BiP/GRP78 is a pro-viral factor for diverse dsDNA viruses that promotes the survival and proliferation of cells upon KSHV infection

  1. Guillermo Najarro
  2. Kevin Brackett
  3. Hunter Woosley
  4. Leah C. Dorman
  5. Vincent Turon-Lagot
  6. Sudip Khadka
  7. Catya Faeldonea
  8. Osvaldo Kevin Moreno
  9. Adriana Ramirez Negron
  10. Christina Love
  11. Ryan Ward
  12. Charles Langelier
  13. Frank McCarthy
  14. Carlos Gonzalez
  15. Joshua E. Elias
  16. Brooke M. Gardner
  17. Carolina Arias

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Abstract

The Endoplasmic Reticulum (ER)-resident HSP70 chaperone BiP (HSPA5) plays a crucial role in maintaining and restoring protein folding homeostasis in the ER. BiP’s function is often dysregulated in cancer and virus-infected cells, conferring pro-oncogenic and pro-viral advantages. We explored BiP’s functions during infection by the Kaposi’s sarcoma-associated herpesvirus (KSHV), an oncogenic gamma-herpesvirus associated with cancers of immunocompromised patients. Our findings reveal that BiP protein levels are upregulated in infected epithelial cells during the lytic phase of KSHV infection. This upregulation occurs independently of the unfolded protein response (UPR), a major signaling pathway that regulates BiP availability. Genetic and pharmacological inhibition of BiP halts KSHV viral replication and reduces the proliferation and survival of KSHV-infected cells. Notably, inhibition of BiP limits the spread of other alpha- and beta-herpesviruses and poxviruses with minimal toxicity for normal cells. Our work suggests that BiP is a potential target for developing broad-spectrum antiviral therapies against double-stranded DNA viruses and a promising candidate for therapeutic intervention in KSHV-related malignancies.

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  1. Version published to 10.1371/journal.ppat.1012660
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    Reply to the reviewers

    __Reviewer #1 (Evidence, reproducibility, and clarity (Required)):____ __ Summary: Viruses exploit host endoplasmic reticulum (ER)-resident chaperones to support new protein synthesis during viral replication. Here, Najarro et al. study the role of the ER-resident HSP70 family member Binding immunoglobulin protein (BiP) during lytic infection by the Kaposi's sarcoma-associated herpesvirus (KSHV). Using the established doxycycline-inducible lytic reactivation infection model cell line iSLK-BAC16, they showed that KSHV reactivation leads to an upregulation of total BiP protein but not RNA, and is independent of the unfolded protein response. siRNA knockdown or pharmacological inhibition by HA15 of BiP significantly reduced global viral gene expression and infectious virus production. The authors attribute this to at least the reduction of levels of the K1 gene which is required for efficient viral replication. Finally, they showed that HA15 has cytostatic activity in KSHV-transformed B cells and cytotoxic effects in KSHV-infected lymphatic endothelial cells arguing for BiP inhibition as a potential therapeutic strategy to treat KSHV-driven malignancies. The manuscript is well-written and the conclusions were generally supported by the data with a few exceptions below.

    Major comments:

    They propose in lines 196-199 that the reduction of K1 from HA15 treatment partially explains the defect in virion production during lytic reactivation. I am not convinced that this statement is fully supported by their data. Reduction of K1 is likely a downstream consequence and not the cause of the inhibition of lytic replication.

    We thank the reviewer for this comment. We conducted a more detailed analysis of our RNAseq data in iSLK.219 cells and confirmed the downregulation of the K1 transcript in latently infected cells treated with HA15 (See Fig 3 and Sup Fig 5). It is likely that the drop in transcript levels results from IRE1-mediated degradation in a recently-described process known as RIDDLE (IRE1-mediated RNA decay lacking endomotif), in which IRE1 depletes mRNAs1*. We have included this hypothesis in the discussion. *

    Unfortunately, we cannot confirm the downregulation of K1 at the protein level in iSLK.219 cells since the antibodies are highly specific for K1 variants in PEL cells. To overcome this technical limitation, we conducted mass spectrometry analysis of the viral proteome from whole cell lysates of latent and lytic cells undergoing HA15 treatment. While we detect the expected global downregulation of viral proteins in lytic cells treated with HA15, we were not able to detect any viral proteins except for LANA in the latently infected cells, and our detection of several lytic proteins was limited. We speculate that the levels of latent viral proteins expressed in iSLK.219 cells are below the limits of detection of our assay, or that extensive modification of some of these viral proteins may hinder their detection. Due to these limitations, we decided not to include these data in the manuscript.

