A cellular pre-mRNA undergoes various post-transcriptional processing events, including capping, splicing and polyadenylation prior to nuclear export. Splicing is particularly important for mRNA nuclear export as two distinct multi-protein complexes, known as human TREX (hTREX) and the exon-junction complex (EJC), are recruited to the mRNA in a splicing-dependent manner. In contrast, a number of Kaposi's sarcoma–associated herpesvirus (KSHV) lytic mRNAs lack introns and are exported by the virus-encoded ORF57 protein. Herein we show that ORF57 binds to intronless viral mRNAs and functions to recruit the complete hTREX complex, but not the EJC, in order assemble an export component viral ribonucleoprotein particle (vRNP). The formation of this vRNP is mediated by a direct interaction between ORF57 and the hTREX export adapter protein, Aly. Aly in turn interacts directly with the DEAD-box protein UAP56, which functions as a bridge to recruit the remaining hTREX proteins to the complex. Moreover, we show that a point mutation in ORF57 which disrupts the ORF57-Aly interaction leads to a failure in the ORF57-mediated recruitment of the entire hTREX complex to the intronless viral mRNA and inhibits the mRNAs subsequent nuclear export and virus replication. Furthermore, we have utilised a trans-dominant Aly mutant to prevent the assembly of the complete ORF57-hTREX complex; this results in a vRNP consisting of viral mRNA bound to ORF57, Aly and the nuclear export factor, TAP. Strikingly, although both the export adapter Aly and the export factor TAP were present on the viral mRNP, a dramatic decrease in intronless viral mRNA export and virus replication was observed in the absence of the remaining hTREX components (UAP56 and hTHO-complex). Together, these data provide the first direct evidence that the complete hTREX complex is essential for the export of KSHV intronless mRNAs and infectious virus production.

HVS (herpesvirus saimiri) is the prototype gamma-2 herpesvirus. This is a subfamily of herpesviruses gaining importance since the identification of the first human gamma-2 herpesvirus, Kaposi's sarcoma-associated herpesvirus. The HVS ORF 57 (open reading frame 57) protein is a multifunctional transregulatory protein homologous with genes identified in all classes of herpesviruses. Recent work has demonstrated that ORF 57 has the ability to bind viral RNA, shuttles between the nucleus and cytoplasm and promotes the nuclear export of viral transcripts. In the present study, we show that ORF 57 shuttles between the nucleus and cytoplasm in a CRM-1 (chromosomal region maintenance 1)-independent manner. ORF 57 interacts with the mRNA export factor REF (RNA export factor) and two other components of the exon junction complex, Y14 and Magoh. The association of ORF 57 with REF stimulates recruitment of the cellular mRNA export factor TAP (Tip-associated protein), and HVS infection triggers the relocalization of REF and TAP from the nuclear speckles to several large clumps within the cell. Using a dominant-negative form of TAP and RNA interference to deplete TAP, we show that it is essential for bulk mRNA export in mammalian cells and is required for ORF 57-mediated viral RNA export. Furthermore, we show that the disruption of TAP reduces viral replication. These results indicate that HVS utilizes ORF 57 to recruit components of the exon junction complex and subsequently TAP to promote viral RNA export through the cellular mRNA export pathway.

The nucleolus is the largest subnuclear structure and is plurifunctional in nature. Here, we demonstrate that nucleolar localization of a key herpesvirus regulatory protein is essential for its role in virus mRNA nuclear export. The herpesvirus saimiri ORF57 protein is a nucleocytoplasmic shuttle protein that is conserved in all herpesviruses and orchestrates the nuclear export of viral intronless mRNAs. We demonstrate that expression of the ORF57 protein induces nucleolar redistribution of human TREX (transcription͞ export) proteins that are involved in mRNA nuclear export. Moreover, we describe a previously unidentified nucleolar localization signal within ORF57 that is composed of two distinct nuclear localization signals. Intriguingly, point mutations that ablate ORF57 nucleolar localization lead to a failure of ORF57-mediated viral mRNA nuclear export. Furthermore, nucleolar retargeting of the ORF57 mutant was achieved by the incorporation of the HIV-1 Rev nucleolar localization signal, and analysis demonstrated that this modification was sufficient to restore viral mRNA nuclear export. This finding represents a unique and fundamental role for the nucleolus in nuclear export of viral mRNA.mRNA export ͉ nucleolus ͉ virus T he eukaryotic cell nucleus is a highly organized environment containing distinct and often dynamic compartments (1). Of these, the nucleolus is the most prominent, and, for many years, its exclusive role was thought to be the site of ribosomal RNA transcription, processing, and assembly into the ribosome subunits (2). Recent studies, however, suggest that it has additional nonclassical roles in many aspects of cell biology, including cell cycle regulation, viral replication, tumorigenesis, and cellular stress responses (3-5). This plurifunctional nature of the nucleolus has been highlighted by extensive proteomic analysis of human nucleoli (6, 7). To date, the nucleolar proteome database archives 728 nucleolar proteins, and functional classification of these proteins reinforces the multiple roles of the nucleolus (8). Furthermore, there is constant dynamic trafficking of nucleolar proteins, and this concomitant dynamic nature of nucleolar structure may provide regulation of nonclassical nucleolar functions.Interestingly, an increasing number of key proteins from both RNA and DNA viruses have been shown to localize to the nucleolus. These proteins include those encoded by viruses such as coronaviruses, influenza, HIV-1, adenoviruses, and herpesviruses (9). Therefore, virus-nucleolar interactions are likely to have important implications in the life cycle of many viruses. However, at present, the precise functional role of these virus-nucleolar colocalizations has not been determined. One such key viral protein that traffics to the nucleolus is the herpesvirus saimiri (HVS) ORF57 protein. HVS is the prototype ␥-2 herpesvirus, or rhadinovirus (10), which has become an important family of viruses since the identification of the first human ␥-2 herpesvirus, the oncogenic Kaposi's sarcoma-associ...

