Construction of SARS-CoV-2 D614G Mutant

One single-nucleotide substitution was introduced into a subclone puc57-CoV-2-F5–7 containing the spike gene of the SARS-CoV-2 wild type (WT) infectious clone^{10 (link)} to convert the 614^th amino acid from aspartic acid (D) to glycine (G) by overlap fusion PCR. The full-length infectious cDNA clone of SARS-CoV-2 D614G was assembled by in vitro ligation of seven contiguous cDNA fragments following the protocol previously described^{10 (link)}. For construction of D614G mNeonGreen SARS-CoV-2, seven SARS-CoV-2 genome fragments (F1 to F5, F6 containing D614G mutation, and F7-mNG containing the mNeonGreen reporter gene) were prepared and in vitro ligated as described previously^{10 (link)}. In vitro transcription was then preformed to synthesize full-length genomic RNA. For recovering the mutant viruses, the RNA transcripts were electroporated into Vero E6 cells. The viruses from electroporated cells were harvested at 40 h post electroporation and served as seed stocks for subsequent experiments. The D614G mutation from the recovered viruses was confirmed by sequence analysis. Viral titers were determined by plaque assay on Vero E6 cells. All virus preparation and experiments were performed in a biosafety level 3 (BSL-3) facilities. Viruses and plasmids are available from the World Reference Center for Emerging Viruses and Arboviruses at the University of Texas Medical Branch.