Herpes simplex virus type 1 (HSV-1) protein ICP27 facilitates the export of viral intronless mRNAs. ICP27 shuttles between the nucleus and cytoplasm, which has been shown to require a leucine-rich nuclear export sequence (NES). ICP27 export was reported to be sensitive to the CRM1 inhibitor leptomycin B (LMB) in HSV- In vitro export assays showed that ICP27 export was not sensitive to LMB but was blocked by a dominant-negative TAP deletion mutant lacking the nucleoporin interaction domain. These data suggest that ICP27 uses the TAP pathway to export viral RNAs. Interestingly, the leucine-rich N-terminal sequence was required for efficient export, even though ICP27 export was LMB insensitive. Thus, this region is required for efficient ICP27 export but does not function as a CRM1-dependent NES.Eukaryotic pre-mRNAs are synthesized in the nucleus by RNA polymerase II, after which they are processed by capping at the 5Ј end, cleavage and polyadenylation to form the 3Ј end, and splicing to remove intervening sequences. After processing, mRNAs must be exported through the nuclear pore complex (NPC) to the cytoplasm for translation. Export of mRNAs requires processing, packaging by RNA-binding proteins into ribonucleoprotein (RNP) complexes, recognition by export factors and translocation through the NPC. Three classes of factors appear to be required for mRNA export: adapter proteins that bind directly to the mRNA, receptor proteins that recognize and bind to adapter proteins, and NPC components termed nucleoporins that mediate export across the nuclear membrane (21, 58).Two pathways have been uncovered that appear to be responsible for the export of mRNA. The first export receptor to be identified was CRM1, which recognizes nuclear export sequences (NES) that consist of short hydrophobic stretches that are leucine-rich. This sequence was first revealed in the HIV-1 export protein Rev and in the cellular protein PKI (7,8). Although Rev interacts with CRM1 to export human immunodeficiency virus (HIV) env mRNA, substantial evidence has shown that CRM1 is not a major contributor to mRNA export in metazoans or yeast (6, 35). The human protein TAP and its yeast ortholog Mex67p are the best candidates for mRNA export receptors because they have been shown to shuttle between the nucleus and cytoplasm, cross-link to poly(A) ϩ RNA, localize at the nuclear pores, and interact directly with nucleoporins (1,2,16,17). TAP was first identified as the cellular factor that interacts with the constitutive transport element (CTE) present in RNAs from type D retroviruses and TAP promotes the export of CTE-containing transcripts (2, 11, 16). In yeast, Mex67p is required for poly(A) ϩ RNA export and cellular viability (45, 47). Further, overexpression of TAP in Xenopus oocytes or mammalian cells stimulates mRNA export (3, 14) and inactivation of TAP in Caenorhabditis elegans by RNA interference blocked nuclear export of poly(A) ϩ RNA (56), indicating a direct role in mRNA export.In metazoans, pre-mRNA splicing is required for rapid and ef...

Infection with some viruses can alter cellular mRNA processing to favor viral gene expression. We present evidence that herpes simplex virus 1 (HSV-1) protein ICP27, which contributes to host shut-off by inhibiting pre-mRNA splicing, interacts with essential splicing factors termed SR proteins and affects their phosphorylation. During HSV-1 infection, phosphorylation of several SR proteins was reduced and this correlated with a subnuclear redistribution. Exogenous SR proteins restored splicing in ICP27-inhibited nuclear extracts and SR proteins isolated from HSV-1-infected cells activated splicing in uninfected S100 extracts, indicating that inhibition occurs by a reversible mechanism. Spliceosome assembly was blocked at the pre-spliceosomal complex A stage. Furthermore, we show that ICP27 interacts with SRPK1 and relocalizes it to the nucleus; moreover, SRPK1 activity was altered in the presence of ICP27 in vitro. We propose that ICP27 modi®es SRPK1 activity resulting in hypophosphorylation of SR proteins impairing their ability to function in spliceosome assembly.

The herpes simplex virus type 1 (HSV-1) regulatory protein ICP27 is a nuclear phosphoprotein required for viral lytic infection, which acts partly at the posttranscriptional level to affect RNA processing and export. In the present study, we show that ICP27 can interact with itself in vivo. Immunofluorescent staining of cells expressing both an ICP27 mutant with a deletion of the major nuclear localization signal and wild-type ICP27 showed that the mutant protein was efficiently imported into the nucleus in the majority of the cotransfected cells, suggesting heterodimer formation between the wild-type and mutant proteins. Coimmunoprecipitation experiments using epitope-tagged wild-type ICP27 and a series of ICP27 mutants with deletions and insertions in important functional regions of the protein revealed that the C-terminal cysteine-histidine-rich zinc-finger-like region of ICP27 was required for the self-association. Furthermore the self-association was also shown in yeast using two-hybrid assays, and again, an intact C-terminal zinc-finger-like region was required for the interaction. This study provides biochemical evidence that ICP27 may function as a multimer in infected cells.

Sexual differentiation in Drosophila is regulated through alternative splicing of doublesex. Female-specific splicing is activated through the activity of splicing enhancer complexes assembled on multiple repeat elements. Each of these repeats serves as a binding platform for the cooperative assembly of a heterotrimeric complex consisting of the SR proteins Tra, Tra2 and 9G8. Using quantitative kinetic analyses, we demonstrate that each component of the enhancer complex is capable of recruiting the spliceosome. Surprisingly, Tra, Tra2 and 9G8 are much stronger splicing activators than other SR protein family members and their activation potential is significantly higher than expected from their serine/arginine content. 9G8 activates splicing not only through its RS domains but also through its RNA-binding domain. The RS domains of Tra and Tra2 are required but not sufficient for efficient complex assembly. Thus, the regulated assembly of the dsx enhancer complexes leads to the generation of an extended activation domain to guarantee the ‘all or none’ splicing switch that is required during Drosophila sexual differentiation.

Pathogenicity of human cytomegalovirus (HCMV) correlates with the immune status of infected individuals. In fully immunocompetent individuals, host antiviral immune responses contain virus replication. In contrast, HCMV can be a serious cause of morbidity and mortality in individuals without an intact immune system (Ho, 1991). Greater understanding of both host antiviral immune responses and mechanisms of viral pathogenesis is required to design strategies which effectively limit HCMV disease. Infection of rhesus macaques (Macacca mulatta) with rhesus CMV