LMP1 association with CD63 in endosomes and secretion...

The EMBO Journal Peer Review Process File - EMBO-2010-75896

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Manuscript EMBO-2010-75896 LMP1 association with CD63 in endosomes and secretion via exosomes limits constitutive NF- κB activation Frederik J. Verweij, Monique A.J. van Eijndhoven, Erik S. Hopmans, Tineke Vendrig, Tom Wurdinger, Ellen Cahir-McFarland, Elliott D. Kieff, Dirk Geerts, Rik van der Kant, Jacques J.C. Neefjes, Jaap M. Middeldorp and D. Michiel Pegtel Corresponding author: D. Michiel Pegtel, VU Medical Center Review timeline: Submission date: 03 August 2010 Editorial Decision: 10 August 2010 Additional Correspondence: 23 August 2010 Additional Correspondence: 24 August 2010 Resubmission: 17 September 2010 Editorial Decision: 02 November 2010 Revision received: 17 January 2011 Editorial Decision: 11 February 2011 Revision received: 04 March 2011 Editorial Decision: 17 March 2011 Revision received: 22 March 2011 Accepted: 25 March 2011 Transaction Report: (Note: With the exception of the correction of typographical or spelling errors that could be a source of ambiguity, letters and reports are not edited. The original formatting of letters and referee reports may not be reflected in this compilation.)

1st Editorial Decision 10 August 2010

Thank you for submitting your manuscript for consideration by The EMBO Journal. After some delay due to the high number of manuscripts we have received over the last few weeks, I have now had the opportunity to read it carefully and to discuss it with the other members of our editorial team. I am afraid that the outcome of these discussions is not a positive one. We appreciate that you were able to provide evidence that the Epstein Barr Virus protein LMP1 is recruited to multivesicular body intraluminal vesicles and finally to exosomes via a mechanism that involves direct interaction with the tetraspanin CD63. Furthermore your data suggest that CD63-dependent recruitment to exosomes attenuates LMP1-induced NF-kappaB activation. However, it is known that EBV LMP1 activates NF-kappaB signalling, and more importantly LMP1 has been detected in exosomes together with CD63 before. Also, tetrasapanins including, CD63, are known to be enriched in exosomes as well as in late endosomal ILVs. Furthermore, a picture appears to be emerging in the literature that CD63 can function as transport regulator of its interaction partners, albeit not in the context of cargo recruitment to the exosomes. Taking together all these considerations we think that the main novelty of this study is thus twofold: LMP1 recruitment to and secretion with exosomes is functionally significant in the context of NF-kappaB signalling and CD63 can recruit a protein to exosomes. We can see and appreciate that these are novel findings.



Still, we are not convinced that the extent of novel biological and mechanistic insight provided by this study in terms of CD63 and/or LMP1 function, reaches the level required for publication in The EMBO Journal. We have thus decided not to send out the paper for in-depth peer review at this point. Please note that we publish only a small percentage of the many manuscripts that we receive at The EMBO Journal, and that we can only subject those manuscripts to external review which are likely to receive enthusiastic responses from our reviewers and readers. As in our carefully considered opinion, this is not the case for the present submission I am afraid to say that our conclusion regarding its publication here cannot be a positive one. Thank you in any case for the opportunity to consider this manuscript. I am sorry that we cannot be more positive on this occasion. Yours sincerely, Editor The EMBO Journal Additional Correspondence 23 August 2010

Resubmission inquiry: We appreciate your thorough evaluation of our submitted manuscript (EMBOJ-2010-75519). In your response you affirm that the data presented in our original manuscript is novel and interesting but that "the extent of novel mechanistic insight does not reach the level required for publication in The EMBO Journal at this point". We now have novel data (please see attachment) indicating that LMP1 recruits CD63 into tetraspannin-rich clusters (protein rafts) in late endosomes (LEs) that are distinct from HLA-DR/CD63 rich domains. HLA-DR/CD63 rich vesicles are repositioned from the cellular periphery to the perinuclear region upon cholesterol accumulation(Lebrand et al., 2002). In contrast, LMP1/CD63-rich vesicles were not repositioned upon cholesterol manipulation, suggesting impaired 'cholesterol sensing' (Rocha et al., 2009). Surprisingly, cholesterol levels in the LMP1/CD63-rich peripheral vesicles were not increased. Based in part on these latest findings we argue that the LMP1 clusters with CD63 in micro-domains through a protein/protein interaction (co-IP) not requiring cholesterol and are thus distinct from conventional lipid rafts. While previous studies suggest that early LMP1 lipid-raft interaction, presumably at the Golgi stage, is important for initiating LMP1 signaling, we propose that LMP1 subsequently attenuates constitutive and potentially oncogenic NF-kB signaling not via common degradation pathways but by taking over CD63 trafficking in cholesterol low domains for secretion through exosomes. We are hopeful that inclusion of these findings into a revised manuscript raises the extent of mechanistic insights into LMP1 function required for publication in EMBO J. Additional Correspondence 24 August 2010

Thank you for your message asking us whether we would be able to consider an extended version of this manuscript. I have now had a chance to look into the matter. Yes, this could certainly be a possibility and we could have a look at it. I would therefore suggest submitting a new extended version as a new submission. I will then take it from there.



