FIG. 5.

(A) Schematic representation of subgenomic HPV-16 expression plasmids pBELM, pT1, pT4, and pT1OPSA. Numbers indicate the nucleotide positions of 5′ splice sites (filled circles) and 3′ splice sites (empty circles) or the positions of pAE and pAL or mark the borders of deletions. Major potential mRNAs produced by these plasmids are indicated below. The optimized 3′ splice site with 21 consecutive pyrimidines (Py₂₁) upstream of the invariable AG dinucleotide of the 3′ splice site is indicated. The L1i mRNA is a previously described mRNA that is spliced directly from SD880 to SA5639 when splicing silencers at SA5639 are inactivated by mutations (61). Splicing directly from SD880 to SA5639 in differentiated W12 epithelial cells has also been observed previously (38). The E4 and L1 probes used for Northern blotting are indicated. Mut, a mutant L1 gene in which splicing silencers have been inactivated (11, 61). (B, C, and D) Northern blots of cytoplasmic RNA extracted from HeLa cells transfected with pBELM, pT1, pT2, pT3, pT4, or pT1OPSA in the absence or presence of a PTB-expressing plasmid. Blots were probed with the E4 probe (B) or the L1 probe (C and D). Gels were also probed for GAPDH expression. The positions of the E4, L1, and L1i mRNAs are indicated. +, present; −, absent.