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.

To discriminate between these possibilities, wild-type and Brts31 RNA synthesis was compared by quantitative RT-PCR. Genomic and subgenomic RNA 7 synthesis was quantified using primers specific to the nsp3-encoding region and across the leader-body junction of subgenomic RNA 7, respectively. Brts31 synthesized somewhat larger amounts of genomic and sub- genomic RNA than the wild type at 33°C, as shown in Fig. 4B . Together, these data demonstrate that efficient RNA production is not necessarily correlated with large or numerous DMVs. DMVs and fitness. To better understand the role of DMV formation in the virus replication cycle, we carried out competitive fitness assays on all the viruses that grew to approximately wildtype titers. In these assays, an equal number of plaque-forming units of wild-type virus and one ts virus were added at low multiplicity to either continuous or primary cells, which were then incubated at 33°C. Fitness was calculated as the ratio of mutant virus to wild-type virus present in the supernatant at the end of the competition. In this assay, a ratio of 1 would indicate that the viruses were equally fit, higher ratios indicate that the mutant virus is fitter than the wild type, and lower ratios indicate that the mutant virus is less fit than the wild type. Viruses were differentiated by means of plaque assays initiated at 33°C for 1 h and then continued by incubation at either 40°C (where only the wild type forms plaques) or 33°C (where both viruses form plaques). The assay was validated by performing differential plaque assays on pure samples of the wild type and Brts105 (Fig. 5A ). The number of plaques formed by the wild type did not differ significantly at the two temperatures. Brts105 formed plaques in 33°C plaque assays but not in plaque assays initiated at 33°C and then continued by incubation at 40°C (Fig. 5A ). This demonstrated that titers of wild-type and ts viruses can be distinguished by differential plaque assays and that the reversion rate of Brts105 was less than 10 Ϫ5 and therefore would not detectably complicate the interpretation of competition experiments, which typically produced more than 10 6 PFU/ml.