Yours sincerely, Editor The EMBO Journal

2nd Editorial Decision 02 November 2010

Thank you for submitting your manuscript for consideration by The EMBO Journal. Let me first of all apologise for the exceptionally long delay in getting back to you with a decision. This was caused by severe difficulties in finding suitable and willing referees for this manuscript. In the meantime your manuscript has now finally been seen by three referees whose comments are shown below. You will see that while referee 1 is clearly not in favour of publication of the manuscript here the other two reviewers are more positive and would support publication after appropriate, but significant revision. We should thus be able to consider a revised manuscript in which the points raised by referees 2 and 3 are addressed in an adequate manner and to their satisfaction, including points 5 and 6 of referee 2 and all major comments of referee 3. I should remind you that it is EMBO Journal policy to allow a single round of revision only and that, therefore, acceptance or rejection of the manuscript will depend on the completeness of your responses included in the next, final version of the manuscript as well as on the final assessment by the referees. When preparing your letter of response to the referees' comments, please bear in mind that this will form part of the Peer Review Process File, and will therefore be available online to the community. For more details on our Transparent Editorial Process initiative, please visit our website: http://www.nature.com/emboj/about/process.html Thank you for the opportunity to consider your work for publication. I look forward to your revision. Yours sincerely, Editor The EMBO Journal REFEREE REPORTS Referee #1 (Remarks to the Author): This is a very mechanistic study about the intracellular trafficking of the Epstein Barr virus (EBV) viral oncogene latent membrane protein 1 (LMP1), and its repercussion on NF-kB activation. The authors of this manuscript show that a significant amount of LMP1 avoids intracellular degradation by entering an exosomal release pathway through its interaction with CD63. Impairment of that exosomal route leads to increased LMP1 levels and the consequent hyperactivation of NF-kB and presumably the tumorigenic potential of LMP1. The experiments presented are logical, well executed from a technical point of view, and their interpretation straightforward and of interest. However, the potential implication of these findings in tumorigenic studies in vivo are missing. Also missing is information on the potential existence of mutations in the CD36-LMP1 interphase in humans that would reproduce the phenotypes reported here in vivo in a more clinically relevant setting. Without that information, it is felt that this is a manuscript more appropriate for a more specialist journal. Referee #2 (Remarks to the Author):



Review to manuscript EMBOJ-2010-75896, Verweij et al. LMP1 of Epstein-Barr virus serves as paradigm of a herpesviral oncoprotein which usurps host cellular signal transduction pathways such as NF-kappaB or JNK for cell transformation. Groundbreaking progress in our understanding of the molecular principles underlying the exploitation of cellular factors by viral proteins has originated from studies on LMP1 signaling. Therefore, this topic is of general interest and well suitable for the EMBO Journal. LMP1 constitutively and very efficiently activates signaling due to autoaggregation of its transmembrane domains. It is well established in the literature that hyperactivity of LMP1 and, thus, LMP1-induced pathways like NF-kappaB is not beneficial for the EBV-infected cell and may contribute to lymphomagenesis. However, it is poorly understood how LMP1 activity is controled at the protein level. In the present manuscript the authors demonstrate fort the first time that regulation of cellular LMP1 trafficking and finally export of LMP1 protein via exosomes rather than protein degradation is an important mechanism of limiting cellular LMP1 activity. Moreover, they show that LMP1 is selectively sorted into exosomes. This is a new and very interesting observation which might have broader relevance also for other viral oncoproteins. The authors also discuss the possibility that exosomal release of LMP1 might contribute to immune evasion of EBV-infected cells because fewer LMP1 peptides would be presented on the surface of the infected cell. Although it is already known that LMP1 can be found in exosomes, biological relevance of this observation has been unclear and a detailed analysis of intracellular trafficking and sorting of LMP1 into exosomes has been lacking. By fluorescence confocal and electron microsocopy the authors show that most LMP1 is located in intraluminal vesicles (ILV) of late multivesicular endosomes (MVE) and that surprisingly little LMP1 can be found in the Golgi or the plasma membrane. The authors identify the tetraspanin CD63 as a novel interaction partner of LMP1 which co-localizes with LMP1 in cholesterol-poor domains of MVEs and is responsible for inclusion of LMP1 into ILVs and, finally, exosomes. Interaction of LMP1 and CD63 seems to be mediated by the C-terminal signaling domain of LMP1, although the precise interaction site has not been mapped. LMP1 colocalizes with CD63 in multivesicular endosomes (MVEs) and accumulates in cholesterol-poor intraluminal vesicles. Notably, knockdown of CD63 leads to a subcellular redistribution of LMP1 and enhanced activation of NF-kappaB by LMP1. The manuscript is well written and the data are clearly presented. The data are of high technical quality and generally support the conclusions drawn. The specific points listed below should be addressed. Specific points: 1. In the present manuscript the authors suggest that sorting of LMP1 into exosomes is a major mechanism of regulating intracellular LMP1 levels. As already mentioned by the authors (page 7, last paragraph), LMP1 protein is very heterogenously expressed between single cells of one lymphoblastoid cell line. Could differentially efficient LMP1 sorting into exosomes be the cause fort his observation? Do LMP1 „low" expressers have more CD63 and vice versa? 2. Figure 4, brightfield images would allow to judge the fluorescence images better. 3. Figure 4C, it is very surprising that almost no LMP1 resides in GM1-rich lipid rafts, which are thought to be the signaling-active sites of LMP1. Where ist the plasma membrane in the picture? Is there some co-localization of LMP1 and GM1 in the PM or in the Golgi? 4. Figure 5A, lysate controls should be shown. 5. CD63 is described as a novel interaction partner of the LMP1 signaling domain. The binding region of CD63 to the LMP1 signaling domain should be defined more precisely. Can the authors narrow down the interaction region by using LMP1 deletion mutants? Can CTAR1 or CTAR2 be excluded as the interaction regions? 6. The authors find that retention of exogenous LMP1-YFP in the Golgi leads to an enhanced



