Symmetry-related clustering of positive charges is a common mechanism for heparan sulfate binding in enteroviruses - PubMed (original) (raw)
Symmetry-related clustering of positive charges is a common mechanism for heparan sulfate binding in enteroviruses
Nigel J McLeish et al. J Virol. 2012 Oct.
Abstract
Coxsackievirus A9 (CAV9), a member of the Picornaviridae family, uses an RGD motif in the VP1 capsid protein to bind to integrin αvβ6 during cell entry. Here we report that two CAV9 isolates can bind to the heparan sulfate/heparin class of proteoglycans (HSPG). Sequence analysis identified an arginine (R) at position 132 in VP1 in these two isolates, rather than a threonine (T) as seen in the nonbinding strains tested. We introduced a T132R substitution into the HSPG-nonbinding strain Griggs and recovered infectious virus capable of binding to immobilized heparin, unlike the parental Griggs strain. The known CAV9 structure was used to identify the location of VP1 position 132, 5 copies of which were found to cluster around the 5-fold axis of symmetry, presumably producing a region of positive charge which can interact with the negatively charged HSPG. Analysis of several enteroviruses of the same species as CAV9, Human enterovirus B (HEV-B), identified examples from 5 types in which blocking of infection by heparin was coincident with an arginine (or another basic amino acid, lysine) at a position corresponding to 132 in VP1 in CAV9. Together, these data show that membrane-associated HSPG can serve as a (co)receptor for some CAV9 and other HEV-B strains and identify symmetry-related clustering of positive charges as one mechanism by which HSPG binding can be achieved. This is a potentially powerful mechanism by which a single amino acid change could generate novel receptor binding capabilities, underscoring the plasticity of host-cell interactions in enteroviruses.
Figures
Fig 1
Infection by some CAV9 isolates is inhibited by agar. Plaque titration assays for several CAV9 isolates were carried out on GMK cells, using an overlay which contains either agarose or agar. The plaque number obtained using agar is expressed as a percentage of that obtained using agarose, which is set at 100%. Values shown are means from three independent experiments, and error bars indicate standard deviation.
Fig 2
Identification of an amino acid position which correlates with the agar inhibition phenotype. Amino acid sequences of the capsid-encoding region of CAV9 isolates CO62, CO79, CO85, CO87, and the prototype strain Griggs were aligned using ClustalW. Numbers refer to P1 position in CAV9 and are equivalent to VP1 positions 123 to 151. CO79 and CO85 (names shown in white on black background), the two isolates inhibited in the presence of agar, have an arginine (R—also shown in white on black background) at VP1 position 132, while the noninhibited viruses (names and corresponding residues all shown on a gray background) have threonine (T).
Fig 3
Heparin inhibits plaque formation of some CAV9 isolates in a dose-dependent manner. CAV9 isolates and the T132R mutant were treated with increasing concentrations of heparin and then subjected to plaque assay on GMK cells. In each case, the number of plaques obtained was expressed as a percentage of the number obtained in a no-heparin control (0 mg/ml). Values are means from three independent experiments, and error bars indicate standard deviations.
Fig 4
Some CAV9 isolates and the T132R mutant can bind to immobilized heparin. Viruses were incubated with agarose or heparin-agarose beads for 1 h and the supernatants subjected to plaque assays on GMK cells. Any bound viruses were eluted using 0.5 M NaCl2 and again analyzed by plaque assays. The results are the means of three experiments, and error bars indicate standard deviations.
Fig 5
GMK cells grown in the presence of sodium chlorate show reduced susceptibility to some CAV9 isolates. Cells were grown for 3 days in medium containing 50 mM sodium chlorate. They were then used for plaque assays using CAV9 isolates and the T132R mutant. In each case, the number of plaques obtained was expressed as a percentage of the number obtained in mock-treated cells. Values are means from three independent experiments, and error bars indicate standard deviations.
Fig 6
Infection with several members of the species HEV-B can be blocked with heparin. Plaque assays were performed on 5 CAV9 and 10 echovirus isolates or variants after treating virus dilutions with 100 mg/ml of heparin or mock treating. Results are shown for large plaque (LP) variants of E11-EVO419B (A) and E7-620 (C), representing nonblocked viruses, and small plaque (SP) variants of E11-EVO419B (B) and E7-620 (D), representing blocked viruses. Two E2 strains, E2-EVO715B (E) (partially blocked) and E2-657 (F) (enhanced infection), are also shown.
Fig 7
HEV-B isolates are polymorphic at the position equivalent to VP1 position 132. The amino acid sequences of part of VP1, including the residue equivalent to position 132 in CAV9, were determined for the viruses tested for heparin blocking and aligned using ClustalW. Numbers refer to P1 position in CAV9 and are equivalent to VP1 positions 127 to 151. Several (names shown in white on a black background) were blocked by heparin and had a basic amino acid at this position (highlighted in white on a black background). Isolates that were not blocked by heparin did not have a basic residue at the corresponding position (on a gray background) apart from the E6 isolates analyzed, which were not blocked by soluble heparin but did have a basic residue. Perfectly conserved residues are indicated by an asterisk (*), and conservation of residues with strongly and weakly similar properties by two dots (:) or by one dot (.), respectively.
Fig 8
Five copies of VP1 position 132 cluster at the 5-fold axis. The location of VP1 position 132 (shown in spacefill) was identified in the three-dimensional structure of CAV9 using Rasmol (16, 39). Two projections are shown to illustrate the clustering (A, top view of the CAV9 pentamer) and the surface exposure (B, side view).
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