Characterization of an anti-apoptotic glycoprotein encoded by Kaposi's sarcoma-associated herpesvirus which resembles a spliced variant of human survivin

Abstract

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).

Characterisation of the anti-apoptotic function of survivin-ΔEx3 during TNFα−mediated cell death

“…pCR3.1-survivin and pCR3.1-survivin-DEx3 expressing haemagglutinin-tagged forms were described previously (Wang et al, 2002). pCR3.1-survivin-DEx3(DBIR) (deleted aa 38 -43) and pCR3.1-survivin-DEx3(DBH2) (deleted aa 101 -107) were created using the QuickChange mutagenesis kit (Stratagene, Amsterdam, The Netherlands) to introduce deletion mutations in pCR3.1-survivin-DEx3.…”

“…The expression of recombinant glutathione S-transferase (GST) fusion proteins, GST pull-down assays, in vivo co-immunoprecipitations, and Western blots were described previously (Wang et al, 2002). The following primary antibodies were used: antisurvivin (6E4 mAb, Cell Signaling, Danvers, USA), anti-HA (BabCo, Cambridge, MA, USA), anti-cytochrome c (Upstate, Chandlers Ford, UK), anti-Bcl-2 (BD Pharmingen, Oxford, UK), anti-caspase-3 (BD Pharmingen).…”

“…We found that vIAP is structurally and functionally related to survivin-DEx3: both proteins contain a disrupted BIR domain, an MTS, and a putative BH2 domain (Wang et al, 2002). However, it is not yet clear, whether survivin-DEx3 also achieves its anti-apoptotic function in a similar way to that of vIAP.…”

“…The protein has a unique Cterminus, and novel anti-apoptotic features could be mediated by this region. We previously suggested that a mitochondrial targeting signal (MTS) and a putative BH2 domain may be located in this region (Wang et al, 2002). Survivin-DEx3 preferentially localises in the nucleus during late G1 to G2 phases of the cell cycle (Fortugno et al, 2002).…”

“…Previously, we showed that a viral protein vIAP (viral inhibitorof-apoptosis protein), which is encoded by ORF K7 of human Kaposi's sarcoma-associated herpesvirus (KSHV), is an adaptor between Bcl-2 and activated caspase-3, thereby, enabling Bcl-2 to inhibit caspase-3 activity (Wang et al, 2002). We found that vIAP is structurally and functionally related to survivin-DEx3: both proteins contain a disrupted BIR domain, an MTS, and a putative BH2 domain (Wang et al, 2002).…”