Evaluation of Cyclin D1 as a Discriminatory Immunohistochemical Biomarker for Idiopathic Pulmonary Fibrosis.

BACKGROUND: Distinction of idiopathic pulmonary fibrosis (IPF) from other chronic fibrosing interstitial pneumonitides, such as hypersensitivity pneumonitis (HP) and connective tissue diseases, is critical due to varied biological and clinical outcomes. However, their histologic overlaps often pose diagnostic challenges. A recent study suggested an association of herpesvirus saimiri infection with IPF. Productive viral infection is associated with coexpression of pirated mammalian protein cyclin D1, shown to be overexpressed by immunohistochemistry (IHC) in the regenerating alveolar epithelium in IPF but not in normal lungs. We evaluated the diagnostic utility of cyclin D1 to discriminate between IPF and other fibrosing interstitial lung diseases. MATERIALS AND METHODS: A retrospective study of cyclin D1 IHC expression in 27 consecutive cases of chronic fibrosing interstitial lung diseases from 2011 to 2017: 12 usual interstitial pneumonia (UIP) pattern; 5 nonspecific interstitial pneumonia pattern; 3 HP pattern; 7 unclassifiable was performed. Five cases of normal lung obtained from lobectomy specimen for malignancy are included as control. Immunoreactivity was graded semiquantitatively on a scale of 0 to 3. RESULTS: Cyclin D1 staining was uniformly strongly positive in all cases evaluated in the study, particularly in proliferating type II pneumocytes in the region of fibrosing areas. There was no statistical difference in the extent of cyclin D1 expression between UIP and non-UIP groups (2.7 vs. 2.5) and IPF versus non-IPF groups (2.7 vs. 2.4). Cyclin D1 expression is lower in control group compared with UIP groups (1.2 vs. 2.7). CONCLUSIONS: Cyclin D1 is not a specific marker of UIP pattern/IPF. The high expression of cyclin D1 in lung tissue of fibrosing interstitial pneumonitides regardless of etiology most likely correlates with proliferation in type II pneumocytes.

Potential of herpesvirus saimiri-based vectors to reprogram a somatic Ewing's sarcoma family tumor cell line.

Herpesvirus saimiri (HVS) infects a range of human cell types with high efficiency. Upon infection, the viral genome can persist as high-copy-number, circular, nonintegrated episomes that segregate to progeny cells upon division. This allows HVS-based vectors to stably transduce a dividing cell population and provide sustained transgene expression in vitro and in vivo. Moreover, the HVS episome is able to persist and provide prolonged transgene expression during in vitro differentiation of mouse and human hemopoietic progenitor cells. Together, these properties are advantageous for induced pluripotent stem cell (iPSC) technology, whereby stem cell-like cells are generated from adult somatic cells by exogenous expression of specific reprogramming factors. Here we assess the potential of HVS-based vectors for the generation of induced pluripotent cancer stem-like cells (iPCs). We demonstrate that HVS-based exogenous delivery of Oct4, Nanog, and Lin28 can reprogram the Ewing's sarcoma family tumor cell line A673 to produce stem cell-like colonies that can grow under feeder-free stem cell culture conditions. Further analysis of the HVS-derived putative iPCs showed some degree of reprogramming into a stem cell-like state. Specifically, the putative iPCs had a number of embryonic stem cell characteristics, staining positive for alkaline phosphatase and SSEA4, in addition to expressing elevated levels of pluripotent marker genes involved in proliferation and self-renewal. However, differentiation trials suggest that although the HVS-derived putative iPCs are capable of differentiation toward the ectodermal lineage, they do not exhibit pluripotency. Therefore, they are hereby termed induced multipotent cancer cells.

The insulator protein CTCF binding sites in the orf73/LANA promoter region of herpesvirus saimiri are involved in conferring episomal stability in latently infected human T cells.

