Identification of the Sialic Acid Structures Recognized by Minute Virus of Mice and the Role of Binding Affinity in Virulence Adaptation

Hyun-Joo Nam,Brittney Gurda-Whitaker,Wand Yee Gan,Shawen Ilaria,Robert Mckenna,Padmaja Mehta,Richard A Alvarez,Mavis Agbandje-Mckenna

doi:10.1074/jbc.m604421200

Hyun-Joo Nam, Brittney Gurda-Whitaker + Show 6 more

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https://doi.org/10.1074/jbc.m604421200

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Abstract

Sialic acid binding is required for infectious cell surface receptor recognition by parvovirus minute virus of mice (MVM). We have utilized a glycan array consisting of approximately 180 different carbohydrate structures to identify the specific sialosides recognized by the prototype (MVMp) and immunosuppressive (MVMi) strains of MVM plus three virulent mutants of MVMp, MVMp-I362S, MVMp-K368R, and MVMp-I362S/K368R. All of the MVM capsids specifically bound to three structures with a terminal sialic acid-linked alpha2-3 to a common Galbeta1-4GlcNAc motif: Neu5Acalpha2-3Galbeta1-4GlcNAcbeta1-4Galbeta1-4GlcNAc (3'SiaLN-LN), Neu5Acalpha2-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc (3'SiaLN-LN-LN), and Neu5Acalpha2-3Galbeta1-4(Fucalpha1-3)-GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)GlcNAc (sLe(x)-Le(x)-Le(x)). In addition, MVMi also recognized four multisialylated glycans with terminal alpha2-8 linkages: Neu5Acalpha2-8Neu5Acalpha2-8Neu5Acalpha ((Sia)(3)), Neu5Acalpha2-8Neu5Acalpha2-3Galbeta1-4Glc (GD3), Neu5Acalpha2-8Neu5Acalpha2-8Neu5Acalpha2-3Galbeta1-4Glc (GT3), and Neu5Acalpha2-8Neu5Acalpha2-3(GalNAcbeta1-4)Galbeta1-4Glc (GD2). Interestingly, the virulent MVMp-K368R mutant also recognized GT3. Analysis of the relative binding affinities using a surface plasmon resonance biospecific interaction (BIAcore) assay showed the wild-type MVMp and MVMi capsids binding with higher affinity to selected glycans compared with the virulent MVMp mutants. The reduced affinity of the virulent MVMp mutants are consistent with previous in vitro cell binding assays that had shown weaker binding to permissive cells compared with wild-type MVMp. This study identifies the sialic acid structures recognized by MVM. It also provides rationale for the tropism of MVM for malignant transformed cells that contain sLe(x) motifs and the neurotropism of MVMi, which is likely mediated via interactions with multisialylated glycans known to be tumor cell markers. Finally, the observations further implicate a decreased binding affinity for sialic acid in the in vivo adaptation of MVMp to a virulent phenotype.

Highlights

Two strains of minute virus of mice (MVM),2 an autonomous member of the Parvoviridae family, have served as models for studies aimed at identifying parvovirus capsid regions involved in cellular host range and pathogenicity determination (9 –11)
To identify the specific sialic acid structures recognized by MVM and biophysically evaluate the individual role of the two virulent MVMp VP mutations I362S and K368R on receptor affinity, recombinant baculoviruses expressing the VP2 of MVMi, MVMp, and the three virulent MVMp mutants were used to produce VLPs for glycan array screening and surface plasmon resonance (SPR) BIAcore assays
Of the ϳ40 sialylated glycans in the array, all of the MVM viruses bound to only three structures with terminal ␣2–3-linked sialic acid, 3ЈSiaLN-linked to Gal-GlcNAc (LN), 3ЈSiaLN-LN-LN, and Neu5Ac␣2–3Gal␤1– 4(Fuc␣1–3)GlcNAc␤1–3Gal␤1– 4(Fuc␣1–3)GlcNAc␤1–3Gal␤1– 4(Fuc␣1–3)GlcNAc (Fig. 3)

Summary

Introduction

Two strains of minute virus of mice (MVM), an autonomous member of the Parvoviridae family, have served as models for studies aimed at identifying parvovirus capsid regions involved in cellular host range (tropism) and pathogenicity determination (9 –11). Pretreatment of the fibroblast cells with neuraminidase inhibited binding by the wt MVMp and mutant viruses [11], confirming previous reports identifying sialic acid as the carbohydrate component required for cellular recognition [17]. Our goal was to identify the specific sialic acid motifs recognized by MVMi and MVMp and three virulent MVMp mutants, MVMp-I362S, MVMp-K368R, and MVMpI362S/K368R, using a glycan array containing ϳ180 carbohydrate motifs. The specificity of the interaction(s) between the glycans was identified and the MVM viruses were confirmed by secondary analysis using a surface plasmon resonance (SPR) biospecific interaction (BIAcore) assay This technique measured the relative binding affinity of MVMi, MVMp, and the virulent MVMp mutants. Our observations point to an infectious mechanism facilitated by recognition of specific sialic acid structures on the cell surface by MVM and further implicate a mechanism involving differ-

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