    • Additionally, we note that the lower levels of K1 detected in latent iSLK.219 and TREx-BCBL-1 cells treated with HA15 may affect viral reactivation, which is consistent with findings from the Damania lab showing K1's crucial role in viral replication2.*

    The quantification of the K1 blots in Fig. 3C only has n=2. With subtle differences by eye, large error bars, and no statistical analysis, it is hard to conclude here with confidence. *

    We agree with the reviewer. We have moved the K1 blot to the Sup. Fig. 3E and adjusted the text accordingly.* *

    Like K1, ORF45, and K8.1 proteins are similarly decreased at 24 h in Fig. 2E, suggesting that the defect is upstream of K1. Does HA15 affect the amount of endogenous and/or transgene copy of RTA being produced (hence the broader effect in early gene expression at 24h?)?

    • **To answer the Reviewer's query, we re-evaluated the impact of HA15 treatment on the activity of dox-inducible RTA. However, we think it is unlikely for HA15 to alter RTA activity since RTA does not enter the secretory pathway. *

    To evaluate the activity of RTA in HA15 treated cells, we measured the expression of the viral episome-encoded RFP reporter, driven by the viral PAN promoter4*, at 24h post-doxycycline treatment of iSLK.219 cells. We compared the response of the PAN promoter to RTA in cells treated with or without HA15 at this early timepoint, to avoid any potential confounding effects stemming from elevated endogenous RTA expression at later times post-reactivation. We demonstrate that the levels of RFP in iSLK.219 cells treated with Dox are identical in presence or absence of HA15. This result, included in Sup. Fig. 3, indicates that the activity of RTA, crucial for initiating the lytic cycle in this context, is unaffected by BiP inhibition at early times post reactivation. *

    K1 levels appear to decrease even during latency. Are the other latent proteins also affected? What about latent genome copies?

    To address this query, we compared the Log2 fold change of latent transcripts (K1, K2, K12, ORF71, ORF72, ORF73) in the iSLK.219 RNAseq data set (Fig 3). Only the K1 transcript is reduced in HA15-treated cells. We include these data in Sup Fig 5A.

    Regarding differences in genome copies, the consistent levels of the viral genome-encoded GFP in HA15 -/+ iSLK-219 cells (Sup Fig 3) indicate no significant changes in the levels of viral genomes at 24h post-treatment (prior to DNA replication). Previous studies by our lab and others show that knockdown of the major latency protein LANA results in episomal loss and lower levels of GFP5*. These results validate the use of GFP fluorescence in iSLK.219 as a proxy for genome copies. *

    Fig. 3C was performed in a PEL cell line which they showed to enter cytostasis upon HA15 treatment (Fig. 5). This cytostasis (rather than K1) may be the root cause of the defect in viral replication as cells could be arrested at a different stage compared to the G2 requirement for lytic replication in PEL cells (Balisteri et al., PLOS Pathogens 2016, PMID: 26891221).

    See point 2. below

    The cytostatic effect in PEL cell lines (Fig. 5) should be demonstrated using more direct methods that measure cell cycle (e.g. PI-BrdU).

    *We thank the reviewer for this comment. While more direct methods to measure the cell cycle stage affected by HA15 treatment will inform on its mechanism of action, these experiments lie outside of the scope of this manuscript and we consider are better suited for future studies on the anticancer properties of HA15. The data presented in Fig. 5 demonstrates that HA15 treatment of PEL cells causes a reduction in cell numbers without cytotoxicity, thus supporting our conclusion of a net negative effect on proliferation rather than cell death. The loss of our LN2 tank and PEL cell lines currently limits *our ability to do these more detailed analyses. At the moment, we do not have an accurate estimate of how long it will take to replace these cell lines for our subsequent studies.

    While having an uninfected B cell as a matched negative control for PEL is challenging, primary peripheral B cells (mostly of mature memory B cell stage) may not be the appropriate negative control. PEL cells are of plasma cell lineage which have unusually high protein translation and overloaded ER. The plasma cell lineage may explain the sensitivity of PEL cells to HA15. It is possible that HA15 may be toxic to plasma cells when used as a therapeutic agent.

