STP and Tip Are Essential for Herpesvirus Saimiri Oncogenicity

Mutant forms of herpesvirus saimiri (HVS) subgroup C strain 488 with deletions in either STP-C488 or Tip were constructed. The transforming potentials of the HVS mutants were tested in cell culture and in common marmosets. Parental HVS subgroup C strain 488 immortalized common marmoset T lymphocytes in vitro to interleukin-2-independent growth, but neither of the deletion mutants produced such growth transformation. Wild-type HVS produced fatal lymphoma within 19 to 20 days of experimental infection of common marmosets, while HVS ΔSTP-C488 and HVS ΔTip were nononcogenic. Virus was repeatedly isolated from the peripheral blood of marmosets infected with mutant virus for more than 5 months. These results demonstrate that STP-C488 and Tip are not required for replication or persistence, but each is essential for transformation in cell culture and for lymphoma induction in common marmosets.     

A Role for Herpesvirus Saimiri orf14 in Transformation and Persistent Infection

The product of open reading frame 14 (orf14) of herpesvirus saimiri (HVS) exhibits significant homology with mouse mammary tumor virus superantigen. orf14 encodes a 50-kDa secreted glycoprotein, as shown previously (Z. Yao, E. Maraskovsky, M. K. Spriggs, J. I. Cohen, R. J. Armitage, and M. R. Alderson, J. Immunol. 156:3260–3266, 1996). orf14 expressed from recombinant baculovirus powerfully induces proliferation of CD4-positive cells originating from several different species. To study the role of orf14 in transformation, a mutant form of HVS (HVS Δorf14) was constructed with a deletion in the orf14 gene. The transforming potential of HVS Δorf14 was tested in cell culture and in common marmosets. Parental HVS subgroup C strain 488 immortalized common marmoset T lymphocytes in vitro to interleukin-2-independent growth, while the HVS Δorf14 mutant did not produce such a growth transformation. In addition, HVS Δorf14 was nononcogenic in common marmosets. In contrast to other nononcogenic HVS mutant viruses which were repeatedly isolated from peripheral blood mononuclear cells of infected marmosets for more than 5 months, HVS Δorf14 did not persist at a high level in vivo. These results demonstrate that orf14 of HVS is not required for replication but is required for transformation and for high-level persistence in vivo.     

STP-C, an oncoprotein of herpesvirus saimiri augments the activation of NF-kappaB through ubiquitination of TRAF6

Herpesvirus saimiri (HVS), a member of the gamma-herpesvirus family, encodes an oncoprotein called Saimiri Transforming Protein (STP) which is required for lymphoma induction in non-human primates. Previous study has shown that STP-C, an oncoprotein of HVS, activates NF-kappaB signaling pathway. However, the detailed mechanism of STP-C-mediated NF-kappaB activation has not been reported yet. We first report that STP-C interacts with TRAF6 protein in vivo and in vitro and further investigation shows that Glu(12) residue of STP-C is critical for binding to TRAF6. Introduction of ubiquitin together with STP-C augments NF-kappaB activity compared to that of STP-C expression alone. STP-C expression further induces ubiquitination of endogenous TRAF6. In addition, either a deubiquitination enzyme, CYLD or a dominant negative E2-conjugation enzyme reduced NF-kappaB activity in spite of the presence of STP-C, supporting that the interaction between STP-C and TRAF6 induces ubiquitination of TRAF6. NF-kappaB activation by STP-C through the ubiquitinated TRAF6 causes the increased production of IL-8, an inflammatory chemokine and the enhanced expression of costimulatory molecule ICAM, which might ultimately contribute cellular transformation by the exposure of HVS-infected cells with inflammatory microenvironment and chronic activation.     

Identification and characterization of the herpesvirus saimiri oncoprotein STP-C488.

