Article Figures & Data
Figures
- 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. An, 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-SDVIM and rMHV-A59-SS 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-HEDVIM, rMHV-A59-SDVIM, and rMHV-A59-HES. 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-HEDVIM; open triangles, rMHV-A59-HES.
- 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.
