Immortalization of human T cells expressing T-cell receptor gamma delta by herpesvirus saimiri.

Herpesvirus saimiri (HVS) has recently been shown to immortalize human CD4+ and CD8+ T cells expressing T-cell receptor alpha beta (TCR-alpha beta) with the maintenance of their original phenotypes and functional properties. However, the immortalization of human T cells expressing TCR-gamma delta by HVS has not been successful. Here we report that HVS can also infect and immortalize human T cells expressing TCR-gamma delta. Two human TCR-gamma delta+ T-cell clones, which continuously proliferated in interleukin-2-containing culture medium without any exogenous stimulation or addition of feeder cells for more than 8 months, were established by HVS infection. Morphologically, the HVS-transformed TCR-gamma delta+ T-cell clones were granular lymphocytes which exhibited wide-range HLA-unrestricted cytotoxicity as untransformed TCR-gamma delta+ T cells. Their phenotypes and cytotoxic activities were not altered during long-term culture. The immortalization of human TCR-gamma delta+ T cells by HVS infection would be useful for functional analysis of this lymphocyte population, which is believed to play an important role in protection against various infectious diseases.     

Herpesvirus ateles Tio can replace herpesvirus saimiri StpC and Tip oncoproteins in growth transformation of monkey and human T cells

Herpesvirus saimiri group C strains are capable of transforming human and simian T-lymphocyte populations to permanent antigen-independent growth. Two viral oncoproteins, StpC and Tip, that are encoded by a single bicistronic mRNA, act in concert to mediate this phenotype. A closely related New World monkey herpesvirus, herpesvirus ateles, transcribes a single spliced mRNA at an equivalent genome locus. The encoded protein, Tio, has sequence homologies to both StpC and Tip. We inserted the tio sequence of herpesvirus ateles strain 73 into a recombinant herpesvirus saimiri C488 lacking its own stpC/tip oncogene. Simian as well as human T lymphocytes were growth transformed by the chimeric Tio-expressing viruses. Thus, a single herpesvirus protein appears to be responsible for the oncogenic effects of herpesvirus ateles.     

The URNA genes of herpesvirus saimiri (strain C488) are dispensable for transformation of human T cells in vitro

The herpesvirus saimiri strain C488 genome contains five genes for small nuclear RNAs, termed herpesvirus saimiri URNAs (or HSURs). Using a cosmid-based approach, all HSURs were precisely deleted from the genome. The mutant virus replicated at levels that were similar to those of wild-type viruses in OMK cells. Although the HSURs are expressed in wild-type virus-transformed human T-cell lines, the deletion does not affect viral transformation in cell culture.     

Inhibition of retromer activity by herpesvirus saimiri tip leads to CD4 downregulation and efficient T cell transformation

The mammalian retromer is an evolutionally conserved protein complex composed of a vacuolar protein sorting trimer (Vps 26/29/35) that participates in cargo recognition and a sorting nexin (SNX) dimer that binds to endosomal membranes. The retromer plays an important role in efficient retrograde transport for endosome-to-Golgi retrieval of the cation-independent mannose-6-phosphate receptor (CI-MPR), a receptor for lysosomal hydrolases, and other endosomal proteins. This ultimately contributes to the control of cell growth, cell adhesion, and cell migration. The herpesvirus saimiri (HVS) tyrosine kinase-interacting protein (Tip), required for the immortalization of primary T lymphocytes, targets cellular signaling molecules, including Lck tyrosine kinases and the p80 endosomal trafficking protein. Despite the pronounced effects of HVS Tip on T cell signal transduction, the details of its activity on T cell immortalization remain elusive. Here, we report that the amino-terminal conserved, glutamate-rich sequence of Tip specifically interacts with the retromer subunit Vps35 and that this interaction not only causes the redistribution of Vps35 from the early endosome to the lysosome but also drastically inhibits retromer activity, as measured by decreased levels of CI-MPR and lower activities of cellular lysosomal hydrolases. Physiologically, the inhibition of intracellular retromer activity by Tip is ultimately linked to the downregulation of CD4 surface expression and to the efficient in vitro immortalization of primary human T cells to interleukin-2 (IL-2)-independent permanent growth. Therefore, HVS Tip uniquely targets the retromer complex to impair the intracellular trafficking functions of infected cells, ultimately contributing to efficient T cell transformation.     

