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. 2003 Jan;77(1):592-9.
doi: 10.1128/jvi.77.1.592-599.2003.

Complement regulation by Kaposi's sarcoma-associated herpesvirus ORF4 protein

O Brad Spiller  1 , Mairi Robinson, Elizabeth O'Donnell, Steven Milligan, B Paul Morgan, Andrew J Davison, David J Blackbourn
Affiliations

Complement regulation by Kaposi's sarcoma-associated herpesvirus ORF4 protein

O Brad Spiller et al. J Virol. 2003 Jan.

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with three types of human tumor: Kaposi's sarcoma, multicentric Castleman's disease, and primary effusion lymphoma. The virus encodes a number of proteins that participate in disrupting the immune response, one of which was predicted by sequence analysis to be encoded by open reading frame 4 (ORF4). The predicted ORF4 protein shares homology with cellular proteins referred to as regulators of complement activation. In the present study, the transcription profile of the ORF4 gene was characterized, revealing that it encodes at least three transcripts, by alternative splicing mechanisms, and three protein isoforms. Functional studies revealed that each ORF4 protein isoform inhibits complement and retains a C-terminal transmembrane domain. Consistent with the complement-regulating activity, we propose to name the proteins encoded by the ORF4 gene collectively as KSHV complement control protein (KCP). KSHV ORF4 is the most complex alternatively spliced gene encoding a viral complement regulator described to date. KCP inhibits the complement component of the innate immune response, thereby possibly contributing to the in vivo persistence and pathogenesis of this virus.

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Figures

FIG. 1.
FIG. 1.
(A) Diagram of human DAF and KSHV KCP. Both human DAF and KSHV KCP contain SCR motifs (60 aa) and have serine/threonine (S/T) regions that are predicted to be highly O glycosylated (-O-). Putative N-glycosylation sites are marked with star symbols. Full-length KCP (KCP-F) has an additional dicysteine motif (C-C). The region demarcated by the hatched box is composed of hydrophobic and neutrally charged amino acids and is a potential transmembrane (TM) domain. (B) Best-fit amino acid alignment of SCR motifs in KCP and human DAF. Essential conserved cysteine residues are indicated (C), as are identical and similar (+) residues.
FIG. 2.
FIG. 2.
(A) ORF4 transcription following induction of lytic cycle replication. Northern blot analysis of mRNA isolated from HBL-6 cells incubated in the presence (+) or absence (−) of PMA for 48 h is shown. The positions of the RNA ladder molecular size markers are given. The upper-panel blot was probed with radiolabeled full-length ORF4 probe. For the lower panel, the blot was stripped of ORF4 probe and hybridized with a radiolabeled cellular gene (glyceraldehyde-3-phosphate dehydrogenase) probe. (B) Splicing of the ORF4 gene. RT-PCR analysis of ORF4 transcripts in BCBL-1 cells treated with phorbol ester revealed the presence of at least three transcript species, ORF-F, ORF4-M, and ORF4-S. Lanes: M, molecular size marker (1-kbp ladder); 1, ORF4 amplification of cDNA from BCBL-1 cells treated with phorbol ester for 48 h; 2, ORF4 amplification of BCBL-1 cells treated as for lane 1, with cDNA synthesis performed in the absence of reverse transcriptase enzyme; 3, p53 amplification of cDNA from BCBL-1 cells treated as for lane 1; 4, p53 amplification of BCBL-1 cells treated as for lane 1, with cDNA synthesis performed in the absence of reverse transcriptase enzyme and template. (C) Transcript map of the ORF4 gene. The locations of the transcript initiation site (identified by 5′-RACE-PCR) and the splice sites (identified by RT-PCR) for each of the transcripts are indicated. Local sequences at the splice sites are shown, with introns bracketed. Five of seven clones yielded position 1113 as the major transcription initiation site, and the two remaining clones indicated transcription start sites at either position 1102 or 1264. Nucleotide positions refer to the published KSHV nucleotide sequence (23). The 3′ ends of the ORF4 transcripts were not mapped.
FIG. 3.
FIG. 3.
(A) Flow cytometry analysis of CHO cells stably transfected with ORF4, human DAF cDNA, or empty vector control. Mean cellular fluorescence (fluor) for triplicate assays is given, with standard deviations. (B) Flow cytometry analysis of KCP and DAF expression on CHO-KCP cells following treatment with trypsin or PIPLC. Mean values for three separate analyses on different days are shown, with standard deviations.
FIG. 4.
FIG. 4.
(A) Western blot analyses of CHO-KCP cells. Blotting was performed with polyclonal anti-KCP or control anti-DAF antibodies on protein extracts prepared from 106 cell equivalents or cell-free supernatant (S/N) from 106 confluent cells as indicated. Molecular size markers are shown to the left. (B) Western blot analyses of KSHV-infected PEL cells. Analyses were performed with affinity-purified anti-KCP antibody lysates on 2.5 × 105 cell equivalents of the negative control BJAB cells (KSHV negative) or KSHV-infected HBL-6, BCBL-1, or JSC-1 PEL cell lines in the presence (+) or absence (−) of PMA. Molecular size markers are shown to the left.
FIG. 5.
FIG. 5.
Confocal microscopic analysis of KSHV-infected PEL cells. Cells were stained with affinity-purified anti-KCP antibody either following phorbol ester treatment for 48 h (A, C, and E) or without phorbol ester treatment (B, D, and F), (A and B) BCBL-1; (C and D) HBL-6; (E and F) JSC-1. Cell nuclei were identified by propidium iodide staining (red). Fields show a mid-cell section through cells which were induced into the lytic cycle (green and red) as well as cells which were not (red only).
FIG. 6.
FIG. 6.
Flow cytometry analysis of CHO cells stably transfected with recombinant forms of the ORF4 gene. Cells were stained with affinity-purified anti-KCP antibody. The cells were transfected with the ORF4-F gene [KCP-F (TM)], a full-length ORF4 gene in which the transmembrane (TM) region was swapped for the human DAF GPI anchor [KCP-F (GPI)], a medium-length ORF4 gene containing the GPI anchor and with the S/T region removed [KCP-M (GPI)], or a short-length ORF4 gene with the S/T region and the dicysteine motif removed [KCP-S (GPI)]. CHO cells transfected with empty vector (control) were also analyzed. Mean cellular fluorescence (fluor) values are given for triplicate analyses, with standard deviations.
FIG. 7.
FIG. 7.
KCP regulates complement activation. Flow cytometry analysis of C3 deposition with a human C3b/iC3b-specific monoclonal antibody following incubation of KCP-transfected or control cells with 10% human serum in the presence or absence of CHO-specific activating rabbit polyclonal antibody (as indicated) is shown. Background refers to the addition of sensitizing antibody and complement, but the anti-C3b antibody was omitted to show that neither complement nor the sensitizing antibody were detected nonspecifically by the PE-conjugated secondary antibody in flow cytometry. An antibody dilution of 0 refers to the addition of complement to the cells in the absence of sensitizing antibodies. Mean data are shown for triplicate assays, with standard deviations. These data represent those from one of four replicate experiments, which gave identical results.
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