Cellular Receptors Involved in KSHV Infection

Round 1

Reviewer 1 Report

This is a concise review of cell-specific receptors involved in KSHV infection. It also emphasizes the important concept that KSHV infects in two steps: binding and cell entry. 

One issue I had was in line 97, "..., results in reduced expression of HS with consequences for KSHV infection." Should it be "... consequences for reduced KSHV infection"? since HS is facilitating the binding of KSHV to target cells? Please clarify.

Author Response

Reviewer 1:

1.   This is a concise review of cell-specific receptors involved in KSHV infection. It also emphasizes the important concept that KSHV infects in two steps: binding and cell entry. One issue I had was in line 97, "..., results in reduced expression of HS with consequences for KSHV infection." Should it be "... consequences for reduced KSHV infection"? since HS is facilitating the binding of KSHV to target cells? Please clarify.

Author reply: We have edited the sentence to improve clarity (lines 104-105): “…resulted in reduced expression of HS which consequently resulted in reduced KSHV infection.”

Reviewer 2 Report

The review paper entitled ‘Cell-specific Receptors Involved in KSHV Infection’ by Meulen and others.  In this review, KSHV infection of specific cell types pertinent to its pathogenesis will be comprehensively summarized with a focus on the specific cell surface binding and entry receptors KSHV exploits to gain access to different cell types. The authors have presented literature on KSHV envelope glycoproteins (gB, gH/gL, K8.1) and host cell receptors (heparin sulfate, DC-SIGN, Eph, Integrins, and xCT) on various cell types (Endothelial, fibroblast, B cells, macrophages, dendritic cells, monocytes, and epithelial cells).

Three recent review papers (listed below and two of them are in the journal Viruses) have extensively covered advances and discoveries made in KSHV interaction with various cell-surface receptors during virus infection and entry in various susceptible cell types (1) Dollery, 2020, Viruses 2019, 11, 1073; doi:10.3390/v11111073. (2) Aalam and Totonchy, 2020. Front. Cell. Infect. Microbiol. 10:607663. doi: 10.3389/fcimb.2020.607663. and (3) Kumar and Chandran, 2016. Viruses. 14;8(11):305. doi: 10.3390/v8110305. This review paper has failed to capture recent developments in KSHV glycoproteins and receptors.  It has not provided a comprehensive presentation of literature and insightful discussion that can support publication in the current state. Thus, the paper needs a lot of improvements with major corrections as outlined below.

1. Mainly this paper lack of in-depth systematic presentation of knowledge and failure to capture and present accurate updated research work. The outline of different sections is repetitive and the absence of compelling discussion with the identification of research gaps and ways to move forward in the research field.

The specific corrections necessary are listed below:

Line 2: The title ‘Cell-specific Receptors Involved in KSHV Infection’ is imprecise (word - Cell-specific Receptors) as the KSHV utilize multiple receptors on a given cell type and those given particular receptors possibly found on different cell-types or not yet determined for the absence of receptor in other KSHV susceptible cell types.

Line 31.  In the introduction author should explain the rationale for the review in the context of what is already known.

Line 21 to 24: These two sentences are provocative in claiming the link between viral receptors with pathogenesis. To the best of my knowledge there is not experimental evidence or convincing argument to support this claim.

Line 42 to 44: The cellular receptor that KSHV binds to is dependent on their ‘occurrence/expression’  rather than cell types...

Line 39: Please provide more details on the KSHV infection of target cells.

Line 54: Section ‘2. KSHV envelope glycoproteins’ and section 3. ‘3. Host cellular binding and entry receptors used by KSHV’ are repetitive and mixed up. In-depth discussion citing seminal papers is lacking.

Line 59: ORF4 is an RSK activator protein and mentioned here as lytic protein is out of context. (Arias et al., 2014. PLoS Pathog 10(1): e1003847. doi:10.1371/) ORF27 and ORF28 are viruses envelope glycoproteins not cited.

Line 62: This section is on KSHV envelope glycoprotein and authors should focus more on those protein structural and functional mechanism aspects like various domains and mutagenesis studies. Here you could include text on herpesvirus fusion dogma in which non-conserved glycoprotein interacts with core fusion glycoproteins leading to virus entry and infection.

Line 87: ORF4 was also found to bind HSPG and this should be included. (Mark et al 2006 Apr;43(10):1665-75.  doi: 10.1016/j.molimm.2005.09.016). Also include and discuss many other HSPGs and KSHV studies (Ex: TerBush J Virol. 2018 Aug 16;92(17):e00803-18. doi: 10.1128/JVI.00803-18).