signaling activity of LMP1 (Figure 8D, suppl. Figure 5), suggesting that LMP1 is primarily active in the Golgi. However, the authors of the present manuscript make the observation that only very little endogenous LMP1 is located to the Golgi (Figure 1 and text, page 5, last paragraph). According to their data most LMP1 seems to be located in the late endosomal compartment and, above that, not in cholesterol-rich lipid rafts, which raises the very important question about the compartment from which endogenous LMP1 is actually signaling. Previous reports demonstrated that LMP1 is primarily located to the Golgi and primarily signals from this compartment involving lipid rafts (Lam and Sugden, EMBO J. 2003; Liu et al., EMBO J. 2006). To clarify this point and to better understand the regulation of LMP1 signaling activity by cellular trafficking, the authors should perform co-stainings of LMP1 with e.g. TRAF2 or TRAF3 to identify the subcellular location of active endogenous LMP1 complexes. 7. Figure 9B is not referenced within the text. I assume that on page 14, line 6 from bottom, the „8B" should actually be 9B? 8. Does CD63 knockdown reduce LMP1 inclusion into exosomes? Referee #3 (Remarks to the Author): The article by Verweij et al describes a very interesting new mechanism of hijacking of intracellular machinery by a viral protein: EBV LMP1 binding to CD63, leading to targeting to internal vesicles of MVB for subsequent release in exosomes. They also show that this mechanism of specific interaction between LMP1 and CD63 allows reduced NF-kB activation, potentially promoting controlled activation of the EBV-infected cells, and limiting the ultimate transformation into tumoral cells The manuscript is generally well written, and the results are very interesting, but in several experiments, some controls and/or quantifications are lacking to really convincingly support the interpretations of the results displayed Major comments : 1) A general major concern is that a large part of the results are presented as immunofluorescence analyses, showing one or maximum 2-3 cells, without any attempt to quantify the representativity of the images shown. The authors must in all experiments provide informations on the percentage of cells displaying the reported morphology, or quantify the actual percentage of co-localisation for each double stainings. This is true for figures 1, 4, 6, 7, 8, 9 2) The information provided on the experimental settings of experiments using anti-LMP1 antibodies do not allow to exclude the possibility of non-specific cross-staining or cross-precipitation of CD63 and LMP1 by the antibodies used. Especially, controls with LMP1-negative cells (stained with the combinations of antibodies to LMP1 and other markers used) should be shown in the immunofluorescence images of Figure 1, and in fig 4A where it is surprising to see several CD63-negative cells, and very similar staining for CD63 and LMP1 in all cells (ie absence of CD63 when absence of LMP1). Similarly, controls of the co-IP experiments presented in figure 5A must be shown to convincingly prove the selectivity of the CD63/LMP1 co-precipitation observed: IP with anti-LMP1 performed on LMP1-negative cells, and signal for anti-HLADR (in the same experiment as with CD63) on the product of anti-LMP1 IP. In addition, fig5D should also show the level of HLADR in LMP1 overexpressing cells and exosomes: it should not be increased, as opposed to CD63, since the authors state that HLADR and LMP1 are not co-localized intracellularly. 3) The effect of shRNA for CD63 on the endogenous level of NF-kB activation in cells that do not express LMP1 must be shown in figure 9C (and/or 9D), to demonstrate that the increased activity upon CD63 knock-down is really linked to its effect on LMP1, rather than to a direct effect of CD63 on general level of NF-kB activity in the cells. Fig9D apparently shows increased NF-kB activity upon transfection with increasing amounts of LMP1-encoding plasmid, but it is described in the text as « transfections of low amount of LMP1 plasmid DNA leading to much higher relative levels of NF-kB activation levels in cells with reduced CD63 levels ». If this figure shows, as stated in the legend, the « relative induction of NF-kB in shRNA CD63 KD cells over control », then this relative induction is stronger upon transfection with higher amounts of LMP1-encoding plasmids, which is



