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.

Northern blotting. RNA for Northern blotting was obtained by in-fecting~10 7 17Cl-1 cells with virus at a multiplicity of 10 PFU per cell for 8 h at 33°C or 6 h at 39.5°C and isolating total RNA using the Trizol reagent (Invitrogen, Paisley, United Kingdom) as described by the manufacturer. The poly(A)-containing RNA was isolated using oligo(dT) 25 Dynabeads (Dynal, Oslo, Norway) as previously described (44) . The RNA was then incubated with formamide (50%) and formaldehyde (2.2 M) at 70°C for 10 min and electrophoresed in a 1% agarose gel containing 20 mM MOPS (3-N-morpholinopropanesulfonic acid) and 600 mM formaldehyde. After electrophoresis, the gel was soaked in 0.05 N NaOH, neutralized, and equilibrated with 20ϫ SSC (3 M NaCl, 0.3 M sodium citrate, 1 mM EDTA) before being vacuum blotted onto a nylon membrane (Optitran BA-S 83; Schleicher and Schuell, Sigma-Aldrich, Poole, United Kingdom). The RNA was cross-linked to the membrane using UV light, and MHV RNAs were detected by hybridization with a 466-bp, ␣-32 P-, random-prime-labeled PCR product corresponding to sequences in the nucleocapsid protein open reading frame of the MHV-A59 genome (45) .