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 data presented here do not necessarily disprove that DMV clusters are needed for intracellular coronavirus RNA synthesis but demonstrate that the link may be looser than anticipated. This finding was anticipated by a recent study that demonstrated that subcellular compartments that contain double-stranded RNA (dsRNA) (i.e., probable DMVs) were not necessarily sites of active RNA replication (38) . We unexpectedly found that viruses that produced larger and more numerous DMVs did not have a consistent fitness advantage in primary or continuous cells under test conditions. However, it is quite possible that further testing in primary macrophages or live animals would reveal a competitive advantage for increased organelle numbers or increased DMV capacity. A previous analysis of coronavirus RNA replication had shown that viral dsRNA colocalizes well with nonstructural proteins early in infection but that the correlation becomes less clear over time (38) . Nevertheless, these results are the first direct evidence to our knowledge that intracellular RNA synthesis can be at least partially decoupled from formation of replicative organelles. We also report here for the first time that viruses with mutations in all 5 known cistrons of the MHV replicase form either slightly or substantially smaller DMVs, suggesting that while DMVs are formed primarily by nsp3, nsp4, and nsp6 mutants (17), mutations in any part of the replicase locus can potentially restrict DMV formation. This suggests that the size and potentially the form of the replicative organelle may be dependent on factors other than the amount of RNA synthesis, such as the efficiency of polyprotein processing.