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.