The role of herpes simplex virus ICP27 protein in mRNA export is investigated by microinjection into Xenopus laevis oocytes. ICP27 dramatically stimulates the export of intronless viral mRNAs, but has no effect on the export of cellular mRNAs, U snRNAs or tRNA. Use of inhibitors shows, in contrast to previous suggestions, that ICP27 neither shuttles nor exports viral mRNA via the CRM1 pathway. Instead, ICP27‐mediated viral RNA export requires REF and TAP/NXF1, factors involved in cellular mRNA export. ICP27 binds directly to REF and complexes containing ICP27, REF and TAP are found in vitro and in virally infected cells. A mutant ICP27 that does not interact with REF is inactive in viral mRNA export. We propose that ICP27 associates with viral mRNAs and recruits TAP/NXF1 via its interaction with REF proteins, allowing the otherwise inefficiently exported viral mRNAs to access the TAP‐mediated export pathway. This represents a novel mechanism for export of viral mRNAs.

The multifunctional herpes simplex virus type 1 (HSV-1) protein IE63 (ICP27) interacts with the essential pre-mRNA splicing factor, spliceosome-associated protein 145 (SAP145), and in infected cells IE63 and SAP145 colocalize. This interaction was reduced or abrogated completely using extracts from cells infected with IE63 viral mutants, with mutations in IE63 KH and Sm homology domains, which do not exhibit host shutoff or inhibit splicing. In the presence of IE63, splicing in vitro was inhibited prior to the first catalytic step and the B/C complex formed during splicing was shifted up in mobility and reduced in intensity. With the use of splicing extracts, IE63 and SAP145 both comigrated with the B/C complex, suggesting that they interact within this complex to inhibit B/C complex formation or conversion. The inhibition of splicing may facilitate the export of viral or cellular transcripts, possibly via other protein partners of IE63. These data provide important new insights into how IE63 influences pre-mRNA processing during HSV-1 infection.Herpes simplex virus type 1 (HSV-1) is a nuclear replicating virus with a large double-stranded DNA genome that encodes some 80 gene products (30). During lytic infection, viral genes are expressed in a temporal manner (48) and are classified as immediate-early (IE), early, or late. The five IE genes do not require prior viral protein synthesis for their expression; four regulate early and late viral gene expression (11,13,15,29,37,50,51,54,67), while one subverts the host cytotoxic T-lymphocyte response (20).IE63 (ICP27), an HSV-1 RNA binding protein (21, 34, 52) essential throughout infection (50), promotes accumulation of a subset of viral early and late mRNAs and is necessary for the switch from early to late virus gene expression (29). IE63 is required for efficient viral DNA replication and stimulates the accumulation of certain viral early and late mRNAs (16,29,31,32,45,50,56,63,65). Itself not a transcriptional activator, IE63 is required for expression of viral early and late genes at both transcriptional and posttranscriptional levels. At the posttranscriptional level, IE63 has effects on the processing of pre-mRNA and mediates nucleocytoplasmic transport of transcripts from intronless viral genes which form the majority of viral RNAs (52). IE63 inhibits splicing of cellular pre-mRNAs to mediate host cell shutoff (18), redistributes splicing factors (39, 53), and increases RNA 3Ј processing at weak virus poly(A) sites (31). In addition, IE63 shuttles between the nucleus and the cytoplasm (35, 40, 52, 59).IE63 contains several functional domains (see Fig. 3B). The N terminus contains an acidic activation domain essential for enhancing early gene expression and hence viral DNA replication (46), a leucine-rich nuclear export signal which promotes nuclear shuttling (52), nuclear and nucleolar localization signals (33), and an RGG box involved in RNA binding (34). Along with a putative zinc finger (66), IE63 contains three KH-like domains homologous to RNA binding r...

Herpes simplex virus type 1 (HSV-1), the prototype ␣-herpesvirus, causes several prominent diseases. The HSV-1 immediate early (IE) protein IE63 (ICP27) is the only regulatory gene with a homologue in every mammalian and avian herpesvirus sequenced so far. IE63 is a multifunctional protein affecting transcriptional and post-transcriptional processes, and it can shuttle from the nucleus to the cytoplasm. To identify interacting cellular proteins, a HeLa cDNA library was screened in the yeast two-hybrid system using IE63 as bait. Several interacting proteins were identified including heterogeneous nuclear ribonucleoprotein K (hnRNP K), a multifunctional protein like IE63, and the ␤ subunit of casein kinase 2 (CK2), a protein kinase, and interacting regions were mapped. Confirmation of interactions was provided by fusion protein binding assays, co-immunoprecipitation from infected cells, and CK2 activity assays. hnRNP K co-immunoprecipitated from infected cells with anti-IE63 serum was a more rapidly migrating subfraction than hnRNP K immunoprecipitated by antihnRNP K serum. Using anti-IE63 serum, both IE63 and hnRNP K were phosphorylated in vitro by CK2, while in immunoprecipitates using anti-hnRNP K serum, IE63 but not hnRNP K was phosphorylated by CK2. These data provide important new insights into how this key viral regulatory protein exerts its functions.The involvement of herpesviruses in a range of prominent medical or veterinary diseases makes them one of the most important virus families. Herpes simplex virus type 1 (HSV-1), 1 a common and effective human pathogen, is capable of establishing both lytic and latent infectious life cycles, and up to 80% of adults in the developed world are seropositive for this virus. HSV-1 is a nuclear replicating virus with a large doublestranded DNA genome that encodes some 80 gene products (1). During lytic infection, virus genes are expressed in a temporal cascade and are categorized as immediate early (IE, ␣), early (␤), or late (␥) based on the time postinfection of their expression (2). The five IE gene products, which do not require prior viral protein synthesis for their expression, regulate early and late gene expression and subvert the host cytotoxic T-lymphocyte response (3, 4). A key IE protein is the 63-kDa IE phosphoprotein IE63 also called ICP27 (5). IE63 is one of two HSV IE proteins essential for lytic virus replication (6) and is the only regulatory gene with a homologue in every herpesvirus of mammals and birds sequenced so far (7), suggesting that aspects of its regulatory role are maintained throughout the herpesvirus family.Studies of IE63 have shown that its expression is required for the switch from early to late virus gene expression (8) and have highlighted the multifunctional nature of this protein that acts both at the transcriptional and post-transcriptional levels (reviewed in Ref. 9). Acting post-transcriptionally, IE63 binds RNA in vivo with a reported specificity for intronless viral transcripts (10), enhances pre-mRNA 3Ј processing (11), in...

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...

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