Dissociation of patching by latent membrane protein-1 of Epstein–Barr virus from its stimulation of NF-{kappa}B activity

Tim Blossb,1, Ajamete Kaykas1 and Bill Sugden1

McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, 1400 University Ave, Madison, WI 53706, USA1

Author for correspondence: Bill Sugden. Fax +1 608 262 2824. e-mail sugden{at}oncology.wisc.edu


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Alterations were made in the amino terminus and the first two transmembrane-spanning regions of the latent membrane protein-1 (LMP-1) of Epstein–Barr virus. These mutant proteins were tested for their abilities to patch and to stimulate NF-{kappa}B activity. A subset of these derivatives retains the wild-type topology of LMP-1 in the plasma membrane, but has lost the ability to patch. Deletion of residues 9–20 of LMP-1, which contain potential SH3-binding motifs, abrogates patching of LMP-1. However, mutation of the prolines within these motifs, which eliminates binding of LMP-1 to SH3 domains in vitro, does not prevent patching by LMP-1. Deletion of the first two transmembrane regions of LMP-1 does prevent it patching. Some of the derivatives of LMP-1 which do not patch do stimulate NF-{kappa}B activity. Patching by LMP-1 appears to be a higher-order assemblage of protein that is compatible with the stimulation of NF-{kappa}B activity but is not necessary for this signalling.


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Latent membrane protein-1 (LMP-1) is a viral gene product necessary for the maintenance of proliferation of B-cells infected by Epstein–Barr virus (EBV) (Kaye et al., 1993 ; Kilger et al., 1998 ). LMP-1 is expressed during the latent phase of the virus life-cycle (Speck & Strominger, 1989 ), and rapidly translocates to the plasma membrane upon its synthesis, where it spans the membrane six times (Fig. 1) (Liebowitz et al., 1986 ). LMP-1 shares several properties with receptors. LMP-1 associates with the cytoskeleton at the plasma membrane, where it also forms aggregates termed patches, which are operationally defined via immunofluorescent assays of fixed cells (Mann et al., 1985 ; Liebowitz et al., 1986 , 1987 ; Mann & Thorley-Lawson, 1987 ). It turns over rapidly (Baichwal & Sugden, 1987 ; Mann & Thorley-Lawson, 1987 ; Martin & Sugden, 1991b ). It also positively regulates cellular transcription factors belonging to the NF-{kappa}B (Hammarskjold & Simurda, 1992 ; Laherty et al., 1992 ; Mitchell & Sugden, 1995 ) and AP-1 families (Kieser et al., 1997 ). Cellular receptors usually require aggregation via ligand-binding for their signalling; for example, CD40 must be aggregated by ligand-binding before it can affect NF-{kappa}B activity (Paulie et al., 1989 ; Rothe et al., 1995 ). LMP-1 also mimics cellular receptors such as CD40 in that its aggregation is necessary for its effect on NF-{kappa}B activity (Gires et al., 1997 ). However, LMP-1 does not appear to require a ligand for its signalling (Martin & Sugden, 1991a ).



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Fig. 1. LMP-1 and its derivatives. LMP-1 is a 386 aa protein that translocates to the plasma membrane and spans the membrane six times. The amino-terminal 25 aa are found in the cytoplasm, as are the carboxy-terminal aa 186–386. Amino acids 26–185 constitute its six transmembrane-spanning regions. 1M HA fuses the first 48 aa of LMP-1 (including the first transmembrane region) to the influenza virus HA epitope, which is fused to aa 232–333 of LMP-1. Amino acids 232–333 of LMP-1 contain LMP-1 epitopes used to detect the protein in antibody staining assays. N{Delta}12–20 deletes aa 12–20, removing the majority of a putative SH3-binding region found between aa 9 and 20. N{Delta}43 removes the first 43 aa of LMP-1, including the first transmembrane region. 6M EE replaces aa 12–20 of LMP-1 with an epitope from the polyomavirus middle T antigen (EYMPMEV). 6M HA replaces aa 3–5 of LMP-1 with the influenza virus HA epitope (YPYDVPDYA). 4M HA fuses the amino-terminal 26 aa of 6M HA to the third transmembrane region of LMP-1, removing transmembrane regions 1 and 2. 12,15,16,19,20 P->A substitutes alanines for prolines at aa 12, 15, 16, 19 and 20, disrupting three PXXP motifs. PXXP motifs are known to mediate SH3-binding for other SH3-binding proteins.