Kaposi's sarcoma-associated herpesvirus (KSHV) expresses numerous intronless mRNAs that are unable to access splicing-dependent cellular mRNA nuclear export pathways. To circumvent this problem, KSHV encodes the open reading frame 57 (ORF57) protein, which orchestrates the formation of an export-competent virus ribonucleoprotein particle comprising the nuclear export complex hTREX, but not the exon-junction complex (EJC). Interestingly, EJCs stimulate mRNA translation, which raises the intriguing question of how intronless KSHV transcripts are efficiently translated. Herein, we show that ORF57 associates with components of the 48S pre-initiation complex and co-sediments with the 40S ribosomal subunits. Strikingly, we observed a direct interaction between ORF57 and PYM, a cellular protein that enhances translation by recruiting the 48S pre-initiation complex to newly exported mRNAs, through an interaction with the EJC. Moreover, detailed biochemical analysis suggests that ORF57 recruits PYM to intronless KSHV mRNA and PYM then facilitates the association of ORF57 and the cellular translation machinery. We, therefore, propose a model whereby ORF57 interacts directly with PYM to enhance translation of intronless KSHV transcripts.

The hTREX complex mediates cellular bulk mRNA nuclear export by recruiting the nuclear export factor, TAP, via a direct interaction with the export adaptor, Aly. Intriguingly however, depletion of Aly only leads to a modest reduction in cellular mRNA nuclear export, suggesting the existence of additional mRNA nuclear export adaptor proteins. In order to efficiently export Kaposi's sarcoma-associated herpesvirus (KSHV) intronless mRNAs from the nucleus, the KSHV ORF57 protein recruits hTREX onto viral intronless mRNAs allowing access to the TAP-mediated export pathway. Similarly however, depletion of Aly only leads to a modest reduction in the nuclear export of KSHV intronless mRNAs. Herein, we identify a novel interaction between ORF57 and the cellular protein, UIF. We provide the first evidence that the ORF57-UIF interaction enables the recruitment of hTREX and TAP to KSHV intronless mRNAs in Aly-depleted cells. Strikingly, depletion of both Aly and UIF inhibits the formation of an ORF57-mediated nuclear export competent ribonucleoprotein particle and consequently prevents ORF57-mediated mRNA nuclear export and KSHV protein production. Importantly, these findings highlight that redundancy exists in the eukaryotic system for certain hTREX components involved in the mRNA nuclear export of intronless KSHV mRNAs.

Platelet microparticle (PMP)-induced angiogenesis plays a key role in tumour metastasis and has been proposed to contribute towards cardiovascular disease by enhancing atherosclerotic plaque vulnerability. However, the mechanisms underlying PMP induced angiogenesis are ill defined. Recent reports demonstrate that PMPs deliver micro-RNAs (miRNAs) to recipient cells, controlling gene expression. We therefore evaluated whether miRNA transfer was a key regulator of PMP-induced angiogenesis. Co-culturing PMPs with human umbilical vein endothelial cells (HUVEC) on extracellular matrix gel induced robust capillary like structure formation. PMP treatment altered the release of angiogenesis modulators from HUVEC, including significantly reducing production of anti-angiogenic thrombospondin-1 (THBS-1). Both functional responses were abrogated by treating PMPs with RNase, suggesting the transfer of PMP-derived RNA was a critical event. PMPs were an abundant source of miRNA Let-7a, which was transferred to HUVEC following co-incubation. Using luciferase reporter assays we have shown that Let-7a directly targets the 3'UTR of the THBS-1 mRNA. HUVEC transfection with a Let-7a anti-sense oligonucleotide reduced the ability of PMPs to inhibit THBS-1 release, and significantly decreased PMP induced in vitro angiogenesis. Antibody neutralisation of THBS-1 reversed the anti-angiogenic effect of let-7a inhibition in PMP treated HUVEC, highlighting Let-7a dependent translational repression of THBS-1 drives angiogenesis. Importantly, plasmid overexpression of Let-7a in HUVEC alone induced robust tubule formation on extracellular matrix gel. These data reveal a new role for Let-7a in promoting angiogenesis and show for the first time PMPs induced angiogenic responses occur through miRNA regulation of HUVEC.

