Identification of an Immunoreceptor Tyrosine-Based Activation Motif of K1 Transforming Protein of Kaposi’s Sarcoma-Associated Herpesvirus

Kaposi’s sarcoma-associated herpesvirus (KSHV) is consistently identified in Kaposi’s sarcoma and body cavity-based lymphoma. KSHV encodes a transforming protein called K1 which is structurally similar to lymphocyte receptors. We have found that a highly conserved region of the cytoplasmic domain of K1 resembles the sequence of immunoreceptor tyrosine-based activation motifs (ITAMs). To demonstrate the signal-transducing activity of K1, we constructed a chimeric protein in which the cytoplasmic tail of the human CD8α polypeptide was replaced with that of KSHV K1. Expression of the CD8-K1 chimera in B cells induced cellular tyrosine phosphorylation and intracellular calcium mobilization upon stimulation with an anti-CD8 antibody. Mutational analyses showed that the putative ITAM of K1 was required for its signal-transducing activity. Furthermore, tyrosine residues of the putative ITAM of K1 were phosphorylated upon stimulation, and this allowed subsequent binding of SH2-containing proteins. These results demonstrate that the KSHV transforming protein K1 contains a functional ITAM in its cytoplasmic domain and that it can transduce signals to induce cellular activation.     

Activation of lymphocyte signaling by the R1 protein of rhesus monkey rhadinovirus

Rhesus monkey rhadinovirus (RRV) is a gamma-2 herpesvirus that exhibits a considerable degree of similarity to the human Kaposi's sarcoma-associated herpesvirus (KSHV). The R1 protein of RRV is distantly related to the K1 protein of KSHV, and R1, like K1, can contribute to cell growth transformation. In this study we analyzed the ability of the cytoplasmic tail of R1 to function as a signal transducer. The cytoplasmic domain of the R1 protein contains several tyrosine residues whose phosphorylation is induced in cells expressing Syk kinase. Expression of a CD8 chimera protein containing the extracellular and transmembrane domains of CD8 fused to the cytoplasmic domain of R1 mobilized intracellular calcium and induced cellular tyrosine phosphorylation in B cells upon stimulation with anti-CD8 antibody. None of the CD8-R1 cytoplasmic deletion mutants tested were able to mobilize intracellular calcium or to induce tyrosine phosphorylation to a significant extent upon addition of anti-CD8 antibody. Expression of wild-type R1 protein activated nuclear factor of activated T lymphocytes (NFAT) eightfold in B cells in the absence of antibody stimulation; expression of the CD8-R1C chimera strongly induced NFAT activity (60-fold) but only upon the addition of anti-CD8 antibody. We conclude that the cytoplasmic domain of R1 is capable of transducing signals that elicit B-lymphocyte activation events. The signal-inducing properties of R1 appear to be similar to those of K1 but differ in that the required sequences are distributed over a much longer stretch of the cytoplasmic domain (>150 amino acids). In addition, the induction of calcium mobilization was considerably longer in duration and stronger with R1 than with K1.     

Identification of the novel K15 gene at the rightmost end of the Kaposi's sarcoma-associated herpesvirus genome

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a distinct open reading frame called K15 at a position equivalent to the gene encoding LMP2A of Epstein-Barr virus (EBV). K15 isolates from body cavity-based lymphoma (BCBL) cells exhibited a dramatic sequence variation and a complex splicing pattern. However, all K15 alleles are organized similarly with the potential SH2 and SH3 binding motifs in their cytoplasmic regions. Northern blot analysis showed that K15 was weakly expressed in latently infected BCBL-1 cells, and the level of its expression was significantly induced by tetradecanoyl phorbol acetate stimulation. K15 encoded 40- to 55-kDa proteins, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was localized at the cytoplasm and plasma membrane. To demonstrate the signal-transducing activity of the K15 protein, we constructed a chimeric protein in which the cytoplasmic tail of the human CD8alpha polypeptide was replaced with that of KSHV K15. While the CD8-K15 chimera was not capable of eliciting cellular signal transduction upon stimulation with an anti-CD8 antibody, it significantly inhibited B-cell receptor signaling, as evidenced by a suppression of tyrosine phosphorylation and intracellular calcium mobilization. This inhibition required the putative SH2 or SH3 binding motif in the cytoplasmic region of K15. Biochemical study of CD8-K15 chimeras showed that the cytoplasmic region of K15 was constitutively tyrosine phosphorylated and that the tyrosine residue within the putative SH2 binding motif of K15 was a primary site of phosphorylation. These results demonstrate that KSHV K15 resembles LMP2A in genomic location, splicing pattern, and protein structure and by the presence of functional signal-transducing motifs in the cytoplasmic region. Thus, KSHV K15 is likely a distant evolutionary relative of EBV LMP2A.     

