IntroductionNoroviruses (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.

ConclusionsIn 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.

Results

Binding characteristics of NoV-LPs to integrin expressing cells

MethodsIn this study, we have incorporated NoV-LPs as the most complimentary model to study human NoV-host protein interactions. The Dijon NoV-LPs used were produced in Sf9 insect cells using the baculovirus system. The VLPs were clarified using ultracentrifugation and sucrose density gradient methods and the purity was confirmed by using SDS-PAGE and Western blot. We also evaluated the binding characteristics of NoV-LPs across various integrin expressing cells using Flow cytometry.

References1.Atmar RL, Estes MK. The epidemiologic and clinical importance of norovirus infection. Gastroenterol Clin North Am 35:275-90, 2006.2. Widdowson, M.A., Monroe, S.S. & Glass, R.I. (2005). Are Noroviruses emerging? Emerg Infect Dis Vol. No. 5 pp. (735-737).3. Xi Jiang and Ming Tan ,Norovirus and its histo-blood group antigen receptors: an answer to a historical puzzle, TRENDS in Microbiology Vol.13 No.6 June 2005.4. Choi, J.M.,Hutson, A.M., Estes, M.K., Prasad, B.V., 2008. Atomic resolution structural characterization of recognition of histo-blood group antigens by Norwalk virus. Proc. Natl. Acad. Sci. U. S. A. 105 (27), 9175–9180.

Acknowledgement Special thanks and recognition to Carin Årdahl my thesis supervisor for teaching me how to plan, perform and evaluate experiments and Professor Niklas Arnberg for providing the opportunity of doing master thesis at the Division of Virology, Umeå university. I would also like to acknowledge the contribution from our collaborators at (Linköping University) LiU.

AimThe 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

Search for cellular attachment receptors used by a GII.4 Norovirus Dijon (30 hp)Gaurav Dutta Dwivedi

Supervisors: Niklas Arnberg and Carin ÅrdahlDivision of Virology, Department of Clinical Microbiology, Umeå University, Umeå -90187

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 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, 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 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-binding motifs plays a role in interactions for binding to integrins and allows virus particles attachment to the host cells.

Detection of produced NoV-LPs. Figure 1 showing the Western blot results of Dijon NoV-LPs 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 (lane 5). Lane 1: Dijon, 2 µg Lane 2: Dijon 10 µg. Lane 3: Dijon 2 µg. Lane 4: Dijon 10 µg. Lane 5: Dijon control (from LiU).

Figure 1

Figure 2 Figure 3

Binding study was accomplished by preincubating cells with NoV-LPs. Binding was confirmed by 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 (αv, α5, β1 and β7). This binding was compared with binding to control cells (CHO-B2 and FHs74Int). Figure 2 and Figure 3 represents the binding percentage of various integrin expressing cells to NoV-LPs (0.4 µg, 2 µg, 10 µg).

Choi et al. 2008 (4)