    We agree with the reviewer on the potential impact of HA15 on plasma cell viability. Indeed, HA15 (>2uM) treatment reduces the viability of plasma cell myeloma lines (NCI-H929 and U266 cells), substantiating its use as a potential anti-cancer drug6. Although HA15 has not been tested as a therapeutic agent in humans, studies in mice have demonstrated tolerability without evident toxicity, measured as normal body weight7*. The potential therapeutic application of HA15 for cancer warrants further investigation and is beyond the scope of our manuscript. *

    Does HA15 have cytostatic effects in uninfected or latently infected iSLK cells?

    We observed no cytostatic or cytotoxic effects in uninfected or latently infected iSLK cells exposed to up to 30uM of HA15. Although HA15 has been tested on various cancer types8*, it has not been evaluated in Renal Carcinoma Cells (RCC), the cellular background of iSLK.219 cells. The mechanism behind the resistance of these cells to HA15 eludes us, but its link to the cellular background of iSLK.219s merits exploration in future studies. *

    Minor comments:

    1. Consider changing the title of line 98 to specify cell type since BiP levels do not increase in BCBL-1 (Supp. Fig. 3).

    Revised in the manuscript

    Fig. 3A may benefit from using z-scores instead of log2TPM so differences are more obvious per gene.

    Since the data have already been collected, can the authors include both latent and lytic cells with and without HA15 treatment in Fig. 3A? It may give more information for the reader. *

    *We have reanalyzed all the RNAseq data and included a z-score plot for all samples in Fig. 3. We also providing three new supplementary tables with the raw counts, the z-scores for viral genes, and the log2 of the normalized counts.

    *Reviewer #1 (Significance (Required)):

    Significance: Here, the authors convincingly demonstrate the proviral role of the ER chaperone BiP during KSHV reactivation. This manuscript will be relevant to researchers in the gammaherpesvirus field. Although the authors did present some interesting data, the scope is narrow, and mechanistic studies were not pursued that would have added more insight in BiP and/or KSHV biology. For instance, how do BiP protein levels increase during reactivation (is this at the level of RNA sequestration/export, translation, or protein stability?)? How does BiP promote lytic replication?

    Field of expertise: KSHV, molecular and cell biology

    __Reviewer #2 (Evidence, reproducibility and clarity (Required)): __ Many viruses have complex relationships with cellular ER proteostasis machinery that remain poorly understood. Here Najarro, et al. report on studies of the oncogenic gammaherpesvirus KSHV. They report that the ER chaperone BiP is upregulated in epithelial cells during KSHV lytic replication. Unexpectedly, BiP upregulation is independent of the unfolded protein response, which stimulates transcriptional activation of BiP to meet the protein folding demand in the ER. Using a combination of genetic and pharmacologic approaches (CRISPRi and selective chemical inhibitor) they demonstrate that BiP inhibition interferes with the replication of diverse enveloped viruses including poxviruses and several herpesviruses, and reduces proliferation of KSHV-infected cells.

    Figure-by-figure:

    Fig. 1: This figure convincingly demonstrates the selective upregulation of BiP at the protein level during the course of KSHV lytic replication, and that KSHV late genes are dispensable for this upregulation. It further demonstrates that BiP is not upregulated at the mRNA level at all during KSHV infection, despite the fact that the UPR-dependent BiP mRNA upregulation pathway (presumably via ATF6 and IRE1) remains functional.

    Fig. 2: This figure convincingly demonstrates that BiP ATPase activity is required to support KSHV lytic replication in both epithelial and B cell models on infection, even though it is also clear that BiP is not upregulated in the B cell model.

    Fig. 3: This data demonstrates that steady-state levels of KSHV lytic gene products are reduced following HA15-treatment, whereas later gene expression was unaffected. As an interesting side note, v-IL6 bucks the trend of HA15-mediated downregulation of viral mRNA levels, suggesting that it may be regulated in a different manner. One thing that the authors may consider is the report from Drs. Yuan Chang and Patrick Moore (PMID: 12434062) that demonstrated that the v-IL6 gene is transactivated by type I interferon. Considering the poor replication of this virus during HA15 treatment, it may be valuable to investigate IFN production by these cells, and the extent to which it is impacted by inhibition of BiP ATPase activity.*

