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. 2005 Dec;79(24):15054-63.
doi: 10.1128/JVI.79.24.15054-15063.2005.

Luxury at a cost? Recombinant mouse hepatitis viruses expressing the accessory hemagglutinin esterase protein display reduced fitness in vitro

A Lissenberg  1 , M M Vrolijk, A L W van Vliet, M A Langereis, J D F de Groot-Mijnes, P J M Rottier, R J de Groot
Affiliations

Luxury at a cost? Recombinant mouse hepatitis viruses expressing the accessory hemagglutinin esterase protein display reduced fitness in vitro

A Lissenberg et al. J Virol. 2005 Dec.

Abstract

Group 2 coronaviruses encode an accessory envelope glycoprotein species, the hemagglutinin esterase (HE), which possesses sialate-O-acetylesterase activity and which, presumably, promotes virus spread and entry in vivo by facilitating reversible virion attachment to O-acetylated sialic acids. While HE may provide a strong selective advantage during natural infection, many laboratory strains of mouse hepatitis virus (MHV) fail to produce the protein. Apparently, their HE genes were inactivated during cell culture adaptation. For this report, we have studied the molecular basis of this phenomenon. By using targeted RNA recombination, we generated isogenic recombinant MHVs which differ exclusively in their expression of HE and produce either the wild-type protein (HE+), an enzymatically inactive HE protein (HE0), or no HE at all. HE expression or the lack thereof did not lead to gross differences in in vitro growth properties. Yet the expression of HE was rapidly lost during serial cell culture passaging. Competition experiments with mixed infections revealed that this was not due to the enzymatic activity: MHVs expressing HE+ or HE0 propagated with equal efficiencies. During the propagation of recombinant MHV-HE+, two types of spontaneous mutants accumulated. One produced an anchorless HE, while the other had a Gly-to-Trp substitution at the predicted C-terminal residue of the HE signal peptide. Neither mutant incorporated HE into virion particles, suggesting that wild-type HE reduces the in vitro propagation efficiency, either at the assembly stage or at a postassembly level. Our findings demonstrate that the expression of "luxury" proteins may come at a fitness penalty. Apparently, under natural conditions the costs of maintaining HE are outweighed by the benefits.