contradictory to the text. Other comments : 1) Figure 7 : the legend does not correspond well to the figure, nor the text describing this figure (Figure7D is referred to in the text, but does not appear in the figure) 2) Conclusions provided in the text on the experiments shown in figure 8 should not mention anything on CD63 since analysis of CD63 distribution and export in exosomes in the cells expressing LMP1-C-YFP were not performed. 1st Revision - authors' response 17 January 2011

Response to Referee #2: Specific points: 1. In the present manuscript the authors suggest that sorting of LMP1 into exosomes is a major mechanism of regulating intracellular LMP1 levels. As already mentioned by the authors (page 7, last paragraph), LMP1 protein is very heterogenously expressed between single cells of one lymphoblastic cell line. Could differentially efficient LMP1 sorting into exosomes be the cause for this observation? Do LMP1 "low" expressers have more CD63 and vice versa? AUTH: We demonstrated that the induction of LMP1 expression in BJAB cells correlated with co-localization and ‘more’ efficient secretion/sorting of CD63 into exosomes (Figure 5). We commented on this on page 9 of the original manuscript, however we did not speculate whether this correlation could be extrapolated to LCL and explain the heterogeneity in LMP1 levels. In the revised manuscript we make the reader aware of this possibility on page 10 (top paragraph). 2. Figure 4, brightfield images would allow to judge the fluorescence images better. LCL are rounded activated lymphoid “blasts” that generally display a pronounced polarized (hand-mirror shape) morphology in culture. After the cytospin procedure and fixation, around 50% of the cells still reveal this morphology i.e. a large part of the cytoplasm extending from one side of the nucleus that contains most intracellular vesicles. This morphology is now clearly demonstrated in figure 4 (and 9E) of the revised manuscript representing an LCL cell simultaneously analyzed by transmission and confocal microscopy showing vesicles with LMP1 (red) and CD63 (green). 3. Figure 4C, it is very surprising that almost no LMP1 resides in GM1-rich lipid rafts, which are thought to be the signaling-active sites of LMP1. Where is the plasma membrane in the picture? Is there some co-localization of LMP1 and GM1 in the PM or in the Golgi? AUTH: We included a zoomed image of the location of the PM (white dotted line) in the revised figure 4F. We found very little evidence that endogenous LMP1 resides in the PM of LCL by any technique we used. It must be mentioned that the images represent internalized GM1 (for details see m&m). The aim was to determine whether endocytosed GM1 localizes to compartments that accumulated LMP1. Clearly our data demonstrates that internalized GM1 traffics to distinct intracellular compartments. In response to a major concern raised by referee 3, we also quantified the percentages of co-localization averaged over multiple cells between endogenous LMP1 vs internalized CTxB and/or CD63. We found significant differences (Pearson correlation) (~0.2 vs. 0.7 respectively, p <0.05). Moreover, LMP1 shows significant co-localization with intracellular CD63 in a large majority of the cells (>60%) while accurate CTxB staining was only observed in a small fraction of cells of which an even smaller fraction showed co-localization, consistent with previous findings (Kaykas et al., 2001;Lam and Sugden, 2003). However a small proportion of endogenous LMP1 co-localized with cis- and trans-Golgi markers. We suspect based on staining patterns that the internalized CTxB-FITC is unlikely to represent Golgi compartments, but instead define endosomal compartments devoid of LMP1. This is consistent with recent findings showing that that MHC II molecules at the surface of B cells are selectively internalized with GM1 (as defined by CTxB) to endosomes (Knorr et al., 2009). 4. Figure 5A, lysate controls should be shown.



