TABLE 1.

E3-6.7/GM-CSF junction sequences

Construct	Sequencea	Description
Wild type	E3-6.7...ccaagATGAttaggtac...E3-gp19	Native sequence showing overlap between the E3-6.7 and gp19 coding sequences
Δgp19a	E3-6.7...ccaagATG........TGA...GM-CSF	GM-CSF added without consideration of effects on E3-6.7 stop codon. A stop codon, in-frame with E3-6.7, is reached after 33 amino acids with human GM-CSF; with the mouse GM-CSF, 9 amino acids are added to E3-6.7
Δgp19b	E3-6.7...ccaagaTAAccATG...GM-CSF	E3-6.7 and GM-CSF sequences do not overlap; start and stop codons separated and Kozak sequence inserted 5′ to GM-CSF
Δgp19c	E3-6.7...ccaagATGA...GM-CSF	Start and stop codons of E3-6.7 and GM-CSF sequences overlap; second codon (fourth nucleotide) of GM-CSF leader sequence mutated
Δgp19d	E3-6.7...ccaagaTGAccATG...GM-CSF	Start and stop codons of E3-6.7 and GM-CSF sequences overlap; two amino acids added to GM-CSF leader sequence
Δgp19/IRES	E3-6.7...ccaagaTGA...IRES...ATG...GM-CSF	Start and stop codons of E3-6.7 and GM-CSF sequences separated by EMC IRES

^aNormal font, wild-type viral sequences; bold, native GM-CSF sequence; italics, nucleotides added as spacer between E3-6.7 and GM-CSF; uppercase, start and stop codons. The Δgp19 a, b, c, and d modifications were introduced by PCR using primers that overlapped the junction between E3-6.7 and GM-CSF. The encephalomyocarditis virus IRES sequence was first attached by PCR to the initial ATG of GM-CSF prior to being joined to E3-6.7. Viruses carrying the mouse GM-CSF cDNA were generated from all of the constructs described. Human GM-CSF viruses were made using the Δgp19 a, b, and c constructs.