    We thank the reviewer for bringing this report to our attention. We also found intriguing the specific transcriptional upregulation of IL6 in IFN-a treated BCP-1 cells. Although we see a dramatic upregulation of the vIL6 in HA15 treated cells, we still detect the expression of most viral genes, albeit at significantly lower levels than in untreated cells, which indicates that the viral transcriptional program in lytic+HA15 iSLK.219 cells is different from the one seen in IFN-treated BCP-1 cells. Preliminary analyses of the host transcriptome from our RNAseq results show the expression of several ISGs (OAS1, 2 and 3, IFI6, IFIT1, IFIT3, IFITM1) in lytic-untreated iSLK.219 cells, but not in those treated with HA15. Together, these observations substantiate the notion that there is no IFN-driven expression of vIL6 in HA15-treated iSLK.219 cells.

    Fig. 4: This figure demonstrates that HA15 has broad, non-cytotoxic, antiviral activity against diverse enveloped viruses.

    Figs. 5/6: These figure shows cytotoxic effects of HA15 on latently infected PEL cells, either solely infected with KSHV or co-infected with KSHV and EBV, whereas normal B cells were unaffected. HA15 was also cytotoxic to KSHV infected lymphatic endothelial cells.

    **Referees cross-commenting**

    I appreciate the insightful comments from Reviewer #1 and Reviewer #3. I think we are largely on the same page. The data is generally supportive of author's conclusions, with a few exceptions that are straightforward to address in revisions. The manuscript is limited in scope, which could also be addressed by additional experimentation if the authors are motivated to explore mechanism in greater depth. Of particular note is the lack of mechanistic insight into how BiP is upregulated at the protein level during lytic replication, if the mRNA is unchanged. The experimental approaches to this are straightforward.

    We appreciate the reviewers' comments on the scope of our study. The mechanism of BiP upregulation remains an outstanding question for the following technical reasons: We hypothesized that the upregulation of BiP may depend on the IRES element present in its 5' UTR9. We tested this hypothesis by transfecting iSLK.219 cells with a bicistronic Renilla-(BiP)IRES-Firefly luciferase reporter from Licursi et. al10*. Unfortunately, for reasons that still elude us, our reactivation rates in transfected cells were consistently low in all of our experiments and therefore, we were not able to measure luciferase changes consistently and reliably. A potential workaround this technical limitation is to use a lentivirus-encoded IRES reporter to a lentiviral vector, as transduction of iSLK.219 cells does not alter viral reactivation, in our experience. At the moment, we do not have access to these reporters due to our lab's move to a different institution, and the first author of our study has started the next stage of their career. Therefore, we will not be able to pursue these experiments in a timely manner. *

    *As for the scope of this manuscript, even when the mechanism of BiP upregulation in KSHV infected cells remains unsolved, we consider that the broad-spectrum antiviral effect of BiP inhibition is an exciting finding that advances the field and benefits the virology community-the proteostasis network has been seldomly explored as a potential node for broad-spectrum antiviral intervention. Our results provide important proof-of-concept to continue the investigation of factors involved in protein synthesis, folding and transport as potential targets for the development of versatile broad-spectrum antivirals. *

    Reviewer #2 (Significance (Required)):

    Strengths: This is a well-written manuscript. The text and figures are clear and accurate and the methods are sufficiently informative that the study can be reproduced. The data generally supports the authors' conclusions. BiP appears to be a druggable target with minimal off-target cytotoxicity in normal, uninfected cells, although this study does not go beyond cell culture studies to validate in vivo.

    Weaknesses: The study is somewhat limited in scope. The authors make the case for UPR transcription-independent upregulation of BiP during KSHV infection, and that late gene synthesis is dispensable, but the mechanism is not investigated further.

    Point by point discussion:

    Could an early KSHV gene product involved in this phenotype be identified by screening an ORF library or viral genome-wide CRISPRi screen?

    The question of the viral protein responsible for the upregulation of BiP during lytic infection is indeed a fascinating one. However, we suspect that the mechanism may be not specifically directed to BiP, but rather general modulation of IRES-related translation. Identifying the gene product(s) affected and corroborating IRES involvement is a major undertaking and a long-term goal requiring considerable effort. These analyses are outside the scope of this manuscript, but we will pursue them in the future.

    Or, beyond implicating viral factors in the mechanism of BiP upregulation, can some simple biochemical studies be performed to investigate BiP protein? Is the BiP mRNA more efficiently spliced and exported in KSHV infected cells?

