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.

The formation of replicative organelles is a conserved strategy among positive-stranded viruses and has led to the assumption that viral organelles play an important and highly conserved role in intracellular RNA replication (1, 2) . Previous studies had shown that newly synthesized viral RNA can be detected as early as 1 h after inoculation (40) , but the earliest time at which DMVs have been detected is 2 h after inoculation (4, 5) , and complete organelles with CM and DMVs were first reported 3 h after inoculation with MHV (5) or 4 h after inoculation with SARS-CoV (4), suggesting that the replicative organelle may be a lagging indicator of viral RNA synthesis. However, other studies have shown that replicative organelles disappear within about 2 h of treatments that strongly inhibit viral RNA synthesis (14, 15) . In this study, we documented viruses with a range of organelle sizes and prevalence that were sufficient to support wild-type levels of viral growth. This included two viruses of the six tested (Brts31 and Brts105) that replicated normally with half the normal complement of organelles or less. These results extend findings that attenuated nsp4 mutant viruses produce fewer organelles than normal (21) . Together, these results suggest that there is a considerable plasticity in the replication complex, since a variety of sizes and numbers of DMVs can support RNA synthesis.