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. 2000 Mar;74(6):2913-9.
doi: 10.1128/jvi.74.6.2913-2919.2000.

Herpes simplex virus ICP27 induces cytoplasmic accumulation of unspliced polyadenylated alpha-globin pre-mRNA in infected HeLa cells

P Cheung  1 , K S Ellison, R Verity, J R Smiley
Affiliations

Herpes simplex virus ICP27 induces cytoplasmic accumulation of unspliced polyadenylated alpha-globin pre-mRNA in infected HeLa cells

P Cheung et al. J Virol. 2000 Mar.

Abstract

Transcripts of most intron-bearing cellular genes must be processed by the splicing machinery in order to efficiently accumulate and gain access to the cytoplasm. However, we found that herpes simplex virus induces cytoplasmic accumulation of both spliced and unspliced polyadenylated alpha-globin RNAs in infected HeLa cells. Accumulation of the unspliced RNA required the immediate-early protein ICP27, and ICP27 was sufficient (in combination with ICP4) to produce this effect in a transient-transfection assay. However, expression of ICP27 did not markedly alter the levels of fully spliced alpha-globin transcripts in infected cells. These data demonstrate that the previously documented effects of ICP27 on the cellular splicing apparatus do not greatly inhibit splicing of alpha-globin RNA and argue that ICP27 induces a splicing-independent pathway for alpha-globin RNA accumulation and nuclear export.

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Figures

FIG. 1
FIG. 1
HSV-1 infection induces multiple α-globin RNA species in HeLa cells. Total RNA was harvested from HeLa cells that were either mock infected, infected with HSV-1 strain KOS, or transfected with an ICP4 expression clone (pBB37) or a plasmid bearing the α2-globin gene (pUCα2). The RNA samples were either treated with RNase H in the presence of oligo(dT) (+RNase H + oligo dT) or left untreated (no RNase H). The α-globin RNA species were examined by Northern blot analysis using a 1.5-kb PstI fragment bearing the human α2-globin gene as a probe. C, control blood RNA; M, RNA size markers (in kilobases).
FIG. 2
FIG. 2
Northern blot analysis of α-globin RNA from HeLa cells infected with various HSV mutants. HeLa cells were infected with the indicated HSV strains in the presence of PAA, and total RNA extracted 6 h postinfection was analyzed by Northern blot as described for Fig. 1. Panels are two different exposures of the same blot. C, control blood RNA; M, RNA size markers (in kilobases).
FIG. 3
FIG. 3
Time course of accumulation of spliced and unspliced α-globin RNA. HeLa cells were infected with KOS or 5dl1.2 in the presence of PAA. Total RNA harvested at the indicated times postinfection was treated with RNase H in the presence of oligo(dT) to remove the poly(A) tails and then analyzed by Northern blot hybridization using 32P-labeled oligonucleotide probes specific for intron 1 (A) or exon 1 (B). Lane C, control blood RNA; lane M, RNA size markers (in kilobases). Signals representing the fully processed (C) and unprocessed (D) α-globin transcripts detected in panel B were quantified by phosphorimager analysis, and normalized data were plotted.
FIG. 4
FIG. 4
Unspliced α-globin RNA accumulates in the cytoplasm. Duplicate dishes of HeLa cells were infected with HSV-1 strain KOS1.1 or d27-1 in the presence of PAA. At 6 h postinfection, total RNA (T) was harvested from one dish and cytoplasmic (C) and nuclear (N) RNA fractions were isolated from the second dish. Cell equivalents of each RNA sample (10 μg of total RNA) were treated with RNase H and oligo(dT) and then analyzed by Northern blot hybridization for the presence of α-globin transcripts (A). As a cell fractionation control, the same samples were analyzed for their content of U3 snoRNA by hybridization to an oligonucleotide probe specific for U3 (B).
FIG. 5
FIG. 5
ICP27 induces accumulation of unspliced α-globin pre-mRNA in transfected cells. HeLa cells were transfected with the ICP4 expression clone (pBB37) in combination with pUC, the control vector pUHD10-3, or the ICP27 expression clone pC27, and total RNA was extracted at 36 h posttransfection. These RNA samples and RNA from KOS-infected cells were treated with RNase H and oligo(dT) and then analyzed for α-globin transcripts by Northern blot hybridization. C, control blood RNA; M, RNA size markers (in kilobases).
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