AUTH: Equal amounts of the same LCL lysate were used for both the IgG control and LMP1 IPs using equal amounts of specific LMP1 and IgG control antibodies. We now include negative results of a co-immunoprecipitation experiment with the LMP1 specific antibody and LMP1-negative lysates that were prepared from wtBJAB cells. These experiments further show the specificity of the LMP1 antibodies and indicate that co-isolation was not due to variable input. For further details see the revised m&m section at page. 5.CD63 is described as a novel interaction partner of the LMP1 signaling domain. The binding region of CD63 to the LMP1 signaling domain should be defined more precisely. Can the authors narrow down the interaction region by using LMP1 deletion mutants? Can CTAR1 or CTAR2 be excluded as the interaction regions? AUTH: Modifications to LMP1s signaling domain disrupted exit from the Golgi and these mutants also failed to co-localize and cluster CD63 (Figures 8 A,C and 9B). Besides the possibility that CD63 interacts directly with LMP1’s signaling domain, tetraspanins can assemble with their associating membrane partners via palmitoylation-mediated lateral associations demonstrated in low stringency detergents (Hemler, 2005). Recent data show that these associations are solely responsible for CD63-mediated Golgi-exit to secretory lysosomes of its transmembrane-associated partner Syt VII (Flannery et al., 2010). We explored the possibility that CD63 can be co-isolated with exogenous LMP1 in a lysis buffer with low stringency detergent and were able to readily detect CD63 (revised Figure 9A). This is consistent with previous findings that LMP1 is palmitoylated (Higuchi et al., 2001) although the biological significance was unresolved at that time. It is considered premature for an extensive mapping of the C terminus and future experiments should include exploration into lateral membrane associations as well. We discuss these insights and the relevant literature in the revised discussion on page 18 at the end of paragraph 1. 6. The authors find that retention of exogenous LMP1-YFP in the Golgi leads to an enhanced signaling activity of LMP1 (Figure 8D, suppl. Figure 5), suggesting that LMP1 is primarily active in the Golgi. However, the authors of the present manuscript make the observation that only very little endogenous LMP1 is located to the Golgi (Figure 1 and text, page 5, last paragraph). According to their data most LMP1 seems to be located in the late endosomal -compartment and, above that, not in cholesterol-rich lipid rafts, which raises the very important question about the compartment from which endogenous LMP1 is actually signaling. Previous reports demonstrated that LMP1 is primarily located to the Golgi and primarily signals from this compartment involving lipid rafts (Lam and Sugden, EMBO J. 2003; Liu et al., EMBO J. 2006). To clarify this point and to better understand the regulation of LMP1 signaling activity by cellular trafficking, the authors should perform co-stainings of LMP1 with e.g. TRAF2 or TRAF3 to identify the subcellular location of active endogenous LMP1 complexes. AUTH: We did interpret our findings in having identified a novel location for active LMP1 signaling complexes because our data show that trafficking and sorting into the late endosomal compartments and secretion of exosomes via protein-based (tetraspanin) rafts antagonizes downstream LMP1 signaling. To better understand the regulation of LMP1 signaling activity by cellular trafficking we performed co-stainings of LMP1 with TRAF3 as suggested by the reviewer. In four B cell types we examined (LCL, EBV-positive RAJI, JY and EBV-negative BJAB-LMP1) we confirmed that the major proportion of endogenous LMP1 resides in peripheral vesicles but that these do not contain endogenous TRAF3 as can be observed in the revised figures 4I and 9E and supplemental 1 that show distinct staining patterns in LCL and little co-localization. In agreement, exogenous wtLMP1 expressed in HEK293 failed to recruit TRAF3 (figure 8…). In contrast LMP1-mutants that are retained in the Golgi showed similar staining pattern with endogenous TRAF3 (figure 8 E+F). Thus LMP1 that resides in endosomal (peripheral vesicles) does not seem to recruit TRAF3. Although these experiments do not rule out the possibility that LMP1 signals from these compartments in a TRAF3-independent manner, our data is consistent with studies suggesting that TRAF3 is largely indispensible for NF-kB activity in LCL (Guasparri et al., 2008). We discuss these new findings and interpretations in the revised discussion on page 18. In addition, we modified figure 9E such that it is more clear that we do not rule out LMP1 signaling from the Golgi in LCL. 7. Figure 9B is not referenced within the text. I assume that on page 14, line



6 from bottom, the "8B" should actually be 9B? AUTH: We apologize for this error which has been corrected in the revision. 8. Does CD63 knockdown reduce LMP1 inclusion into exosomes? AUTH: We performed this experiment as suggested by the reviewer and expressed wtLMP1 in CD63KD and control HEK293 cells. We isolated exosomes 48hrs post transfection and measured LMP1 protein levels in each exosome fraction. We did not measure a significant impact on LMP1 levels in the total exosome fraction, although we observed some increase in cellular LMP1 levels (Supplemental Figure 6). One explanation is that compensatory mechanisms for LMP1 secretion via exosomes may exist through alternative tetraspanins as many tetraspanins are present in exosome populations that could co-operate with CD63 (Escola et al., 1998;Pols and Klumperman, 2009). Response to Referee #3: Major comments : 1) A general major concern is that a large part of the results are presented as immunofluorescence analyses, showing one or maximum 2-3 cells, without any attempt to quantify the representativity of the images shown. The authors must in all experiments provide informations on the percentage of cells displaying the reported morphology, or quantify the actual percentage of co-localisation for each double stainings. This is true for figures 1, 4, 6, 7, 8, 9. AUTH: To reduce concerns for selecting 2-3 cells, we provide additional information on the percentages of co-localization including statistics and/or the percentage of the reported morphology in Figure 1 B. We now provide new overview images of double stainings performed on LCL in the supplemental data. In the revision we opted to use LAMP1 as a lysosomal maker (as opposed to Lysotracker Red) because we observed that LAMP1 (a lysosomal protein) was more homogenously expressed in the LCL and more readily detectable compared to Lysotracker Red. The result is similar in that little endogenous LMP1 localizes to LAMP1–positive lysosomes. 2) The informations provided on the experimental settings of experiments using anti-LMP1 antibodies do not allow to exclude the possibility of non-specific cross-staining or cross-precipitation of CD63 and LMP1 by the antibodies used. Especially, controls with LMP1-negative cells (stained with the combinations of antibodies to LMP1 and other markers used) should be shown in the immunofluorescence images of Figure 1, and in fig 4A where it is surprising to see several CD63-negative cells, and very similar staining for CD63 and LMP1 in all cells (ie absence of CD63 when absence of LMP1).

