Fig. 2. K7 is a lytic viral gene. (A) Schematic representation of the structure of the K7 gene. The cDNA probe used in northern blot analysis and the K7-specific primers used in RT–PCRs are shown. (B) Northern blot analyses of K7 RNAs. The KSHV latently infected PEL cell line BCP-1 and the KSHV-negative Ramos cell line were lytically induced by TPA, and total RNA was extracted at the indicated time points. Blots were first probed against K7 mRNA, then reprobed with a GAPDH probe or with another KSHV gene, PAN, to indicate the induction of viral lytic cycle. (C) RT–PCR analysis for K7 transcripts. RNA from TPA-treated, KSHV-infected PEL cells (BC3) was collected at the indicated time points. M, 100-bp DNA fragment markers. (+), RNA from TPA-treated BCP-1 cells (lane 2) and cDNA of K7 (lane 3) were used as positive controls.

Fig. 4. K7 localizes to the mitochondria and ER. (A) Confocal microscopy of HeLa cells transfected with HA-tagged K7. Transfected cells were stained with an anti-HA mAb at 48 h post-transfection. (B) Colocalization of K7 and mitochondria. K7-transfected HeLa cells were stained with antibody to HA (green), and mitochondria were stained with CMXRos (red). The cell nucleus was stained with Hoechst 33258 dye (blue). Partial colocalization is seen upon overlay of these images. (C) Subcellular fractionation of HeLa cells transfected with K7. Transfected cells were separated into nucleoplasmic (NP), cytoplasmic (C), mitochondria (M) and ER fractions. The fractions were probed by western blots with antibodies to HA (for tagged K7), human Bcl-2 and Bax. (D) The N-terminal 70 amino acids of K7 is sufficient and essential for mitochondrial targeting. HeLa cells transfected with the indicated plasmids were subjected to subcellular fractionation analysis. The fractions were probed by western blots with a polyclonal antibody to GST to detect each fusion protein.

Fig. 6. K7 interacts with Bcl-2 and activated caspase-3 via its BH2 and BIR domains, respectively. (A) K7 and Bax do not bind in vitro. HeLa cell extracts, which contain endogenous Bax, were incubated with GST or GST-K7-CT recombinant proteins as described in Figure 33E. Specifically retained proteins were blotted with either anti-Bax Ab (upper panel) or anti-GST Ab (bottom panel). (B) Bax does not bind to K7 in vivo. HeLa cells were transfected with Bax plus the indicated expression plasmids and then subjected to GST pull-down assays 48 h post-transfection. Asterisks indicate the expressed GST fusion proteins. (C) K7 heterodimerizes with Bcl-2 in vivo. HeLa cells were cotransfected with equal amounts of Bcl-2 and the indicated plasmids. Transfected cells were harvested and GST pull-down assays were performed to detect coprecipitated Bcl-2. (D) The C-terminal BH2 domain of K7 is essential for Bcl-2 interaction. HeLa cell extracts were subjected to in vitro GST pull-down assays with the indicated K7 mutants. Specifically bound Bcl-2 was detected by an anti-Bcl-2 mAb. The GST–VDAC3 recombinant protein served as a positive control. (E) K7 interacts with active caspase-3. HEK 293 cells were treated for 8 h with 10 ng/ml TNF-α plus 1 µg/ml CHX, and cell lysates were then mixed with the indicated recombinant proteins. Bounded caspase-3 was analysed by an anti-caspase-3 mAb, which recognizes both pro- and activated caspase-3. (F) K7 and active caspase-3 interact in vivo through the BIR domain. Cell extracts were made 48 h post-transfection from HeLa cells cotransfected with Bax and wild-type or BIR mutant of K7. Anti-HA mAb and isotype control were used to pull down K7, followed by western blot analysis with anti-caspase-3 mAb. (G) Bcl-2 precipitates active caspase-3 in the presence of K7. HeLa cells were cotransfected with 15 µg each of plasmids encoding K7 (or empty vector) and Bax. Cell extracts were prepared 2 days later and immunoprecipitations were performed using anti-Bcl-2 mAb. Immune complexes were analysed by western blot with anti-caspase-3 mAb. (H) K7 is an inhibitor of activated caspase-3. Fluorescent substrate (Sub) and recombinant activated caspase-3 (rCas3) were mixed with RIPA buffer or with protein extracts from K7-transfected or non-transfected HeLa cells. Reaction mixtures were incubated for 1 h at 37°C before determinion of fluorescence. A caspase-3 inhibitor was used as a positive control.

