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. 2006 Feb;80(3):1280-9.
doi: 10.1128/JVI.80.3.1280-1289.2006.

Palmitoylations on murine coronavirus spike proteins are essential for virion assembly and infectivity

Edward B Thorp  1 , Joseph A Boscarino, Hillary L Logan, Jeffrey T Goletz, Thomas M Gallagher
Affiliations

Palmitoylations on murine coronavirus spike proteins are essential for virion assembly and infectivity

Edward B Thorp et al. J Virol. 2006 Feb.

Abstract

Coronavirus spike (S) proteins are palmitoylated at several cysteine residues clustered near their transmembrane-spanning domains. This is achieved by cellular palmitoyl acyltransferases (PATs), which can modify newly synthesized S proteins before they are assembled into virion envelopes at the intermediate compartment of the exocytic pathway. To address the importance of these fatty acylations to coronavirus infection, we exposed infected cells to 2-bromopalmitate (2-BP), a specific PAT inhibitor. 2-BP profoundly reduced the specific infectivities of murine coronaviruses at very low, nontoxic doses that were inert to alphavirus and rhabdovirus infections. 2-BP effected only two- to fivefold reductions in S palmitoylation, yet this correlated with reduced S complexing with virion membrane (M) proteins and consequent exclusion of S from virions. At defined 2-BP doses, underpalmitoylated S proteins instead trafficked to infected cell surfaces and elicited cell-cell membrane fusions, suggesting that the acyl chain adducts are more critical to virion assembly than to S-induced syncytial developments. These studies involving pharmacologic inhibition of S protein palmitoylation were complemented with molecular genetic analyses in which cysteine acylation substrates were mutated. Notably, some mutations (C1347F and C1348S) did not interfere with S incorporation into virions, indicating that only a subset of the cysteine-rich region provides the essential S-assembly functions. However, the C1347F/C1348S mutant viruses exhibited relatively low specific infectivities, similar to virions secreted from 2-BP-treated cultures. Our collective results indicate that the palmitate adducts on coronavirus S proteins are necessary in assembly and also in positioning the assembled envelope proteins for maximal infectivity.