AUTH: We performed stainings with LMP1-negative BJAB cells in combination with endogenous CD63 as control that were placed in the supplemental data of the original manuscript (Supplemental Figure 2B). We agree with the referee that from the original pdf in figures 1 and 4 endogenous expression of CD63 is not clearly visible in the “LMP1-negative” cells. Differences in signal intensities between endogenous CD63 (low) and LMP1 (high) in the B cell backgrounds seems to have caused this discrepancy. Endogenous CD63 in (LMP1 transfected and non-transfected) HEK293 cells and HELA cells (Figures 7 and 9 and supplemental 3 of the original manuscript) is more clear. We improved the staining procedures for LCL and BJAB cells and now show clear endogenous CD63 in LCL and BJAB cells in the absence of LMP1. In the revised figure 4 we demonstrate this in showing that endogenous CD63 is present in BJAB cells and LCL cells while LMP1 is absent. Specifically in figure 4B of the revised manuscript multiple LCL cells show a variable degree of endogenous LMP1 levels while all cells contain CD63-positive vesicles being more prominent in LMP1 “high” cells. Additionally, we included supplemental movies (z-stack animation) highlighting the vesicular nature (intracellular) of LMP1 and CD63 (Supplemental Video S1). -Similarly, controls of the co-IP experiments presented in figure 5A must be shown to convincingly prove the selectivity of the CD63/LMP1 co-precipitation observed: IP with anti-LMP1 performed on LMP1-negative cells,



AUTH: To reduce the concerns of cross-reactivity further we performed additional co-IP experiments as proposed by the referee with LMP1-negative (BJAB) cells and included this data in the revised figure 5A. CD63 is only precipitated with our specific anti-LMP1 antibody in LMP1-positive LCL cells but not in LMP1-negative,CD63-positive B (BJAB) cell lysates. -In addition, fig5D should also show the level of HLADR in LMP1 overexpressing cells and exosomes: it should not be increased, as opposed to CD63, since the authors state that HLADR and LMP1 are not co-localized intracellularly. AUTH: We performed these experiments and added the results of HLA-DR levels in figure 5D. We now show that HLA-DR levels are not increased in exosomes of LMP1 overexpressing cells as opposed to CD63. 3) The effect of shRNA for CD63 on the endogenous level of NF-kB activation in cells that do not express LMP1 must be shown in figure 9C (and/or 9D), to demonstrate that the increased activity upon CD63 knock-down is really linked to its effect on LMP1, rather than to a direct effect of CD63 on general level of NF-kB activity in the cells. AUTH: This is an important control experiment and included this data in figure 9C of the revised manuscript. We now demonstrate that the increased NF-kB activity in CD63 knock-down cells is specifically mediated via LMP1, rather than via a direct effect of CD63 on general level of NF-kB activity. Fig9D apparently shows increased NF-kB activity upon transfection with increasing amounts of LMP1-encoding plasmid, but it is described in the text as « transfections of low amount of LMP1 plasmid DNA leading to much higher relative levels of NF-kB activation levels in cells with reduced CD63 levels ». If this figure shows, as stated in the legend, the « relative induction of NF-kB in shRNA CD63 KD cells over control », then this relative induction is stronger upon transfection with higher amounts of LMP1-encoding plasmids, which is contradictory to the text. AUTH: The reviewer is right and we apologize for the confusion and changed the wording. The relative induction of NF-kB is stronger upon transfection with higher amounts of LMP1-encoding plasmids. This data is aimed to highlight that CD63KD cells are “sensitized” for LMP1-mediated NFkB activation in that transfection of low amounts of LMP1 plasmid (20ng) also significantly (3-fold) induce NFkB activity in these cells. Other comments : 1) Figure 7 : the legend does not correspond well to the figure, nor the text describing this figure (Figure7D is referred to in the text, but does not appear in the figure) AUTH: We have corrected the legend and the text 2) Conclusions provided in the text on the experiments shown in figure 8 should not mention anything on CD63 since analysis of CD63 distribution and export in exosomes in the cells expressing LMP1-C-YFP were not performed. AUTH: The reviewer is correct and we corrected the wording accordingly which and now reads; “Thus the modifications to the LMP1 C-terminus impair intracellular LMP1 trafficking and export via exosomes which increases downstream NF-κB signaling.”