 
We have characterized the ability of LMP-1 to patch through mutational analyses. Patched LMP-1 molecules were first identified by their punctate pattern of staining detected in immunofluorescent analyses of cells expressing the protein (Mann et al., 1985 ; Liebowitz et al., 1986 ). Large deletions within the amino terminus and transmembrane regions abrogated this activity, resulting in a diffuse pattern of staining (Martin & Sugden, 1991b ; Izumi et al., 1994 ). Smaller deletions within the amino terminus did not affect patching (Izumi et al., 1994 ). We have mapped regions within LMP-1 required for its patching, and determined whether patching is required for its stimulation of NF-{kappa}B activity. We have found that elements within the first 25 amino acids (aa) of LMP-1 as well as the first two transmembrane-spanning regions are necessary for patching; that the functional SH3-binding region found within the first 25 aa of LMP-1 is not necessary for patching; and that stimulation of NF-{kappa}B activity by LMP-1 does not require patching. These findings indicate that patching is likely to represent an organized assemblage of multiple LMP-1 molecules, perhaps with cellular proteins, at the plasma membrane. This assemblage differs from the minimal aggregation LMP-1 requires to signal (Gires et al., 1997 )

Mutations in LMP-1 were generated that disrupt the amino terminus and transmembrane regions of the protein (Fig. 1). N{Delta}43 removes the first 43 aa of LMP-1, including the first transmembrane region. N{Delta}12–20 removes aa 12–20 of LMP-1, deleting a large portion of the putative SH3-binding region PPGPRRPPRGPP. SH3-binding regions bind to proteins that contain SH3 domains, often through PXXP motifs found within the region (Feng et al., 1994 ). Proteins that contain SH3 domains are typically found to be involved in cell signalling pathways (e.g. c-Src, c-Abl) (Koch et al., 1991 ; Mayer & Baltimore, 1993 ). 12,15,16,19,20 P->A disrupts PXXP motifs within this region by replacing prolines with alanines at aa 12, 15, 16, 19 and 20. A subset of mutants used in these studies was constructed, members of which possessed an epitope tag within the amino termini of the resulting proteins, such that the topologies of these proteins within the plasma membrane could be determined via immunofluorescent cell analysis (see below). 6M EE contains the six transmembrane regions of LMP-1 and an epitope from polyomavirus middle T antigen (EE) (Grussenmeyer et al., 1985 ; Wilson & Bourne, 1995 ) in place of aa 12–20. 6M HA contains the six transmembrane regions of LMP-1 and the influenza virus haemagglutinin epitope (HA) (Wilson et al., 1984 ; Arden et al., 1995 ) in place of aa 3–5. 4M HA has transmembrane regions 1 and 2 of LMP-1 deleted, but retains the HA tag in the same context as in 6M HA. These mutants all contain the wild-type carboxy terminus of LMP-1 (aa 200–386), which in turn contains the epitopes of LMP-1 recognized by our rabbit antiserum.

