FIG. 7.

(A) Schematic representation of subgenomic HPV-16 expression plasmids pBSplice, pBSpliceM, p2xpAL, pBSpMD4288-4530, and pBSpMDL1. 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 pBSpliceM. E4* indicates a short mRNA encoding E4 and E5 that is unspliced due to the absence of SD880 and other 5′ splice sites upstream of the major E4 3′ splice site SA3358 in the pBSplice-derived plasmids. The E4 and L1 probes used for Northern blotting are indicated. Gray bars represent RT-PCR primers 3455s, 3575s, and L1a(M) (44, 61). Mut, a mutant L1 gene in which splicing silencers have been inactivated (11, 61). (B, D, E, and F) Northern blots of cytoplasmic RNA extracted from HeLa cells transfected with pBSplice, pBSpliceM, p2xpAL, pBSpMD4288-4530, and pBSpMDL1in the absence or presence of a PTB-expressing plasmid. Blots were probed with the E4 probe (B, right panel), the L1 probe (B, left panel, and D, E, and F), and a GAPDH probe. The positions of the alternative E4 mRNA named E4* and the L2/L1 and L1 mRNAs are indicated. +, present; −, absent. (C) Results from RT-PCR with primers 3455s or 3575s and L1a(M) (see panel A) (44, 61) on the same cytoplasmic RNA that was analyzed in the blot in panel B. GAPDH cDNA was amplified as a control.