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9. Pols MS and Klumperman J (2009) Trafficking and function of the tetraspanin CD63. Exp Cell Res, 315, 1584-1592.

*As indicated by the editor, we ignored the comments of reviewer #1. 3rd Editorial Decision 11 February 2011

Thank you for sending us your revised manuscript. Referees 2 and 3 have now seen it again and support publication here. Nevertheless, they raise a number of remaining minor issues (see below) that need to be addressed before we can ultimately accept your manuscript. Furthermore, there are a number of editorial issues that need further attention. First, please adjust the format of the 'References' section to EMBOJ format (i.e. no numbering; please see 'guide to the authors'). Second, please add the statistical details to the legends of figures: 4G; 5C; 8D; 9B,C,D; S3B, S4E; S6; S7C. Third, prior to acceptance of every paper we perform a final check for figures containing lanes of gels that are assembled from cropped lanes. We recognise that cropping and pasting may be considered acceptable practices in some cases (please see Rossner and Yamada, JCB 166, 11-15, 2004). Still, according to our editorial policies, there needs to be a proper indication as well as an explanation in the figure legend where such processing has been done. Please note that it is our standard procedure to ask for the original scans when images have been assembled from cropped lanes without adequate indication and/or explanation. Along these lines, I would like to ask you to indicate in the legend of figure 3A and 3C that all lanes that are compared directly ('cell' versus 'exo') come from the same gel. We also need to see the original scans for these panels. I am sorry to have to be insistent on this at this late stage. However, we feel that it is in your as well as in the interest of our readers to present high quality figures in the final version of the paper. Thank you very much for your cooperation. Yours sincerely, Editor The EMBO Journal



REFEREE REPORTS Referee #2 (Remarks to the Author): Comments to revised version of manuscript by Verweij et al. The revised version of the manuscript by Verweij et al. has been significantly improved versus the initial version. The authors provide a large set of additional data and/or informations to satisfactorily address the reviewer's comments and questions. In summary the manuscript is suitable for publication in the EMBO Journal after the authors have addressed the following two points: 1. As requested (point 6, referee 2), the authors now provide data demonstrating that TRAF3 is not co-localized with LMP1 in peripheral vesicles (Figures 4 and 9) and that LMP1-YFP, which is precluded from secretion through exosomes, colocalizes with TRAF3 in GM130-positive Golgi. However, data presentation should be improved to make them clearer to the reader as follows: - Figure 4I is too small. It should have approx. the size of Figures 4A-C and overlays should be shown. - Figures 8 E, F are confusing and difficult to understand. Even the Figure legend does not help much. What was transfected, what was the staining, what do we see in each image? For instance, why is the bottom image of Figure 8E labeled with TRAF3, although it was stained with GM130 according to the legend? Figure legend and labeling of the images must be improved. Moreover, the images of Figure 8F are too small. 2. As requested (point 8, referee 2), the authors have performed CD63 knockdown (KD) experiments in HEK293 cells and tested whether LMP1 sorting into exosomes is reduced, which is an important control experiment. Surprisingly, they found that there was only marginal effect on cellular LMP1 levels and even no effect of CD63 KD on sorting of LMP1 into exosomes (the latter information is only given in the rebuttle latter, but not in the paper). These observations are a certain problem for conclusions made in the paper, see e.g. the title "LMP1 association with CD63 in the endosomal-exosomal pathway limits NF-kB activation" or the interpretation (page 18, bottom paragraph) "... it is tempting to speculate that formation of endosomal subdomains with closely associated LMP1 and CD63 may drive the sorting and secretion through exosomes..." which imply that CD63 is required for LMP1 showing up in exosomes and, thus, NF-kB regulation. This reviewer, however, acknowledges the convincing data showing that CD63 KD has striking effects on subcellular localization of LMP1 and NF-kB activation (Figures 9B, C), demonstrating that CD63 is crucial for LMP1 trafficking and regulation of signaling which are key observations of this study. However, it might be that CD63 is essential for sorting of LMP1 into MVEs but not for exosome secretion, although both can be found together in exosomes. The authors suggest in the rebuttle letter that other tetraspanins could compensate for CD63 loss in exosome inclusion of LMP1. On the other hand it is surprising that LMP1 accumulates in perinuclear regions after CD63 KD (Figure 9B), whereas at the same time there are no effects on exosomal secretion of LMP1. How does this work and how does LMP1 get into exosomes in this situation? The authors should inform the reader about the missing effects of the CD63 KD on LMP1's localization into exosomes and discuss possible implications. Statements implying that CD63 is essential for exosome sorting of LMP1 should be weakened unless the authors can come up with data demonstrating this. Referee #3 (Remarks to the Author): the authors have succesfully addressed my previous concerns (although they should ahve provided quantification of the confocal experiments of figure 7 in addition to figures 1 and 4). But Legends of some of the modified figures have not been accordingly modified (fig5D: HLA-DR is not mentioned, Fig8E-F is not clearly explained). In addition, one answer to the other reviewer puzzles me: the authors say that CD63 KD does not decrease LMP1 secretion in exosomes, and explain that CD63 is probably compensated by other tetraspanins. But if this is true, how do they explain that nfkB signaling via LMP1 is increased by CD63 KD and that LMP1 is relocalized to the Golgi: this should decrease LMP1 secretion on exosomes! This somehow weakens the connexion that the authors propose between secretion in