Herpesviruses establish latency in suitable cells of the host organism after a primary lytic infection. Subgroup C strains of herpesvirus saimiri (HVS), a primate gamma-2 herpesvirus, are able to transform human and other primate T lymphocytes to stable growth in vitro. The viral genomes persist as nonintegrated, circular, and histone-associated episomes in the nuclei of those latently infected T cells. Epigenetic modifications of episomes are essential to restrict the transcription during latency to selected viral genes, such as the viral oncogenes stpC/tip and the orf73/LANA. In this study, we describe a genome-wide chromatin immunoprecipitation-on-chip (ChIP-on-chip) analysis to profile the occupancy of CTCF on the latent HVS genome. We then focused on two distinct, conserved CTCF binding sites (CBS) within the orf73/LANA promoter region. Analysis of recombinant viruses harboring deletions or mutations within the CBS indicated that the lytic replication of such viruses is not substantially influenced by CTCF. However, T cells latently infected with CBS mutants were impaired in their proliferation abilities and showed a significantly reduced episomal maintenance. We detected a reduced transcription of the orf73/LANA gene in the T cells, corresponding to the reduced viral genomes; this might contribute to the loss of HVS episomes, as LANA is central in the maintenance of viral episomes in the dividing T cell populations. These data demonstrate that the episomal stability of HVS genomes in latently infected human T cells is dependent on CTCF.

Mutation of herpesvirus Saimiri ORF51 glycoprotein specifically targets infectivity to hepatocellular carcinoma cell lines.

Herpesvirus saimiri (HVS) is a gamma herpesvirus with several properties that make it an amenable gene therapy vector; namely its large packaging capacity, its ability to persist as a nonintegrated episome, and its ability to infect numerous human cell types. We used RecA-mediated recombination to develop an HVS vector with a mutated virion protein. The heparan sulphate-binding region of HVS ORF51 was substituted for a peptide sequence which interacts with somatostatin receptors (SSTRs), overexpressed on hepatocellular carcinoma (HCC) cells. HVS mORF51 showed reduced infectivity in non-HCC human cell lines compared to wild-type virus. Strikingly, HVS mORF51 retained its ability to infect HCC cell lines efficiently. However, neutralisation assays suggest that HVS mORF51 has no enhanced binding to SSTRs. Therefore, mutation of the ORF51 glycoprotein has specifically targeted HVS to HCC cell lines by reducing the infectivity of other cell types; however, the mechanism for this targeting is unknown.

Episomal replication timing of gamma-herpesviruses in latently infected cells.

This study addresses the timing of gammaherpesviral episomal DNA replication with respect to the cell cycle. For the first time we analyzed a rhadinovirus, the prototype Herpesvirus saimiri (HVS), and compared it to the lymphocryptovirus Epstein-Barr virus (EBV). Newly synthesized DNA of latently infected B- or T-cells was first BrdU-labeled; then we sorted the cells corresponding to cell cycle phases G(0/1), G(2/M), and S (4 fractions S(1)-S(4)) and performed anti-BrdU chromatin immunoprecipitation. Next, DNA of different viral gene loci was quantitatively detected together with cellular control genes of known replication time. The sensitive technique is further enhanced by an internal coprecipitation standard for increased precision. Both gammaherpesviruses replicated very early in S-phase, together with cellular euchromatin. Our work suggests that early S-phase DNA replication is a general characteristic of episomal herpesviral genomes.

Vesicle traffic to the immunological synapse: a multifunctional process targeted by lymphotropic viruses.

The site of contact between T lymphocytes and antigen-presenting cells becomes, upon antigen recognition, an organized junction named the immunological synapse. Various T cell organelles polarize, together with microtubules, toward the antigen-presenting cell. Among them, intracellular vesicular compartments, such as the Golgi apparatus, the recycling endosomal compartment, or cytotoxic granules help to build the immunological synapse and ensure effector functions, such as polarized secretion of cytokines by helper T cells, or exocytosis of lytic granules by cytotoxic T cells. Lymphotropic retroviruses, such as the human immunodeficiency virus type 1, the human T cell leukemia virus type 1, or the Herpesvirus saimiri, can subvert some of the vesicle traffic mechanisms impeding the generation and function of the immunological synapses. This review focuses on the polarization of vesicle traffic, its regulation, and its role in maintaining the structure and function of the immunological synapse. We discuss how some lymphotropic viruses target the vesicle traffic in T lymphocytes, inhibiting the formation of immunological synapses and modulating the response of infected T cells.

Transformation efficiency by Herpesvirus saimiri is not a limiting factor in clonal CD8pos T cell outgrowth.