Identification of the R1 oncogene and its protein product from the rhadinovirus of rhesus monkeys

Rhesus monkey rhadinovirus (RRV) is a gamma-2 herpesvirus that is most closely related to the human Kaposi's sarcoma-associated herpesvirus (KSHV). We have identified a distinct open reading frame at the left end of RRV and designated it R1. The position of the R1 gene is equivalent to that of the saimiri transforming protein (STP) of herpesvirus saimiri (HVS) and of K1 of KSHV, other members of the gamma-2 or rhadinovirus subgroup of herpesviruses. The R1 sequence revealed an open reading frame encoding a product of 423 amino acids that was predicted to contain an extracellular domain, a transmembrane domain, and a C-terminal cytoplasmic tail reflective of a type I membrane-bound protein. The predicted structural motifs of R1, including the presence of immunoreceptor tyrosine-based activation motifs, resembled those in K1 of KSHV but were distinct from those of STP. R1 sequences from four independent isolates from three different macaque species revealed 0.95 to 7.3% divergence over the 423 amino acids. Variation was located predominantly within the predicted extracellular domain. The R1 protein migrated at 70 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was extensively glycosylated. Tagged R1 protein was localized to the cytoplasmic and plasma membranes of transfected cells. Expression of the R1 gene in Rat-1 fibroblasts induced morphologic changes and focus formation, and injection of R1-expressing cells into nude mice induced the formation of multifocal tumors. A recombinant herpesvirus in which the STP oncogene of HVS was replaced by R1 immortalized T lymphocytes to interleukin-2-independent growth. These results indicate that R1 is an oncogene of RRV.     

Dual requirement for the Ig alpha immunoreceptor tyrosine-based activation motif (ITAM) and a conserved non-Ig alpha ITAM tyrosine in supporting Ig alpha beta-mediated B cell development

Surface Ig (sIg) expression is a critical checkpoint during avian B cell development. Only cells that express sIg colonize bursal follicles, clonally expand, and undergo Ig diversification by gene conversion. Expression of a heterodimer, in which the extracellular and transmembrane domains of murine CD8alpha or CD8beta are fused to the cytoplasmic domains of chicken Igalpha (chIgalpha) or Igbeta, respectively (murine CD8alpha (mCD8alpha):chIgalpha + mCD8beta:chIgbeta), or an mCD8alpha:chIgalpha homodimer supported bursal B cell development as efficiently as endogenous sIg. In this study we demonstrate that B cell development, in the absence of chIgbeta, requires both the Igalpha ITAM and a conserved non-ITAM Igalpha tyrosine (Y3) that has been associated with binding to B cell linker protein (BLNK). When associated with the cytoplasmic domain of Igbeta, the Igalpha ITAM is not required for the induction of strong calcium mobilization or BLNK phosphorylation, but is still necessary to support B cell development. In contrast, mutation of the Igalpha Y3 severely compromised calcium mobilization when expressed as either a homodimer or a heterodimer with the cytoplasmic domain of Igbeta. However, coexpression of the cytoplasmic domain of Igbeta partially complemented the Igalpha Y3 mutation, rescuing higher levels of BLNK phosphorylation and, more strikingly, supporting B cell development.     

