Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the VP8* carbohydrate-binding protein of the human rotavirus strain Wa

Abstract

Rotaviruses exhibit host-specificity and the first crystallographic information on a rotavirus strain that infects humans is reported here. Recognition and attachment to host cells, leading to invasion and infection, is critically linked to the function of the outer capsid spike protein of the rotavirus particle. In some strains the VP8* component of the spike protein is implicated in recognition and binding of sialic-acid-containing cell-surface carbohydrates, thereby enabling infection by the virus. The cloning, expression, purification, crystallization and initial X-ray diffraction analysis of the VP8* core from human Wa rotavirus is reported. Two crystal forms (trigonal P3 2 21 and monoclinic P2 1 ) have been obtained and X-ray diffraction data have been collected, enabling determination of the VP8* 64-223 structure by molecular replacement.

Insight into Host Cell Carbohydrate-recognition by Human and Porcine Rotavirus from Crystal Structures of the Virion Spike Associated Carbohydrate-binding Domain (VP8*)

“…The significant hydrogen-bonding scheme in which the arginine residues participate suggests that it would be unlikely for this to occur, though cannot be completely eliminated based on this crystal structure of Wa, since a symmetry-related molecule is packed directly above this region. Strongly supportive of their determined orientation being prevalent is that in both our Wa VP8* structures solved in a different crystal form (initial data published) 24 where this region is solvent exposed, and also in the human rotavirus strain DS-1 VP8* structure 17 a similar arrangement of the arginine residues straddling the cleft and secured by hydrogen bonds with the opposing β-strand occurs. This cleft appears nonoptimal for binding sialic acids and neither our structures from two different crystal forms of Wa VP8* both crystallised in the presence of Neu5Ac-α2Me, nor our STD NMR analysis of this ligand with the Wa VP8* (unpublished results) provide any evidence for Wa VP8* binding this particular sialic acid derivative.…”

“…X-ray crystal structure determination of CRW-8 VP8* 64-224 and Wa VP8* 64-223 in respective space groups P2 1 2 1 2 1 and P3 2 21 were done via molecular replacement. 24,26 We report here refinement (REFMAC 28 ) and analysis of these structures. Solvent-flattening and noncrystallographic symmetry averaging (implemented in CCP4 29 ) was done as appropriate.…”

“…24,26 Homology models (MODELLER 27 ) were based on a template of the crystallographic structure of RRV VP8* 62-224 (PDB code 1KQR 12 and 72% (CRW-8) and 45% (Wa) amino acid sequence identity. X-ray crystal structure determination of CRW-8 VP8* 64-224 and Wa VP8* 64-223 in respective space groups P2 1 2 1 2 1 and P3 2 21 were done via molecular replacement.…”

Revisiting the role of histo-blood group antigens in rotavirus host-cell invasion

“…However, commonly used bacterial and viral sialidases remove only terminal sialic acids from sialylglycoconjugates. Sialic acid recognition by sialidasesensitive animal rotaviruses via VP8* is structurally well characterized through a series of crystallographic studies 13,[15][16][17][18][19][20][21] . Our recent studies using nuclear magnetic resonance (NMR) spectroscopy and cell-based assays identified GM1 as a ganglioside receptor for the human rotavirus strains Wa (P [8]) and RV-3 (P [6]), and revealed that the internal Nacetylneuraminic acid (sialic acid) residue of GM1 plays a key role in VP8* recognition 12,22 .…”

Relative Roles of GM1 Ganglioside, N -Acylneuraminic Acids, and α2β1 Integrin in Mediating Rotavirus Infection

“…The wild-type CRW-8 VP8* containing Pro rather than Ser at position 157 was produced (38). The Wa VP8* core (aa 64 to 223) was expressed as described before (59). RV-3 VP8*64-223 was cloned, expressed, and purified as described for the Wa VP8* core.…”