The routine transformation of human CD8(pos) T cells by Herpesvirus saimiri has so far not been achieved in the case of pre-expanded antigen-specific CTLs. Here we transformed 73% of polyclonal EBV-specific CD8(pos) T cell cultures using an optimized culture medium supplemented with IL-2, IL-7, IL-12, and TGF-beta(1). Still, antigen-specific cytotoxicity was frequently lost and analysis of the TCR Vbeta-chain repertoire revealed a variable outgrowth of several initially subdominant populations. Limiting dilution cloning of cells in the presence of high titers of HVS did not result in clonal transformation but in the rapid loss of the viral genome in outgrowing clones. In summary, our data suggest that transformation of CD8(pos) T cells out of bulk cultures can be routinely achieved, while viral transformation itself remains an infrequent event on a per cell basis. The practical use of the improved immortalization of antigen-expanded CD8(pos) T cell lines, however, is limited by the arbitrary outgrowth of subdominant populations of unpredictable specificity.

Uncoupling of hTREX demonstrates that UAP56 and hTHO-complex recruitment onto herpesvirus saimiri intronless transcripts is required for replication.

Herpesvirus saimiri (HVS) ORF57 nucleocytoplasmic shuttle protein binds viral RNA and interacts with the cellular nuclear export adaptor protein, Aly, to access the TAP-mediated nuclear export pathway. This enables the efficient nuclear export of HVS intronless mRNAs. Herein, we extend these studies and demonstrate that ORF57 recruits several members of hTREX, namely Aly, UAP56 and hTHO-complex proteins, onto the viral mRNAs to assemble an export-competent ribonucleoprotein particle. Moreover, using a transdominant form of Aly which inhibits UAP56 and hTHO-complex association with viral intronless mRNA, we show that complete hTREX recruitment is required for efficient HVS mRNA nuclear export and replication.

Herpesvirus saimiri episomal persistence is maintained via interaction between open reading frame 73 and the cellular chromosome-associated protein MeCP2.

Herpesvirus saimiri (HVS) is the prototype gamma-2 herpesvirus, which naturally infects the squirrel monkey Saimiri sciureus, causing an asymptomatic but persistent infection. The latent phase of gamma-2 herpesviruses is characterized by their ability to persist in a dividing cell population while expressing a limited subset of latency-associated genes. In HVS only three genes, open reading frame 71 (ORF71), ORF72, and ORF73, are expressed from a polycistronic mRNA. ORF73 has been shown to be the only gene essential for HVS episomal maintenance and can therefore be functionally compared to the human gammaherpesvirus latency-associated proteins, EBNA-1 and Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA). HVS ORF73 is the positional homologue of KSHV LANA and, although it shares limited sequence homology, has significant structural and functional similarities. Investigation of KSHV LANA has demonstrated that it is able to mediate KSHV episomal persistence by tethering the KSHV episome to host mitotic chromosomes via interactions with cellular chromosome-associated proteins. These include associations with core and linker histones, several bromodomain proteins, and the chromosome-associated proteins methyl CpG binding protein 2 (MeCP2) and DEK. Here we show that HVS ORF73 associates with MeCP2 via a 72-amino-acid domain within the ORF73 C terminus. Furthermore, we have assessed the functional significance of this interaction, using a variety of techniques including small hairpin RNA knockdown, and show that association between ORF73 and MeCP2 is essential for HVS chromosomal attachment and episomal persistence.

Activation of non-canonical NF-kappaB pathway mediated by STP-A11, an oncoprotein of Herpesvirus saimiri.

Although Saimiri Transforming Protein (STP)-A11, an oncoprotein of Herpesvirus saimiri, has been known to activate NF-kappaB signaling pathway, the detailed mechanism has not been reported yet. We herein report that STP-A11 activates non-canonical NF-kappaB pathway, resulting in p100 processing to p52. In addition, translocation of p52 protein (NF-kappaB2) into the nucleus is observed by the expression of STP-A11. STP-A11-mediated processing of p100 to p52 protein requires proteosome-mediated proteolysis because MG132 treatment clearly blocked p52 production in spite of the expression of STP-A11. Analysis of STP-A11 mutants to activate NF-kappaB2 pathway discloses the requirement of TRAF6-binding site not Src-binding site for STP-A11-mediated NF-kappaB2 pathway. Blockage of STP-A11-mediated p52 production using siRNA against p52 enhanced a chemotherapeutic drug-mediated cell death, suggesting that p52 production induced by the expression of STP-A11 would contribute to cellular transformation, which results from a resistance to cell death.

