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. 2004 Jul;78(14):7369-78.
doi: 10.1128/JVI.78.14.7369-7378.2004.

Coronavirus Spike Glycoprotein, Extended at the Carboxy Terminus With Green Fluorescent Protein, Is Assembly Competent

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Free PMC article

Coronavirus Spike Glycoprotein, Extended at the Carboxy Terminus With Green Fluorescent Protein, Is Assembly Competent

Berend Jan Bosch et al. J Virol. .
Free PMC article

Abstract

Due to the limited ultrastructural information about the coronavirion, little is known about the interactions acting at the interface between nucleocapsid and viral envelope. Knowing that subtle mutations in the carboxy-terminal endodomain of the M protein are already lethal, we have now probed the equivalent domain of the spike (S) protein by extending it terminally with a foreign sequence of 27 kDa: the green fluorescent protein (GFP). When expressed individually in murine cells, the S-GFP chimeric protein induced the formation of fluorescent syncytia, indicating that it was synthesized and folded properly, trimerized, and transported to the plasma membrane, where it exhibited the two key S protein functions, i.e., interaction with virus receptor molecules and membrane fusion. Incorporation into virus-like particles demonstrated the assembly competence of the chimeric spike protein. The wild-type S gene of mouse hepatitis coronavirus (MHV) was subsequently replaced by the chimeric construct through targeted recombination. A viable MHV-SGFP was obtained, infection by which could be visualized by the fluorescence induced. The efficiency of incorporation of the chimeric protein into particles was, however, reduced relative to that in wild-type particles which may explain, at least in part, the reduced infectivity produced by MHV-SGFP infection. We conclude that the incorporation of spikes carrying the large GFP moiety is apparently impaired by geometrical constraints and selected against during the assembly of virions. Probably due to this disadvantage, deletion mutants, having lost the foreign sequences, rapidly evolved and outcompeted the chimeric viruses during virus propagation. The fluorescent MHV-SGFP will now be a convenient tool to study coronaviral cell entry.

