Identification of a spliced gene from Kaposi's sarcoma-associated herpesvirus encoding a protein with similarities to latent membrane proteins 1 and 2A of Epstein-Barr virus

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV-8) is a novel herpesvirus implicated as the causative agent of Kaposi's sarcoma (KS), primary effusion lymphoma, and some cases of multicentric Castleman's disease. KSHV persists in the majority of KS spindle (endothelial tumor) cells and lymphoid cells in a latent form, with only a limited set of viral genes expressed in a tissue-specific manner. Here, we report the identification of a family of alternatively-spliced transcripts of approximately 7.5 kb expressed in latently infected body cavity-based lymphoma (BCBL) cell lines which are predicted to encode membrane proteins with similarities to the LMP2A and LMP1 proteins of Epstein-Barr virus. In two highly divergent sequence variants of the right end of the KSHV genome, alternative splicing of eight exons located between KSHV ORF 75 and the terminal repeats yields transcripts appropriate for proteins with up to 12 transmembrane domains, followed by a hydrophilic C-terminal, presumably cytoplasmic, domain. This C-terminal domain contains several YxxI/L motifs reminiscent of LMP2A and a putative TRAF binding site as in LMP1. In latently (persistently) infected BCBL cells the predominant transcript utilizes all eight exons, whereas in phorbol-ester-induced cells, a shorter transcript, lacking exons 4 and 5, is also abundant. We also found evidence for an alternative use of exon 1. Transfection of an epitope-tagged cDNA construct containing all exons indicates that the encoded protein is localized on cell surface and intracellular membranes, and glutathione S-transferase pull-down experiments indicate that its cytoplasmic domain, like that of LMP1, interacts with TRAF1, -2, and -3. Two of 20 KS patients had antibodies to the hydrophilic C-terminal domain, suggesting that the protein is expressed in vivo.

FIG. 1

Location of exons for the KSHV membrane protein in the two major KSHV sequence variants represented by HBL-6 and GK18/BCP-1. (Top) Schematic diagram of conserved genomic regions (open boxes) and genes found only in KSHV or closely related gamma-2 herpesviruses (black boxes). The orientation of the black boxes reflects their transcriptional orientation. The position and organization of a cosmid clone (Cos 83) obtained from a classic KS case (GK18; see text) is shown enlarged. (Bottom) Location of exons 1 to 8 for KSHV K15/LAMP in HBL-6 and GK18/BCP-1. The location of the 4.3-kb probe that was generated from GK18 and which was used to screen a latent HBL-6 cDNA library is indicated. Short horizontal lines mark the locations of primers used in this study. A bracket above the HBL-6 diagram indicates the position of the ORF K15 initially predicted by Russo et al. (56). An additional T residue within exon 1 of the HBL-6 sequence results in the entire exon 1 being in frame with exons 2 to 8 (see text).

FIG. 2

(a) Northern blot analysis of two KSHV cell lines. A 4.3-kb genomic probe generated from GK18 (Fig. 1) was used to probe total cellular RNA as described in Materials and Methods. Total RNA isolated from the RAJI cell line was used as a negative control. This probe detects both the 7.5-kb class II K15/LAMP transcript and the class I 4.5-kb LT3 transcript (see text and reference 58). The equal intensity of the (noninducible) class I transcript serves as a loading control. (b) The same blot was analyzed with a probe generated from exons 2 to 5 of the HBL-6 version of the spliced transcript described here which detects only the 7.5-kb K15/LAMP transcript (see text). I, induced; U, uninduced.

