Choi J K, Lee B S, Shim S N, Li M, Jung J U
Department of Microbiology and Molecular Genetics, New England Regional Primate Research Center, Harvard Medical School, Southborough, Massachusetts 01772, USA.
J Virol. 2000 Jan;74(1):436-46.
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.
卡波西肉瘤相关疱疹病毒（KSHV）在一个与爱泼斯坦-巴尔病毒（EBV）编码LMP2A的基因相当的位置编码一个名为K15的独特开放阅读框。从体腔淋巴瘤（BCBL）细胞中分离出的K15表现出显著的序列变异和复杂的剪接模式。然而，所有K15等位基因在其细胞质区域具有相似的组织方式以及潜在的SH2和SH3结合基序。Northern印迹分析表明，K15在潜伏感染的BCBL-1细胞中弱表达，其表达水平在十四烷酰佛波醇乙酸酯刺激下显著诱导。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定，K15编码40至55 kDa的蛋白质，并且定位于细胞质和质膜。为了证明K15蛋白的信号转导活性，我们构建了一种嵌合蛋白，其中人CD8α多肽 的细胞质尾巴被KSHV K15的细胞质尾巴取代。虽然CD8-K15嵌合体在用抗CD8抗体刺激时不能引发细胞信号转导，但它显著抑制B细胞受体信号传导，酪氨酸磷酸化的抑制和细胞内钙动员证明了这一点。这种抑制需要K15细胞质区域中假定的SH2或SH3结合基序。对CD8-K15嵌合体的生化研究表明，K15的细胞质区域组成性酪氨酸磷酸化，并且K15假定的SH2结合基序内的酪氨酸残基是磷酸化的主要位点。这些结果表明，KSHV K15在基因组位置、剪接模式和蛋白质结构以及细胞质区域中存在功能性信号转导基序方面类似于LMP2A。因此，KSHV K15可能是EBV LMP2A的远缘进化亲属。