The topologies of the 6M EE, 6M HA and 4M HA mutants within the membrane were established by determining the positions of the epitope tags found on the amino and carboxy termini of these proteins as being inside or outside the cells. 293 cells were transfected with mutant DNA constructs via calcium phosphate precipitation (Sambrook et al., 1989 ). Various times after transfection, the cells were harvested and resuspended in impermeable cell wash [1x PBS, 10% bovine calf serum (BCS), 0·05% sodium azide (NaAz)], and distributed into two sets of tubes. Cells in one set of tubes remained resuspended in impermeable cell wash and were defined as the impermeable set. Cells in the second set of tubes were pelleted and resuspended in permeable cell wash (1x PBS, 10% BCS, 0·05% NaAz, 0·01% saponin), and defined as the permeable set. Primary antibody was added to both sets. The EE epitope was detected with mouse monoclonal anti-EE antibody (Grussenmeyer et al., 1985 ); the HA epitope was detected by mouse monoclonal anti-HA antibody (HA-11, BAbCO); the LMP-1 epitopes were detected by affinity-purified polyclonal rabbit anti-LMP-1 antibody (Baichwal & Sugden, 1987 ). Both sets of cells were washed and the appropriate fluorescein-conjugated secondary antibody was added, goat anti-rabbit IgG (Jackson) or goat anti-mouse IgG (Boehringer Mannheim Biochemical). Both sets of cells were washed and resuspended in 1x PBS. The cells were fixed with 2% paraformaldehyde, washed in 1x PBS, resuspended in PBS and analysed for fluorescence using an EPICS Profile Analyser.

The results of the immunofluorescent cell analyses indicate that the insertion of either the EE or HA epitope into the amino terminus does not alter the wild-type topology of the resulting protein. Table 1(a) shows the percentage of cells scored as specifically stained by antibody for either permeable or impermeable cells. For both 6M EE and 6M HA, the detection of amino- or carboxy-terminal epitopes is more efficient in cells that are permeable than cells that are impermeable, consistent with these regions of the proteins being found on the inside of the cells. Similar results using 4M HA in these assays indicate that the removal of transmembrane regions 1 and 2 does not disrupt the wild-type topology of the resulting protein (Table 1a). The topologies of these proteins were interpreted based on the positions of both the amino and carboxy termini, and these topologies are indicated in Fig. 1. As a control, 1M HA (Fig. 1) was used to determine our ability to detect epitopes found on the outside of the cell. 1M HA is predicted to place both an HA epitope and LMP-1 epitopes on the outside of the cell. The results using 1M HA show that the HA epitope can be detected on impermeable cells and the LMP-1 epitopes can be detected on both permeable and impermeable cells, consistent with the epitopes being placed on the outside of the cell.


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Table 1. Results of immunofluorescent cell analysis of 293 cells expressing LMP-1 derivatives

 

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6M EE, 6M HA and 4M HA were each tested for their abilities to patch (Fig. 2). 293 cells were plated onto coverglass slips and grown to 30–50% confluence in DMEM–10% foetal bovine serum–200 µg/ml streptomycin–200 U/ml penicillin. DNAs encoding derivatives of LMP-1 were transfected via calcium phosphate precipitation, the medium was removed after 24–48 h, and the cells were washed in 1x PBS. The cells were dried and fixed with cold acetone–methanol (1:1), washed in 1x PBS–2% BCS, and polyclonal rabbit anti-LMP antibody was added. The cells were incubated, washed in 1x PBS–2% BCS, and fluorescein-conjugated goat anti-rabbit antibody (Jackson) was added. They were incubated and washed in 1x PBS–2% BCS. The coverglass slips were mounted onto glass slides using glycerol–0·4% propyl-gallate, and fluorescent cells were counted and scored for their phenotypes without foreknowledge of the DNAs with which they had been transfected.



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Fig. 2. Immunofluorescence of nonpatched and patched cells. For patching assays, vectors expressing LMP-1 or its 4M HA-derivative were transfected into 293 cells, and the cells were analysed via immunofluorescent cell staining to determine the distribution of LMP-1. The cells transfected with 4M HA show a uniform expression of staining at the plasma membrane and are defined as nonpatched (left panel); those transfected with wild-type LMP-1 display a punctate pattern of protein expression at the plasma membrane and are defined as patched (right panel).