    Do alternative translation initiation mechanisms like eIF2A play a role in boosting BiP levels during infection?

    What is the normal BiP protein turnover mechanism, and is this hindered during KSHV lytic replication? Is BiP AMPylation/de-AMPylation by FICD affected (PMID: 36041787)? These kinds of mechanistic studies are well within reach and would help extend the impact and interest to a broad audience.

    We agree on the putative involvement of translation initiation factors like eIF2A on promoting the translation of BiP (see discussion). We tested the effect of siRNA-mediated KD of eIF2A on BiP expression and found that, interestingly, the levels of BiP rose above those of controls in latent iSLK.219 cells (Data included in the manuscript and the discussion has been modified accordingly). This finding aligns with previous reports suggesting that eIF2A may suppress IRES-mediated translation in yeast cells and in mammalian in vitro translation assays. Moreover, Starck et. al11, observed a 50% increase of endogenous BiP levels in HeLa cells transfected with siRNAs against eIF2A, supporting the IRES-suppressor role for eIF2A in mammalian cells. Future work will be required to address the role of eIF2A on BiP translation. These analyses are beyond the scope our manuscript.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    The manuscript by Najarro et al. investigates the contribution of BiP/GRP78 to double-stranded DNA virus infection, primarily focusing on the oncogenic gammaherpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV). The authors observe that BiP expression is increased in lytic iSLK.219 cells as well as in KSHV-infected LECs. Interestingly, the authors data suggest a post-translational regulation of BiP in the iSLK.219 cells. Using various knockdown approaches and chemical inhibitors the authors demonstrate that inhibition of BiP impacts KSHV reactivation in multiple cells lines. Importantly, the authors also find that BiP inhibition can selectively kill KSHV-infected cells, while sparing primary B cells. Overall, this is a very well controlled and presented manuscript. My comments for the manuscript are minor, and largely cosmetic to aid the presentation of the data.

    • Fig 1C, It would be ideal to show that PAA treatment did indeed prevent the virus from entering the late stage of gene expression.

    *We have included an immunoblot for K8.1 in Figure 1C to confirm the effect of PFA on arresting the KSHV lytic cycle. *

    Sup Fig2, should show KD efficiency of XBP1, same goes for ATF6.

    Sup. Fig. 2D shows the expression of XBP1s in NS vs. XBP1KD cells in the presence or absence of Tg. In Sup Fig. 2G we have also included a bar graph showing the efficiency of downregulation of ATF6 mRNA in the presence of the targeting sgRNA.

    Sup Fig 3. It is interesting that the authors do not see increased BiP in TREx-BCBL1-RTA cells. A potential caveat is that lytic reactivation in TREx-BCBL1-RTA cells is not as efficient as in iSLK.219 cells. Therefore, it may simply be a result of the reduced population entering the lytic cycle. It may be worth adding a comment regarding this.

    Images of the microscopy for Figure 4 would be useful.

    Images have been included in Fig. 4

    • Add label of the cell types for Figure 5.

    DONE

    • Does HSV1, HCMV, or VacV increase BiP expression compared to mock-infected cells?

    Yes, we have included a comment on this in the discussion

    Reviewer #3 (Significance (Required)):

    Overall, this is a very well controlled and presented manuscript.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #3

    Evidence, reproducibility and clarity

    The manuscript by Najarro et al. investigates the contribution of BiP/GRP78 to double-stranded DNA virus infection, primarily focusing on the oncogenic gammaherpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV). The authors observe that BiP expression is increased in lytic iSLK.219 cells as well as in KSHV-infected LECs. Interestingly, the authors data suggest a post-translational regulation of BiP in the iSLK.219 cells. Using various knockdown approaches and chemical inhibitors the authors demonstrate that inhibition of BiP impacts KSHV reactivation in multiple cells lines. Importantly, the authors also find that BiP inhibition can selectively kill KSHV-infected cells, while sparing primary B cells. Overall, this is a very well controlled and presented manuscript. My comments for the manuscript are minor, and largely cosmetic to aid the presentation of the data.

