Fig. 4.

Acute toxicity assay in HeLa and 293 cells transiently transfected with ORF2 putative phosphorylation mutant plasmids. a HeLa cells were cotransfected with a Neo^R expression vector and the indicated ORF2 putative phosphorylation mutant plasmid. b 293 cells were cotransfected with a Neo^R expression vector and the indicated ORF2 putative phosphorylation mutant plasmid. In both panel a and b ORF2 is the functional protein and ORF2 EN-RT- is a non-functional protein containing mutations in the endonuclease (D205A) and reverse transcriptase (D702A) domains. Control indicates cells transfected with an empty vector and the Neo^R expression vector. Colony formation was assayed after 2 weeks under G418 selection (Y-axis) and used as a measure of toxicity as previously described [26, 42]