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Figures

FIG. 1.
FIG. 1.
Effect of 2-BP on virus infectivities. 17cl1 murine fibroblast monolayers (105 cells/cm2) were inoculated with the indicated RNA viruses at 0.1 PFU per cell. At 1 h postinfection, media were aspirated and replaced with fresh growth media containing the indicated concentrations of 2-BP. Fifteen hours later, media were removed, and infectivities were determined by plaque titration using 17cl1 cell monolayers as indicator cells.
FIG. 2.
FIG. 2.
Effect of 2-BP on MHV-specific infectivities. MHV-infected 17cl1 cells (multiplicity of infection = 2) were incubated from 1 to 16 hpi with the indicated 2-BP doses. Media were then removed and replaced with 35S-labeling media containing the same 2-BP concentrations. Four hours later, media were collected and 35S-labeled virions purified on sucrose density gradients. Banded 35S-labeled virions were quantitated by scintillation spectrometry and radioactive virus yields plotted relative to controls. Specific infectivities of purified virions (PFU/35S cpm) were determined by plaque assay on 17cl1 cells and plotted relative to control values. In the absence of 2-BP, A59- and JHMX-specific infectivities were 2.06 and 1.55 PFU/35S cpm, respectively.
FIG. 3.
FIG. 3.
Effect of 2-BP on MHV-induced syncytia. MHV (strain JHMX)-infected 17c1l cells were incubated from 1 to 16 hpi with the indicated micromolar concentrations of 2-BP and photographed by phase-contrast microscopy. Inset in panel A depicts uninfected cells.
FIG. 4.
FIG. 4.
Effect of 2-BP on the distribution of S proteins between cells and media. MHV (strain A59)-infected 17cl1 cells were incubated with the indicated concentrations of 2-BP from 1 to 9 hpi, and then media were collected and cells dissolved in PBS containing 0.5% NP-40. S proteins in clarified media and cell lysates were immunoprecipitated, and S proteins captured from 104 cell equivalents were then electrophoresed and visualized by Western immunoblotting. S-unc, uncleaved S; MW, molecular weights in thousands.
FIG. 5.
FIG. 5.
Electrophoretic profiles of 35S-labeled virion proteins. Virions were metabolically radiolabeled with 35S amino acids and purified on density gradients as described in the legend to Fig. 2. Equal 35S radioactivities were collected from each purified virion preparation, electrophoresed on SDS gels, and detected by autoradiography (left panel). Quantification of exposed film densities was obtained by densitometry, and values for S and M were plotted relative to those for control virions produced in the absence of 2-BP (right panel). MW, molecular weights in thousands.
FIG. 6.
FIG. 6.
Effect of 2-BP on the abundance of coimmunoprecipitating S-M complexes. 17c11 cells infected with JHMX (multiplicity of infection = 0.1) or A59 (multiplicity of infection = 1) were cultured with the indicated 2-BP doses from 1 to 9 hpi and then dissolved in NP-40/DOC buffer as described in Materials and Methods. Clarified extracts were mixed overnight with magnetic protein A-NCEA-Fc beads, which bound the S proteins. Proteins on magnet-captured beads were eluted with SDS, electrophoresed, and analyzed by immunoblotting using a cocktail of S- and M-specific MAbs. ori, origin of resolving gel; S unc, uncleaved S; MW, molecular weights in thousands.
FIG. 7.
FIG. 7.
Effect of 2-BP on incorporation of [3H]palmitate onto S proteins. 17cl1 cells infected with A59 (multiplicity of infection = 2) were incubated from 1 to 16 hpi with the indicated 2-BP doses and then pulse labeled with [3H]palmitate from 16 to 20 hpi, maintaining the indicated 2-BP concentrations throughout. Cells were dissolved in NP-40 buffer, and S proteins were immunoprecipitated from clarified lysates using magnetic protein A-NCEA-Fc beads. Radioactive S proteins were imaged by fluorography after electrophoresis on SDS-polyacrylamide gels. S-agg, aggregated S oligomers; S-unc, uncleaved S; *, S proteolytic fragments; MW, molecular weights in thousands.
FIG. 8.
FIG. 8.
S-protein association with detergent-resistant membranes and effect of 2-BP on distribution of S proteins in the plasma membrane. (A-C) HeLa-CEACAM cells were infected with MHV strain JHMX (A), vaccinia recombinants vTF7.3 and vTM1-S-JHMX (14), or VSV (C). At 8 hpi, infected cultures were dissolved in cold Triton X-100 and lysates subjected to flotation in sucrose density gradients, as described in Materials and Methods. Gradients were fractionated from air-gradient interfaces, and proteins were recognized by Western immunoblotting using S- and VSV G-specific antibodies. (D-F) 17cl1 cells were infected with MHV strain JHMX, and at 1 hpi, 0 μM (D), 10 μM (E), or 25 μM (F) 2-BP doses were added. At 16 hpi, sulfo-NHS-LC-biotin was briefly applied and the unreacted reagent quenched with glycine. Cells were then dissolved in cold Triton X-100 buffer and lysates centrifuged to equilibrium in sucrose density gradients. S proteins in the gradient fractions were immunoprecipitated, and the biotinylated subpopulations were recognized on Western blots using streptavidin-horseradish peroxidase conjugate and a colorimetric assay for the immobilized horseradish peroxidase. Sunc, uncleaved S.
FIG. 9.
FIG. 9.
Isolation and characterization of C1347F and C1347F/C1348S MHV mutants. (A) A S-protein trimer is depicted with transmembrane span and cysteine-rich residues illustrated in the context of the virion membrane. Mutations chosen for introduction into recombinant viruses are indicated by the asterisks. (B) Growth kinetics of wild-type JHM-G310S (26), -C1347F, and -C1347F/C1348S. 17c11 cells were infected with recombinant JHM isolates at 0.01 PFU/cell. Output titers were determined in culture supernatants collected at the indicated hours postinfection, using HeLa-CEACAM cells as plaque indicators. (C) Syncytial expansion of JHM-G310S, -C1347F, and -C1347F/C1348S infections. HeLa-CEACAM cells were infected with JHM isolates at 0.01 PFU/cell, and photographs were taken at the indicated hours postinfection (numbers in center panels). (D) Electrophoretic profiles of rJHM-G310S (lane 1) and rJHM-G310S with the C1347F and C1348S mutations (lane 2). Infected 17cl1 cells were radiolabeled with 35S amino acids from 20 to 28 hpi, media were collected, and virions were purified by two cycles of equilibrium sucrose density gradient centrifugation. Virion proteins were electrophoresed on SDS gels and visualized by fluorography. Specific infectivities of the virion preparations (PFU/35S cpm) are indicated beneath each lane. MW, molecular weights in thousands.
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