In an effort to derive monoclonal antibodies (mAb) which can induce production of macrophage-activating factor (MAF) by cloned murine cytolytic T lymphocyte (CTL) lines, we have fused spleen cells from a rat immunized with a CTL clone with the nonsecreting mouse myeloma X63-Ag8.653. Three mAb (designated I-22, III-5 and V-8) were found to stimulate MAF production by the immunizing CTL clone and (with a single exception) two other unrelated CTL clones. However, none of these mAb inhibited the cytolytic activity of the clones. Immunoprecipitation studies indicated that the three mAb reacted primarily with a 25-30-kDa protein which could not be distinguished from that precipitated by either a reference anti-Thy-1.2 mAb or a polyclonal rabbit anti-Thy-1 antiserum. Moreover, competition binding experiments demonstrated that the three mAb competed with each other and with the reference anti-Thy-1.2 mAb. Flow cytofluorometric analysis of the strain distribution of the molecules defined by the mAb revealed that two of the antibodies (I-22 and III-5) were directed against nonpolymorphic determinants of Thy-1, whereas V-8 mAb reacted only with Thy-1.2+ lymphocytes. One of the mAb (III-5) was also able to stimulate proliferation and interleukin 2 secretion by normal splenic T cells. Since mAb directed against a number of other surface structures on CTL clones did not stimulate MAF production, it thus appears that Thy-1 (or molecules associated with Thy-1) may play a functional role in T lymphocyte triggering.

Abstract. The latent membrane protein 1 (LMP1) encoded by the Epstein-Barr virus acts like a constitutively activated receptor of the tumor necrosis factor receptor (TNFR) family and is enriched in lipid rafts. We showed that LMP1 is targeted to lipid rafts in transfected HEK 293 cells, and that the endogenous TNFR-associated factor 3 binds LMP1 and is recruited to lipid rafts upon LMP1 expression. An LMP1 mutant lacking the C-terminal 55 amino acids (CD55) behaves like the wild-type (WT) LMP1 with respect to membrane localization. In contrast, a mutant with a deletion of the 25 N-terminal residues (ND25) does not concentrate in lipid rafts but still binds TRAF3, demonstrating that cell localization of LMP1 was not crucial for TRAF3 localization. Moreover, ND25 inhibited WT LMP1-mediated induction of the transcription factors NF-kB and AP-1. Morphological data indicate that ND25 hampers WT LMP1 plasma membrane localization, thus blocking LMP1 function.Key words. LMP1; lipid rafts; TRAF3; TRAF2.Epstein-Barr virus (EBV) is a ubiquitous lymphotropic herpesvirus that causes infectious mononucleosis and is consistently associated with nasopharyngeal carcinoma and large cell lymphomas of the immunocompromised host. EBV infection of B lymphocytes is mostly nonlytic. It results in the expression of a limited number of nuclear (Epstein-Barr nuclear antigens, EBNAs) or membrane (latent membrane proteins, LMPs) proteins, and in perpetual cell proliferation [1]. Although it has a well-known tropism for B lymphocytes and epithelial cells, EBV also infects T lymphocytes, monocytes and granulocytes [2]. The virus-encoded LMP1 is a major driving force in the process of neoplastic transformation. LMP1 is the only EBV product that has transforming effects on non-lymphoid cells [3,4]. In lymphoid cells, LMP1 induces most of the phenotypic changes observed during EBV infection, including activation of NF-kB and Bcl-2, as well as up-regulation of adhesion molecules such as LFA-1 and ICAM-1 [5 -7]. LMP1 is a constitutively active receptor-like molecule that alters cell growth [8]. In addition, it mimics CD40 [9] and engages signaling proteins of the tumor necrosis factor (TNF) family [10]. Finally, LMP1 is a powerful inducer of NF-kB-mediated transcription [11]. Two motifs are critical for cell transformation and activation of NF-kB. The first, designated as the C-terminal activation region 1 (CTAR1), spans residues 199 -231. It interacts with the TNF receptor-associated factors TRAF1, TRAF2, TRAF3 and TRAF5 [7,12,13]. TRAF2 activates NF-kB by targeting the NF-kB-inducing kinase (NIK) followed by activation of the IkBa and IkBb kinases [14].

Glycosyl-inositolphospholipid (GPL) anchoring structures are incorporated into GPL-anchored proteins immediately posttranslationally in the rough endoplasmic reticulum, but the biochemical and cellular constituents involved in this "glypiation" process are unknown. To establish whether glypiation could be achieved in vitro, mRNAs generated by transcription of cDNAs encoding two GPL-anchored proteins, murine Thy-i antigen and human decay-accelerating factor (DAF), and a conventionally anchored control protein, polymeric-immunoglobulin receptor (IgR), were translated in a rabbit reticulocyte lysate. Upon addition of dog pancreatic rough microsomes, nascent polypeptides generated from the three mRNAs translocated into vesicles. Dispersal of the vesicles with Triton X-114 detergent and incubation of the hydrophobic phase with phosphatidylinositol-specific phospholipases C and D, enzymes specific for GPL-anchor structures, released Thy-1 and DAF but not IgR protein into the aqueous phase. The selective incorporation of phospholipase-sensitive anchoring moieties into Thy-1 and DAF but not IgR translation products during in vitro translocation indicates that rough microsomes are able to support and regulate glypiation.

The latent membrane protein 1 (LMP1) encoded by the Epstein-Barr virus functions as a constitutively activated receptor of the tumor necrosis factor receptor family. LMP1 is a short-lived protein that is ubiquitinated and degraded by the proteasome. We have previously shown that LMP1 recruits the adapter protein tumor necrosis factor receptor-associated factor 3 (TRAF3) to lipid rafts. To test if TRAFs are involved in LMP1's ubiquitination, we have mutated the LMP1 CTAR1 site that has been identified as a TRAF binding site. We show that the CTAR1 mutant (CTAR1 À ) is expressed after transfection at a similar level to wild-type LMP1, and behaves as wildtype LMP1 with respect to membrane localization. However, CTAR1 À does not bind TRAF3. We demonstrate that ubiquitination of CTAR1 À is significantly reduced when compared to wild-type LMP1. In addition, the expression of wild-type LMP1 induces the ubiquitination, an effect that is significantly reduced when the CTAR1 À is expressed. Taken together, our results suggest that TRAF proteins are involved in the ubiquitination of LMP1, and that their binding to LMP1 may facilitate their own ubiquitination.

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