Basic fibroblast growth factor (bFGF) induces cell death in cells of the Ewing's sarcoma family of tumors in vivo and in vitro.In this study we demonstrate that this is dependent on the rapid and sustained activation of (ESFT) 1 encompasses a group of malignancies, including Ewing's sarcoma, Askin's tumor of the chest wall, and peripheral primitive neuroectodermal tumor, which are thought to be of neural histogenesis (1-3). ESFT exhibit a common genetic rearrangement involving fusion of the 5Ј end of the EWS gene on chromosome 22 to the 3Ј portion of members of the Ets gene family of transcription factors. In over 90% of cases the Ets gene family member is fused to the Fli1 gene located on chromosome 11 (4). This results in the generation of a fusion gene, the protein product of which has been implicated in development of the transformed ESFT phenotype (5-7). ESFT typically arise in the bone or soft tissue of adolescents and young adults, ϳ15-30% of patients presenting with metastatic disease. The outcome for this group of patients is particularly poor despite the use of aggressive therapeutic regimes, emphasizing the need for new therapeutic strategies.A role for autocrine and/or paracrine growth factor survival loops in ESFT is well documented; the blockade of insulin-like growth factor/insulin-like growth factor receptor 1 (8 -11) or stem cell factor (SCF)/c-Kit (12, 13) circuits results in a decrease in ESFT cell number in both in vitro and in vivo models. Previous studies (14) have also suggested that a basic fibroblast growth factor (bFGF)/fibroblast growth factor receptor autocrine/paracrine survival loop may be important for the survival and proliferation of ESFT. However, we have found no evidence of such a survival loop, and we demonstrated recently that treatment of ESFT with bFGF results in the up-regulation of the death receptor p75NTR and induction of cell death (15, 16). The intracellular signaling pathways leading to the induction of cell death following exposure of ESFT cells to bFGF are unknown. Although our preliminary results have shown that incubation of ESFT cells with bFGF causes phosphorylation of fibroblast growth factor receptor 1 and activation of the downstream signaling molecules Ras and ERK (16), whether these events are important effectors of bFGF-induced cell death is not clear. Following receptor activation, phosphorylated tyrosines function as binding sites for a number of downstream adapter and signaling proteins, including the docking protein FRS2 that recruits several signal transduction molecules leading to activation of the mitogen-activated protein kinase (MAPK) cascade and the phosphatidylinositol 3-kinase-AKT anti-apoptotic pathway (17, 18). Recruitment of guanine nucleotide exchange factors (e.g. hSOS) leads to the conversion of the small GTPase Ras from an inactive GDP-bound state to an active GTP-bound state and activation of the extracellular signal-regulated kinase (ERK) pathway (19). Activation of the Ras-ERK pathway has been shown to mediate such diverse cellular...

Herpesvirus saimiri (HVS) is the prototype gamma-2 herpesvirus and is a useful model to study the basic mechanisms of lytic replication in this herpesvirus subfamily. This review focuses upon the role of an essential lytic protein, ORF57, which is functionally conserved in all classes of herpesviruses. ORF57 is a multidomain, multifunctional protein responsible for both activation and repression of viral gene expression at a post-transcriptional level. ORF57-mediated repression of gene expression is determined by mRNA processing signals, in particular the presence of an intron within the target gene. This may also be linked to the ability of ORF57 to redistribute SC-35 and U2 splicing factors into specific nuclear domains. ORF57 also plays a pivotal role in transactivating viral gene expression by specifically mediating the nuclear export of HVS intronless transcripts. ORF57 has the ability to shuttle between the nucleus and the cytoplasm, bind viral RNA and recruit cellular nuclear export proteins, such as hTREX components and TAP, onto the viral mRNA. This enables the efficient nuclear export and cytoplasmic accumulation of virus intronless mRNA.