Fig 6.

Construction of the ΔPS and ΔPS-PS2 mutants. (A) Ribbon diagram and molecular surface rendering of the MHV nsp15 monomer (PDB accession code 2GTH) (33) generated with PyMol (http://pymol.org). The nsp15 amino and carboxy termini are labeled N and C, respectively. The dotted line represents part of the surface loop connecting the amino- and carboxy-terminal domains of the protein. (B) Alignment of the central region of nsp15 of MHV with that of representative coronaviruses: lineage A betacoronaviruses BCoV and HCoV-HKU1, lineage B betacoronavirus SARS-CoV, alphacoronavirus TGEV, and gammacoronavirus IBV. GenBank accession numbers for the sequences shown are as follows: MHV, AY700211; BCoV, U00735; HCoV-HKU1, AY597011; SARS-CoV, AY278741; TGEV, AJ271965; and IBV, AJ311317. Boxed residues in the MHV sequence are those that are encoded by the PS and that were deleted in the ΔPS mutant. The bar above the alignment indicates flexible loop residues (Y195 through L216) that are missing from the crystal structure of MHV nsp15 (33). Dotted lines connect deleted and missing residues to their corresponding positions in the structure in panel A. At the bottom of the alignment, the five heterologous amino acids encoded by the substituted sequence in the ΔPS mutant are underlined. (C) Representation of the unstructured 15-nt sequence substituted for the PS in the ΔPS mutant compared to the wild-type PS. (D) Schematics of the relevant genomic regions of the constructed ΔPS mutant, the ΔPS-PS2 mutant, and their isogenic wild-type counterpart.