Characterisation of G serotype dependent non-antibody inhibitors of rotavirus in normal mouse serum

150

170

Most mammalian rotaviruses contain tripeptide amino acid sequences in outer capsid proteins VP4 and VP7 which have been shown to act as ligands for integrins ␣2␤1 and ␣4␤1. Peptides containing these sequences and monoclonal antibodies directed to these integrins block rotavirus infection of cells. Here we report that SA11 rotavirus binding to and infection of K562 cells expressing ␣2␤1 or ␣4␤1 integrins via transfection is increased over virus binding to and infection of cells transfected with ␣3 integrin or parent cells. The increased binding and growth were specifically blocked by a monoclonal antibody to the transfected integrin subunit but not by irrelevant antibodies. In our experiments, integrin activation with phorbol ester did not affect virus binding to cells. However, phorbol ester treatment of K562 parent and transfected cells induced endogenous gene expression of ␣2␤1 integrin, which was detectable by flow cytometry 16 h after treatment and quantitatively correlated with the increased level of SA11 virus growth observed after this time. Virus binding to K562 cells treated with phorbol ester 24 h previously and expressing ␣2␤1 was elevated over binding to control cells and was specifically blocked by the anti-␣2 monoclonal antibody AK7. Virus growth in ␣4-transfected K562 cells which had also been induced to express ␣2␤1 integrin with phorbol ester occurred at a level approaching that in the permissive MA104 cell line. We therefore have demonstrated that two integrins, ␣2␤1 and ␣4␤1, are capable of acting as cellular receptors for SA11 rotavirus.Rotaviruses, members of the family Reoviridae, are the major etiological agents of severe acute gastroenteritis in infants and young children worldwide and are important pathogens in most mammalian species. It is anticipated that the longawaited introduction of the first vaccine against human rotavirus into use in North America in 1998 will lead to a reduction in the most severe human illness associated with this virus, but other vaccination and therapeutic approaches are required. Novel strategies may be devised following the identification of cellular receptors for rotavirus and from an understanding of the process of viral entry into cells, particularly into intestinal epithelial cells. These are essential steps for productive rotavirus infection and major determinants of host cell tropism.The nonenveloped, icosahedral rotavirus particle consists of a genome of 11 segments of double-stranded RNA (48) in a triple-layered protein capsid (66). The outermost layer of each virion is composed of trimers of the 37-kDa glycoprotein VP7 and 60 spikes of the 88-kDa protein VP4, probably as dimers, which extend about 12 nm above the VP7 surface and interact extensively with VP7 throughout the outer surface (47,65,66).Both VP4 and VP7 independently elicit neutralizing, protective antibodies and are virulence determinants (26, 46). VP4 is an important determinant of host cell tropism (27), receptor binding, and cell penetration (37). Proteolytic cleavage of VP4 into two...

mentioning

confidence: 99%

“…VP7 appears also to play a role in rotavirus cell attachment, since it has been identified as the infected cell lysate protein which bound to MA104 cell monolayers (52), and it may interact with VP4 to influence receptor-binding specificity (6,37,43).…”

mentioning

confidence: 99%

Integrins α2β1 and α4β1 Can Mediate SA11 Rotavirus Attachment and Entry into Cells

Hewish

Takada

Coulson

2000

150

170

“…Rotaviruses may enter either through an endocytotic pathway or through direct penetration. Although earlier studies implicated VP7 in cell attachment (19,46), recent studies have increasingly indicated that VP4 is the major player in the entry process (11,26,30) while VP7 may modulate the functions of VP4 (3,10,35,49).VP4 is susceptible to proteolysis and is cleaved by trypsin into VP8 ‫ء‬ (26 kDa) and VP5 ‫ء‬ (60 kDa). The two trypsin cleavage products remain associated with the virion, although the precise topographical locations of VP5 ‫ء‬ and VP8 ‫ء‬ in the VP4 spike are still uncertain.…”

mentioning

confidence: 99%

mentioning

confidence: 99%

Trypsin Cleavage Stabilizes the Rotavirus VP4 Spike

Crawford¹,

Mukherjee²,

Estes³

et al. 2001

126

103

Trypsin enhances rotavirus infectivity by an unknown mechanism. To examine the structural basis of trypsin-enhanced infectivity in rotaviruses, SA11 4F triple-layered particles (TLPs) grown in the absence (nontrypsinized rotavirus [NTR]) or presence (trypsinized rotavirus [TR]) of trypsin were characterized to determine the structure, the protein composition, and the infectivity of the particles before and after trypsin treatment. As expected, VP4 was not cleaved in NTR particles and was cleaved into VP5‫ء‬ and VP8 ‫ء‬ in TR particles. However, surprisingly, while the VP4 spikes were clearly visible and well ordered in the electron cryomicroscopy reconstructions of TR TLPs, they were totally absent in the reconstructions of NTR TLPs. Biochemical analysis with radiolabeled particles indicated that the stoichiometry of the VP4 in NTR particles was the same as that in TR particles and that the VP8 ‫ء‬ portion of NTR, but not TR, particles is susceptible to further proteolysis by trypsin. Taken together, these structural and biochemical data show that the VP4 spikes in the NTR TLPs are icosahedrally disordered and that they are conformationally different. Structural studies on the NTR TLPs after trypsin treatment showed that spike structure could be partially recovered. Following additional trypsin treatment, infectivity was enhanced for both NTR and TR particles, but the infectivity of NTR remained 2 logs lower than that of TR particles. Increased infectivity in these particles corresponded to additional cleavages in VP5 ‫ء‬ , at amino acids 259, 583, and putatively 467, which are conserved in all P serotypes of human and animal group A rotaviruses and also corresponded with a structural change in VP7. These biochemical and structural results show that trypsin cleavage imparts order to VP4 spikes on de novo synthesized virus particles, and these ordered spikes make virus entry into cells more efficient.Rotaviruses are the leading cause of severe gastroenteritis in young children worldwide (16). Structural and biochemical analyses show that rotaviruses are large, icosahedral particles having a complex architecture consisting of three concentric capsid layers surrounding a genome of 11 segments of doublestranded RNA (41, 42). The innermost capsid layer, composed of VP2, encloses the genomic double-stranded RNA. A significant portion of the genomic RNA, particularly that in close contact with the inner surface of the VP2 layer, is icosahedrally ordered (43). Anchored to the inner surface of VP2 at the icosahedral vertices are two proteins, VP1 and VP3, involved in transcription of the genome within the intact particle. The intermediate capsid layer is composed of trimers of VP6 organized on a Tϭ13 (levo) icosahedral lattice (44). The outermost layer in the infectious virus is composed of the major capsid glycoprotein VP7 and the hemagglutinin spike protein VP4 (16). VP7 is present as 780 molecules grouped as 260 trimers at the local and strict threefold axes of a Tϭ13 left-handed icosahedral lattice (44, 51). The VP7 ...

“…The origins, cultivation in MA104 cells, and infectivity assay of human rotavirus strains Wa (G1P1A [8]), RV-5 (G2P1B [4]), RV-3 (G3P2A [6]), and S12/85 (G3P2A [6]); monkey rotavirus RRV (G3P5B [3]); bovine rotaviruses NCDV (G6P6 [1]) and UK (G6P7 [5]); and porcine rotaviruses CRW-8 (G3P9 [7]) and TFR-41 (G5P9 [7]) have been described previously (15,(45)(46)(47)(48). Reassortant rotaviruses 12-1 and 28-1 were produced and characterized previously (49).…”

Section: Methodsmentioning

confidence: 99%

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

Fleming

Böhm

Dang

et al. 2014

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.