Induction of a novel cellular homolog of interleukin-10, AK155, by transformation of T lymphocytes with herpesvirus saimiri

Although herpesvirus saimiri-transformed T lymphocytes retain multiple normal T-cell functions, only a few changes have been described. By subtractive hybridization, we have isolated a novel cellular gene, ak155, a sequence homolog of the interleukin-10 gene. Specifically herpesvirus saimiri-transformed T cells overexpress ak155 and secrete the protein into the supernatant. In other T-cell lines and in native peripheral blood cells, but not in B cells, ak155 is transcribed at low levels. AK155 forms homodimers similarly to interleukin-10. As a lymphokine, AK155 may contribute to the transformed phenotype of human T cells after infection by herpesvirus saimiri.     

Ptr ToxA requires multiple motifs for complete activity

Ptr ToxA was the first proteinaceous necrosis-inducing toxin identified and cloned from the wheat pathogen, Pyrenophora tritici-repentis. How this protein causes necrosis in sensitive wheat cultivars is not known. In an effort to understand the structural features of Ptr ToxA required for induction of necrosis, we employed a combination of site-directed mutagenesis and peptide inhibition studies. Mutagenesis was carried out on conserved motifs within the active domain of Ptr ToxA. Proteins with mutations of potential casein kinase 2 phosphorylation sites but not protein kinase C phosphorylation sites have significantly reduced activity. Additionally, mutations in a region with high homology to amino acids surrounding and including the RGD cell attachment motif of vitronectin result in proteins with significantly less activity than Ptr ToxA. The importance of the vitronectin-like motif was confirmed by a decrease of Ptr ToxA-induced activity when coinfiltrated with peptides corresponding to amino acids within this motif. Reduction in Ptr ToxA activity by competition with mutant proteins demonstrates the necessity of multiple motifs for Ptr ToxA activity.     

Phenotypic and functional consequences of herpesvirus saimiri infection of human CD8+ cytotoxic T lymphocytes.

Herpesvirus saimiri (HVS) was used to infect and transform human CD8+ cytotoxic T lymphocytes (CTL), and the phenotypic and functional consequences of HVS infection of CD8+ T lymphocytes were investigated. HVS-transformed CTL no longer require antigen restimulation yet maintain their phenotype and HLA-restricted cytolytic function and specificity. The ability of HVS to transform CTL may have an important role in the functional analysis of human antigen-specific CTL.     

Deletion mutants of herpesvirus saimiri define an open reading frame necessary for transformation.

Analysis of a 5,549-base-pair sequence at the left end of herpesvirus saimiri unique (L-) DNA revealed two open reading frames and genes for five small nuclear U RNAs (herpesvirus saimiri U RNAs). Replication-competent deletion mutants were constructed in order to assess the importance of these genetic features for transformation by this oncogenic herpesvirus. Although not required for replication, one of the open reading frames was found to be required for immortalization of marmoset T lymphocytes into continuously growing cell lines. The protein predicted by this reading frame (STP; saimiri transformation-associated protein) has a highly hydrophobic stretch of 26 amino acids sufficient for a membrane-spanning domain near its carboxy terminus; this domain is immediately preceded by a sequence appropriate for formation of a metal-binding domain (His X2 His X6 Cys X2 Cys, where Xs are other amino acids). One of two poly(A)+ RNAs that could encode STP is bicistronic, while the other has a long 5' untranslated region (approximately 1.5 kilobases). Although some analogies can be drawn between STP and LMP (lymphocyte membrane protein) of Epstein-Barr virus, STP is not related in sequence to LMP.     

In vitro immortalization of marmoset cells with three subgroups of herpesvirus saimiri

Sequences within the rightmost 7 kilobases of the unique L DNA of herpesvirus saimiri are required for oncogenicity of the virus. The same DNA region has been found to be highly variable among different strains of herpesvirus saimiri. On the basis of this variability, herpesvirus saimiri strains were classified into groups A, B, and non-A, non-B. Herpesvirus saimiri strains representing the three groups were used successfully for in vitro immortalization of phytohemagglutinin-activated, interleukin 2 (IL-2)-expanded peripheral blood lymphocytes of common marmosets (Callithrix jacchus). Peripheral blood leukocytes could be immortalized from only a subset of common marmosets (5 of 13). All of the immortalized cell lines contained covalently closed circular viral DNA molecules and initially showed a low level of virus production. Cells immortalized by group A and group non-A, non-B strains did not require IL-2 in the medium. However, the only group B immortalized cell line, 473-SMHI, did not grow well in the absence of IL-2. The different characteristics of cell lines immortalized by herpesvirus saimiri strains belonging to different groups may help to elucidate some functions coded by the highly variable DNA region which is involved in the oncogenic process.     

A herpesvirus saimiri protein required for interleukin-2 independence is associated with membranes of transformed T cells.

