Fig. 2

Expression of viral PKR antagonist protein Vaccinia E3L or Ebola VP35 inhibits excision of HIV rev/tat intron. A Vector encoding the 3’ half of the HIV genome. Vector pcDNA-3’HIV carries the 3’ portion of the HIV-1 genome expressed under the constitutive cytomegalovirus (CMV) promoter. The viral sequences start downstream of the rev AUG translation start codon to prevent Tat and Rev production. The construct retains splice donor D4 (5’ss #4) and splice acceptor A7 (3’ss #7), allowing for a single splicing event of the rev/tat intron (bottom). B, C E3L and VP35 inhibit splicing of rev/tat mRNA. BHK-21 cells were cotransfected with 1 µg of pcDNA-3’HIV DNA together with 1 µg pBS empty vector (EV), E3L expression vector (E3L) or VP35 expression vector (VP35). Total RNA was isolated at 18 h post-transfection. Unspliced pre-mRNA (413 nt) and spliced rev/tat mRNA (140 nt) were determined by RNase protection analysis (B). The top autoradiogram (pre-mRNA) underwent a longer exposure. Band intensity was quantitated and the ratio of spliced over unspliced RNA within each lane, which reflects splicing efficiency, is plotted in bar graph (C) (error bars, SEM; n = 3). A representative experiment is shown. D E3L and VP35 inhibit splicing of rev/tat mRNA. In independent transfections, performed as in B, total RNA was isolated at 12 h; spliced and unspliced rev/tat transcripts were determined by qRT-PCR. Splicing efficiency is expressed as mRNA/pre-mRNA ratio (error bars, SEM; n = 3). E Whereas rev/tat intron excision is inhibited by co-expression of E3L, it is stimulated by co-expression of PKR. BHK-21 cells were cotransfected with 1 µg of pcDNA-3’HIV DNA together with 1 µg pBS empty vector (EV) or with vector expressing E3L or human PKR. Total RNA was isolated at 12 h post-transfection. Spliced and unspliced rev/tat transcripts were determined by qRT-PCR. Splicing efficiency is expressed as mRNA/pre-mRNA ratio (error bars, SEM; n = 3). F Splicing of rev/tat mRNA is blocked by expression of K296R trans-dominant negative mutant PKR. BHK-21 cells were cotransfected with 1 µg of pcDNA-3’HIV DNA together with 1 µg pBS empty vector (EV) or with vector expressing mutant K296R or human PKR. Total RNA was isolated at 12 h post-transfection. Spliced and unspliced rev/tat transcripts were determined by qRT-PCR. Splicing efficiency is expressed as mRNA/pre-mRNA ratio (error bars, SEM; n = 3)