Histone modification pattern of the T-cellular Herpesvirus saimiri genome in latency.

Herpesvirus saimiri (HVS) subgroup C strains are able to growth transform human T lymphocytes in vitro. The stably persisting and nonintegrating HVS episome represents an optimal prerequisite for the investigation of the epigenetic state of latent herpesvirus genomes in vitro. Quantitative chromatin immunoprecipitation experiments using seven different histone acetylation- or methylation-specific antibodies revealed repressive marks at four lytic gene promoters and a variable pattern at the weakly transcribed LANA/orf73 promoter. The constitutive stpC/tip promoter regulating the viral oncoproteins and, more interestingly, the noncoding repetitive H-DNA elements flanking the coding region, showed a permissive chromatin structure. This study provides an appropriate model for the analysis of epigenetic herpesvirus genome modifications and their dynamics in T cells.

Growth transformation of human T cells by herpesvirus saimiri requires multiple Tip-Lck interaction motifs.

Lymphoma induction and T-cell transformation by herpesvirus saimiri strain C488 depends on two viral oncoproteins, StpC and Tip. The major interaction partner of Tip is the protein tyrosine kinase Lck, a key regulator of T-cell activation. The Lck binding domain (LBD) of Tip comprises two interaction motifs, a proline-rich SH3 domain-binding sequence (SH3B) and a region with homology to the C terminus of Src family kinase domains (CSKH). In addition, biophysical binding analyses with purified Lck-SH2 domain suggest the phosphorylated tyrosine residue 127 of Tip (pY127) as a potential third Lck interaction site. Here, we addressed the relevance of the individual binding motifs, SH3B, CSKH, and pY127, for Tip-Lck interaction and for human T-cell transformation. Both motifs within the LBD displayed Lck binding activities and cooperated to achieve a highly efficient interaction, while pY127, the major tyrosine phosphorylation site of Tip, did not enhance Lck binding in T cells. Herpesvirus saimiri strain C488 recombinants lacking one or both LBD motifs of Tip lost their transforming potential on human cord blood lymphocytes. Recombinant virus expressing Tip with a mutation at position Y127 was still able to transform human T lymphocytes but, in contrast to wild-type virus, was strictly dependent on exogenous interleukin-2. Thus, the strong Lck binding mediated by cooperation of both LBD motifs was essential for the transformation of human T cells by herpesvirus saimiri C488. The major tyrosine phosphorylation site Y127 of Tip was particularly required for transformation in the absence of exogenous interleukin-2, suggesting its involvement in cytokine signaling pathways.

gamma-2 Herpes virus post-transcriptional gene regulation.

gamma-2 herpes viruses, which include Kaposi's sarcoma-associated herpes virus, are an important subfamily of herpes virus because of their oncogenic potential. Herpes virus saimiri (HVS) is the prototype gamma-2 herpes virus and is a useful model to study the basic mechanisms of lytic replication in this subfamily. Like all herpes viruses, HVS has two distinct life cycles, latent persistence and lytic replication. Analysis of herpes virus genomes has demonstrated that, in contrast to cellular genes, most virus genes that are expressed lytically do not have introns. Herpes viruses replicate in the nucleus of the host cell, and therefore require that the viral intron-lacking mRNAs are exported from the nucleus to allow virus mRNA translation. This review focuses upon the role of HVS ORF 57, a post-transcriptional regulatory protein, which is conserved in all herpes viruses. HVS ORF 57 is a multifunctional protein involved in both trans-activation and trans-repression of target mRNAs. The major role of the ORF 57 protein in mediating viral mRNA export is considered, and the ORF 57-host cell interactions that are required for this function are discussed.

Association of herpesvirus saimiri tip with lipid raft is essential for downregulation of T-cell receptor and CD4 coreceptor.

