Herpes Simplex Virus ICP27 Induces Cytoplasmic Accumulation of Unspliced Polyadenylated α-Globin Pre-mRNA in Infected HeLa Cells

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 ␣-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 ␣-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 ␣-globin RNA and argue that ICP27 induces a splicing-independent pathway for ␣-globin RNA accumulation and nuclear export. [L]) are sequentially activated in a regulatory cascade driven by viral gene products, while expression of most host cell genes is strongly suppressed. The HSV lytic program is initiated by the virion transactivator VP16, which acts in combination with host factors to stimulate transcription of the five IE genes (reviewed in references 16 and 60). Four of the IE proteins (ICP0, ICP4, ICP22, and ICP27) then play key roles in orchestrating expression of the viral E and L genes (10,30,48,49,55,59,63).Early studies demonstrated that the HSV lytic cascade involves both transcriptional and posttranscriptional controls of mRNA metabolism (64). Mounting evidence suggests that the immediate-early (IE) protein ICP27 contributes to the posttranscriptional component of the viral regulatory program (57; reviewed in reference 51). ICP27 is an essential regulator that stimulates accumulation of a subset of viral E and L mRNAs and is required for efficient viral DNA replication (12,30,34,43,44,48,56,62). A clue to the mechanism of ICP27 action was provided by Sandri-Goldin and Mendoza (53), who showed that ICP27 enhances expression of constructs bearing a synthetic and incomplete polyadenylation signal and inhibits expression of reporter genes bearing certain introns in transient-cotransfection assays. A complementary series of studies by Clements and colleagues demonstrated that HSV infection alters the specificity of the host polyadenylation machinery in an ICP27-dependent fashion, thereby allowing more efficient use of a subset of viral polyadenylation signals (31-33). These findings indicate that ICP27 modulates intranuclear processing of pre-mRNAs and suggest that it inhibits splicing of at least some intron-bearing transcripts (53).The hypothesis that ICP27 impairs RNA splicing has been supported by a considerable amount of data showing the following: (i) unspliced pre-RNA derived from the intron-bearing HSV genes encoding ICP0 and UL15 accumulate in the nuclei of some cell types infected with wild-type HSV but not with an ICP27 mutant (20,21,40), (ii) ICP27 colocalizes with and redistributes snRNPs in HSV-infected cell nuclei (38), (iii) ICP2...

Herpes Simplex Virus ICP27 Increases Translation of a Subset of Viral Late mRNAs

“…Nucleic acids from this gel were transferred to a nylon membrane for Northern analysis. The membrane was first probed with an oligonucleotide specific for U3 snRNA (6,46) to quantify the amount of nuclear RNA contamination in the cytoplasm (Fig. 2C).…”

“…The DNA templates used to make various DNA probes were as follows: for the VP16 (U L 48) gene, the 437-bp template was amplified by PCR from viral DNA by using the primers 5Ј-TGGGCAGCGTTGATAGGAAT-3Ј and 5Ј-GTTTGGGGGTTTTCTCTTCC-3Ј; for the ICP5 (U L 19) gene, the 203-bp probe was amplified by PCR from viral DNA by using the primers 5Ј-CTTAGCACGATCGAGGT-3Ј and 5Ј-GTTCATGTAGGCCAAGCT-3Ј; and for the gD (U S 6) gene, the 342-bp probe was amplified by PCR from viral DNA by using the primers 5Ј-CCGTGATTTTGTTTGTCGTCATAGTGGG C-3Ј and 5Ј-CAAGCGATGGTCAGGTTGTAGGGTTGTTTC-3Ј. To probe for the U3 snRNA, radiolabeled oligonucleotide 5Ј-ACCACTCAGACCGCGTTC TCTCCCTCTCAC-3Ј was used as a probe (6). All oligonucleotides were obtained from Invitrogen.…”

The herpes simplex virus 1 U L 41 gene-dependent destabilization of cellular RNAs is selective and may be sequence-specific

“…The electrophoretic mobility of the untreated GADD45␤ RNA suggested a relatively uniform distribution of RNA sizes that did not change between 3 and 7 h after infection (Fig. 3 Lower, lanes [11][12][13][14][15][16][17][18][19][20]. The results indicate that the GADD45␤ RNA is relatively stable and does not undergo rapid deadenylation characteristic of IEX-1 RNA.…”

“…Analysis of mRNA deadenylation. HeLa cells were mock-infected (lanes 1-10) or infected with 10 pfu of HSV-1(F) per cell (lanes [11][12][13][14][15][16][17][18][19][20], and cytoplasmic RNA was extracted at the indicated hours after infection. Poly(A) Ϫ RNA was prepared in vitro by treating the RNA samples with oligo(dT) and RNase H as described in Materials and Methods.…”

A comparison of herpes simplex virus type 1 and varicella-zoster virus latency and reactivation

“…HSV-1 immediateearly protein ICP22 targets RNA polymerase II activity to virus promoters through altered phosphorylation of the C-terminal domain of the large polymerase subunit (Poffenberger et al, 1993;Rice et al, 1995;Lin et al, 2010). Most HSV-1 genes are not spliced and HSV-1 immediate-early protein ICP27 binds unspliced mRNA facilitating nuclear export and translation of low-abundance HSV-1 mRNA amongst excess cellular transcripts (SandriGoldin, 1998;Cheung et al, 2000). In summary, HSV-1 immediate-early proteins efficiently inactivate host antivirus defence, commandeer the transcription apparatus and establish an environment conducive to continued virus gene expression.…”