Investigation Into the Sialic-Acid Binding Domain, Vp8*, Associated with Rotavirus Host-Cell

INVESTIGATION INTO THE SIALIC-ACID BINDING DOMAIN, VP8*, ASSOCIATED WITH ROTAVIRUS HOST-CELL RECOGNITION

Stacy A. Scott1, Mark J. Kraschnefski1, Barbara Coulson2, Milton J Kiefel1,

Mark von Itzstein1 and Helen Blanchard1*

1 Institute for Glycomics, Griffith University (Gold Coast Campus) Queensland 9726, Australia.

2Department of Microbiology and Immunology, University of Melbourne, Parkville, VIC 3052, Australia

*Corresponding Author: E-mail:

Rotavirus is the major cause of severe acute dehydrating gastroenteritis in children under 5 years of age. Rotavirus infection is responsible for an estimated 20-50% of all hospitalizations associated with severe diarrhea and approximately 440,000 children, primarily in the developing world, die annually as a result of the infection1. The tremendous incidence of rotavirus disease, and associated economic burden underscores the urgent need for the development of new methods targeting rotavirus infection.

Rotaviruses have a very specific cell tropism, infecting only the mature enterocytes on the tip of the intestinal villi, which suggests the existence of a specific host cell receptor2. Although not completely defined, the attachment and invasion of the rotavirus virion is a complex process involving multiple cell-surface interactions, some being species specific3. Rotavirus virions do bind to glycoconjugates on the host cell surface, but the exact involvement and dependency on sialic acids for rotavirus infection remains controversial4,5. It is however, proven that for host-cell attachment to occur a functional outer capsid spike protein, VP4, is required5; and that tryptic cleavage of VP4 produces the N-terminal fragment VP8* which is a sialic-acid binding domain. The X-ray crystallographic structure of Rhesus VP8* bound to a sialic acid6 confirms the ability of this domain to bind carbohydrate ligands.

In order to understand rotavirus host-specificity and the associated recognition of host-cell carbohydrates we are working on the determination and analysis of structures of the VP8* domain from various rotavirus strains as well as complexes with carbohydrate ligands. We have successfully expressed, purified and done analysis of homogeneity via dynamic light scattering, the VP8* protein from human (Wa) and porcine rotavirus (CRW8) strains. We have produced three different crystal forms of apo Wa, and two crystal forms of CRW8 in complex with sialic acid. We have collected X-ray diffraction data at the European Synchrotron Radiation Facility (ESRF) sufficient to enable the determination of the structure of CRW8, and refinement of the structure is in progress. This work is focused on providing an increased understanding of the rotavirus that will potentially enable the design of potent rotavirus inhibitors and assist in combating the devastating effects caused by rotavirus infection.

References: [1] Fischer, T. K.; Bresee, J. S.; Glass, R. I. Vaccine22 Suppl 1: S49-54, (2004). [2] Ciarlet, M.; Estes, M. Current Opinion in Microbiology4, 435-441, (2001). [3] Mendez, E.; Lopez, S.; Cuadras, M. A.; Romero, P.; Arias, C. F. Virology263, 450-459, (1999). [4] Kiefel, M. J.; von Itzstein, M. Methods in Enzymology363, 395-412, (2003). [5] Estes, M. In Fields Virology; 4 ed.; Knipe, D. M.; Howley, P. M.; Eds.; Lippincott Williams & Wikins: Philadelphia, 2001; Vol. 2, 1747-1785. [6] Dormitzer, P. R.; Sun, Z.-Y. J.; Wagner, G.; Harrison, S. C. EMBO Journal21, 885-897, (2002).