Lipid rafts are membrane microdomains that are proposed to function as platforms for both receptor signaling and trafficking. Our previous studies have demonstrated that Tip of herpesvirus saimiri (HVS), which is a T-lymphotropic tumor virus, is constitutively targeted to lipid rafts and interacts with cellular Lck tyrosine kinase and p80 WD repeat-containing endosomal protein. Through the interactions with Lck and p80, HVS Tip modulates diverse T-cell functions, which leads to the downregulation of T-cell receptor (TCR) and CD4 coreceptor surface expression, the inhibition of TCR signal transduction, and the activation of STAT3 transcription factor. In this study, we investigated the functional significance of Tip association with lipid rafts. We found that Tip expression remarkably increased lipid raft fractions in human T cells by enhancing the recruitment of lipid raft-resident proteins. Genetic analysis showed that the carboxyl-terminal transmembrane, but not p80 and Lck interaction, of Tip was required for the lipid raft localization and that lipid raft localization of Tip was necessary for the efficient downregulation of TCR and CD4 surface expression. Correlated with this, treatment with Filipin III, a lipid raft-disrupting agent, effectively reversed the downregulation of CD3 and CD4 surface expression induced by Tip. On the other hand, Tip mutants that were no longer present in lipid rafts were still capable of inhibiting TCR signaling and activating STAT3 transcription factor activity as efficiently as wild-type (wt) Tip. These results indicate that the association of Tip with lipid rafts is essential for the downregulation of TCR and CD4 surface expression but not for the inhibition of TCR signal transduction and the activation of STAT3 transcription factor. These results also suggest that the signaling and targeting activities of HVS Tip rely on functionally and genetically separable mechanisms, which may independently modulate T-cell function for viral persistence or pathogenesis.

The Herpesvirus saimiri replication and transcription activator acts synergistically with CCAAT enhancer binding protein alpha to activate the DNA polymerase promoter.

The open reading frame (ORF) 50 gene product, also known as the replication and transcription activator (Rta), is an immediate-early gene which is well conserved among all gamma-2 herpesviruses and plays a pivotal role in regulating the latent-lytic switch. Herpesvirus saimiri (HVS) ORF 50a functions as a sequence-specific transactivator capable of activating delayed-early (DE) gene expression via binding directly to an ORF 50 response element (RE) within the respective promoter. Analysis of the ORF 50 REs have identified two distinct types within HVS gene promoters. The first comprises a consensus sequence motif, CCN(9)GG, the second an AT-rich sequence. Here we demonstrate that ORF 50a is capable of transactivating the DE ORF 9 promoter which encodes the DNA polymerase. Deletion analysis of the ORF 9 promoter mapped the ORF 50 RE to a 95-bp region situated 126 bp upstream of the initiation codon. Gel retardation analysis further mapped the RE to a 28-bp fragment, which was able to confer ORF 50 responsiveness on an enhancerless simian virus 40 minimal promoter. Furthermore, sequence analysis identified multiple CCAAT enhancer binding protein alpha (C/EBPalpha) binding sites within the ORF 9 promoter and specifically two within the close vicinity of the AT-rich ORF 50 RE. Analysis demonstrated that the HVS ORF 50a and C/EBPalpha proteins associate with the ORF 9 promoter in vivo, interact directly, and synergistically activate the ORF 9 promoter by binding to adjacent binding motifs. Overall, these data suggest a cooperative interaction between HVS ORF 50a and C/EBPalpha proteins to activate the DNA polymerase promoter during early stages of the lytic replication cycle.

Open reading frame 73 is required for herpesvirus saimiri A11-S4 episomal persistence.

Herpesvirus saimiri (HVS) establishes a latent infection in which the viral genome persists as a non-integrated episome. Analysis has shown that only open reading frames (ORFs) 71-73 are transcribed in an in vitro model of HVS latency. ORF73 also colocalizes with HVS genomic DNA on host mitotic chromosomes and maintains the stability of HVS terminal-repeat-containing plasmids. However, it is not known whether ORF73 is the only HVS-encoded protein required for episomal maintenance. In this study, the elements required for episomal maintenance in the context of a full-length HVS genome were examined by mutational analysis. A recombinant virus, HVS-BAC delta71-73, lacking the latency-associated genes was unable to persist in a dividing cell population. However, retrofitting an ORF73 expression cassette into the recombinant virus rescued episomal maintenance. This indicates that ORF73 is the key trans-acting factor for episomal persistence and efficient establishment of a latent infection.

T-cell growth transformation by herpesvirus saimiri is independent of STAT3 activation.