A region of the herpesvirus saimiri genome encoding an mRNA with two open reading frames (ORFs) has been identified to be essential for transformation of T cells. Deletion of either ORF resulted in the loss of transforming ability. ORF-1 has been shown to code for a collagen-like oncoprotein. This study shows for the first time that the bicistronic mRNA can translate a 32-kDa protein from ORF-2. Polyclonal serum to ORF-2 was generated by using a glutathione fusion protein. Using this antiserum, ORF-2 was localized in cell membranes and is expressed on the outer cell membrane. The half-life of this membrane protein was found to be about 5.5 h. Limited sequence similarity was found between ORF-2 and interleukin-11; however, no secretion of ORF-2 protein was detected in supernatants from transformed cells. Further studies are required to investigate the potential interaction with the interleukin-11 receptor.     

Downregulation of Lck-mediated signal transduction by tip of herpesvirus saimiri.

A protein, called tip, of herpesvirus saimiri associates with Lck in transformed T cells. To investigate the effects of complex formation on cellular signal transduction, we constructed human Jurkat-T-cell lines expressing tip. The expression of tip in Jurkat-T cells dramatically suppressed cellular tyrosine phosphorylation and surface expression of lymphocyte antigens. The expression of tip also blocked the induction of tyrosine phosphorylation by anti-CD3 stimulation. The expression of tip in fibroblast cells suppressed the transforming activity of oncogenic F505 Lck. Binding assays showed that the SH3 domain of Lck is sufficient to form a stable complex with tip in vitro. These results demonstrate that tip acts at an early stage of the T-cell signal transduction cascade by associating with Lck and downregulating Lck-mediated activation. Inhibition of Lck-mediated signal transduction by tip in T cells appears to be analogous to the inhibition of Lyn/Syk-mediated signal transduction in B cells by LMP2A of the B-cell-tropic Epstein-Barr virus.     

Rapid signaling to B cells by antigen-specific T cells requires CD18/CD54 interaction.

This study reports early B and T cell signaling events during cognate interactions between a human B cell line pulsed with peptide and an Ag-specific T cell clone. As has been previously reported, peptide in the context of the appropriate class II molecule stimulated a rise in intracellular calcium [Ca2+]i in the Ag-specific T cell clone. The activation of the T cell clone was associated with a reciprocal rise in [Ca2+]i in the B cells. Engagement of receptors on the B cell surface by the T cell also was associated with inositol phospholipid turnover comparable to that elicited by stimulation through sIg. Early signaling events in B cells can therefore be stimulated in cognate interactions with Ag-specific T cells, without the direct engagement of Ig receptors. A class II deficient B lymphoblastoid mutant, 6.1.6, which was incapable of presenting peptide to the T cell clone, could be stimulated to produce a rise in [Ca2+]i if the T cell clone was activated by monoclonal antibodies to CD3. Therefore, the interaction of class II molecules on the B cell with the TCR and/or the CD4 accessory molecule was not essential for T-dependent B cell activation. However, T-dependent signalling of B cells was profoundly inhibited by mAb to CD18 (beta-chain of LFA-1) on the T cell or CD54 (ICAM-1) on the B cell, demonstrating the importance of this pair of adhesion molecules in early T-B cell interactions.     

Primary structure of the herpesvirus saimiri genome.

This report describes the complete nucleotide sequence of the genome of herpesvirus saimiri, the prototype of gammaherpesvirus subgroup 2 (rhadinoviruses). The unique low-G + C-content DNA region has 112,930 bp with an average base composition of 34.5% G + C and is flanked by about 35 noncoding high-G + C-content DNA repeats of 1,444 bp (70.8% G + C) in tandem orientation. We identified 76 major open reading frames and a set of seven U-RNA genes for a total of 83 potential genes. The genes are closely arranged, with only a few regions of sizable noncoding sequences. For 60 of the predicted proteins, homologous sequences are found in other herpesviruses. Genes conserved between herpesvirus saimiri and Epstein-Barr virus (gammaherpesvirus subgroup 1) show that their genomes are generally collinear, although conserved gene blocks are separated by unique genes that appear to determine the particular phenotype of these viruses. Several deduced protein sequences of herpesvirus saimiri without counterparts in most of the other sequenced herpesviruses exhibited significant homology with cellular proteins of known function. These include thymidylate synthase, dihydrofolate reductase, complement control proteins, the cell surface antigen CD59, cyclins, and G protein-coupled receptors. Searching for functional protein motifs revealed that the virus may encode a cytosine-specific methylase and a tyrosine-specific protein kinase. Several herpesvirus saimiri genes are potential candidates to cooperate with the gene for saimiri transformation-associated protein of subgroup A (STP-A) in T-lymphocyte growth stimulation.     

Identification of proteins specific for human herpesvirus 6-infected human T cells.