exosomes and inhibition of nfkb signaling, maybe they should amend the proposed model?... 2nd Revision - authors' response 04 March 2011

Response to the editor We have dealt with the three editorial issues as mentioned in your e-mail. 1st) the literature format has been adjusted 2nd) the statistical details are included in the figure (legends) as requested. 3rd) The full scans of the images in figure 2 are included (below) and the legend now informs the reader on the figures content. Response to the referees; Referee II mentions the small size of figure 4I, and asked for an overlay. We changed the size of these figures according to the size of figure 4B and included an overlay, as suggested by the referee. We removed the single stainings for figure 4G, since they do not provide extra information to both the overlay and the correlation graph already shown in figure 4G. Both referees mention the lack in clarity of figures 8E and F and what they represent. We adjusted figure 8E and the legend which now reads; (E) HEK293 cells transfected with LMP1-YFP were analyzed by CLSM showing similar (co) localization of LMP1-YFP and TRAF3 in the perinuclear region, presumably Golgi compartments. (F) CLSM analysis of HEK293 cells that express YFP-LMP1, or LMP1-YFP or wtLMP1 (control) were fixed and stained with the cis-Golgi marker GM130. Represented in each image are 2 individual cells. Note the ‘fuzzy’ GM130-Golgi staining in the LMP1-YFP expressing cells (right image). Both referees mentioned the marginal, and therefore somewhat disappointing effects on LMP1 levels we observed in the CD63 KD cells. In the LMP1-inducible BJAB cells we observed that newly synthesized LMP1 was rapidly secreted with CD63 through exosomes. We repeated the CD63 KD experiments and decided to measure LMP1 levels in both the CD63 KD and control cells as well as the exosome fraction at earlier time points. Our new findings clearly demonstrate a sizable increase in LMP1 levels in CD63 KD vs control cells after 24 hours. Accordingly, LMP1 levels in the exosome fraction at this time point are markedly reduced (Figure 9E). We concluded based upon reduced HSP70 levels in the exosome fraction, that general exosome production maybe reduced in the CD63 KD cells compared to control. We comment on these results in the revised result section at pages 15-16 and in the discussion at page 18.



Primary Scans for Figure 2



4th Editorial Decision 17 March 2011

Thank you for sending us your re-revised manuscript. Referee 2 has now seen it again and thinks that you have now addressed all remaining issues in an adequate manner (see below). Still, prior to publication, there are a number of remaining editorial issues that need to be addressed: primary scans to figure 3A: the primary scans for HLA-DR and HSP70 appear to be different blots than those show in the figure. Could you check this once more and send us the correct scans, please? statistical details for figure 9: the number of independent experiments that were performed (n=?) needs to be included into the figure legend for each plot. statistical details for figure S3: two independent experiments (n=2) are not sufficient to calculate standard deviations and error bars. In this case, it would be better to either reproduce the experiment once more (n=3) or to show the two individual points in a vertical arrangement rather than column diagram and to indicate this in the figure legend. Finally, according to our updated guidelines for authors, I need to ask you to include an author contribution section into the manuscript text. Thank you for your cooperation. Yours sincerely, Editor The EMBO Journal REFEREE REPORTS



Referee #2 (Remarks to the Author): Review to 2nd revised version of Verweij et al. The authors have again improved the paper and satisfactorily addressed all important issues raised by the reviewers. The data regarding shCD63 effects on LMP1 signaling, expression and exosome inclusion are sound and convincing now. The manuscript in its present form represents a novel, beautiful and convincing body of work and should be published in the EMBO J. 3rd Revision - authors' response 22 March 2011

We were delighted with the kind response of the referee concerning our latest results. We believe we have dealt adequately with the remaining 2 editorial issues as mentioned in your e-mail.

LMP1 association with CD63 in endosomes and secretion...

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