Line 106: This section should discuss receptor structure and its function in the uninfected cell life cycle. Some sentences are repetitive or out of context. Specific interaction details of various KSHV glycoproteins and receptor is missing (like gB binding specific domains on DC-SIGN, gH/gL Domain 4 binding EPhA2) .

Line 118: Discuss different types of Eph receptors and their normal functional relationship in the cell cycle.

Line 123: Discuss different types of integrins and their normal functional relationship in the cell cycle.

Line 126: This sentence is repeated, see Line 65.

Line 1: Endothelial introduction part of section 4.1.  Endothilial cells and Fibroblast’, ‘4,.2. B cells, macrophages, dendritic cells and monocytes’ and ‘4.3. Epithelial cells’ could be summarized/put  together to give readers a compressive understanding of steps in virus infection of various cell types in a single place.

Line 164: The author should identify the gaps and discuss future research directions (like to identify the KSHV glycoprotein interacting with xCT and others). If with the current information available the authors still believe that this is a receptor.

Line 170: Authors did not mention in which cell type the virus infection experiment were performed.

Line 178: It has now been identified as specific amino acid residues of EphA2 interaction with gH/gL. The author's own work on the EphA2 sequence (reference 64) could be more discussed in the context of sequence-functional interactions.

Line 206: The author could discuss the role of gH/gL interaction with Eph receptor in B cell infection (see Muniraju et al., J Virol. 2019 Jul 30;93(16):e00630-19. doi: 10.1128/JVI.00630-19.

Line 276: Absence of compelling discussion. Authors should identify the research gaps and ways to advance the understanding of KSHV glycoproteins and cellular receptors interactions in virus infection and in turn developing virus infection intervention strategies.

Author Response

Reviewer 2: 

1.     The review paper entitled ‘Cell-specific Receptors Involved in KSHV Infection’ by Meulen and others.  In this review, KSHV infection of specific cell types pertinent to its pathogenesis will be comprehensively summarized with a focus on the specific cell surface binding and entry receptors KSHV exploits to gain access to different cell types. The authors have presented literature on KSHV envelope glycoproteins (gB, gH/gL, K8.1) and host cell receptors (heparin sulfate, DC-SIGN, Eph, Integrins, and xCT) on various cell types (Endothelial, fibroblast, B cells, macrophages, dendritic cells, monocytes, and epithelial cells).Three recent review papers (listed below and two of them are in the journal Viruses) have extensively covered advances and discoveries made in KSHV interaction with various cell-surface receptors during virus infection and entry in various susceptible cell types (1) Dollery, 2020, Viruses 2019, 11, 1073; doi:10.3390/v11111073. (2) Aalam and Totonchy, 2020. Front. Cell. Infect. Microbiol. 10:607663. doi: 10.3389/fcimb.2020.607663. and (3) Kumar and Chandran, 2016. Viruses. 14;8(11):305. doi: 10.3390/v8110305. This review paper has failed to capture recent developments in KSHV glycoproteins and receptors.  It has not provided a comprehensive presentation of literature and insightful discussion that can support publication in the current state. Thus, the paper needs a lot of improvements with major corrections as outlined below. Mainly this paper lack of in-depth systematic presentation of knowledge and failure to capture and present accurate updated research work. The outline of different sections is repetitive and the absence of compelling discussion with the identification of research gaps and ways to move forward in the research field. The specific corrections necessary are listed below.

Author reply: We thank the reviewer for their thorough and helpful corrections below. Having addressed these (see below for specific feedback), we have improved the collation of updated research work in this review, the logical flow and clarity of the manuscript and the identification of research gaps in the literature.

2.     Line 2: The title ‘Cell-specific Receptors Involved in KSHV Infection’ is imprecise (word - Cell-specific Receptors) as the KSHV utilize multiple receptors on a given cell type and those given particular receptors possibly found on different cell-types or not yet determined for the absence of receptor in other KSHV susceptible cell types.

Author reply: We have rectified the imprecision noted by the reviewer by editing our title as follows, “Cellular Receptors involved in KSHV Infection”.

3.     Line 31.  In the introduction authors should explain the rationale for the review in the context of what is already known.