The protein encoded by herpesvirus saimiri transforming gene STP-C488 was identified and characterized. Antibodies were produced in rabbits by immunization with keyhole limpet hemocyanin-conjugated synthetic peptides specific for the predicted sequence of STP-C488. STP-C488-encoded protein was detected in recombinant Escherichia coli, transformed Rat-1 cells, transfected COS-1 cells, and in common marmoset T lymphocytes immortalized by herpesvirus saimiri strain 488. STP-C488 protein was sensitive to treatment by bacterial collagenase, consistent with the 18 uninterrupted collagenlike repeats predicted by the DNA sequence. The apparent molecular size of STP-C488 in sodium dodecyl sulfate (SDS)-polyacrylamide gels (20 to 22 kDa) was considerably larger than that predicted from the DNA sequence (9.9 kDa). Using indirect immunofluorescence tests and subcellular fractionation, STP-C488 was found to be membrane bound, primarily in perinuclear compartments. The 18 uninterrupted collagenlike repeats, sensitivity to collagenase, location in the cell, and anomalous migration through SDS-polyacrylamide gels suggest an unusual, membrane-associated, fibrous structure for this transforming herpesvirus oncoprotein.     

Association of the viral oncoprotein STP-C488 with cellular ras.

The STP-C488 oncogene of herpesvirus saimiri has transforming activity independent of the rest of the viral genome. We now demonstrate that STP-C488 associates with cellular ras in transformed cells. Mutations that disrupted this association with ras disrupted the transforming ability of the STP-C488 oncogene. Binding assays showed that STP-C488 was capable of competing with raf-1 for binding to ras. Expression of STP-C488 activated the ras signaling pathway as evidenced by a two- to fourfold increase in the ratio of ras-GTP to ras-GDP and by the constitutive activation of mitogen-activated protein kinase. Consistent with an activation of signaling through ras, STP-C488 expression induced ras-dependent neurite outgrowth in PC12 cells. STP-C488 is the first virus-encoded protein shown to achieve oncogenic transformation via association with cellular ras.     

The collagen repeat sequence is a determinant of the degree of herpesvirus saimiri STP transforming activity

Herpesvirus saimiri (HVS) is divided into three subgroups, A, B, and C, based on sequence divergence at the left end of genomic DNA in which the saimiri transforming protein (STP) resides. Subgroup A and C strains transform primary common marmoset lymphocytes to interleukin-2-independent growth, whereas subgroup B strains do not. To investigate the nononcogenic phenotype of the subgroup B viruses, STP genes from seven subgroup B virus isolates were cloned and sequenced. Consistent with the lack of oncogenic activity of HVS subgroup B viruses, STP-B was deficient for transforming activity in rodent fibroblast cells. Sequence comparison reveals that STP-B lacks the signal-transducing modules found in STP proteins of the other subgroups, collagen repeats and an authentic SH2 binding motif. Substitution mutations demonstrated that the lack of collagen repeats but not an SH2 binding motif contributed to the nontransforming phenotype of STP-B. Introduction of the collagen repeat sequence induced oligomerization of STP-B, resulting in activation of NF-kappaB activity and deregulation of cell growth control. These results demonstrate that the collagen repeat sequence is a determinant of the degree of HVS STP transforming activity.     

Association of TRAF1, TRAF2, and TRAF3 with an Epstein-Barr virus LMP1 domain important for B-lymphocyte transformation: role in NF-kappaB activation.

The Epstein-Barr virus (EBV) transforming protein LMP1 appears to be a constitutively activated tumor necrosis factor receptor (TNFR) on the basis of an intrinsic ability to aggregate in the plasma membrane and an association of its cytoplasmic carboxyl terminus (CT) with TNFR-associated factors (TRAFs). We now show that in EBV-transformed B lymphocytes most of TRAF1 or TRAF3 and 5% of TRAF2 are associated with LMP1 and that most of LMP1 is associated with TRAF1 or TRAF3. TRAF1, TRAF2, and TRAF3 bind to a single site in the LMP1 CT corresponding to amino acids (aa) 199 to 214, within a domain which is important for B-lymphocyte growth transformation (aa 187 to 231). Further deletional and alanine mutagenesis analyses and comparison with TRAF binding sequences in CD40, in CD30, and in the LMP1 of other lymphycryptoviruses provide the first evidence that PXQXT/S is a core TRAF binding motif. The negative effects of point mutations in the LMP1(1-231) core TRAF binding motif on TRAF binding and NF-kappaB activation genetically link the TRAFs to LMP1(1-231)-mediated NF-kappaB activation. NF-kappaB activation by LMP1(1-231) is likely to be mediated by TRAF1/TRAF2 heteroaggregates since TRAF1 is unique among the TRAFs in coactivating NF-kappaB with LMP1(1-231), a TRAF2 dominant-negative mutant can block LMP1(1-231)-mediated NF-kappaB activation as well as TRAF1 coactivation, and 30% of TRAF2 is associated with TRAF1 in EBV-transformed B cells. TRAF3 is a negative modulator of LMP1(1-231)-mediated NF-kappaB activation. Surprisingly, TRAF1, -2, or -3 does not interact with the terminal LMP1 CT aa 333 to 386 which can independently mediate NF-kappaB activation. The constitutive association of TRAFs with LMP1 through the aa 187 to 231 domain which is important in NF-kappaB activation and primary B-lymphocyte growth transformation implicates TRAF aggregation in LMP1 signaling.     