    • Fig 1C, It would be ideal to show that PAA treatment did indeed prevent the virus from entering the late stage of gene expression.
    • Sup Fig2, should show KD efficiency of XBP1, same goes for ATF6.
    • Sup Fig 3. It is interesting that the authors do not see increased BiP in TREx-BCBL1-RTA cells. A potential caveat is that lytic reactivation in TREx-BCBL1-RTA cells is not as efficient as in iSLK.219 cells. Therefore, it may simply be a result of the reduced population entering the lytic cycle. It may be worth adding a comment regarding this.
    • Images of the microscopy for Figure 4 would be useful.
    • Add label of the cell types for Figure 5.
    • Does HSV1, HCMV, or VacV increase BiP expression compared to mock-infected cells?

    Significance

    Overall, this is a very well controlled and presented manuscript.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    Many viruses have complex relationships with cellular ER proteostasis machinery that remain poorly understood. Here Najarro, et al. report on studies of the oncogenic gammaherpesvirus KSHV. They report that the ER chaperone BiP is upregulated in epithelial cells during KSHV lytic replication. Unexpectedly, BiP upregulation is independent of the unfolded protein response, which stimulates transcriptional activation of BiP to meet the protein folding demand in the ER. Using a combination of genetic and pharmacologic approaches (CRISPRi and selective chemical inhibitor) they demonstrate that BiP inhibition interferes with the replication of diverse enveloped viruses including poxviruses and several herpesviruses, and reduces proliferation of KSHV-infected cells.

    Figure-by-figure:

    Fig. 1: This figure convincingly demonstrates the selective upregulation of BiP at the protein level during the course of KSHV lytic replication, and that KSHV late genes are dispensable for this upregulation. It further demonstrates that BiP is not upregulated at the mRNA level at all during KSHV infection, despite the fact that the UPR-dependent BiP mRNA upregulation pathway (presumably via ATF6 and IRE1) remains functional.

    Fig. 2: This figure convincingly demonstrates that BiP ATPase activity is required to support KSHV lytic replication in both epithelial and B cell models on infection, even though it is also clear that BiP is not upregulated in the B cell model.

    Fig. 3: This data demonstrates that steady-state levels of KSHV lytic gene products are reduced following HA15-treatment, whereas later gene expression was unaffected. As an interesting side note, v-IL6 bucks the trend of HA15-mediated downregulation of viral mRNA levels, suggesting that it may be regulated in a different manner. One thing that the authors may consider is the report from Drs. Yuan Chang and Patrick Moore (PMID: 12434062) that demonstrated that the v-IL6 gene is transactivated by type I interferon. Considering the poor replication of this virus during HA15 treatment, it may be valuable to investigate IFN production by these cells, and the extent to which it is impacted by inhibition of BiP ATPase activity.

    Fig. 4: This figure demonstrates that HA15 has broad, non-cytotoxic, antiviral activity against diverse enveloped viruses.

    Figs. 5/6: These figure shows cytotoxic effects of HA15 on latently infected PEL cells, either solely infected with KSHV or co-infected with KSHV and EBV, whereas normal B cells were unaffected. HA15 was also cytotoxic to KSHV infected lymphatic endothelial cells.

    Referees cross-commenting

    I appreciate the insightful comments from Reviewer #1 and Reviewer #3. I think we are largely on the same page. The data is generally supportive of author's conclusions, with a few exceptions that are straightforward to address in revisions. The manuscript is limited in scope, which could also be addressed by additional experimentation if the authors are motivated to explore mechanism in greater depth. Of particular note is the lack of mechanistic insight into how BiP is upregulated at the protein level during lytic replication, if the mRNA is unchanged. The experimental approaches to this are straightforward.

    Significance

    Strengths: This is a well-written manuscript. The text and figures are clear and accurate and the methods are sufficiently informative that the study can be reproduced. The data generally supports the authors' conclusions. BiP appears to be a druggable target with minimal off-target cytotoxicity in normal, uninfected cells, although this study does not go beyond cell culture studies to validate in vivo.

    Weaknesses: The study is somewhat limited in scope. The authors make the case for UPR transcription-independent upregulation of BiP during KSHV infection, and that late gene synthesis is dispensable, but the mechanism is not investigated further. Could an early KSHV gene product involved in this phenotype be identified by screening an ORF library or viral genome-wide CRISPRi screen? Or beyond implicating viral factors in the mechanism of BiP upregulation, can some simple biochemical studies be performed to investigate BiP protein? Is the BiP mRNA more efficiently spliced and exported in KSHV infected cells? Do alternative translation initiation mechanisms like eIF2A play a role in boosting BiP levels during infection? What is the normal BiP protein turnover mechanism, and is this hindered during KSHV lytic replication? Is BiP AMPylation/de-AMPylation by FICD affected (PMID: 36041787)? These kinds of mechanistic studies are well within reach and would help extend the impact and interest to a broad audience.