Figures

FIG. 1.
FIG. 1.
(A) Schematic diagram of the MHV S and S-GFP protein. The arrowhead indicates the cleavage site, where posttranslational cleavage of S into two subunits (S1 and S2) takes place. In S-GFP the GFP protein is fused to the carboxy terminus of the S protein. The dashed lines indicate the position of the transmembrane region. (B) Plasmid constructs, targeted recombination, and recombinant viruses. The plasmid pMH54 (described previously [25]) and pXHSGFP (see Materials and Methods) were used to transcribe the defective RNAs in vitro by using T7 polymerase. The arrow at the left end of the vectors indicates the T7 promoter; the solid circle represents the polylinker between the 5′-end segment of the MHV genome (labeled 5′/1) and the HE gene, followed by the structural and group specific genes, the 3′ untranslated region (UTR) and the polyadenylate segment (together labeled as 3′/U). The positions of the numbered sequences shown in panel C correspond to the numbered arrowhead positions. At the top, a scheme is shown for targeted recombination by using the interspecies chimeric fMHV, which grows only in feline cells. Recombinant viruses generated by the indicated crossover event can be selected on the basis of their ability to grow in murine cells. The genomes of these viruses are represented at the right. (C) Sequences of the numbered junctions shown in panel B. The numbers correspond to those under the arrowheads in panel B. The amino acids encoded by the 3′ end of the S and the GFP gene, by the linker sequence and by the 5′ end of the GFP gene are indicated. The asterisk denotes the stop codon. The MHV TRS belonging to gene 4 is boxed.
FIG. 2.
FIG. 2.
CLSM of OST7-1 cells expressing the S (A and B) and S-GFP (C and D) proteins (×40 magnification). Cells were fixed at 8 h p.i., permeabilized, and processed for immunofluorescence by using the anti-MHV serum (1:400) and the red fluorescent Cy5 secondary antibody (1:200) and excited at 488 nm (GFP; B and D) or 568 nm (Cy5; A and C).
FIG. 3.
FIG. 3.
Coimmunoprecipitation of the M protein by S-GFP fusion protein. (A) Coexpression in murine cells of the MHV M and E proteins either in the absence or in combination with the S or S-GFP protein. Cells were labeled with 35S-labeled amino acids from 5 to 6 h p.i., followed by a 2-h chase. Immunoprecipitations were carried out on the cell lysates with the anti-MHV serum (α-MHV) or the anti-S MAb (α-S) and analyzed by SDS-PAGE. (B) SDS-PAGE analysis of VLPs affinity purified from the culture medium with α-MHV and α-S. The molecular mass markers are indicated on the left. Arrows on the right indicate the positions of the expressed proteins.
FIG. 4.
FIG. 4.
CLSM of MHV-SGFP-infected LR7 cells grown in the presence (A) or absence (B) of membrane fusion inhibitor HR2 (×40 magnification).
FIG. 5.
FIG. 5.
Growth kinetics of MHV-SGFP and MHV-WT. LR7 cells were infected with either MHV-WT or with the independent MHV-SGFP clones A or B at an MOI of 1. Viral infectivities in the culture media were determined at different times postinfection by a quantal assay on LR7 cells, and the TCID50 values were calculated.
FIG. 6.
FIG. 6.
(A) PCR analysis of recombinant MHV-SGFP. RT-PCR was performed on purified genomic RNA of MHV-SGFP from passage 2 and 6 (P2 and P6, respectively) to analyze the presence of the GFP sequence. A water control (H2O) is also shown. As additional controls, a PCR was performed on the transcription vectors pXHSGFP (C1) and pMH54 (C2). PCR products were analyzed by electrophoresis in 1% agarose gels and stained with ethidium bromide. Primers used in the experiment, their location on the genome, and the predicted sizes of the PCR products are indicated at the right. In the RT step, primer 1261 was used, whereas primers 935 and 1290 were used in the PCR step. The sizes of relevant DNA fragments of the 1-kb DNA marker ladder (M; Invitrogen) are indicated on the left. (B) Sequence analysis of passage 6 MHV-SGFP. The RT-PCR product obtained from MHV-SGFP clone A passage 6 was cloned into pGEMT-Easy, and plasmid DNA from two colonies was sequenced. The nucleotide sequences of the 3′ region of the spike gene of the two sequenced clones, as well as of MHV-WT, are shown. The translated amino acid sequences corresponding to the S ORF are indicated under the nucleotide sequence. Deletions in the nucleotide sequence are represented by dashed lines. The asterisks mark the stop codons. The TRS sequence of gene 4 is boxed.
FIG. 7.
FIG. 7.
Viral proteins in the cell lysates and culture media of MHV-SGFP-infected cells. LR7 cells were either mock infected (lanes m), infected with MHV-WT (lanes WT), or infected with MHV-SGFP clones A and B (lanes A and B). (A) At 5 h p.i., cells were labeled for 1 h with 35S-labeled amino acids and lysed immediately. Some cultures were further incubated with chase medium for 2 h, after which the culture supernatant was harvested and diluted with lysis buffer. Immunoprecipitations were performed with the anti-MHV serum (α-MHV), the anti-S MAb (α-S), or the anti-GFP serum (α-GFP) from either the cell lysate (A) or the culture medium (B and C [panel C is a longer exposure of a section of panel B]) and analyzed by SDS-PAGE. The molecular mass marker is indicated on the left. Arrows on the right indicate the positions of the expressed proteins.
FIG. 8.
FIG. 8.
Protein quantification of affinity-purified coronavirus particles. LR7 cells were either mock infected, infected with MHV-WT, or infected with MHV-SGFP clone B. At 5 h p.i. cells were labeled for 1 h with 35S-labeled amino acids and chased for another 2 h. Virus particles were affinity purified from the cleared culture medium with either the anti-M MAb (α-M MAb) or the anti-S MAb (α-S MAb) and analyzed by SDS-PAGE. The amounts of radioactivity in the M, S (total, i.e., S+S-GFP), and N proteins in the dried gels were determined by phosphorimager scanning. The ratios of the amounts of M to N, S to M, and S to N in affinity-purified MHV-SGFP relative to those in MHV-WT were calculated.
FIG. 9.
FIG. 9.
CLSM on MHV-SGFP and MHV-WT. Virus obtained from ∼107 infected LR7 cells was pelleted through a 20% sucrose cushion and resuspended in 100 μl of PBS. A 10-μl aliquot of either MHV-WT (A) or MHV-SGFP (B) was examined by CLSM (×100 magnification).

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