Mutation of the Lck-Binding Motif of Tip Enhances Lymphoid Cell Activation by Herpesvirus Saimiri

The proline-rich SH3-binding (SH3B) motif of the tyrosine kinase-interacting protein (Tip) of herpesvirus saimiri (HVS) is required for binding to the cellular Src family kinase Lck. We constructed a mutant form of HVS in which prolines in the SH3B motif of Tip were altered to alanines. This mutant form of Tip was incapable of binding to Lck. The mutant virus, HVS/Tip mSH3B, retained its ability to immortalize common marmoset lymphocytes in culture. In fact, common marmoset lymphocytes immortalized by the HVS/Tip mSH3B mutant displayed increased expression of HLA-DR lymphocyte activation marker, an altered pattern of tyrosine phosphorylation, increased expression of the tyrosine kinase Lyn, and a shift in electrophoretic mobility of Lck compared to cells immortalized by wild-type HVS. Experimental infection of common marmosets resulted in fulminant lymphoma with both HVS/Tip mSH3B and wild-type HVS. However, HVS/Tip mSH3B produced greater infiltration of affected organs by proliferating lymphoid cells compared to wild-type HVS. These results demonstrate that Tip binding to Lck is not necessary for transformation and that abrogation of Tip binding to Lck alters the characteristics of transformed cells and the severity of the pathologic lesions.     

Herpesvirus saimiri oncogene STP-C488 encodes a phosphoprotein.

The STP-C488 open reading frame of herpesvirus saimiri encodes an oncoprotein that has transforming and tumor-inducing activities independent of the rest of the herpesvirus genome. STP-C488 protein has an unusual, membrane-associated, fibrous structure and is located primarily in perinuclear compartments. We now report that STP-C488 is phosphorylated in vivo. The phosphorylated form, which accounted for about 15% of STP-C488 in transformed cells, migrated slightly more slowly through sodium dodecyl sulfate-polyacrylamide gels than unphosphorylated STP-C488. A serine residue near the amino terminus was shown to be the site of phosphorylation. However, phosphorylation was not required for transformation of Rat-1 cells by STP-C488.     

Unique signaling properties of CTAR1 in LMP1-mediated transformation

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) gene is considered the EBV oncogene as it is necessary for EBV-mediated transformation of B lymphocytes and itself transforms rodent fibroblasts. LMP1 activates the NF-kappaB, phosphatidylinositol 3-kinase (PI3K)-Akt, mitogen-activated protein kinase, and Jun N-terminal protein kinase signaling pathways through its two signaling domains, carboxyl-terminal activating regions 1 and 2 (CTAR1 and CTAR2). CTAR1 and CTAR2 induce signal transduction pathways through their direct (CTAR1) or indirect (CTAR2) recruitment of tumor necrosis factor receptor-associated factors (TRAFs). CTAR1 is necessary for LMP1-mediated transformation as well as activation of PI3K signaling and induction of cell cycle markers associated with G(1)/S transition. In this study, activation of PI3K-Akt signaling and deregulation of cell cycle markers were mapped to the TRAF-binding domain within CTAR1 and to the residues between CTAR1 and CTAR2. LMP1 CTAR1 also activated the MEK1/2-extracellular signal-regulated kinase 1/2 signaling pathway, and this activation was necessary for LMP1-induced transformation of Rat-1 fibroblasts. Dominant-negative forms of TRAF2 and TRAF3 inhibited but did not fully block LMP1-mediated transformation. These findings identify a new signaling pathway that is uniquely activated by the TRAF-binding domain of LMP1 and is required for transformation.     

