Attachment of mouse hepatitis virus to O-acetylated sialic acid is mediated by hemagglutinin-esterase and not by the spike protein

Abstract

The members of Betacoronavirus phylocluster A possess two types of surface projections, one comprised of the spike protein (S) and the other of hemagglutinin-esterase (HE). Purportedly, these viruses bind to O-acetylated sialic acids (O-Ac-Sias) primarily through S, with HE serving merely as receptor-destroying enzyme. Here, we show that, in apparent contrast to human and ungulate host range variants of Betacoronavirus-1, murine coronaviruses actually bind to O-Ac-Sias via HE exclusively. Apparently, expansion of group A betacoronaviruses into new hosts and niches was accompanied by changes in HE ligand and substrate preference and in the roles of HE and S in Sia receptor usage.

FIG. 1.

Construction of recombinant MHV viruses via targeted RNA recombination. (A) Genome organization of MHV-A59, MHV-DVIM, MHV-S, and recombinant MHV-A59 derivatives. White boxes, MHV-A59 genes; black boxes, MHV-DVIM genes; shaded boxes, MHV-S genes. Genes for the polymerase polyproteins (ORF1a and ORF1b) and the 2a, HE, S, small envelope (E), membrane (M), and nucleocapsid (N) proteins are indicated. A_n, poly(A) tail. Note that in MHV-A59, the HE gene is interrupted by a nonsense mutation at codon 15 (indicated by a shortened box) and that consequently MHV-A59 and derivates rMHV-A59-S^DVIM and rMHV-A59-S^S do not express the HE protein. Numbers on the right are the relative specific infectivity (RSI) values as calculated for the recombinant viruses from the PFU-to-genome copy ratio, with that of MHV-A59 set at 100%. Plaque assays in LR7 cells and TaqMan RT-PCR assays were performed in triplicate; standard deviations are given in parentheses. (B) mRNA profiles of MHV-A59 and recombinant viruses rMHV-A59-HE^DVIM, rMHV-A59-S^DVIM, and rMHV-A59-HE^S. Intracellular viral RNAs were [³H]uridine labeled and separated in formaldehyde-0.8% agarose gels as previously described (17). mRNA species are numbered according to convention; mRNA2b is indicated by an arrowhead. (C) SDS-PAGE analysis of virus particles, metabolically labeled and immunopurified as described previously (17). Molecular masses (in kDa) are given at the left; bands corresponding to the structural proteins M, N, HE, and S are indicated at the right.

FIG. 2.

Attachment of wild-type and recombinant MHVs to O-Ac-Sias as measured by solid-phase whole-virion binding assay. (A) HE is sufficient to mediate MHV virion binding to natural sialoglycoconjugates. BSM and HSG, applied to 96-well Maxisorp plates (Nunc), were either mock treated or treated with purified 9-O-acetylesterase of porcine torovirus strain Markelo HE (9-O-AE) (16, 31), purified 4-O-acetylesterase of MHV strain S HE (4-O-AE) (22), or Arthrobacter ureafaciens neuraminidase (NA) (Roche Applied Science). The solid-phase assay was performed as described previously (12, 44) with equal amounts of virus particles (equivalent to 1.0 × 10⁷ PFU of MHV-A59). Binding of viruses was measured by fluorophotometric detection (excitation and emission wavelengths of 330 nm and 445 nm, respectively) of 4-methylumbelliferone released from the synthetic substrate 4-methylumbelliferyl acetate (4-MUAc) by virion-associated HE O-acetylesterase activity. The data are presented as bar charts, with the fluorescence measured for MHV-DVIM bound to BSM and for MHV-S bound to HSG set at 100%. (B) HEs of MHV-S and MHV-DVIM hydrolyze 4-MUAc at comparable rates. Equal amounts of virus particles (equivalent to 2.0 × 10⁶ PFU of MHV-A59) in 100 μl phosphate-buffered saline (PBS) with 0.2 mM 4-MUAc were incubated at ambient temperature, and hydrolysis of the substrate was followed over time. Black squares, MHV-DVIM; black dots, MHV-S; open diamonds, rMHV-A59-HE^DVIM; open triangles, rMHV-A59-HE^S.

FIG. 3.

Attachment of wild-type and recombinant MHVs to O-Ac-Sias as measured by hemagglutination assay. Rat erythrocytes (Rattus norvegicus strain Wistar) were mock treated or treated with 9-O-acetylesterase (9-O-AE), 4-O-acetylesterase (4-O-AE), or A. ureafaciens neuraminidase (NA) prior to hemagglutination assay with twofold serial dilutions of virus. Assays were performed in PBS with final dilution of erythrocytes to 0.2% and with equal amounts of virus particles (starting amounts equivalent to 1.0 × 10⁷ PFU of MHV-A59). The arrow indicates the direction from low to high virus dilutions. Gray circles indicate wells displaying hemagglutination.