Author reply: The reviewer has pointed out three recent reviews on the topic of KSHV infection and asked us to place our review into the context of these and other literature. Our review is unique in its focus on cellular receptors rather than viral glycoproteins (as in the Dollery, 2019 review) in diverse cell types involved in KSHV pathogenesis (additionally to the focus on B cells in the Aalam and Totonchy, 2020 review) and as an update to this particular aspect also covered in the extensive review of Kumar and Chandran, 2016, published five years ago. The final sentence of the introduction has been amended to speak to this (lines 54-57): “This review comprehensively summarises the current knowledge on cellular receptors involved in KSHV infection of diverse cell types implicated in its pathogenesis to supplement recent reviews covering viral glycoproteins involved in KSHV infection [16] and infection in the lymphocyte compartment [17].”

4.     Line 21 to 24: These two sentences are provocative in claiming the link between viral receptors with pathogenesis. To the best of my knowledge there is not experimental evidence or convincing argument to support this claim.

Author reply: We have rephrased as follows (lines 22-24): “Several molecules involved in KSHV entry have been well characterized, particularly those postulated to be associated with KSHV-associated pathologies such as Kaposi’s Sarcoma (KS).” and have removed the second sentence which was provocative as suggested by the reviewer.

5.     Line 42 to 44: The cellular receptor that KSHV binds to is dependent on their ‘occurrence/expression’ rather than cell types...

Author reply: We have rephrased the sentence as follows (lines 44-46): “The cellular receptor(s) that KSHV binds to is dependent on its abundance or expression level on a particular cell, allowing KSHV its broad host cell tropism.”

6.     Line 39: Please provide more details on the KSHV infection of target cells.

Author reply: Details on KSHV infection of target cells is provided in the sentences that follow (lines 42-53): “Herpesviruses typically engage multiple cell surface receptors with their envelope glycoproteins to gain access. Some of these host molecules are required for binding to concentrate the virus on the cell surface, while others facilitate entry [11]. The cellular receptor(s) that KSHV binds to is dependent on its abundance or expression level on a particular cell, allowing KSHV its broad host cell tropism.

Following binding and making use of cellular signaling molecules, KSHV enters cells utilising diverse endocytic pathways including clathrin- and caveolin-mediated endocytosis, macropinocytosis and undefined endocytic entry pathways, depending on the cellular context [12]. Thereafter, the viral envelope fuses with the membrane of the endosome, likely triggered by low pH as in other herpesviruses, and the capsid is released into the perinuclear region. The KSHV genome enters the nucleus via nuclear pores where the linear genome rapidly undergoes circularization into an episome [13-16].”

7.    Line 54: Section ‘2. KSHV envelope glycoproteins’ and section ‘3. Host cellular binding and entry receptors used by KSHV’ are repetitive and mixed up. In-depth discussion citing seminal papers is lacking.

Author reply: This section has been thoroughly revised to avoid repetitions and to include aspects on glycoprotein function during viral fusion (65-75): “The glycoproteins considered essential to KSHV entry are K8.1, gB and the gH-gL heterodimer, and their engagement with specific cellular receptors is covered in section 3. The particular repertoire of cellular receptors available to engage with specific glycoproteins eventually leads to a concerted series of molecular events culminating in fusion of the viral envelope with the host cell membrane. It is widely accepted that gB, which is comprised of five functional domains typical for type III fusion glycoproteins, is the initial cell binding protein [29] and key fusogen leading to virus entry and infection, and that low pH may facilitate gB-mediated KSHV fusion [16]. A study of individuals from diverse geographical locations infected with KSHV showed that gB was highly conserved [30] and that KSHV infectivity could be neutralised by rabbit anti-gB antibodies [29]. Besides gB, the gH-gL heterodimer is required for fusion [31], and is hypothesized to play an important role specifically in the post-binding steps of KSHV infection…”

See also comment 9.

8.     Line 59: ORF4 is an RSK activator protein and mentioned here as lytic protein is out of context. (Arias et al., 2014. PLoS Pathog 10(1): e1003847. doi:10.1371/) ORF27 and ORF28 are viruses envelope glycoproteins not cited

Author reply: The sentence has been rephrased to reflect this and the reference has been added (lines 63-65): “The glycoproteins gpK8.1A and B, ORF4, ORF27, ORF28 and ORF68, associated with the lytic cycle, and ORF45, an RSK activator protein, are unique to KSHV [19, 21-28].”

9.     Line 62: This section is on KSHV envelope glycoprotein and authors should focus more on those protein structural and functional mechanism aspects like various domains and mutagenesis studies. Here you could include text on herpesvirus fusion dogma in which non-conserved glycoprotein interacts with core fusion glycoproteins leading to virus entry and infection.

Author reply: This section has been revised as outlined above (see comment 7), and the role of KSHV envelope glycoproteins during fusion has been highlighted; however, since the focus of this review is on cellular receptors used by KSHV rather than KSHV glycoproteins, this section has been kept rather concise.

