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

Abstract

N-acetyl-and N-glycolylneuraminic acids (Sia) and ␣2␤1 integrin are frequently used by rotaviruses as cellular receptors through recognition by virion spike protein VP4. The VP4 subunit VP8*, derived from Wa rotavirus, binds the internal N-acetylneuraminic acid on ganglioside GM1. Wa infection is increased by enhanced internal Sia access following terminal Sia removal from main glycan chains with sialidase. The GM1 ligand cholera toxin B (CTB) reduces Wa infectivity. Here, we found sialidase treatment increased cellular GM1 availability and the infectivity of several other human (including RV-3) and animal rotaviruses, typically rendering them susceptible to methyl ␣-D-N-acetylneuraminide treatment, but did not alter ␣2␤1 usage. CTB reduced the infectivity of these viruses. Aceramido-GM1 inhibited Wa and RV-3 infectivity in untreated and sialidasetreated cells, and GM1 supplementation increased their infectivity, demonstrating the importance of GM1 for infection. Wa recognition of ␣2␤1 and internal Sia were at least partially independent. Rotavirus usage of GM1 was mapped to VP4 using virus reassortants, and RV-3 VP8* bound aceramido-GM1 by saturation transfer difference nuclear magnetic resonance (STD NMR). Most rotaviruses recognizing terminal Sia did not use GM1, including RRV. RRV VP8* interacted minimally with aceramido-GM1 by STD NMR. Unusually, TFR-41 rotavirus infectivity depended upon terminal Sia and GM1. Competition of CTB, Sia, and/or aceramido-GM1 with cell binding by VP8* from representative rotaviruses showed that rotavirus Sia and GM1 preferences resulted from VP8*-cell binding. Our major finding is that infection by human rotaviruses of commonly occurring VP4 serotypes involves VP8* binding to cell surface GM1 glycan, typically including the internal N-acetylneuraminic acid. IMPORTANCERotaviruses, the major cause of severe infantile gastroenteritis, recognize cell surface receptors through virus spike protein VP4. Several animal rotaviruses are known to bind sialic acids at the termini of main carbohydrate chains. Conversely, only a single human rotavirus is known to bind sialic acid. Interestingly, VP4 of this rotavirus bound to sialic acid that forms a branch on the main carbohydrate chain of the GM1 ganglioside. Here, we use several techniques to demonstrate that other human rotaviruses exhibit similar GM1 usage properties. Furthermore, binding by VP4 to cell surface GM1, involving branched sialic acid recognition, is shown to facilitate infection. In contrast, most animal rotaviruses that bind terminal sialic acids did not utilize GM1 for VP4 cell binding or infection. These studies support a significant role for GM1 in mediating host cell invasion by human rotaviruses.

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

“…To validate protein activity, an equimolar concentration of GM1a glycan, a confirmed receptor for the DS-1-like P [4] rotavirus strain RV-5 and for RV-3 (ref. 22) was added to the A-tri complex of VP8* protein from DS-1 or RV-3.…”

“…Binding by GST-VP8* from HAL1166, K8 and Wa rotaviruses to MA104 cells was assayed in the same experiment at 4°C as previously described 22 . The optimal (saturating) concentration of each GST-VP8* was initially determined from its dose-dependent cell-binding curve.…”

“…or DMEM was reacted for 1 H at 4°C with the optimal level of GST-VP8* for HAL1166 (150 mg ml À 1 ), K8 (600 mg ml À 1 ) or Wa (300 mg ml À 1 ), then added to cells for 45 min. Stained single cells were analysed by flow cytometry as previously described 22 . The results provided are representative of those obtained in three independent experiments.…”

“…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 . We concluded that human sialidase-insensitive strains such as Wa and RV-3, and most probably other human strains, require the presence of sialic acidcontaining receptors for efficient infection 12,22 .…”

Human Milk Contains Novel Glycans That Are Potential Decoy Receptors for Neonatal Rotaviruses

“…Recent observations show that not all rotaviruses require a sialic acid determinant for binding (49,74,93). Based on the studies reported here, it is important that the so-called neuraminidase-insensitive or sialic-acid-independent rotaviruses be analyzed for their glycan binding specificities on similar shotgun glycan microarrays comprising milk glycans or glycans released from cells or tissues susceptible to rotavirus infection.…”