Characterization of the roles of TNF receptor-associated factor 6 in CD40-mediated B lymphocyte effector functions

Signaling through CD40 in B cells leads to B cell proliferation, Ig and IL-6 secretion, isotype switching, and up-regulation of surface molecules. TNF receptor-associated factor (TRAF) proteins associate with the cytoplasmic tail of CD40 and act as adapter molecules. Of the six TRAFs identified to date, TRAFs 2, 3, 5, and 6 are reported to associate directly with the cytoplasmic tail of CD40, but previous studies have principally examined transient overexpression of TRAF6 in cells that do not normally express CD40. Thus, we examined the role of TRAF6 in CD40-mediated B lymphocyte effector functions using two approaches. We produced and stably expressed in mouse B cell lines a human CD40 molecule with two cytoplasmic domain point mutations (hCD40EEAA); this mutant fails to bind TRAF6, while showing normal association with TRAFs 2 and 3. We also inducibly expressed in B cells a transfected "dominant-negative" TRAF6 molecule which contains only the C-terminal TRAF-binding domain of TRAF6. Using both molecules, we found that TRAF6 association with CD40 is important for CD40-induced IL-6 and Ig secretion, and that TRAF6 mediates its effects on CD40-stimulated Ig secretion principally through its effects on IL-6 production by the B cell. TRAF6 association with CD40 was also found to be important for B7-1 up-regulation, but not for up-regulation of other surface molecules. Interestingly, however, although we could show TRAF6-dependent CD40-mediated activation of NF-kappaB in 293 kidney epithelial cells, no such effect was seen in B cells, suggesting that TRAF6 has cell-type-specific functions.     

Recruitment of CD40 and tumor necrosis factor receptor-associated factors 2 and 3 to membrane microdomains during CD40 signaling

Signals delivered to antigen-presenting cells through CD40 are critical for the activation of immune responses. Intracellular tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are key elements of the signal transduction pathways of many TNF receptor family members, including CD40. We show for the first time that engagement of CD40 in intact B cells induces the rapid translocation of TRAF2 from the cytoplasm to the plasma membrane. We found that CD40 engagement also results in its recruitment, together with TRAF2 and TRAF3, to membrane microdomains, regions of the plasma membrane enriched in signaling molecules such as the Src family kinases. Using a membrane-permeable chelator of zinc or a mutant TRAF2 molecule, we show that the putative zinc-binding domains of TRAFs contribute to their recruitment to microdomains and to the downstream activation of c-Jun N-terminal kinase. We suggest that the zinc RING and zinc finger domains of TRAFs are required for communication between CD40 and microdomain-associated signaling molecules and may serve a similar role in the signal transduction pathways of other TNF receptor family members.     

TNFR-associated factor family protein expression in normal tissues and lymphoid malignancies

TNFR-associated factors (TRAFs) constitute a family of adapter proteins that associate with particular TNF family receptors. Humans and mice contain six TRAF genes, but little is known about their in vivo expression at the single cell level. The in vivo locations of TRAF1, TRAF2, TRAF5, and TRAF6 were determined in human and mouse tissues by immunohistochemistry. Striking diversity was observed in the patterns of immunostaining obtained for each TRAF family protein, suggesting their expression is independently regulated in a cell type-specific manner. Dynamic regulation of TRAFs was observed in cultured PBLs, where anti-CD3 Abs, mitogenic lectins, and ILs induced marked increases in the steady-state levels of TRAF1, TRAF2, TRAF5, and TRAF6. TRAF1 was also highly inducible by CD40 ligand in cultured germinal center B cells, whereas TRAF2, TRAF3, TRAF5, and TRAF6 were relatively unchanged. Analysis of 83 established human tumor cell lines by semiquantitative immunoblotting methods revealed tendencies of certain cancer types to express particular TRAFs. For example, expression of TRAF1 was highly restricted, with B cell lymphomas consistently expressing this TRAF family member. Consistent with results from tumor cell lines, immunohistochemical analysis of 232 non-Hodgkin lymphomas revealed TRAF1 overexpression in 112 (48%) cases. TRAF1 protein levels were also elevated in circulating B cell chronic lymphocytic leukemia specimens (n = 49) compared with normal peripheral blood B cells (p = 0.01), as determined by immunoblotting. These findings contribute to an improved understanding of the cell-specific roles of TRAFs in normal tissues and provide evidence of altered TRAF1 expression in lymphoid malignancies.     