10.  Line 87: ORF4 was also found to bind HSPG and this should be included. (Mark et al 2006 Apr;43(10):1665-75.  doi: 10.1016/j.molimm.2005.09.016). Also include and discuss many other HSPGs and KSHV studies (Ex: TerBush J Virol. 2018 Aug 16;92(17):e00803-18. doi: 10.1128/JVI.00803-18).

Author reply: The suggested study by Mark et al. (2006) has been included which now reads (lines 91-92): “There is also evidence that the KSHV complement control protein (KCP), encoded by ORF4, binds to HSPGs [38].” The recommended paper by TerBush et al. (2018) did not immediately focus on HSPGs and was therefore not included here.

11.  Line 106: This section should discuss receptor structure and its function in the uninfected cell life cycle. Some sentences are repetitive or out of context. Specific interaction details of various KSHV glycoproteins and receptor is missing (like gB binding specific domains on DC-SIGN, gH/gL Domain 4 binding EPhA2).

Author reply:  Section 3.2. serves as a short introductory section into the different entry receptors that can be potentially engaged by KSHV. It has been revised to avoid repetitions, and more detail has been provided in the individual sections under “4. Cell-type specific expression of surface receptors used by KSHV for cellular entry”, including the gB binding specific domain on DC-SIGN (lines 230-232): “DC-SIGN, a C-type lectin receptor primarily involved in innate immune recognition, has been found to bind KSHV gB through its carbohydrate recognition domain (CRD) [48]; and high-resolution structures of the gH/gL dimer binding to EPHA2 (lines 202-206): “A recent high-resolution structural investigation into the complex formation between KSHV gH-gL and the ligand binding domain (LBD) of EPHA2 revealed primarly gL protein binding to LBD [74]. It was further revealed that many amino acids of EPHA2’s LBD are potentially recognized by other γ-herpesviruses, thereby providing the structural basis of EPHA2 recognition by γ-herpesvirus gHgL [74].”

12.  Line 118: Discuss different types of Eph receptors and their normal functional relationship in the cell cycle.

Author reply:  More general information on Eph receptors has been added, with mentioning of the 2 classes of Eph receptors and their specific physiological roles, including involvement in KSHV infection. It now reads as follows (lines 130-134): “There are 14 different Eph receptors, divided into EphA and EphB classes by the homology of their extracellular domains. Ephrins A2, A4, A5 and A7 have been implicated in KSHV infection. Under normal physiological conditions EphA2 is known to play a key role in several developmental processes, cell migration, adhesion and trafficking, while EphA4, EphA5 and EphA7 are largely involved in the development of the nervous system [54].

13.  Line 123: Discuss different types of integrins and their normal functional relationship in the cell cycle.

Author reply:  More general information on integrins have been provided, with focus on those that are potentially engaged by KSHV. The revised section reads as follows (lines 138-145): “They function as transmembrane linkers between the ECM and the actin cytoskeleton of animal cells, and often cooperate with other receptors to promote cell growth, cell survival, and cell proliferation. For example, α3β1 plays an important role in establishing and maintaining epithelial tissues [57], while the αV integrins such as αVβ3 and αVβ5 bind a broad range of cell adhesion proteins such as vibronectin and play a role in angiogenesis [58, 59]. Integrin α5β1, also known as the fibronectin receptor, has functions in angiogenesis, particularly in endothelial cells during development. It may also act as a regulator of angiogenic signals [60].”

14.  Line 126: This sentence is repeated, see Line 65.

Author reply:  This sentence has been removed earlier and left in the “integrins” section, which now reads (lines 147-149): “… and indeed, KSHV gB was found to possess an integrin binding Arginine-Glycine-Aspartate (RGD) motif, potentially interacting with the integrins α3β1, αVβ3 and αVβ5, and α5β1 [63].

15.  Line 1: Endothelial introduction part of section 4.1.  Endothilial cells and Fibroblast’, ‘4,.2. B cells, macrophages, dendritic cells and monocytes’ and ‘4.3. Epithelial cells’ could be summarized/put together to give readers a compressive understanding of steps in virus infection of various cell types in a single place.

Author reply: As each of these sections (“Endothelial cells and Fibroblasts”, “B cells, macrophages, dendritic cells and monocytes” and “Epithelial cells”) is followed by subsections detailing the receptors utilised in these cell types, we feel that combining the sections would create confusion in the text and therefore will keep the sections discrete.

