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.

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.

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.

CD2-mediated autocrine growth of herpes virus saimiri-transformed human T lymphocytes.

Herpes virus saimiri (HVS) immortalizes T lymphocytes from a variety of primates and causes acute T cell lymphomas and leukemias in nonnatural primate hosts. Here we have analyzed the requirements for growth of three HVS-transformed human T cell lines. The cells expressed the phenotype of activated T cells: two were CD4+, and one was CD8+. All three cells responded to all allogeneic human cell lines tested with enhanced proliferation, production of interleukin 2 (IL-2), and increased expression of the IL-2 receptor. Binding of CD2 to its ligand CD58 was the critical event mediating stimulation because: (a) monoclonal antibodies (mAbs) to CD2 and to CD58, but not to a variety of other surface structures, blocked induced and spontaneous proliferation and IL-2 production; (b) only anti-CD2 mAbs were stimulatory if crosslinked; (c) a nonstimulatory cell was rendered stimulatory by CD58 transfection; and (d) the cells responded specifically to CD58 on sheep red blood cells. Growth of the cells required activation because cyclosporin A and FK506 blocked stimulator cell-induced IL-2 production and proliferation as well as the spontaneous growth of the lines. Antibodies to the IL-2 receptor reduced proliferation of the cells and blocked IL-2 utilization. Taken together, these results show that HVS-transformed T cells proliferate in response to CD2-mediated contact with stimulator cells or with each other in an IL-2-dependent fashion. They suggest that HVS transforms human T cells to an activation-dependent autocrine growth.

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.

Modulation of T-cell receptor signal transduction by herpesvirus signaling adaptor protein.

Because of its central regulatory role, T-cell receptor (TCR) signal transduction is a common target of viruses. We report here the identification of a small signaling protein, ORF5, of the T-lymphotropic tumor virus herpesvirus saimiri (HVS). ORF5 is predicted to contain 89 to 91 amino acids with an amino-terminal myristoylation site and six SH2 binding motifs, showing structural similarity to cellular LAT (linker for activation of T cells). Sequence analysis showed that, despite extensive sequence variation, the myristoylation site and SH2 binding motifs were completely conserved among 13 different ORF5 isolates. Upon TCR stimulation, ORF5 was efficiently tyrosine phosphorylated and subsequently interacted with cellular SH2-containing signaling proteins Lck, Fyn, SLP-76, and p85 through its tyrosine residues. ORF5 expression resulted in the marked augmentation of TCR signal transduction activity, evidenced by increased cellular tyrosine phosphorylation, intracellular calcium mobilization, CD69 surface expression, interleukin-2 production, and activation of the NF-AT, NF-kappa B, and AP-1 transcription factors. Despite its structural similarity to cellular LAT, however, ORF5 could only partially substitute for LAT function in TCR signal transduction. These results demonstrate that HVS utilizes a novel signaling protein, ORF5, to activate TCR signal transduction. This activation probably facilitates viral gene expression and, thereby, persistent infection.

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.

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.

Herpesvirus saimiri-immortalized human lymphocytes: novel hosts for analyzing HIV type 1 in vitro neutralization.

Herpesvirus saimiri-immortalized CD4(+) T lymphocytes (HVS T cells) are activated memory cells that support efficient replication of primary R5 strains of HIV-1, which predominate in virus transmission. Being continuous, they are phenotypically more stable and technically less demanding than peripheral blood mononuclear cells (PBMCs). Here we present the first report using HVS T cells to assay HIV-1 neutralization in vitro. Neutralization sensitivities of paired viruses isolated from individuals in both HVS T cells (CN-2 cells) and PBMCs were similar, with homologous and heterologous plasma/sera in both CN-2- and PBMC-based assays. Analysis of V3 loop and CD4-binding site (CD4-BS) sequences showed that changes present in CN-2 isolates were neither more numerous nor more significant than those selected in their PBMC counterparts. Neutralization profiles of CN-2/PBMC virus pairs were similar again when V3- and CD4-binding site (BS)-specific monoclonal antibodies, whose mapped epitopes were conserved or of similar sequence in the virus pairs, were tested. Unlike other T cell line isolates, CN-2 isolates were not more sensitive to neutralization than their PBMC counterparts. We also show that HVS T cells do not appear to exert significant biological selection pressures on primary isolates. Paired viruses have a similar phenotype with respect to syncytium formation, cell tropism, and coreceptor usage. Thus CN-2 cells are suitable hosts for assaying neutralization and could be useful in standardizing neutralization assays performed in different laboratories.

Herpesvirus saimiri transformation of HIV type 1 suppressive CD8+ lymphocytes from an HIV type 1-infected asymptomatic individual.