IL-1 induced chemokine production through the association of Syk with TNF receptor-associated factor-6 in nasal fibroblast lines.

The fibroblasts stimulated by cytokines released the chemokine and recruited the infiltrating cells, including eosinophils, that play a key role in the pathogenesis of airway disease. We established the human fibroblast lines showing high Syk expression and the lines showing low Syk expression from pieces of nasal polyp. IL-1 induces the interaction of TNFR-associated factor (TRAF) 6 with IL-1R-associated kinase, which is rapidly recruited to the IL-1R after IL-1 induction, whereas TRAF2 participates in TNF-alpha-signaling. In the present study, we found that Syk played a different role in IL-1- and TNF-alpha-induced chemokine production through a signaling complex involving Syk and TRAF6. Overexpression of wild-type Syk by gene transfer enhanced RANTES production from nasal fibroblasts stimulated with IL-1. The decrease of Syk expression by the administration of Syk antisense inhibited RANTES production in response to IL-1. However, the change of Syk expression did not affect RANTES production by TNF-alpha stimulation. We concluded that Syk is required for the IL-1-induced chemokine production through the association with TRAF-6 in fibroblasts of nasal polyps.     

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.     

LMP1 protein from the Epstein-Barr virus is a structural CD40 decoy in B lymphocytes for binding to TRAF3

Epstein-Barr virus is a human herpesvirus that causes infectious mononucleosis and lymphoproliferative malignancies. LMP1 (latent membrane protein-1), which is encoded by this virus and which is essential for transformation of B lymphocytes, acts as a constitutively active mimic of the tumor necrosis factor receptor (TNFR) CD40. LMP1 is an integral membrane protein containing six transmembrane segments and a cytoplasmic domain at the C terminus that binds to intracellular TNFR-associated factors (TRAFs). TRAFs are intracellular co-inducers of downstream signaling from CD40 and other TNFRs, and TRAF3 is required for activation of B lymphocytes by LMP1. Cytoplasmic C-terminal activation region 1 of LMP1 bears a motif (PQQAT) that conforms to the TRAF recognition motif PVQET in CD40. In this study, we report the crystal structure of this portion of LMP1 C-terminal activation region-1 (204PQQATDD210) bound in complex with TRAF3. The PQQAT motif is bound in the same binding crevice on TRAF3 where CD40 is bound, providing a molecular mechanism for LMP1 to act as a CD40 decoy for TRAF3. The LMP1 motif is presented in the TRAF3 crevice as a close structural mimic of the PVQET motif in CD40, and the intermolecular contacts are similar. However, the viral protein makes a unique contact: a hydrogen bond network formed between Asp210 in LMP1 and Tyr395 and Arg393 in TRAF3. This intermolecular contact is not made in the CD40-TRAF3 complex. The additional hydrogen bonds may stabilize the complex and strengthen the binding to permit LMP1 to compete with CD40 for binding to the TRAF3 crevice, influencing downstream signaling to B lymphocytes and contributing to dysregulated signaling by LMP1.     

Differential regulation of CD40-mediated TNF receptor-associated factor degradation in B lymphocytes

Engagement of CD40 on murine B cells by its ligand CD154 induces the binding of TNFR-associated factors (TRAFs) 1, 2, 3, and 6, followed by the rapid degradation of TRAFs 2 and 3. TRAF degradation occurs in response to signaling by other TNFR superfamily members, and is likely to be a normal regulatory component of signaling by this receptor family. In this study, we found that receptor-induced TRAF degradation limits TRAF2-dependent CD40 signals to murine B cells. However, TRAFs 1 and 6 are not degraded in response to CD40 engagement, despite their association with CD40. To better understand the mechanisms underlying differential TRAF degradation, mixed protein domain TRAF chimeras were analyzed in murine B cells. Chimeras containing the TRAF2 zinc (Zn) domains induced effective degradation, if attached to a TRAF domain that binds to the PXQXT motif of CD40. However, the Zn domains of TRAF3 and TRAF6 could not induce degradation in response to CD40, regardless of the TRAF domains to which they were attached. Our data indicate that TRAF2 serves as the master regulator of TRAF degradation in response to CD40 signaling, and this function is dependent upon both the TRAF Zn domains and receptor binding position.