or small animal disease model. In this
study we look for additional cellular attachment receptors used by NoV Like Particles (NoV-LPs) for their binding with various integrin expressing cells and compare with control cells.
Aim
The aim of this study was to explore cellular receptors used by the GII.4 Human Norovirus Dijon VLPs and analyze its binding characteristics across various integrin expressing cells to aid in design of attachment
inhibitors.
Background Results Conclusions
Choi et al. 2008 (4)
In conclusion, the production and characterization of GII.4 NoV-LPs
in insect cells was validated in this study. Western blot detected
NoV-LPs using NoVs raised antibodies from rabbit sera. This receptor
binding study indicates approximately 2-6 times increased binding of
NoV-LPs to various integrin expressing cells signifying the
importance of integrins as candidate receptors for NoV.
Figure 1
a) This figure displays the icosahedral capsid structure (presenting only the backbone
atoms) of Norwalk virus formed by 180 molecules (90 dimers) of the capsid viral protein Detection of produced NoV-LPs. Figure 1 showing the Western blot results of Dijon NoV-LPs
1 (VP1).The S domain (amino acids 1–225) and the P1 (amino acids 225–278 and 406–
519) and P2 (amino acids 279–405) subdomains (shown in blue, red, and yellow,
in various concentrations probed with primary antibody (rabbit-anti-NoV, serum) and
secondary antibody (HRP-conjugated-swine-anti-rabbit) which is compared with the control
Acknowledgement
respectively. (b) This is a ribbon form of VP1 dimer from the capsid structure with S, P1,
and P2 domains colored as in a. (c) The organisation of the P domain makes (amino acids (lane 5). Lane 1: Dijon, 2 µg Lane 2: Dijon 10 µg. Lane 3: Dijon 2 µg. Lane 4: Dijon 10 µg. Lane Special thanks and recognition to Carin Årdahl my thesis supervisor
225–519).The P1 and P2 subdomains are colored as in b. The N and C termini in b and c
are indicated. Unpublished observations indicate that the presence of specific integrin-
5: Dijon control (from LiU). for teaching me how to plan, perform and evaluate experiments and
binding motifs plays a role in interactions for binding to integrins and allows virus
particles attachment to the host cells. Binding characteristics of NoV-LPs to integrin expressing cells
Professor Niklas Arnberg for providing the opportunity of doing
Figure 2 Figure 3
master thesis at the Division of Virology, Umeå university. I would
also like to acknowledge the contribution from our collaborators at
Methods (Linköping University) LiU.
In this study, we have incorporated NoV-LPs as the
most complimentary model to study human NoV-
host protein interactions. The Dijon NoV-LPs used
References
1.Atmar RL, Estes MK. The epidemiologic and clinical importance of norovirus infection.
were produced in Sf9 insect cells using the Gastroenterol Clin North Am 35:275-90, 2006.
baculovirus system. The VLPs were clarified using 2. Widdowson, M.A., Monroe, S.S. & Glass, R.I. (2005). Are Noroviruses emerging? Emerg
ultracentrifugation and sucrose density gradient Binding study was accomplished by preincubating cells with NoV-LPs. Binding was confirmed by Infect Dis Vol. No. 5 pp. (735-737).
3. Xi Jiang and Ming Tan ,Norovirus and its histo-blood group antigen receptors: an
methods and the purity was confirmed by using using primary antibody (rabbit-anti-NoV, serum) and secondary antibody (goat-anti-rabbit Alexa
Flour 647). Flow cytometry was used to detect the binding of NoV-LPs to integrin expressing cells answer to a historical puzzle, TRENDS in Microbiology Vol.13 No.6 June 2005.
SDS-PAGE and Western blot. We also evaluated the 4. Choi, J.M.,Hutson, A.M., Estes, M.K., Prasad, B.V., 2008. Atomic resolution structural
(αv, α5, β1 and β7). This binding was compared with binding to control cells (CHO-B2 and FHs74Int).
binding characteristics of NoV-LPs across various Figure 2 and Figure 3 represents the binding percentage of various integrin expressing cells to NoV- characterization of recognition of histo-blood group antigens by Norwalk virus. Proc. Natl.
integrin expressing cells using Flow cytometry. LPs (0.4 µg, 2 µg, 10 µg). Acad. Sci. U. S. A. 105 (27), 9175–9180.