Figure 6

Impact of R14K and D111N mutations on the CA-NTD structure. (A) CCS Combined chemical shift (CCS) changes of R14K and WT CA-NTD (blue), and D111N and WT (red). Helices 1 - 6 are annotated as h1 - h6. (B) Residues with the largest CCS caused by the R14K mutation: green - R14; orange: T3, E4, T5, V6 in β-hairpin; blue: W52, D57 in loop connecting helices 2 and 3; yellow: Q113, M114, Q115, Y116, D117, G119, F121, A122 in helices 5, 6, and the connecting loop. The asterisk indicates the internal cleavage site between A43 and I44 (red). The WT M-PMV CA-NTD structure (PDB ID: 2KGF) was used for this model. (C) Western blot of CA related proteins in released WT and CA-NTD mutant M-PMV particles detected with rabbit antibodies raised against M-PMV CA. VLPs released from the of HEK 293T cells 48 h post-transfection were centrifuged through a 20% sucrose cushion. (D) HEK 293T cells were co-transfected with WT or mutant pSARM-EGFP and pTMO vectors. At 48 h post-transfection, the virus was filtered from culture media and normalized by quantitative Western blotting for CA. At 48 h post-infection with equivalent amounts of virions, the HEK 293T cells were harvested and the GFP-positive cells were counted using flow cytometry. The mean percentage of three independent infectivity measurements for each mutant relative to WT is shown. (E) Electron micrographs of thin sections of E. coli expressing Q115A and Q115E M-PMV CANC protein. (F, G) Coomassie blue stained (F) or western blot analysis (G) of R14K CA-NTD and WT CA-NTD proteins cleavage in vitro. M-PMV R14K CA-NTD and WT CA-NTD purified proteins in storage buffer (lanes 1 and 4, respectively) were diluted into M-PMV protease cleavage buffer (lanes 2 and 5). M-PMV protease was added to R14K CA-NTD (lane 3) and WT CA-NTD (lane 6) and incubated for 4 h at 37°C.