Coronavirus particle assembly: primary structure requirements of the membrane protein

Abstract

Coronavirus-like particles morphologically similar to normal virions are assembled when genes encoding the viral membrane proteins M and E are coexpressed in eukaryotic cells. Using this envelope assembly assay, we have studied the primary sequence requirements for particle formation of the mouse hepatitis virus (MHV) M protein, the major protein of the coronavirion membrane. Our results show that each of the different domains of the protein is important. Mutations (deletions, insertions, point mutations) in the luminal domain, the transmembrane domains, the amphiphilic domain, or the carboxy-terminal domain had effects on the assembly of M into enveloped particles. Strikingly, the extreme carboxy-terminal residue is crucial. Deletion of this single residue abolished particle assembly almost completely; most substitutions were strongly inhibitory. Site-directed mutations in the carboxy terminus of M were also incorporated into the MHV genome by targeted recombination. The results supported a critical role for this domain of M in viral assembly, although the M carboxy terminus was more tolerant of alteration in the complete virion than in virus-like particles, likely because of the stabilization of virions by additional intermolecular interactions. Interestingly, glycosylation of M appeared not essential for assembly. Mutations in the luminal domain that abolished the normal O glycosylation of the protein or created an N-glycosylated form had no effect. Mutant M proteins unable to form virus-like particles were found to inhibit the budding of assembly-competent M in a concentration-dependent manner. However, assembly-competent M was able to rescue assembly-incompetent M when the latter was present in low amounts. These observations support the existence of interactions between M molecules that are thought to be the driving force in coronavirus envelope assembly.

FIG. 1

Overview of mutant M proteins. In the middle is a schematic representation of the structure of the M protein, with the three transmembrane (TM) domains indicated. Amino acid sequences of the amino-terminal and carboxy-terminal domains, and of the mutants with mutations in these domains, are shown above and below the diagram, respectively. Dashes represent unchanged residues, gaps represent deletions. The domain deleted in mutant ΔC (residues E₁₂₁ through D₁₉₅) is indicated by a horizontal line. The abilities of the different M proteins to support VLP assembly is indicated at the right. The ratio of the amount of M present in the culture medium to that in cells was taken as a measure for VLP assembly by using WT M as a reference. The semiquantitative scores ++, +, +/−, and − indicate efficient, moderately efficient, inefficient, and nonexistent VLP synthesis, respectively.

FIG. 2

Effects of deletions in the M cytoplasmic domain on VLP assembly. OST7-1 cells infected with recombinant vaccinia virus vTF7-3 were transfected with a plasmid containing the WT or mutant M gene either alone or in combination with a plasmid containing the E protein gene, each gene behind a T7 promoter. Cells were labeled for 3 h with ³⁵S-labeled amino acids. Both cells (upper panel) and the culture medium (lower panel) were prepared and used for immunoprecipitation, and the precipitates were analyzed by SDS-PAGE. The different M genes expressed are indicated above each set.

FIG. 3

Indirect immunofluorescence analysis of mutant M proteins. vTF7-3 infected BHK-21 cells were transfected with plasmids encoding WT M (A) or the ΔC (B), Δ18 (C), ΔN (D), A₄A₅ (E), A₈A₁₀ (F), or A₈A₁₀C₁₄ (G) mutant or were mock transfected (H and I). Cells were fixed at 5 h postinfection and processed for immunofluorescence with the anti-MHV serum k134 (A through H) or a polyclonal rabbit serum against the resident Golgi protein α-mannosidase II (I) (a kind gift from K. Moremen [50]).

FIG. 4

Effects of mutations in the M carboxy-terminal domain on VLP assembly. Expression of M and E genes was performed as described in the legend to Fig. 2. The different M genes tested are indicated above the gels.

FIG. 5

Effects of mutations in the amino-terminal domain of M on VLP assembly. M and E genes were expressed as described in the legend to Fig. 2.

FIG. 6

Assembly of an N-glycosylated form of the MHV M protein into VLPs. OST7-1 cells infected with recombinant vaccinia virus vTF7-3 were transfected with a plasmid containing the WT or the S₂N mutant M gene, either alone or in combination with a plasmid containing the E protein gene, each gene behind a T7 promoter. Cells were labeled for 3 h with ³⁵S-labeled amino acids. In some cases, cells were treated with 1 mM DMJ (lanes 3 and 4, 9 to 12, and 16); in other cases, cells were treated with 5 μg of tunicamycin (TM)/ml (lanes 13 and 14). Both cells (upper panel) and the culture medium (lower panel) were prepared and used for immunoprecipitation, and the precipitates were analyzed by SDS-PAGE. Prior to gel electrophoresis, some immunoprecipitates were treated with glyco F to remove N-linked sugars (lanes 7, 8, 11, and 12).

FIG. 7

Inhibition of VLP formation by mutant M proteins with carboxy-terminal deletions. After infection of OST7-1 cells with recombinant vaccinia virus vTF7-3, cells were transfected with plasmid DNA encoding WT M and E (5 μg of each) together with 1 or 5 μg of plasmid DNA encoding mutant M. The different M mutants expressed are indicated above the gels. Cells were labeled for 3 h with ³⁵S-labeled amino acids. Both cells (upper panel) and the culture medium (lower panel) were prepared and used for immunoprecipitation, and the precipitates were analyzed by SDS-PAGE.

FIG. 8

Quantitation of the concentration-dependent inhibition of VLP formation by mutant M protein Δ18. The relative amounts of glycosylated WT or unglycosylated Δ18 mutant M protein present in cells and media for each transfection condition shown in Fig. 6 were determined by using a phosphorimager. The relative amount of WT M secreted (i.e., VLPs in media) is also shown.

FIG. 9

Rescue of carboxy-terminal deletion mutant M proteins into VLPs. After infection of OST7-1 cells with recombinant vaccinia virus vTF7-3, cells were transfected with plasmid DNA encoding mutant M protein A₂A₃ and E protein (5 μg of each). In some cases, these were cotransfected with 1 μg of plasmid DNA encoding the indicated mutant or WT M protein. Cells were labeled for 3 h with ³⁵S-labeled amino acids. Both cells (upper panel) and the culture medium (lower panel) were prepared and used for immunoprecipitation, either with the polyclonal anti-MHV serum k134 or with the monoclonal antibody J1.3, and the precipitates were analyzed by SDS-PAGE.

FIG. 10

Incorporation of M protein cytoplasmic-domain mutations into the MHV genome. (A) Schematic for construction of M mutants by targeted recombination between donor synthetic RNA and the N-gene deletion mutant Alb4. Each donor RNA containing a codon 228 mutation in the M gene (denoted by a star) was transcribed from a vector derived from pCFS8 (16), which includes the entire portion of the MHV genome 3′ to the start of the S gene and is tagged with a 19-base marker in gene 4 (denoted by a triangle). In the case shown, the MHV mutant, generated by a single upstream crossover, has inherited the gene 4 tag, the constructed M mutation, and the region repairing the N-gene deletion. (B) Sequence of the relevant region of genomic RNA isolated from passage-3 purified virions of one mutant of each type. For each codon 228 mutation, recombinants were obtained that contained and that lacked the upstream gene 4 tag. The particular mutants shown are all positive for the gene 4 tag, except for Alb206 and Alb200. For Alb200, a BCV-MHV chimeric M protein mutant, the donor RNA was derived from pFV1 (16) and did not contain the gene 4 tag.