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 ϳ180 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 ␣2-3 to a common Gal␤1-4GlcNAc motif: Neu5Ac␣2-3Gal␤1-4GlcNAc␤1-4Gal␤1-4GlcNAc (3SiaLN-LN), Neu5Ac␣2-3Gal␤1-4GlcNAc␤1-3Gal␤1-4GlcNAc␤1-3Gal␤1-4GlcNAc (3SiaLN-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 (sLe x -Le x -Le x ). In addition, MVMi also recognized four multisialylated glycans with terminal ␣2-8 linkages: Neu5Ac␣2-8Neu5Ac␣2-8Neu5Ac␣ ((Sia) 3 ), Neu5Ac␣2-8Neu5Ac␣2-3Gal␤1-4Glc (GD3), Neu5Ac␣2-8Neu5Ac␣2-8Neu5Ac␣2-3Gal␤1-4Glc (GT3), and Neu5Ac␣2-8Neu5Ac␣2-3(GalNAc␤1-4)Gal␤1-4Glc (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.Attachment to a cell surface receptor is an essential first step in the life cycle of many viruses. The initial viral attachment frequently determines the tissue tropism, and subsequent interactions determine the pathogenic outcome. In many virushost interactions, glycan chains often function as initial recognition molecules, with or without a protein component. Glycans are a major element of living systems, with Ͼ50% of all proteins being glycosylated. They are abundantly present in the outer cell surface and thus serve as convenient targets for viral attachment. The recognition of specific glycan motifs by viruses can be mediated by a specific viral surface protein component, such as the hemagglutinin of influenza virus (1), or specific regions of the viral capsid itself, as is proposed for members of the single-stranded DNA Parvoviridae family (2-8). The initial attachment of viruses to host cells is followed by an internalization process that often involves concerted actions of many factors, such as co-receptors and/or the endocytot...