  5. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    Viruses exploit host endoplasmic reticulum (ER)-resident chaperones to support new protein synthesis during viral replication. Here, Najarro et al. study the role of the ER-resident HSP70 family member Binding immunoglobulin protein (BiP) during lytic infection by the Kaposi's sarcoma-associated herpesvirus (KSHV). Using the established doxycycline-inducible lytic reactivation infection model cell line iSLK-BAC16, they showed that KSHV reactivation leads to an upregulation of total BiP protein but not RNA, and is independent of the unfolded protein response. siRNA knockdown or pharmacological inhibition by HA15 of BiP significantly reduced global viral gene expression and infectious virus production. The authors attribute this to at least the reduction of levels of the K1 gene which is required for efficient viral replication. Finally, they showed that HA15 has cytostatic activity in KSHV-transformed B cells and cytotoxic effects in KSHV-infected lymphatic endothelial cells arguing for BiP inhibition as a potential therapeutic strategy to treat KSHV-driven malignancies. The manuscript is well-written and the conclusions were generally supported by the data with a few exceptions below.

    Major comments:

    1. They propose in lines 196-199 that the reduction of K1 from HA15 treatment partially explains the defect in virion production during lytic reactivation. I am not convinced that this statement is fully supported by their data. Reduction of K1 is likely a downstream consequence and not the cause of the inhibition of lytic replication. Consider revising this statement in light of my comments below:
      • a. The quantification of the K1 blots in Fig. 3C only has n=2. With subtle differences by eye, large error bars, and no statistical analysis, it is hard to draw conclusions from here with confidence.
      • b. Like K1, ORF45 and K8.1 proteins are similarly decreased at 24 h in Fig. 2E suggesting that the defect is upstream of K1. Does HA15 affect the amount of endogenous and/or transgene copy of RTA being produced (hence the broader effect in early gene expression at 24h?)?
      • c. K1 levels appear to decrease even during latency. Are the other latent proteins also affected? What about latent genome copies?
      • d. Fig. 3C was performed in a PEL cell line which they showed to enter cytostasis upon HA15 treatment (Fig. 5). This cytostasis (rather than K1) may be the root cause of the defect in viral replication as cells could be arrested at a different stage compared to the G2 requirement for lytic replication in PEL cells (Balisteri et al., PLOS Pathogens 2016, PMID: 26891221).
    2. The cytostatic effect in PEL cell lines (Fig. 5) should be demonstrated using more direct methods that measure cell cycle (e.g. PI-BrdU).
    3. While having an uninfected B cell as a matched negative control for PEL is challenging, primary peripheral B cells (mostly of mature memory B cell stage) may not be the appropriate negative control. PEL cells are of plasma cell lineage which have unusually high protein translation and overloaded ER. The plasma cell lineage may explain the sensitivity of PEL cells to HA15. It is possible that HA15 may be toxic to plasma cells when used as a therapeutic agent.
    4. Does HA15 have cytostatic effects in uninfected or latently infected iSLK cells?

    Minor comments:

    1. Consider changing the title of line 98 to specify cell type since BiP levels do not increase in BCBL-1 (Supp. Fig. 3).
    2. Fig. 3A may benefit from using z-scores instead of log2TPM so differences are more obvious per gene.
    3. Since the data have already been collected, can the authors include both latent and lytic cells with and without HA15 treatment in Fig. 3A? It may give more information for the reader.

    Significance

    Here, the authors convincingly demonstrate the proviral role of the ER chaperone BiP during KSHV reactivation. This manuscript will be relevant to researchers in the gammaherpesvirus field. Although the authors did present some interesting data, the scope is narrow, and mechanistic studies were not pursued that would have added more insight in BiP and/or KSHV biology. For instance, how do BiP protein levels increase during reactivation (is this at the level of RNA sequestration/export, translation, or protein stability?)? How does BiP promote lytic replication?

    Field of expertise: KSHV, molecular and cell biology

  6. Version published to 10.1101/2023.09.29.560238v1 on bioRxiv