Negatively charged residues in the endodomain are critical for specific assembly of spike protein into murine coronavirus

Abstract

Coronavirus spike (S) protein assembles into virions via its carboxy-terminus, which is composed of a transmembrane domain and an endodomain. Here, the carboxy-terminal charge-rich motif in the endodomain was verified to be critical for the specificity of S assembly into mouse hepatitis virus (MHV). Recombinant MHVs exhibited a range of abilities to accommodate the homologous S endodomains from the betacoronaviruses bovine coronavirus and human SARS-associated coronavirus, the alphacoronavirus porcine transmissible gastroenteritis virus (TGEV), and the gammacoronavirus avian infectious bronchitis virus respectively. Interestingly, in TGEV endodomain chimeras the reverting mutations resulted in stronger S incorporation into virions, and a net gain of negatively charged residues in the charge-rich motif accounted for the improvement. Additionally, MHV S assembly could also be rescued by the acidic carboxy-terminal domain of the nucleocapsid protein. These results indicate an important role for negatively charged endodomain residues in the incorporation of MHV S protein into assembled virions.

Fig. 1

Strategy to test S assembly by construction of MHV recombinants bearing mutated Tm and Endo domains. (A) The upper part of the diagram shows the introduction of mutations (blue triangle) into the S gene, resulting in production of S protein with mutated Tm and Endo domains (blue rectangles). (B) The lower part of the diagram shows the introduction of mutations (blue triangle) into the HK gene, which is substituted for the nonessential 2a/HE genes, resulting in production of HK protein with mutated Tm and Endo domains (blue rectangles), while the wild-type S gene and protein remain unaltered.

Fig. 2

Effects on assembly caused by the replacement of the C-terminus of MHV S protein with C-termini from different coronaviruses. (A) Alignment of C-terminal transmembrane domain (bold) and endodomain amino-acid sequences of the S proteins of alphacoronavirus TGEV (AJ271965), gammacoronavirus IBV (AJ311317), and betacoronaviruses MHV-A59 (AY700211), BCoV (U00735), and SARS-CoV Urbani (AY278741) (GenBank accession numbers given in parentheses). (B) Plaque assay of MHV S protein recombinants. The C-terminus of MHV S protein was replaced with the homologous sequence from TGEV, BCoV, IBV, or SARS-CoV. Mouse L2 cells were infected with recombinant viruses for 2 h, overlaid with agar for 40 h and then stained for 8 h with neutral red. Mock infection was conducted with sterile media. (C) Immunoblotting analysis of substituted HK proteins incorporated into MHV recombinant. Top panel, HK expressed by recombinant MHVs in 17Cl1 cells (lysates). Middle panel, HK incorporated into purified recombinant MHV virions. Bottom panel, N protein as a control for normalization of virions (Coomassie-stained). In the top and middle panels, HK was detected with mouse mAb to HA tag and HRP-conjugated goat anti-mouse IgG.

Fig. 3

Dominant role of the charge-rich motif in S assembly. (A) Amino acid sequences of the chimeric C-termini in S or HK MHV recombinants. Tm are shown in bold and Endo are divided into the cysteine-rich motif and the charge-rich motif (underlined). The upper two sequences are from wild-type S (WT) or a charged-rich motif-truncated mutant (Mut-30). In the remaining four chimeric mutant sequences, the first, second, and third letters represent the source (MHV, SARS-CoV, or TGEV) of the Tm, cysteine-rich motif, and charge-rich motif, respectively. Plaque assays of recombinant MHV S protein mutants and immunoblotting analysis of mutant HK proteins incorporated into recombinant virions are shown in panels (B) and (C), as described in Fig. 2.

Fig. 4

Reverting mutations arising in TGEV chimeric S proteins. (A) Plaque assay of the original TGEV S protein Endo recombinants (Mut-TGEV and Mut-MMT) and their corresponding purified revertants (TGEV-R1, TGEV-R2, MMT-R1, and MMT-R2); wild-type (WT) and Mut-30 viruses served as positive and negative controls, respectively. (B) Alignment of mutated Endo sequences of revertants compared to their parents and controls; the newly generated heptapeptide terminus of TGEV-R1 and MMT-R1 is underlined.

Fig. 5

The charged-rich motif produced by the MMT-R1 reverting mutation restored S assembly into virions. (A) Alignment of C-terminal sequences of S or HK MHV recombinants with reverting mutations. Mut-70 and Mut-71 were constructed with sequences from reverting mutant MMT-R1. WT and two terminally-truncated mutants (Mut-69 and Mut-30) served as controls. Tm are shown in bold, and the heptapeptide TENLNNL is underlined. Plaque assays of recombinant MHV S protein mutants and immunoblotting analysis of mutant HK proteins incorporated into recombinant virions are shown in panels (B) and (C), as described in Fig. 2.

Fig. 6

N protein domain 3 could partially rescue S assembly into virions. (A) Alignment of C-terminal sequences of S or HK MHV recombinants in which the charge-rich motif was replaced with N protein domain 3 from either MHV (Mut-MN) or BCoV (Mut-BN), or else with HE Endo (Mut-HE). WT, truncated mutant (Mut-69), and MMT-R1 mimicking mutant (Mut-70) served as controls. Tm are shown in bold, and the heptapeptide TENLNNL is underlined. Plaque assays of recombinant MHV S protein mutants and immunoblotting analysis of mutant HK proteins incorporated into recombinant virions are shown in panels (B) and (C), as described in Fig. 2.