The K15 protein of Kaposi's sarcoma-associated herpesvirus recruits the endocytic regulator intersectin 2 through a selective SH3 domain interaction

Kaposi's sarcoma-associated herpesvirus, also known as human herpesvirus 8, is closely associated with several cancers including Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. The rightmost end of the KSHV genome encodes a protein, K15, with multiple membrane-spanning segments and an intracellular carboxy-terminal tail that contains several conserved motifs with the potential to recruit interaction domains (i.e., SH2, SH3, TRAF) of host cell proteins. K15 has been implicated in downregulating B cell receptor (BCR) signaling through its conserved motifs and may thereby play a role in maintaining viral latency and/or preventing apoptosis of the infected B cells. However, K15's mode of action is largely unknown. We have used mass spectrometry, domain and peptide arrays, and surface plasmon resonance to identify binding partners for a conserved proline-rich sequence (PPLP) in the K15 cytoplasmic tail. We show that the PPLP motif selectively binds the SH3-C domain of an endocytic adaptor protein, Intersectin 2 (ITSN2). This interaction can be observed both in vitro and in cells, where K15 and ITSN2 colocalize to discrete compartments within the B cell. The ability of K15 to associate with ITSN2 suggests a new role for the K15 viral protein in intracellular protein trafficking.     

Immunoreceptor tyrosine-based activation motif-dependent signaling by Kaposi's sarcoma-associated herpesvirus K1 protein: effects on lytic viral replication

The Kaposi's sarcoma-associated herpesvirus (KSHV) K1 gene encodes a polypeptide bearing an immunoreceptor tyrosine-based activation motif (ITAM) that is constitutively active for ITAM-based signal transduction. Although ectopic overexpression of K1 in cultured fibroblasts can lead to growth transformation, in vivo this gene is primarily expressed in lymphoid cells undergoing lytic infection. Here we have examined function of K1 in the setting of lytic replication, through the study of K1 mutants lacking functional ITAMs. Expression of such mutants in BJAB cells cotransfected with wild-type K1 results in dramatic inhibition of K1 signal transduction, as judged by impaired activation of Syk kinase and phospholipase C-gamma2 as well as by diminished expression of a luciferase reporter gene dependent upon K1-induced calcium and Ras signaling. Thus, the mutants behave as dominantly acting inhibitors of K1 function. To assess the role of K1 in lytic replication, we introduced these K1 mutants into BCBL-1 cells, a B-cell lymphoma line latently infected with KSHV, and induced lytic replication by ectopic expression of the KSHV ORF50 transactivator. Expression of lytic cycle genes was diminished up to 80% in the presence of a K1 dominant negative mutant. These inhibitory effects could be overridden by tetradecanoyl phorbol acetate treatment, indicating that inhibition was not due to irreversible cell injury and suggesting that other signaling events could bypass the block. We conclude that ITAM-dependent signaling by K1 is not absolutely required for lytic reactivation but functions to modestly augment lytic replication in B cells, the natural reservoir of KSHV.     

Identification of the DNA sequence interacting with Kaposi's sarcoma-associated herpesvirus viral interferon regulatory factor 1

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma. The open reading frame (K9) of KSHV encodes viral interferon regulatory factor 1 (vIRF1), which functions as a repressor of interferon-mediated signal transduction. The amino-terminal region of vIRF1 displays significant homology to the DNA-binding domain of cellular interferon regulatory factors, supporting the theory that the protein interacts with specific DNA sequences. Here, we identify the consensus sequence of vIRF1-binding sites from a pool of random oligonucleotides. Moreover, our data show that vIRF1 interacts with the K3:viral dihydrofolate reductase:viral interleukin 6 promoter region in the KSHV genome.     

Tyrosine residues direct the ubiquitination and degradation of the NY-1 hantavirus G1 cytoplasmic tail