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Figures

FIG. 1.
FIG. 1.
Construction of recombinant viruses via targeted RNA recombination. (A) Schematic representation of the genome organization of MHV strain A59, the novel acceptor virus fMHVΔ2aHE, and its recombinant derivatives rMHV-HE+, -HE0, and -HE− (rMHV-HE). Also shown schematically are the structures of the synthetic transcripts, derived from transfer vectors pFMΔ2aHE and the pMH54HE series, which were used to generate fMHVΔ2aHE and the various rMHV-HE viruses, respectively. Open boxes represent the various genes, with those for the polymerase polyproteins (POL1a and POL1b) and the 2a, HE, S, E, M, and N proteins indicated. An, poly(A) tail. The jagged line in the POL1ab fusion gene in pFMΔ2aHE and pMH54HE indicates the border between ORF1a- and ORF1b-derived sequences. Shaded boxes represent S sequences derived from FIPV strain 79-1146 and the HE gene of MHV strain S. (B) mRNA profiles of MHV A59 (A59), rMHV-HE+ (HE+), rMHV-HE0 (HE0), and rMHV-HE− (HE−). Viral RNAs, [3H]uridine labeled in the presence of actinomycin D, were extracted from infected cells, separated in 0.8% agarose-MOPS-formaldehyde gels, and visualized by fluorography. Numbers indicate the MHV genome (1) and the various subgenomic mRNAs (2 through 7). The arrowhead indicates RNA2b, which is produced in cells infected with the rMHV-HE viruses but is absent from those infected with the parental virus, MHV-A59.
FIG. 2.
FIG. 2.
Expression of HE protein in cells infected with recombinant viruses rMHV-HE+ and rMHV-HE0. (A) Analysis of intracellular viral proteins. Cells which were mock infected (m) or infected with MHV A59 (A59), rMHV-HE+ (HE+), rMHV-HE0 (HE0), rMHV-HE− (HE−), or MHV-S (S) were metabolically labeled from 5 to 6 h p.i. with 35S in vitro cell-labeling mix (Amersham). Cell lysates were subjected to RIPA with antiserum K135 (α A59), antiserum UP3 (α 2a), or MAb 4G12-2F9 (α HE). Proteins were analyzed by electrophoresis in SDS-polyacrylamide gels followed by fluorography. Only the relevant parts of the gels are shown; bands corresponding to the various proteins (S, N, M, 2a, and HE) are indicated. (B) The HE protein expressed by rMHV-HE+ is enzymatically active. Samples from tissue culture supernatants of MHV-infected cells (designations as described above) were separated in nonreducing 7.5% SDS-PAGE gels. Gels were soaked in PBS at room temperature to allow for protein refolding and then stained for acetylesterase activity, with α-naphthyl acetate as a substrate.
FIG. 3.
FIG. 3.
Recombinant MHVs incorporate the HE protein into their envelopes. (A) Radiolabeled preparations of MHV-A59, rMHV-HE0, rMHV-HE+, and MHV-S were subjected to equilibrium density gradient centrifugation in 20 to 50% (wt/vol) linear sucrose gradients. The gradients were fractionated from the bottom up in 25 fractions. Fractions were analyzed for (i) infectivity by end-point dilution (solid line, open squares), (ii) the presence of the M protein by SDS-PAGE, with quantitation with a STORM PhosphorImager/Fluorimager 860 (Molecular Dynamics) and ImageQuant software (broken line, open circles), and (iii) the presence of enzymatically active HE by an in-gel O-acetylesterase assay (insets in panels HE+ and S). (B) Analysis of proteins in sucrose gradient-purified virus preparations. Samples from peak fractions 5 were separated in 15% SDS-PAGE gels. Molecular masses are indicated on the left (in kDa). The locations of the HE, M, and N proteins are indicated. The S protein could not be identified unambiguously. (C). Electron micrographs of negatively stained virions of rMHV-HE− and rMHV-HE+ (courtesy of Jean Lepault, Laboratoire de Virologie Moléculaire et Structurale, Gif-sur-Yvette, France).
FIG. 4.
FIG. 4.
Immunopurified particles of rMHV-HE+ and rMHV-HE0 contain HE. Supernatants of metabolically labeled cells which had been mock infected or infected with MHV-A59 (A59), rMHV-HE+ (HE+), rMHV-HE0 (HE0), rMHV-HE− (HE−), or MHV-S (S) were subjected to immunopurification with MAb J1.3 (α M) or MAb J1.7 (α S). Precipitates were analyzed in 15% SDS-PAGE gels. Molecular masses (in kDa) are given on the left, and bands corresponding to the structural proteins M, N, HE, and S (c, cleaved; u, uncleaved) are indicated on the right.
FIG. 5.
FIG. 5.
In vitro growth properties of rMHV-HE+, -HE0, and -HE−. (A) Single-step growth kinetics of MHV recombinants. LR7 cells were infected with MHV-A59 or with each of the recombinant MHVs at an MOI of 10. Viral infectivity present in the culture medium at different times postinfection was determined by titration in LR7 cells by end-point dilution, and titers (50% tissue culture infective doses/ml [TCID50/ml]) were calculated. (B and C) Relative fitness of recombinant MHVs, as measured in mixed propagation-competition assays. rMHV-HE+ was serially passaged in LR7 cells either alone or in combination with rMHV-HE0 or rMHV-HE−, mixed at the indicated ratios. The propagation-competition experiments shown were performed with three different sets of independently isolated rMHVs (indicated by open and solid squares and triangles). Monolayers were inoculated at an MOI of 0.01. Tissue culture supernatants were harvested at 16 h p.i. and analyzed by plaque assays and in situ esterase staining. The graphs show the percentage of acetylesterase-positive plaques (y axis) in each of the passages (x axis). For graph B, at least 200 plaques were counted for each sample after every passage (average, 440 ± 170), and for graph C, at least 700 plaques were counted for each sample (average, 990 ± 340).
FIG. 6.
FIG. 6.
Spontaneous acetylesterase-deficient mutants of rMHV-HE+ produce defective HE proteins which are not incorporated into viral particles. (Upper panel) Analysis of intracellular viral proteins. Cells were mock infected (m) or infected with rMHV-HE+ (HE+), rMHV-HE− (HE−), or rMHV-HE+ mutant type 1 or 2. The cells were metabolically labeled from 5 to 6 h p.i. with 35S in vitro cell-labeling mix (Amersham), and cell lysates were subjected to RIPA with MAb 4G12-2F9 (α HE) or antiserum K135 (α A59). Precipitates were analyzed by electrophoresis in SDS-polyacrylamide gels followed by fluorography. Bands corresponding to the structural proteins M, N, HE, and S (u, uncleaved; c, cleaved) are indicated. MAb 4G12-2F9 bound a 30-kDa host cell protein in addition to the HE protein. (Lower panel) Protein content of immunopurified virions. Supernatants of metabolically labeled cells which had been mock infected or infected with rMHV-HE+ (HE+), rMHV-HE− (HE−), or mutant type 1 or 2 were subjected to immunopurification with MAb J1.3 (α M) or with MAb J1.7 (α S). Precipitates were analyzed in 15% SDS-PAGE gels. Bands corresponding to the structural proteins M, N, HE, and S (u, uncleaved; c, cleaved) are indicated.
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