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. 2021 Aug 18;10(1):1626–1637. doi: 10.1080/22221751.2021.1964385

Figure 1.

Figure 1.

Construction and characterization of MHV nsp14 N7-MTase mutants. (A) Structure of MHV-A59 genome. The mutation sites in nsp14 N7-MTase domain are shown. (B) N7-MTase activity assay for nsp14 and its mutants in vitro. The N7-MTase activity of WT MHV nsp14 was set as 100%. Means with SD are shown (n = 3). (C) Plaque morphology of mutant D330A, Y414A and WT virus at passage 10 (P10). Viruses were passaged on Neuro 2a cells for 10 rounds, and the resulting P10 viruses were examined by plaque assay. The diameters of representative plaques were measured and are shown as means with SD (n = 10). (D) The D330A and Y414A mutants were maintained after 10 rounds of passage. The cDNA fragments encoding nsp14 of P10 viruses were amplified by RCR and the products were sequenced. (E) One-step replication curves of D330A, Y414A, and WT virus were measured on Neuro 2a cells and L2 cells at an MOI of 0.01. Viral titre in cell supernatant were measured by plaque assay. Means with SD are shown (n = 3). ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 (unpaired Student’s t-test).