Competitive Fitness in Coronaviruses Is Not Correlated with Size or Number of Double-Membrane Vesicles under Reduced-Temperature Growth Conditions

Al-Mulla, Hawaa M. N.; Turrell, Lauren; Smith, Nicola M.; Payne, Luke; Baliji, Surendranath; Züst, Roland; Thiel, Volker; Baker, Susan C.; Siddell, Stuart G.; Neuman, Benjamin W.

We next compared the growth of the panel of viruses at 33°C on continuous 17Clone-1 fibroblasts (17Cl-1), primary mouse embryo fibroblasts (MEFs), adipose-derived mesenchymal stem cells (ADMSC), and bone marrow-derived stem cells (BMSC) ( Table 1 ). All of the mutants except Albts22 grew to approximately wild-type levels at 33°C in every cell type tested. Albts16 grew slightly slower than the wild type in 17Cl-1 cells but grew to wildtype levels in MEFs. Albts22 consistently produced about one-FIG 2 Characterization of Brts105 growth. Viruses were inoculated at a multiplicity of 10 PFU/cell on primary MEFs (mouse embryo fibroblasts) at 33°C for 1 h and then incubated at either 33°C (A) or 40°C (B) for the duration of the experiment. Viral growth was measured by plaque assay at 33°C. The average of three replicates is shown. Dotted lines indicate the lower limit of detection for the plaque assay. (C) Northern blots show the effect of temperature on viral RNA production by the wild type and Brts105. Bands corresponding to viral subgenomic RNAs 4 to 7 are indicated. (D to F) The relative severity of ts phenotypes was characterized by a growth stop/resume assay. Growth was stopped 4.5 h after inoculation by incubating ts virus at 40°C or by treating wild-type virus with 10 g/ml cycloheximide (CHX). After 3 h of arrested growth, the inhibition was either maintained to the end of the experiment (Stopped) or released (Resumed). Virus growth was measured by plaque assay, and the results for Brts105 are shown in panel E. The averages for 6 replicates are shown. The titer ratio (resumed to stopped) from panel E is shown in comparison to other mutants and CHX treatment in panel F. Statistically significant increases in titer after resumption are indicated (*, P Յ 0.05; ns, not significant). tenth as many infectious progeny as wild-type virus. These results showed that, except for Albts22, the panel of mutants displayed growth characteristics similar to those of wild-type virus at 33°C. These results were consistent with previous results, which showed that Wüts18 (37) and Brts31 (14) grow to wild-type levels at 33°C, while growth of Albts22 is attenuated even at 30°C (23) . Aberrant DMV formation at 33°C. Cells were infected at high multiplicity with wild-type or mutant viruses and then examined by electron microscopy to investigate the effects of mutations on replicative organelle formation at 33°C. One randomly oriented ultrathin section per cell was examined in order to compare the sizes and numbers of intracellular organelles virions, as described previously (17) .