The hantavirus G1 protein contains a long C-terminal cytoplasmic tail of 142 residues. Hantavirus pulmonary syndrome-associated hantaviruses contain conserved tyrosine residues near the C terminus of G1 which form an immunoreceptor tyrosine activation motif (ITAM) and interact with Src and Syk family kinases. During studies of the G1 ITAM we observed that fusion proteins containing the G1 cytoplasmic tail were poorly expressed. Expression of G1 cytoplasmic tail constructs were dramatically enhanced by treating cells with the proteasome inhibitor ALLN, suggesting that the protein is ubiquitinated and degraded via the 26S proteasome. By using a 6-His-tagged ubiquitin, we demonstrated that the G1 cytoplasmic tail is polyubiquitinated and degraded in the absence of proteasome inhibitors. Expression of only the ITAM-containing domain also directed protein ubiquitination and degradation in the absence of upstream residues. Deleting the C-terminal 51 residues of G1, including the ITAM, stabilized G1 and blocked polyubiquitination and degradation of the protein. Site-directed mutagenesis of both ITAM tyrosines (Y619 and Y632) to phenylalanine also blocked polyubiquitination of G1 proteins and dramatically enhanced G1 protein stability. In contrast, the presence of Y627, which is not part of the ITAM motif, had no effect on G1 stability. Mutagenesis of just Y619 enhanced G1 stability, inhibited G1 ubiquitination, and increased the half-life of G1 by threefold. Mutating only Y632 had less of an effect on G1 protein stability, although Y619 and Y632 synergistically contributed to G1 instability. These findings suggest that Y619, which is conserved in all hantaviruses, is the primary signal for directing G1 ubiquitination and degradation. Collectively these findings indicate that specific conserved tyrosines within the G1 cytoplasmic tail direct the polyubiquitination and degradation of expressed G1 proteins and provide a potential means for down-regulating hantavirus G1 surface glycoproteins and cellular proteins that interact with G1.     

Kaposi's sarcoma-associated herpesvirus cytotoxic T lymphocytes recognize and target Darwinian positively selected autologous K1 epitopes

Kaposi's sarcoma-associated herpesvirus (KSHV) is the infectious cause of Kaposi's sarcoma (KS) and certain lymphoproliferations particularly in the context of human immunodeficiency virus (HIV) type 1-induced immunosuppression. The introduction of effective therapies to treat HIV has led to a decline in the incidence of KS, suggesting that immune responses may play a role in controlling KSHV infection and pathogenesis. Cytotoxic-T-lymphocyte (CTL) activity against KSHV proteins has been demonstrated; however, the identification of KSHV CTL epitopes remains elusive and problematic. Although the herpesvirus genomic layout is generally conserved, KSHV encodes a unique hypervariable protein, K1, with intense biological selection pressure at specific amino acid sites. To investigate whether this variability is partly driven by cellular immunity, we designed K1 peptides that match only the unique viral sequence for every individual studied here (autologous peptides). We identified functional CTL epitopes within K1's most variable areas, and we show that a given individual responds only to autologous peptides and not to peptides from other individuals. Furthermore, these epitopes are highly conserved sequences within KSHV isolates from a specific strain but are not conserved between different strains. We conclude that CTL recognition contributes to K1, and therefore to KSHV, evolution.     

Kaposi's sarcoma-associated herpesvirus K7 protein targets a ubiquitin-like/ubiquitin-associated domain-containing protein to promote protein degradation

Pathogens exploit host machinery to establish an environment that favors their propagation. Because of their pivotal roles in cellular physiology, protein degradation pathways are common targets for viral proteins. Protein-linking integrin-associated protein and cytoskeleton 1 (PLIC1), also called ubiquilin, contains an amino-terminal ubiquitin-like (UBL) domain and a carboxy-terminal ubiquitin-associated (UBA) domain. PLIC1 is proposed to function as a regulator of the ubiquitination complex and proteasome machinery. Kaposi's sarcoma-associated herpesvirus (KSHV) contains a small membrane protein, K7, that protects cells from apoptosis induced by various stimuli. We report here that cellular PLIC1 is a K7-interacting protein and that the central hydrophobic region of K7 and the carboxy-terminal UBA domain of PLIC1 are responsible for their interaction. Cellular PLIC1 formed a dimer and bound efficiently to polyubiquitinated proteins through its carboxy-terminal UBA domain, and this activity correlated with its ability to stabilize cellular I kappa B protein. In contrast, K7 interaction prevented PLIC1 from forming a dimer and binding to polyubiquitinated proteins, leading to the rapid degradation of I kappa B. Furthermore, K7 expression promoted efficient degradation of the p53 tumor suppressor, resulting in inhibition of p53-mediated apoptosis. These results indicate that KSHV K7 targets a regulator of the ubiquitin- and proteasome-mediated degradation machinery to deregulate cellular protein turnover, which potentially provides a favorable environment for viral reproduction.