Proteins specific for human herpesvirus 6 (HHV-6)-infected human T cells (HSB-2) were examined by using polyclonal rabbit antibodies and monoclonal antibodies against HHV-6-infected cells and human sera. More than 20 proteins and six glycoproteins specific for HHV-6-infected cells were identified from [35S]methionine- and [3H]glucosamine-labeled total-cell extracts. Polyclonal rabbit antibodies immunoprecipitated 33 [35S]methionine-labeled HHV-6-specific polypeptides with approximate molecular weights ranging from 180,000 to 31,000. In immunoprecipitation and Western immunoblot reactions, a patient's serum also recognized more than 30 HHV-6-specific proteins and seven glycoproteins. In contrast, sera from individuals with high-titered antibodies against other human herpesviruses reacted with fewer HHV-6-infected cell proteins, and only a 135,000-Mr polypeptide was prominent. Monoclonal antibodies to HHV-6-infected cells reacted with single and multiple polypeptides specific for virus-infected cells and immunoprecipitated three distinct sets of glycoproteins, which were designated gp105k and gp82k, gp116k, gp64k, and gp54k, and gp102k.     

Expression of collagenlike sequences by a tumor virus, herpesvirus saimiri.

Sequencing demonstrates that the oncogenic regions of a group A strain and a group C strain of herpesvirus saimiri are nonhomologous. A bicistronic viral mRNA from this region is transcribed in tumor cells transformed by a highly oncogenic group C virus. The first open reading frame is homologous to collagen; no such sequences were found in group A or B strains. This is the first report that a virus encodes for sequences similar to those of a connective tissue protein.     

Analysis of gene expression in a human cell line stably transduced with herpesvirus saimiri

Herpesvirus saimiri (HVS) is the prototype gamma-2 herpesvirus; it has significant homology to the human gammaherpesviruses Kaposi's sarcoma-associated virus and Epstein-Barr virus and the murine gammaherpesvirus murine herpesvirus 68. HVS causes a persistent asymptomatic infection in its natural host, the squirrel monkey. Both subgroups A and C possess the ability to immortalize common marmoset T lymphocytes to interleukin-2-independent proliferation. However, only subgroup C is capable of transforming human, rabbit, and rhesus monkey lymphocytes in vitro. In addition, HVS can stably transduce a variety of human cell lines where the virus persists as a nonintegrating circular episome. In this study, we have developed a system in which the HVS DNA is stably maintained as a nonintegrated circular episome in the human lung carcinoma cell line A549. Virus production can be reactivated using chemical inducing agents, including tetradecanoyl phorbol acetate and n-butyrate, suggesting that the infection in human A549 cells is latent. To analyze virus gene expression in these stably transduced cells, Northern blot analysis was performed using a series of probes produced from restriction fragments spanning the entire coding region of the HVS genome. This demonstrated that an adjacent set of genes containing open reading frames (ORFs) 71 to 73 are expressed in this stably transduced cell line. Moreover, these genes are transcribed as a polycistronic mRNA species produced from a common promoter upstream of ORF 73. This model may serve as a useful tool in the further analysis of the role of ORFs 71 to 73 in gamma-2 herpesvirus latency.     

Selectable recombinant herpesvirus saimiri is capable of persisting in a human T-cell line.

Strategies were developed to insert the neo resistance marker into the junction between L DNA and the right terminal repetitive H DNA of herpesvirus saimiri. Recombinant viruses were selectable in permissive epithelioid cultures. The human T-cell line Jurkat could be infected persistently with the Neor virus; the cells contained episomal viral DNA in high copy number. This selectable vector should be generally applicable for gene expression in human T cells.     

Viral gene expression patterns in human herpesvirus 6B-infected T cells

Herpesvirus gene expression is divided into immediate-early (IE) or alpha genes, early (E) or beta genes, and late (L) or gamma genes on the basis of temporal expression and dependency on other gene products. By using real-time PCR, we have investigated the expression of 35 human herpesvirus 6B (HHV-6B) genes in T cells infected by strain PL-1. Kinetic analysis and dependency on de novo protein synthesis and viral DNA polymerase activity suggest that the HHV-6B genes segregate into six separate kinetic groups. The genes expressed early (groups I and II) and late (groups V and VI) corresponded well with IE and L genes, whereas the intermediate groups III and IV contained E and L genes. Although HHV-6B has characteristics similar to those of other roseoloviruses in its overall gene regulation, we detected three B-variant-specific IE genes. Moreover, genes that were independent of de novo protein synthesis clustered in an area of the viral genome that has the lowest identity to the HHV-6A variant. The organization of IE genes in an area of the genome that differs from that of HHV-6A underscores the distinct differences between HHV-6B and HHV-6A and may provide a basis for further molecular and immunological analyses to elucidate their different biological behaviors.