Fig. 1. K7 has sequence and predictive structural similarities to survivin-ΔEx3. (A) Schematic representation of the structure of K7. The MTS, the transmembrane (TM) domain, the BIR-like domain (N-BIR) and the putative BH2 domain of K7 are indicated. Several putative post-translational modification sites of K7, such as the N-myristoylation site (N-myr), SH3 motif, protein kinase C phosphorylation sites (PKC) and the N-glycosylation site (N-Gly), are also shown. (B). Comparison between K7 and survivin BIR domains. Sequence alignment of K7 and survivin BIR domains. (Upper panel) The BIR domain of survivin is underlined. (Bottom left) Ribbon representation of the K7 BIR domain. The α-helices, β-strands and turns are represented as red coils, yellow arrows and blue loops, respectively, and the corresponding amino acids are shown in the upper panel. (Bottom right) Superimposed image of K7 and survivin BIR domains. K7 is represented by red strands and survivin by blue strands. (C) Endogenous expression of survivin and survivin-ΔEx3, and the structures of these two isoforms. The kinetochore-binding domain of survivin, which is essential for chromosome segregation and cytokinesis, is indicated. R/K-rich, Arg/Lys-rich region. PCR analysis for cDNAs of survivin isoforms in a human fetal cDNA library is also shown (bottom panel). (D) Alignment of different BIR domains. The species designations used for the alignment, and the primary accession number or database entry number for each sequence are as follows, with the indicated accession numbers: K7, AAC57096. Human BIR domain proteins: survivin, 2315863; XIAP (X-linked IAP), P98170; cIAP1, NP_001157; cIAP2, NP_001156. Mouse: BRUCE (BIR repeat containing ubiquitin-conjugating enzyme), CAA76720; TIAP, BAA28266; NAIP (neuronal apoptosis inhibitory protein), AAB69223. Drosophila: Deterin, XP_081836. Schizosaccharomyces pombe BIR protein SpIAP, NP_587866. Caenorhabditis elegans BIR protein CeIAP, NP_505949. Numbers to the left of the sequences indicate the positions of the amino acids in each protein. Residues conserved in the majority of the sequences are in black boxes, whereas similar sequences are in grey. (E) Phylogenic relationship of different BIR domains. The alignment data shown in (D) was used to construct a Phylogenic tree, which was derived by maximum likelihood searching by the TreeView program. The divergence scale of the across-page branches is indicated. (F) Alignment of the BH2 domain of Bcl-2 family members with K7 and survivin-ΔEx3. The following human and viral proteins are used in this alignment: Bax-β (AAA03620), Bax-α (AAA03619), Bcl-2 (AAA35591), Bcl-XL (CAA80661), Bcl-W (NP_004041), EBV (Epstein–Barr virus Bcl-2; CAA01638), HVP (herpesvirus papio Bcl-2; AAF99596), KSHV (KSHV Bcl-2; AAB62596), RHV (Rhesus herpesvirus Bcl-2; AAD21342), HVS (herpesvirus simiri Bcl-2; CAA45639), AHV3 (Ateline herpesvirus 3 Bcl-2; NP_047987), BHV4 (bovine herpesvirus 4 Bcl-2; AAD34361), Bak (Q16611), and ASFV (African swine fever virus Bcl-2; NP_042735). The R/K-rich region in survivin-ΔEx3 is underlined. (G) Phylogenic tree for BH2 domain-containing apoptotic regulators. The same BH2 domains shown in Figure 13F were used to construct this tree.

Fig. 3. K7 expressed as a predicted 16 kDa protein and as a 19 kDa glycoprotein. Cells were transfected with the indicated plasmids, and protein extracts were collected 48 h after transfection and probed by western blots with an anti-HA mAb. (A) K7 expression pattern. Two different forms of K7 were detected in transfected cells. (B) The 19 kDa form of K7 is a glycoprotein. (Left panel) HeLa cells transfected with K7 were treated with different doses of tunicamycin to inhibit N-glycosylation for 24 h before extraction. (Right panel) A point mutation (N108Q) on the N-glycosylation site of K7 could prevent K7 from modification. (C) The N-glycosylation of K7 is increased after TNF-α treatment. HeLa cells transfected with a K7-expressing plasmid or with vector alone were exposed 24 h post-transfection to 10 ng/ml TNF-α plus 1 µg/ml CHX. (D) K7 has a short half-life. K7-transfected HeLa cells were treated with 10 µg/ml CHX at 48 h post-transfection to block protein synthesis. Cell extracts were collected at indicated time points. (Lower panel) Same blot stained with Coomassie Blue to show equal loading of protein in each lane. (E) K7 forms homodimers. HeLa cell extracts, which contain transfected K7, were incubated with GST, GST-K7-CT or GST-K7-BIR. Specifically bound proteins were eluted from beads with SDS–PAGE sample buffer before analysis.

Fig. 5. K7 inhibits apoptosis. (A) K7 protects cells from death receptor mediated apoptosis. HeLa cells transfected with a K7-expressing plasmid or with vector alone were exposed 24 h post-transfection to 10 ng/ml TNF-α or 100 ng/ml anti-Fas mAb plus 1 µg/ml CHX. Apoptotic cells were double stained with Annexin V–FITC/PI and counted by flow cytometry. Results are expressed as the mean ± SD of three independent experiments. (B) K7 rescues Bax-induced apoptosis. HeLa cells were transfected with 100 ng of Bax-expression plasmid plus 400 ng (4:1), 900 ng (9:1) or 1800 ng (18:1) of the indicated constructs. Forty-eight hours post-transfection, all cells, both adherent and in suspension, were pooled, washed and stained with the mitochondrial membrane-specific dye CMXRosamine to assess the mitochondrial membrane potential (ΔΨ_m), and were then analysed by fluorescence activated cell sorting (FACS). Inhibition percentage was calculated as follows: (% apoptosis in Bax-transfected cells) – (% apoptosis in the indicated DNA-cotransfected cells)/(% apoptosis in Bax-transfected cells), where % apoptosis is the percentage of apoptotic cells relative to transfected cells. (C) Both the putative BH2 and the BIR domains of K7 appear to be essential for inhibition of TNF-α induced cell death. HeLa cells transfected with the indicated expression plasmids were treated with TNF-α plus CHX. Twenty-four hours after treatment, cell viability was monitored by PI staining and by FACS counting (top panel). (Bottom panel) The expression of wild or mutant types is shown.

Fig. 7. Proposed model of K7 (vIAP) function. One function of K7 (vIAP) may be as a molecular adaptor to present activated caspases to anti-apoptotic proteins, such as Bcl-2.