Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is the infectious cause of KS and is also linked to the pathogenesis of certain lymphoproliferations (4,14). It is proposed that KSHV latent proteins are directly involved in modulating signal transduction pathways and cellular circuits leading to uncontrolled cell proliferation (2).At the far right-hand end of the KSHV genome, open reading frame (ORF) K15 encodes a putative transmembrane protein in the same genomic location as the Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) (5,7,12,39). K15 resembles LMP2A not only in genomic location but also in its splicing pattern and predicted protein structure. Two highly divergent forms of K15 have been identified: the predominant (P) and minor (M) forms (5, 12, 39). These two alleles possess only 33% amino acid identity yet retain 12 membrane-spanning domains and a putative cytoplasmic signal-transducing carboxyl terminus (C terminus) (5). The C terminus of K15 has potential signaling motifs, including Src homology 2 and 3 binding domains (SH2-B and SH3-B, respectively) (12). A CD8-K15 C-terminal chimeric protein was shown to be constitutively tyrosine phosphorylated within the SH2-B motif (5). Like LMP2A, this CD8-K15 chimeric protein modulates B-cell receptor (BCR) signal transduction. The mechanism(s) of signal transduction is unknown but appears to be distinct from that of LMP2A and does not involve intracellular free calcium mobilization (5).In addition, the C terminus of K15 has sequences similar to those found in EBV LMP1, including a putative tumor necrosis factor receptor-associated factor (TRAF) binding site. K15 therefore appears to be a hybrid of a distant evolutionary relative of both EBV LMP1 and LMP2A (13). The putative C terminus of K15 has been shown to interact with the TRAFs (12), and we have also shown that K15 can indeed activate NF-B via this putative TRAF binding site (unpublished data). By way of activating NF-B, LMP1 of EBV plays an essential role in EBV-induced transformation of B lymphocytes (3,16,21). NF-B activation also appears to be essential for the proliferation potential of KSHV positive primary effusion lymphoma (PEL) cells (22), but whether all of this NF-B activity in PEL cells is due to K15 expression is not yet known.Although K15 mRNA has been demonstrated in PEL cells (5, 12, 39), it is not known whether the K15 protein is actually expressed in latently infected tumor cells. The size of endogenous protein, its exact subcellular localization, and its cellular binding partners have not previously been determined.We generated a monoclonal antibody (MAb) against K15 and show here that when K15 cDNA is ectopically expressed we detect the predicted 50-kDa form as well as a series of smaller proteolytically cleaved forms, of which the 35-and 23-kDa species are predominant. Deletion of the initiator AUG of the K15 ORF abolished protein expression, suggesting that the 50-kDa form of K15 is a precursor which is subsequently proteolytically processed into smaller species. We ...

We have investigated the expression and function of a novel protein encoded by open reading frame (ORF) K7 of Kaposi's sarcoma-associated herpesvirus (KSHV). Computational analyses revealed that K7 is structurally related to survivin-DEx3, a splice variant of human survivin that protects cells from apoptosis by an unde®ned mechanism. Both K7 and survivinDEx3 contain a mitochondrial-targeting sequence, an N-terminal region of a BIR (baculovirus IAP repeat) domain and a putative BH2 (Bcl-2 homology)-like domain. These suggested that K7 is a new viral antiapoptotic protein and survivin-DEx3 is its likely cellular homologue. We show that K7 is a glycoprotein, which can inhibit apoptosis and anchor to intracellular membranes where Bcl-2 resides. K7 does not associate with Bax, but does bind to Bcl-2 via its putative BH2 domain. In addition, K7 binds to active caspase-3 via its BIR domain and thus inhibits the activity of caspase-3. The BH2 domain of K7 is crucial for the inhibition of caspase-3 activity and is therefore essential for its anti-apoptotic function. Furthermore, K7 bridges Bcl-2 and activated caspase-3 into a protein complex. K7 therefore appears to be an adaptor protein and part of an anti-apoptotic complex that presents effector caspases to Bcl-2, enabling Bcl-2 to inhibit caspase activity. These data also suggest that survivin-DEx3 might function by a similar mechanism to that of K7. We denote K7 as vIAP (viral inhibitorof-apoptosis protein).

Survivin is an oncogenic protein involved in cell division and acts as an anti-apoptotic factor. It is highly expressed in most cancers and is associated with chemotherapy resistance, increased tumour recurrence, and shorter patient survival. This makes anti-survivin therapy an attractive cancer treatment strategy. These functions are mediated by several survivin spliced variants, whose expression may correlate with cancer progression. One of the spliced variants, survivin-DEx3, is known to inhibit apoptosis, through undefined mechanisms. Here, we characterised these mechanisms upon TNFaÀmediated apoptosis, and showed that survivin-DEx3 acts as an adaptor, allowing the formation of a complex between Bcl-2 and activated caspase-3. The Bcl-2/survivin-DEx3 complex, but not survivin-DEx3 itself, inhibits the activity of caspase-3. Bcl-2 is therefore linked to the postmitochondrial apoptotic machinery by survivin-DEx3. Thus, survivin-DEx3 plays a key role in the inhibition of caspase-3 activity, and in the control of the mitochondrial checkpoint of apoptosis. This study suggests that targeting survivin-DEx3, rather than survivin alone, could be relevant for treating human cancers.