16.  Line 164: The author should identify the gaps and discuss future research directions (like to identify the KSHV glycoprotein interacting with xCT and others). If with the current information available the authors still believe that this is a receptor.

Author reply: A concluding sentence to this section has been included (lines 186-187): “However, it still needs to be determined which KSHV glycoprotein interacts with xCT to conclusively elucidate its function as an entry receptor.”

17.  Line 170: Authors did not mention in which cell type the virus infection experiment were performed.

Author reply: This has been added (lines 194-197): “In agreement, a separate study using HMVEC-d cells found that pre-treatment of KSHV with soluble EPHA2, using short hairpin RNA (shRNA) against EPHA2 or treating the endothelial cells with monoclonal antibodies towards EPHA2 significantly reduced KSHV internalisation and viral gene expression [72].”

18.  Line 178: It has now been identified as specific amino acid residues of EphA2 interaction with gH/gL. The author's own work on the EphA2 sequence (reference 64) could be more discussed in the context of sequence-functional interactions.

Author reply: A comment on gH/gL interaction with EPHA2 has been inserted (lines 202-206): “A recent high-resolution structural investigation into the complex formation between KSHV gH-gL and the ligand binding domain (LBD) of EPHA2 revealed primarly gL protein binding to LBD [74]. It was further revealed that many amino acids of EPHA2’s LBD are potentially recognized by other γ-herpesviruses, thereby providing the structural basis of EPHA2 recognition by γ-herpesvirus gHgL [74].” Also, more detail on the identified EPHA2 variants in our own clinical cohort has been provided (lines 207-209): “Recently, an investigation of EPHA2 sequence variants in the protein coding region of the gene in South African HIV positive individuals revealed that certain variants, primarily located in the functionally important tyrosine kinase domain, …”

19.  Line 206: The author could discuss the role of gH/gL interaction with Eph receptor in B cell infection (see Muniraju et al., J Virol. 2019 Jul 30;93(16):e00630-19. doi: 10.1128/JVI.00630-19.

Author reply: Information on Eph receptors in B cells has been added as follows (lines 258-262): “Using the human lymphoma B cell line MC116, a role for gH in infection could not be conclusively established; it was therefore suggested that KSHV entry into B cells is mediated through the K8.1 envelope glycoprotein (see also 4.2.4.). Indeed, MC116 cells were shown to express EPHA4 and EPHA7 which have been implicated in KSHV infection in both cell-free and cell-to-cell settings [81, 82].” However, we refrained from including the study by Muniraju et al., 2019, as it did not focus on B cells but rather showed the role of gH for KSHV infection of epithelial, endothelial, and fibroblast cells.

20.  Line 276: Absence of compelling discussion. Authors should identify the research gaps and ways to advance the understanding of KSHV glycoproteins and cellular receptors interactions in virus infection and in turn developing virus infection intervention strategies.

Author reply: We have amended the manuscript to more clearly propose research gaps in the understanding of cellular receptors utilised by KSHV for infection. The following additions have been made:

-       Abstract (lines 26-29): “Gaps in the current literature needed to advance understanding of the interactions between KSHV glycoproteins and cellular receptors in virus infection are identified to lead to the development of virus infection intervention strategies.

-       See comment 17. Discussion (lines 186–187): “However, it still needs to be determined which KSHV glycoprotein interacts with xCT to conclusively elucidate its function as an entry receptor.” 

-       Discussion (lines 212–213): “However, it remains to be understood how this receptor mechanistically contributes to pathogenesis of KSHV infection and/or tumorigenesis following KSHV infection.. Additionally, incomplete reduction of infection upon EPHA2 knock out in endothelial cells and fibroblasts suggests that additional receptors are used by KSHV for entry, likely other EPH receptors. While EPHA2 has garnered interest as a target for therapeutic intervention, the role of other receptors in KSHV entry would need to be teased apart before such a development.”

-       Discussion (lines 262–265): “Again, the role of EPH receptors in KSHV infection of B cells and subsequent pathogenesis remains only partially understood and it remains to be seen if the abundance of EPH expression on primary B cells correlates with permissiveness to KSHV infection and PEL development.”

-       Discussion (lines 284–286): However, little is known about in vivo infection of epithelial cells in this environment, representing an important arena for future study, particularly relevant for the development of prophylactic interventions to initial KSHV infection.

-       Additionally, section 4.2.4 highlights “Unidentified KSHV receptors on B cells” as a research gap and the conclusion (particularly lines 327–338) highlights our parting thoughts on the future direction of this research field.