Download An Investigation into Glycoproteins Associated with Rotaviral Infection

AN INVESTIGATION INTO GLYCOPROTEINS ASSOCIATED WITH
ROTAVIRAL INFECTION
Mark Kraschnefski1, Thomas Haselhorst1, Barbara Coulson2, Milton Kiefel1, Mark von
Itzstein1 and Helen Blanchard1
1. Institute for Glycomics, Griffith University, Gold Coast, QLD 4215, Australia
2. Department of Microbiology and Immunology, University of Melbourne, Parkville,
VIC 3052, Australia
Rotaviruses are the most significant cause of gastroenteritis in the young [1]. Globally,
rotaviral infection afflicts an estimated 111 million children per year, regardless of their
socioeconomic or environmental conditions with approximately 440,000 children dying
annually as a result of the infection [2]. Rotavirus is also an important veterinary pathogen
causing disease in cattle, sheep, swine, horses, dogs, cats, and poultry [1]. The tremendous
incidence of rotavirus disease, as well as the economic burden underscores the urgent need
for the development of new methods to treat the disease.
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 receptor [3].
The cellular recognition site(s) for rotavirus are not clearly defined despite the efforts of
several research groups. It has been shown that the binding and penetration of the rotavirus
virion is a complex process with the existence of multiple cell surface interactions, some of
which are species specific [4]. It is now accepted that rotavirus virions bind to
glycoconjugates on the host cell surface, although there are differing opinions as to their
chemical components, primarily concerning the postulated involvement of sialic acids [5].
For host cell attachment to occur a functional outer capsid spike protein, VP4, is required
[1]. An activating tryptic cleavage of VP4 produces the N-terminal fragment VP8* whose
structure bound to a sialic acid was recently determined by X-ray crystallography [6].
We present here our recent research towards gaining a better understanding of the
carbohydrate-binding interactions of VP8*. We have successfully expressed, purified and
crystallised the VP8* protein from a rhesus (RRV) and human strain (Wa). Additionally,
site-directed mutants of VP8* have been designed and these proteins are currently being
expressed, purified and crystallised. The X-ray crystallographic structure determination of
a RRV mutant VP8* protein has already provided novel insights into the carbohydratebinding specificities of VP8*. Within our group, the combination of X-ray crystallographic
structure determination, STD-NMR and structure analysis techniques are enabling the
assessment of the important interactions for sialic acid binding and ligand specificity. An
increased understanding of these interactions will enable the design of targeted rotavirus
inhibitors with the aim being to develop a potent, in vivo, inhibitor.
References
[1] M. Estes, In Fields Virology 4 ed., D. M. Knipe and P. M. Howley Eds., Lippincott Williams &
Wilkins, Philadelphia, Vol. 2, pg 1747, (2001).
[2] U. Parashar, E. J. B. Hummelman and M. R. G. Miller, Emerging Infectious Diseases 9, 565, (2003).
[3] M. Ciarlet and M. Estes, Current Opinion in Microbiology 4, 435, (2001).
[4] E. Mendez, S. Lopez, M. A. Cuadras, P. Romero and C. F. Arias, Virology 263, 450, (1999).
[5] M. J. Kiefel, and M. von Itzstein, Methods in Enzymology 363, 395, (2003).
[6] P. R. Dormitzer, Z. -Y. J. Sun, G. Wagner and S. C. Harrison, EMBO Journal 21, 885, (2002).