 
The results of these assays indicate that the aa 12–20 region and the first two transmembrane regions are all necessary for patching. Table 1 shows that replacement of aa 12–20 by the EE epitope (6M EE) results in a protein that cannot patch, even though it maintains a wild-type topology. Restoration of aa 12–20 in 6M HA restores patching ability. 4M HA retains aa 12–20 and a wild-type topology, but does not possess transmembrane regions 1 and 2. This protein cannot patch, indicating that the first two transmembrane regions are also necessary for patching of LMP-1.

One possible explanation for the role of aa 12–20 in patching results from the fact that it contains a putative SH3-binding region located between aa 9 and 20. This putative SH3-binding region has three PXXP motifs (PPGP, PRRP, PRGP), which might facilitate patching of LMP-1 through associations with cytoskeletal or adaptor proteins (some of which are known to contain SH3 domains) (Mayer & Baltimore, 1993 ). We performed filter-binding assays according to Cicchetti et al. (1992) and demonstrated that the amino terminus of LMP-1 bound to the SH3 domain of c-Abl fused to glutathione S-transferase (GST) 4·5-fold more than GST alone, while a positive control, 3BP1-10 fused to GST (Ren et al., 1993 ), bound the SH3 domain of c-Abl 3·5-fold more than GST alone in these assays. Disruption of the SH3-binding region of LMP-1 in these assays decreased binding to background levels.

To test the importance of the putative SH3-binding region of LMP-1 for patching activity, mutants N{Delta}12–20 and 12,15,16,19,20 P->A were tested in patching assays. Removal of aa 12–20 resulted in a protein that could not patch, while disruption of putative SH3-binding in this region did not affect patching (Table 1b). Thus, while the region between residues 12 and 20 of LMP-1 contributes to patching, the SH3-binding activity within this region is not required for patching.

Results of assays that measured the stimulation of NF-{kappa}B activity by mutants of LMP-1 indicate that this stimulation is not dependent on the ability of LMP-1 to patch. DNAs expressing LMP-1 or a derivative were cotransfected into 293 cells along with a DNA encoding a luciferase reporter gene under the control of NF-{kappa}B-responsive elements (Mitchell & Sugden, 1995 ). These cells were grown for 48 h, harvested, lysed and the luciferase was activity measured. The level of stimulation of NF-{kappa}B activity by each derivative was normalized for the level of protein expressed via quantitative Western blotting (Sandberg et al., 1997 ). The results of these assays are shown in Table 1(b), and show that stimulation of NF-{kappa}B activity, as measured by luciferase activity, is not dependent on patching of LMP. By way of examples, 6M EE, which does not patch, has a wild-type topology within the plasma membrane and stimulates NF-{kappa}B to near wild-type levels. 4M HA, which also does not patch but assumes a wild-type topology, upregulates NF-{kappa}B to greater than wild-type levels. N{Delta}43 neither stimulates NF-{kappa}B activity nor patches. These findings show that stimulation of NF-{kappa}B activity by LMP-1 and its patching are genetically separable.

We propose that patching of LMP-1 reflects a higher-order assemblage that is not required for its stimulation of NF-{kappa}B activity, but is compatible with this stimulation (Table 1b). Patching may be mediated by association of LMP-1 with cellular molecules, for example, an association with glycosphingolipid-rich membrane domains (Clausse et al., 1997 ), but this association does not impede LMP-1’s stimulation of NF-{kappa}B activity. The contribution patching by LMP-1 makes during the life-cycle of EBV is likely to be important because the two regions of LMP-1 that we have identified to be required for patching are highly conserved in different strains of EBV (Miller et al., 1994 ; Sandvej et al., 1997 ). The conservation of the sequences of the first two transmembrane-spanning regions in particular is consistent with these regions contributing a specific activity to patching beyond that of being hydrophobic.


   Footnotes
 
b Present address: Department of Molecular, Cellular and Developmental Biology and Neuroscience Research Institute, University of California, Santa Barbara, CA 93106, USA.


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Received 10 May 1999; accepted 3 August 1999.