Identification of the Novel K15 Gene at the Rightmost End of the Kaposi's Sarcoma-Associated Herpesvirus Genome

Abstract

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 CD8␣ 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.DNA sequences of a novel member of the herpesvirus group, called Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8, have been widely identified in Kaposi's sarcoma (KS) tumors from human immunodeficiency virus-positive and -negative patients (4,5,21,32,35). KSHV has also been identified in body cavity-based lymphoma (BCBL) and some forms of Castleman's disease (4, 5, 28). These are principally or exclusively of B-cell origin. Cell lines have been derived from some of the BCBL, and while some harbor both Epstein-Barr virus (EBV) and KSHV, others harbor KSHV only. The genomic sequence indicates that KSHV is a gammaherpesvirus that is closely related to herpesvirus saimiri (HVS) (25, 29) and the recently isolated rhesus monkey rhadinovirus (7,33).In primary lymphocytes, cross-linking the B-cell receptor (BCR) or T-cell receptor (TCR) leads to an intricate signal cascade including the recruitment and activation of the src family tyrosine kinases; the subsequent activation and recruitment of other kinases, phosphatases, or adapter proteins; the hydrolysis of phospholipids; the mobilization of intracellular calcium; the activation of protein kinase C; ...

Multi-transmembrane protein K15 of Kaposi's sarcoma-associated herpesvirus targets Lyn kinase in the membrane raft and induces NFAT/AP1 activities

“…We previously demonstrated that the cytoplasmic region of K15 inhibited the proximal signal transduction of BCR to cellular tyrosine phosphorylation and calcium mobilization. Based on these results, we hypothesized that K15 is not only a structural homolog but also a functional homolog to EBV LMP2A protein (Choi et al, 2000). However, detailed molecular mechanism remained unclear.…”

“…The preparation of CD-delta ( = CD8-∆) and CD8-K15 chimera constructs is described in Choi et al (2000).…”

“…Full length K15 cDNA from BCBL1 was cloned into pCS4-3xflag (BamHI and NcoI sites) and a kozak sequence (GCCACC) was placed in front of the initiation methionine as described in Choi et al (2000). The preparation of CD-delta ( = CD8-∆) and CD8-K15 chimera constructs is described in Choi et al (2000).…”

“…KSHV K15 open reading frame exhibits a dramatic sequence variation among different isolates of PEL cells in addition to a complex splicing pattern (Glenn et al, 1999;Poole et al, 1999;Choi et al, 2000). We previously demonstrated that the cytoplasmic region of K15 inhibited the proximal signal transduction of BCR to cellular tyrosine phosphorylation and calcium mobilization.…”

Modulation of T-Cell Receptor Signal Transduction by Herpesvirus Signaling Adaptor Protein

“…To do this, lymphotropic gammaherpesviruses, particularly Epstein-Barr virus, KSHV, and HVS, have been shown to deregulate lymphocyte signal transduction (8,27,34). Two HVS proteins, ORF14 and Tip, have been previously found to target and alter TCR signal transduction.…”