Host-selected amino acid changes at the sialic acid binding pocket of the parvovirus capsid modulate cell binding affinity and determine virulence

Abstract

The role of receptor recognition in the emergence of virulent viruses was investigated in the infection of severe combined immunodeficient (SCID) mice by the apathogenic prototype strain of the parvovirus minute virus of mice (MVMp). Genetic analysis of isolated MVMp viral clones (n = 48) emerging in mice, including lethal variants, showed only one of three single changes (V325M, I362S, or K368R) in the common sequence of the two capsid proteins. As was found for the parental isolates, the constructed recombinant viruses harboring the I362S or the K368R single substitutions in the capsid sequence, or mutations at both sites, showed a large-plaque phenotype and lower avidity than the wild type for cells in the cytotoxic interaction with two permissive fibroblast cell lines in vitro and caused a lethal disease in SCID mice when inoculated by the natural oronasal route. Significantly, the productive adsorption of MVMp variants carrying any of the three mutations selected through parallel evolution in mice showed higher sensitivity to the treatment of cells by neuraminidase than that of the wild type, indicating a lower affinity of the viral particle for the sialic acid component of the receptor. Consistent with this, the X-ray crystal structure of the MVMp capsids soaked with sialic acid (N-acetyl neuraminic acid) showed the sugar allocated in the depression at the twofold axis of symmetry (termed the dimple), immediately adjacent to residues I362 and K368, which are located on the wall of the dimple, and approximately 22 A away from V325 in a threefold-related monomer. This is the first reported crystal structure identifying an infectious receptor attachment site on a parvovirus capsid. We conclude that the affinity of the interactions of sialic-acid-containing receptors with residues at or surrounding the dimple can evolutionarily regulate parvovirus pathogenicity and adaptation to new hosts.

FIG. 1.

Sequence analysis of MVMp evolution in SCID mice. (Top) Amino acid substitutions in the VP gene of plaque-isolated MVMp clones obtained from main organs (B, brain; K, kidney; L, liver) of SCID mice (no. 1 to 5) sequenced across the entire VP gene (nt 2240 to 4687). (Bottom) Distribution of amino acid changes in the entire collection of MVMp clones. A VP region (nt 3710 to 4200) of the MVMp genome was sequenced in viral clones (n = 48) obtained from the indicated mice (no. 1 to 7) and organs. The amino acid changes at residues 325, 362, and 368 of the VP2 sequence are outlined. n, number of clones with identical genotypes in this region.

FIG. 2.

Phenotypic effects of amino acid capsid changes selected in mice on MVMp plaque morphology and primary receptor interaction. (A) Plaque morphologies of recombinant viruses in NB324K cells compared with the wt. (B) Capsid affinity for a productive fibroblast receptor. Shown are kinetics of interaction at 4°C of the indicated viruses with A9 and NB324K cells in suspension. Upon virus binding, the cells were plated and cultured for 10 days. Percentages of surviving colonies for each binding time compared to those of the mock-infected cells are indicated. The plots show representative results from three independent experiments. Values for the I362S mutant in A9 cells are from a previous study (66).

FIG. 3.

Differential affinities of MVMp mutants for the sialic acid portion of the receptor. (A) Binding of ³⁵S-labeled wild-type and I362S capsids to neuraminidase-treated NB324K cells. Cells in suspension (2 × 10⁶ cells/ml) were treated with the indicated doses of neuraminidase (1 h at 37°C) prior to binding (1 h at 4°C) to the labeled capsids. Cell-associated radioactivity was determined by sedimentation and scintillation counting (100% binding was approximately 2 × 10³ cpm). One representative result from three independent experiments is shown. (B) Monolayers of 2 × 10⁵ NB324K cells per 60-mm dish were treated with the indicated doses of neuraminidase for 1 h at 37°C prior to virus binding for 1 h at 4°C, and the productive interactions were developed by a PFU assay. The plots show the average results and standard errors from at least four independent assays. The V325M mutant corresponds to one of the three clones isolated from the liver of mouse no. 2 containing this mutation (Fig. 1, bottom).

FIG. 4.

Single capsid amino acid changes confer virulence by the natural oronasal route on MVMp. The infectious virus titers in organs of SCID mice after intranasal inoculation with purified stocks (10⁶ PFU/mouse) of the indicated recombinant mutants are outlined. The bars represent the mean titers (two independent determinations) with standard errors of four mice, with the exception of the data from the I362S/K368R infection at 8 weeks p.i., which are from six mice. Titers in three organs (B, K, and L) for the I362S mutant are from a previous study (66). DL, detection limit of the assay.

FIG. 5.

Mortality of SCID mice upon intranasal inoculation with recombinant MVMp mutants. Mice were inoculated by the oronasal route with the indicated viruses (10⁶ PFU/mouse) and monitored for survival for 30 weeks p.i. (n = 10 for single mutants and n = 6 for the double-mutant virus). The results were accumulated from two independent inoculations. The data were scored daily.

FIG. 6.

Sialic acid binds in the dimple of the MVMp capsid, surrounded by residues involved in virulence. (A and B) Surface representations of a close-up of the depression at the icosahedral twofold axes of the MVMp capsid showing a reference VP2 monomer (ref, in gray), and icosahedrally related twofold (2f, in magenta), threefold (3f1 and 3f2, in orange and green), and fivefold (5f, in cyan) monomers. The surface positions of residues I362 and K368 are highlighted in yellow and blue, respectively. Residue V325 is not surface accessible. The SA model (colored according to atom type) is shown inside a 2F₀ − F_c map, in blue, contoured at 1.8 σ in the two possible orientations of the carboxyl and N-acetyl groups of the SA molecule. (C) Coil representations of the ref, 2f, 3f1, 3f2, and 5f VP2 monomers, colored as in panels A and B. The positions and side chain atoms of residues I362, K368 (in the reference monomer), and V325 (in a threefold-related monomer) are shown colored according to atom type. The SA molecule is shown as in panel A, with the carboxyl group pointing down from the ring and the N-acetyl group pointing upward. (D) Close-up of the SA molecule (as in panel C) and residues on the wall of the twofold depression close to the binding pocket that either differ between MVMp and MVMi or confer fibrotropism on MVMi. The approximate location of the icosahedral twofold axes is shown in the filled oval. The figure was prepared using the programs PyMol (24) (for panels A and B) and BOBSCRIPT (27) (for panels C and D) and the MVMp coordinates; Protein Data Bank accession no. 1Z14.