Interaction between Herpes Simplex Virus Type 1 IE63 Protein and Cellular Protein p32

Abstract

The herpes simplex virus type 1 (HSV-1) immediate-early gene IE63 (ICP27), the only HSV-1 regulatory gene with a homologue in every mammalian and avian herpesvirus sequenced so far, is a multifunctional protein which regulates transcriptional and posttranscriptional processes. One of its posttranscriptional effects is the inhibition of splicing of viral and cellular transcripts. We previously identified heterogeneous nuclear ribonucleoprotein (hnRNP) K and casein kinase 2 (CK2) as two protein partners of IE63 (H. Bryant et al., J. Biol. Chem. 274:28991-28998, 1999). Here, using a yeast two-hybrid assay, we identify another partner of IE63, the cellular protein p32. Confirmation of this interaction was provided by coimmunoprecipitation from virusinfected cells and recombinant p32 binding assays. A p32-hnRNP K-CK2 complex, which required IE63 to form, was isolated from HSV-1-infected cells, and coimmunoprecipitating p32 was phosphorylated by CK2. Expression of IE63 altered the cytoplasmic distribution of p32, with some now colocalizing with IE63 in the nuclei of infected and transfected cells. As p32 copurifies with splicing factors and can inhibit splicing, we propose that IE63 together with p32, possibly with other IE63 partner proteins, acts to disrupt or regulate pre-mRNA splicing. As well as contributing to host cell shutoff, this effect could facilitate splicing-independent nuclear export of viral transcripts.A key regulatory protein of herpes simplex type 1 (HSV-1) lytic infection is the 63-kDa nuclear phosphoprotein IE63 (also known as ICP27). IE63 is essential for viral replication (23,(36)(37)(38)(39) and is required for the switch from early to late virus gene expression (21). It has been shown to perform multiple functions at both transcriptional and posttranscriptional levels (reviewed in reference 33). Acting posttranscriptionally, IE63 binds RNA in vivo with a reported specificity for intronless viral transcripts (40), enhances pre-mRNA 3Ј processing (22), and contributes to the shutoff of host protein synthesis by inhibiting splicing of viral and cellular transcripts (11,12). IE63 colocalizes with nuclear antigens such as snRNPs (31) and causes the nuclear retention of intron-containing viral transcripts (34). More recently, IE63 has been shown to be capable of shuttling from the nucleus to the cytoplasm (24,32,46) and may facilitate the nuclear export of intronless RNAs, which form the majority of viral transcripts (40). IE63 mediates the export of some viral RNAs via a Crm-1-dependent pathway, whereas other viral RNAs are exported via a Crm-1-independent pathway (47).In HSV-1-infected cells, IE63 interacts with heterogeneous nuclear ribonucleoprotein (hnRNP) K and with casein kinase 2 (CK2), the latter activity being able to phosphorylate both IE63 and hnRNP K, possibly to alter their activities (4). Here, we show that, consistent with its multiple functions, IE63 interacts with another cellular protein, p32. First isolated as a protein tightly associated with ASF/SF2 purified from HeLa cells (16...

The Herpesvirus Saimiri Open Reading Frame 73 Gene Product Interacts with the Cellular Protein p32

“…Similar observations have been demonstrated for other p32-interacting viral proteins, including EBV EBNA-1, HSV-1 ICP27, and adenovirus protein V (11,35,52,54). At present, the mechanism of p32 nuclear redistribution is unknown.…”

“…Through its association with the splicing factors ASF/SF2 and SR30pc, p32 has also been implicated in control of gene expression at a posttranscriptional level (38). This specific function of p32 has been implicated in the mechanism by which both HIV-1 Rev and HSV-1 ICP27 inhibit cellular splicing (11,34,50).…”

“…First isolated associated with the splicing factor SF2/ASF (32), p32 interacts with a number of additional cellular and viral proteins, including human immunodeficiency virus type 1 (HIV-1) Rev (34,50) and Tat (56,57), herpes simplex virus type 1 (HSV-1) ORF P (10) and ICP27 (11), adenovirus core protein V (35), rubella virus capsid protein (4), and EBV EBNA-1 protein (13,52,54). Although the function of p32 is as yet undetermined, it has been suggested to play roles in splicing (11,32,38,50), nucleocytoplasmic transport (9,11,35), maintenance of oxidative phosphorylation (36), transcriptional activation (52,54,56,57), and possibly latent-cycle DNA replication (13,52). To address the implications of this interaction, we demonstrate that ORF 73 and p32 have a synergistic effect on the transactivation of a number of heterologous promoters and its own promoter.…”

Association of Herpes Simplex Virus Type 1 ICP8 and ICP27 Proteins with Cellular RNA Polymerase II Holoenzyme

“…. The ratio of cell lysate loaded on the gel to the amount of lysate used in immunoprecipitation was 1:13 for lanes 6,8,10,12, and 14 and 1:26 for lanes 7, 9, 11, 13, and 15. 1x, cell lysate was treated with 25 U of RNase A/ml and 1,000 U of RNase T 1 /ml at 30°C for 30 min; 3x, cell lysate was treated with 75 U of RNase A/ml and 3,000 U of RNase T 1 /ml at 30°C for 30 min; DNase, cell lysate was treated with 10 g of DNase I/ml.…”

“…ICP27 promotes transcription of at least some late genes, e.g., the gC and U L 47 genes, in infected Vero cells (39). Evidence for posttranscriptional effects of ICP27 include the observations that ICP27 binds RNA via its RGG motif (7,38,53,76), regulates pre-mRNA 3Ј processing (33,51,52), stabilizes labile 3Ј ends of mRNA (7), interacts with cellular protein p32 (8) and spliceosome-associated protein 145 (9), inhibits the splicing of both viral and cellular transcripts (34), induces retention of intron-containing transcripts in the nucleus (61) but shuttles viral intronless transcripts from the nucleus to the cytoplasm (60,76,83), and regulates distribution of host small nuclear ribonucleoproteins (snRNPs) (59,78). In addition, ICP27 has been found to interact with ICP0 and ICP4 (54,57), to colocalize with ICP4 within HSV replication compartments in infected cells (23) and transfected cells (102), and to affect the posttranslational modification and DNA-binding ability of ICP4 (57,97,98).…”