FIG. 3

Relative abundance of spliced mRNAs. (A) Southern blot of RT-PCR products of BCP-1. RNA was reverse transcribed and amplified by using primers ex8arev and ex1afor. Resulting products were analyzed by Southern blotting with a probe derived from exon 8 and were also cloned into pGEM-T and sequenced to obtain the splicing patterns. Lanes 1 and 2, TPA-treated BCP-1 cells, lanes 3 and 4, untreated BCP-1 cells; lane 5, genomic BCP-1 DNA; lane 6, water control. RT was added during first-strand synthesis in lanes 1 and 3 but not in lanes 2 and 4. The positions of the two most abundant transcripts containing all eight exons (i) or lacking exons 4 and 5 (ii) are indicated, as are two minor transcripts lacking exons 2, 3, and 5 (iii) or exons 2, 3, 4, and 5 (iv). The splicing patterns of these transcripts are shown in the lower panel. (B) Southern blot of RT-PCR of BCP-1 showing alternative splice within exon 1. cDNA was synthesized from uninduced BCP-1 RNA by using primer LAMParev, followed by PCR amplification of the region encompassing exons 1 and 2 with primers LRH4rev and ex1afor. Lane 1, BCP-1 DNA; lanes 2 and 3, BCP-1 RNA, respectively, with and without RT during first-strand synthesis. The conventional splice yields a product of 294 bp (i), while the alternative splice yields a product of 115 bp (ii), and unspliced RNA yields a product of 404 bp. The splicing patterns of the conventional splice (i) and the alternative splice (ii) are shown in the lower panel. Because of the locations of the primers used, we cannot infer which exons downstream of exon 2 are present in transcripts utilizing this alternative splice site. Hence, these are indicated by a dotted line in the lower panel.

FIG. 4

Sequence alignment of LAMP proteins. Protein sequence alignment of the 8-exon form of LAMP from the two KSHV sequence variants represented by BCP-1/GK18 and HBL-6. The position of predicted membrane-spanning domains is indicated by dotted lines above and below the sequence, and that of individual exons is marked above the sequence by arrows. Conserved sequence motifs reminiscent of YxxI/L motifs in LMP-2A are double underlined, and the putative TRAF binding site reminiscent of CTAR-1 of LMP-1 is shown in bold (see text).

FIG. 5

Western blot of recombinant cytoplasmic terminal domain of LAMP. A recombinant MBP-exon 8 fusion of BCP-1 was expressed in Escherichia coli, affinity purified, and reacted with sera from a KS patient as described in Materials and Methods. Lane 1, reactivity of a serum from a patient with classic KS with the uncleaved fusion protein, the expected size of which is 57 kDa. Lane 2, reactivity of the same serum with factor Xa-digested protein. The expected size of the free cytoplasmic domain of LAMP is 15 kDa. The same serum did not react with control MBP protein (lane 3) or with control protein plus factor Xa (lane 4). A serum sample from a healthy subject was used as a control on uncleaved fusion protein (lane 5), fusion protein plus factor Xa (lane 6), control MBP (lane 7), or MBP plus factor Xa (lane 8). Molecular size markers (in kilodaltons) are shown on the left, and the position of the uncleaved MBP-fusion protein and the cleaved C-terminal domain of K15/LAMP are indicated by arrowheads.

FIG. 6

Transfection of K15/LAMP expression constructs into 293 cells. Immunofluorescence assay of 293 cells transfected with mammalian expression constructs for K15/LAMP. 293 cells were transfected with either a genomic (A) or a cDNA (B) myc-tagged construct of the ORF for the membrane protein, and expression was analyzed by immunofluorescence with an anti-myc antibody on permeabilized cells.

FIG. 7

In vitro binding of TRAF1, TRAF2, and TRAF3 to the carboxy-terminal domain of K15/LAMP. FLAG-tagged TRAF1, TRAF2, or TRAF3 expression vectors were transiently transfected into HEK 293 cells, and the interaction between the carboxy-terminal domain of K15/LAMP of GK18/BCP-1 and the TRAF proteins was examined by pull-down assays as described in Materials and Methods. Lanes 1 to 3, TRAF1 transfected cells; lanes 4 to 6, TRAF2-transfected cells; lanes 7 to 9, TRAF-3-transfected cells. Lanes 1, 4, and 7, proteins bound to GST-LAMP cytoplasmic domain (GK18/BCP-1); Lanes 2, 5, and 7, proteins bound to GST; lanes 3, 6, and 9, total cell lysates. Similar results were obtained with GST-C-terminal fusion protein of the HBL-6 sequence (data not shown).