Fig. 6.

Investigating growth of P323 and L323 in cell culture. A Representative images of plaques formed by two viruses created through reverse genetics that have the Wuhan-Hu-1 background (NC_045512) and an engineered D614G substitution in the spike protein, and either P323 or L323 in NSP12 (termed Wuhan/G614/P323 and Wuhan/G614/L323 respectively). B Relative RNA levels of genomic or N subgenomic RNA with Wuhan/G614/P323 or Wuhan/G614/L323 from RT-qPCR on ACE2-A549 cells infected with either virus at 24h. Error bars show standard deviation. Unpaired t-tests without Welch’s correction, p=0.0181 and p=0.0393 respectively, for n=3 biological replicates. C Western blot analysis of the abundance of nucleoprotein produced in either mock infected, or cells infected with Wuhan/G614/P323 or Wuhan/G614/L323. This is an exemplar western blot for an experiment that was done in triplicate; GAPDH is shown as a protein loading control. D Mean viral titers (pfu/ml, n=3 biological replicates ± standard deviation) at 24hpi in Vero E6, Vero/hSLAM, and ACE2-A549 cells infected with either Wuhan/G614/P323 or Wuhan/G614/L323. E,F Proportion of amino acid P323/L323 in NSP12 (E) or D614/G614 in the Spike protein (F) in the Victoria/01/2020 isolate serially passaged through cells over 13 sequential passages (coverage filtered at 20×)