Relative expression of precursor proteins and cleaved gLuc after the truncation of the viral sequence upstream of AUG. (A) Schematic representation of AUG2-M, AUG3-M, AUG4-M, or AUG5-M, which express a single precursor from the 2nd, 3rd, 4th, or the 5th AUG, respectively. AUG2-M lacks the 3rd, 4th, and 5th AUGs, AUG3-M lacks the 4th and 5th AUGs, and AUG4-M lacks the 5th AUG. The mutant plasmids encode a common 20 nucleotide viral sequence upstream of the 1st AUG (5′-ACACAAAGACGGUGCACGAGAUG (initiation codon is underlined)); (B) 293 cells were co-transfected with pSV40-CLuc (transfection control), and pCAGGS-PreGn-gLuc-SF or the mutant plasmids. At 36 h post transfection, culture supernatants were collected, and the gLuc and cLuc activities were measured. Then, the ratio of gLuc to cLuc was normalized to that of parental pCAGGS-PreGn-gLuc-SF plasmid. The graph represents the mean + the standard error of three independent experiments. Asterisks represent statistically significant differences (one-way ANOVA followed by Tukey’s multiple comparisons test, * p < 0.05, ** p < 0.01).