Herpesvirus saimiri (saimirine herpesvirus 2) (HVS), a T-lymphotropic tumor virus, induces lymphoproliferative disease in several species of New World primates. In addition, strains of HVS subgroup C are able to transform T cells of Old World primates, including humans, to permanently growing T-cell lines. In concert with the Stp oncoprotein, the tyrosine kinase-interacting protein (Tip) of HVS C488 is required for T-cell transformation in vitro and lymphoma induction in vivo. Tip was previously shown to interact with the protein tyrosine kinase Lck. Constitutive activation of signal transducers and activators of transcription (STATs) has been associated with oncogenesis and has also been detected in HVS-transformed T-cell lines. Furthermore, Tip contains a putative consensus YXPQ binding motif for the SH2 (src homology 2) domains of STAT1 and STAT3. Tip tyrosine phosphorylation at this site was required for binding of STATs and induction of STAT-dependent transcription. Here we sought to address the relevance of STAT activation for transformation of human T cells by introducing a tyrosine-to-phenylalanine mutation in the YXPQ motif of Tip of HVS C488. Unexpectedly, the recombinant virus was still able to transform human T lymphocytes, but it had lost its capability to activate STAT3 as well as STAT1. This demonstrates that growth transformation by HVS is independent of STAT3 activation.

Herpesvirus saimiri immortalization of Aotus T lymphocytes specific for an immunogenically modified peptide of Plasmodium falciparum merozoite surface antigen 2.

The Plasmodium merozoite surface antigen 2 (MSA2) is one of several candidates for a protective vaccine against malaria. Previous studies have shown that antibodies directed against the MSA2 variable region are not protective and that constant regions are non-immunogenic. However, modified peptides derived from constant regions can be rendered immunogenic and partially protective in Aotus monkeys. In this study, we reveal the establishment, using in vitro Herpesvirus samiri (HVS) infection, of an Aotus monkey T-cell line (AnTMSA2) specific for a modified immunogenic and partially protective peptide derived from a constant and highly conserved region of MSA2 (SKYSNTFINNAYNMSIRRSM). AnTMSA2 is a CD4 T lymphocyte expressing high levels of MHC class II molecules, CD58 and CD2, which are important for proliferation and growth. AnTMSA2 proliferates specifically in response to the modified monomeric MSA2 peptide sequence. It is also capable of specific antigen recognition after glycine-cysteine-polymerized sequence processing and presentation by autologous APC. Interestingly, AnTMSA2 presents cross-reactivity with D-peptide analogues in which residues in positions 8 and 9 were changed for NDID residues. Therefore, at least for this particular sequence, polymerized D-peptides could be used for immunizing animals without losing the immunogenic epitope. AnTMSA2 presents a cytokine profile corresponding to a Th0-like pattern, which suggests that as a result of HVS immortalization AnTMSA2 is in transit from a Th2 to a Th1 pattern. Taken together our results suggest that Th2 T-cell induction and/or T-cell cross-reactivity generation by the modified peptide could be responsible for the immunogenic conversion observed in Aotus monkeys and that D-peptide analogues with longer half-lives could provide an alternative for inducing protective immunity.

Defective CD3zeta chain expression in Herpesvirus saimiri (HVS)-derived T-cell lines in gastric adenocarcinoma.

Low expression of the CD3zeta chain has been reported in patients with cancer and it has been suggested that tumor-derived factors are involved in its downregulation. The expression of CD3zeta chain was measured in T-cell lines from patients with gastric adenocarcinoma and healthy volunteers and grown in vitro for several months and, hence, in the absence of any tumor-derived factors. T-cell lines of mucosal origin were obtained by Herpesvirus saimiri transformation from gastric cancer patients. The expression of CD3zeta and CD3epsilon was measured by flow cytometry and Western-blot analysis. Calcium mobilization and apoptosis rate were also measured. The levels of CD3zeta, but not CD3epsilon, chain on the cell surface were significantly reduced in T-cell lines derived from patients with gastric cancer when cultured in the absence of IL-2. Western-blot analysis of total cell extracts or lipid raft fractions confirmed this finding. Calcium mobilization, a measure of signal transduction, was reduced in T cell lines from patients with gastric cancer. We conclude that T cells from patients with cancer express lower levels of CD3zeta. This downregulation is not caused by a direct effect of tumor-derived factors but, rather, it appears to be inherent to the patient cells. The low CD3zeta expression would render T lymphocytes unable to control the growth of tumor cells.

The prototype gamma-2 herpesvirus nucleocytoplasmic shuttling protein, ORF 57, transports viral RNA through the cellular mRNA export pathway.

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.