CD8+ T lymphocytes from HIV+ individuals can potently suppress HIV-1 replication in a noncytolytic manner. This suppression appears to be multifactorial and the molecules contributing have not been fully elucidated. As an approach to this question we used herpesvirus saimiri (HVS) to transform CD8+ T lymphocytes from an HIV+ asymptomatic donor to a continuously growing, activation-independent, IL-2-dependent phenotype. The transformed cell population, termed CD8(HVS), had an activated phenotype, contained HVS sequences, did not shed infectious HVS virus, and was polyclonal. The CD8(HVS) cells, despite the absence of detectable CTL activity, potently suppressed HIV-1 production by both autologous and heterologous CD4+ cells from infected donors. The CD8(HVS) cells in coculture also suppressed virus production from PBMCs acutely infected with syncytium-inducing (SI) strains or NSI primary isolates of HIV-1. The supernatants from the CD8(HVS) cells and their concentrates derived from these supernatants were suppressive to NSI primary isolates of HIV-1 but not to SI strains. Fractionation of these concentrates showed that the suppressive activity was associated with low molecular mass (6500- to 19,300-Da) protein species. Western blotting and ELISA indicated that the CC chemokines MIP-1alpha, MIP-1beta, and RANTES were present in these fractions. Antibody-blocking studies with antibodies to the CC chemokines indicated that a significant portion of the soluble HIV-suppressive activity was due to these molecules. However, these experiments also suggested the inhibitory activity of the CD8(HVS) cells in coculture is not due exclusively to the CC chemokines. The HVS-transformed cells provide a useful tool for the study of noncytolytic CD8+ T lymphocyte-mediated suppression of HIV-1.

Enhanced replication of M-tropic HIV-1 strains in Herpesvirus saimiri immortalised T-cells which express CCR5.

A better characterisation of mononuclear cell-tropic (M-tropic) HIV-1 is central to disease control as these viruses predominate in disease transmission. M-tropic viruses do not replicate in conventional T-cell lines, and virus titres obtained in peripheral blood mononuclear cells (PBMC) are low. Human T-lymphocytes which have been immortalised by Herpesvirus saimiri strain C488 (HVS T-cells) are highly permissive to the replication of T-cell tropic strains of HIV. This study aimed to determine if HVS T-cells support replication of M-tropic HIV isolates that have not been adapted to conventional T-cell lines. A panel of PBMC low passage/primary field isolates and their molecular clones was used. Results show that infection in HVS T-cells was longer lived than in PBMC. In terms of peak virus titre and duration of productive infection, the two HVS T-cell lines studied were superior to PBMC, and one supported enhanced replication of all M-tropic isolates. This is important for generating M-tropic virus pools of sufficient titre for further biological studies such as virus neutralisation, co receptor usage and testing of antivirals. Phenotypic analysis showed that HVS T-cells are CD4+-activated memory cells expressing both CXCR-4 and CCR5 co receptors. Thus, HVS immortalisation appears to select for the T-cell subset targeted by HIV-1 in vivo.

Mechanisms of cell transformation by Herpesvirus saimiri.

Herpesvirus saimiri is a virus capable of inducing oncogenic transformation of T lymphocytes of New World primates and immortalizing human T cells in vitro. T lymphocytes immortalized by H. saimiri demonstrate functional biological responses to their antigens. Therefore, H saimiri-induced transformation of T cells emerges as a very powerful tool of T-cell biology. The mechanism of this transformation remains to be elucidated. However, it is apparent that H. saimiri proteins Tip and StpC are crucial for T-cell transformation. Potential mechanisms mediating the effects of Tip and StpC, as well as the possible role of other H. saimiri proteins, are discussed in this review.

Up-regulation of telomerase activity in Herpesvirus saimiri immortalized human T-lymphocytes.

Human T-lymphocytes can be transformed to unlimited growth by Herpesvirus saimiri (HVS). We studied the telomerase activity of a recently established HVS immortalized human CD4 T cell clone in comparison to peripheral blood lymphocytes (PBL) and unstimulated or phytohemagglutinin (PHA)-stimulated CD4 T-cells by a Telomeric Repeat Amplification-Protocol (TRAP) -Assay. Telomerase activity in PHA-stimulated CD4 T-cells was seven-fold and in HVS-infected CD4 T-cells 14-fold higher than in untreated CD4 T-cells. The HVS immortalized T-cell clone provides a useful tool for studying the regulation of telomerase activity during carcinogenesis and for testing of telomerase-inhibitory drugs.

Characterization of four herpesviruses isolated from owl monkeys and their comparison with Herpesvirus saimiri type 1 (Herpesvirus tamarinus) and herpes simplex virus type 1.

Four herpesviruses were previously isolated from four outbreaks of lethal disease in owl monkeys. All four isolates have been shown to be antigenically closely related to each other and to Herpesvirus saimiri 1 (HVS-1) by kinetic neutralizations. The owl monkey strains also share similarities to HVS-1 and to each other with respect to host range, growth cycles and molecular weights of peptides and of DNA fragments generated by restriction endonuclease (R.E.) digestion. R.E. analysis, however, can differentiate strains by the use of certain enzymes. All four isolates share a common G-C ratio percentage with HVS-1 of 67 per cent. On the basis of these findings, we believe that these owl monkey virus isolates are strains of HVS-1.

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.

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.

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.