Crimean–Congo haemorrhagic fever virus uses LDLR to bind and enter host cells

Climate change and population densities accelerated transmission of highly pathogenic viruses to humans, including the Crimean–Congo haemorrhagic fever virus (CCHFV). Here we report that the Low Density Lipoprotein Receptor (LDLR) is a critical receptor for CCHFV cell entry, playing a vital role in CCHFV infection in cell culture and blood vessel organoids. The interaction between CCHFV and LDLR is highly specific, with other members of the LDLR protein family failing to bind to or neutralize the virus. Biosensor experiments demonstrate that LDLR specifically binds the surface glycoproteins of CCHFV. Importantly, mice lacking LDLR exhibit a delay in CCHFV-induced disease. Furthermore, we identified the presence of Apolipoprotein E (ApoE) on CCHFV particles. Our findings highlight the essential role of LDLR in CCHFV infection, irrespective of ApoE presence, when the virus is produced in tick cells. This discovery holds profound implications for the development of future therapies against CCHFV.

1. Characterize the suitability to use pseudotyped viruses to study CCHFV entry (i.e.Gn and Gc have the correct conformation).2. Provide information on the suitability to use blood vessel organoids your to study CCHFV 3. Investigate the role of Gn during viral entry via LDLR 4. Use orthogonal approaches to confirm LDLR as entry receptor and to quantify virus entry for CCHFV via LDLR as suggested by the reviewers.5. Adjust sample numbers, time to analyze post-infection, include additional assays to look at pathogenesis and define disease scoring, in your vivo experiments 6. Confirm expression levels of LDLR in KD and mutant setups.
The remaining issues should be straightforward to address.
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Should further experimental data allow you to address these criticisms, we would be happy to look at a revised manuscript.

Answers to reviewers
Referee #1 (Remarks to the Author): In this manuscript by Monteil et al., the authors propose that Low Density Lipoprotein Receptor (LDLR) is a critical receptor for Crimean-Congo hemorrhagic fever virus (CCHFV).Using genetic screening in murine haploid cells deploying both pseudotype VSV-CCHFV and wild-type CCHFV they identify LDLR as a receptor for CCHFV.Subsequently, binding was confirmed in African green monkey cells and human lung epithelial cells (reference lab strain and clinical strain of CCHFV).Using biosensor experiments in living cells shows that soluble Gc from CCHFV can bind to LDLR, and soluble LDLR can block CCHFV infection.The findings are corroborated by infecting human blood vessel organoids and LDLR knockout mice.The data presented here is clearly an important finding.The experimental approach is a tried-and-true approach and yielded fruitful results with live CCHF virus.Nevertheless, the authors need to temper the language as LDLR is one important entry factor out of multiple.Most importantly, although the data presented here indicate that LDLR plays a role in virus entry, the data needs to be more rigorously investigated to define and quantify the role of LDLR as a crucial receptor in CCHFV infection.For instance, using an uncharacterized pseudotype virus or for CCHFV-filed new organoid system to justify some of this approach seems haphazard.A better comparison should have been done with either viral-like particles or viral replicon particles for CCHFV.Better cell lines, specifically those that rely on canonical LDL receptors for function, such as liver cells, would have been much better alternatives for study, both for the receptor and for viral tropism.The in vivo mouse studies have major experimental design and interpretation flaws.Thus, additional experiments and controls must be conducted to discern if LDLR is just a cofactor.General comments on the manuscript: • The authors focus solely on Gc in their studies and not both glycoproteins Gn and Gc, which form a unit on the virion surface.Curiously, the authors did not look at Gn first, as presumably it is the attachment protein and Gc is the fusion protein.Was Gn also tested, and if not, why?Recent studies in the field have revealed new findings on the surface of the virion.For example, GP38 is also supposedly incorporated in the virion, and Gn/Gc can be former dimers or trimers.By exclusively focusing on the Gc, I am concerned that vital tertiary structures on the virion and its glycoproteins relevant for receptor binding are overlooked (see also below, Gc binding to the receptor).
Response: We agree.We included data on Gn, Gn+Gc as well as GP38 in revised MS (Figure 3 and extended data figure 3 -MS lines 172-212).
• Throughout the manuscript, the authors use "level of infection" as a readout of virus entry.First of all, I have an issue with how this is measured."Level of Infection" as a readout unit (relative quantification using ΔΔ analysis) when it is measuring viral RNA, which is basically genome copies.Few CCHFV publications have used the ΔΔ analysis, which is rather unusual.Why was not an absolute RNA quantification in the form of genome copies used just like it was used in animal studies?

Response: This has been addressed in revised MS line 98
Pseudovirus studies: • 317: This pseudotype has not been adequately characterized to show that conformationally dependent Gc is incorporated on the surface of the virion.Additionally, characterization is needed for this model.The authors state in line 323 that the VSV-pseudotype was previously published (ref. 56).But this is the wrong reference it does not describe the VSV-Gc pseudotype.Response: In revised MS, A neutralization data has been added as Extended data figure

And the reference was corrected line 480
• VSV should have been used as a positive control for all in vitro experiments once the authors determined it was LDLR.LDL receptor and its family members are cellular receptors for the vesicular stomatitis virus.Danit Finkelshtein 1, Ariel Werman, Daniela Novick, Sara Barak, Menachem advised.The experiment here it's not thorough enough to characterize the system as a tool for the infectivity of CCHFV.
• 189: you need citations to support this claim.It is unclear if the tropism is for the virus for CCHFV, or if the pathologies induced by hemorrhagic fevers, in general, elicit the roles of blood vessels.The author states that the blood vessels are key target cells for viral tropism involved in hemorrhaging, and I agree with them; however, for many hemorrhagic fever viruses, it is still unclear if the hemorrhage is due to viral injury or injury due to pro-inflammatory cytokines or both (Hawman andFeldmann, 2018, doi: 10.12688/f1000research.16189).Furthermore, very little data is available for CCHFV infections of endothelial cells, especially in humans (e.g., what percent become infected).Conversely, a vast body of literature characterizes other target cells, such as hepatocytes, macrophages, dendritic cells, and monocytes.A quick review in the NCBI database (Gene ID: 3949) also revealed to me that hepatocytes, besides lung cells, have the highest level of LDLR expression of any cell type in the body why were those cells not chosen since there is much more information available?
Response: Hepatocytes could be an excellent system.However, complete silence of the gene in these cells are VERY complicated if NOT close to impossible.We would like to see the specificity of LDLR in more complex system, that's why we used human blood vessels organoids as endothelial cells are a target for CCHFV (Burt F. The authors state that "the blood vessel organoids are sometimes detrimental to infection," and thus, the cells were cultured as monolayers.Does this mean there was infectivity variation in the cell culture?Furthermore, isn't a 3D culture of cells more representative than a monolayer?In Figure 5b, the authors use "Level of Infection" as a readout unit (relative quantification using Delta Dealta analysis) when measuring viral RNA, which is basically genome copies.Why was not an absolute RNA quantification in the form of genome copies used just like it was used in the animal studies in the same figure panel?The data show an RNA difference between WT and KO cells; nevertheless, the KO cells still had a significant amount of RNA.I believe the crux of the whole paper is reflected in the author's statement in line 200: "…though another entry route appears to be operational".If the virion has multiple modes of getting into the cell, and one of those modes is not accessible/knockout, it is conceivable that the entry is not as efficient.Thus, looking at days 1 and 3 post-infection and extending the timespan would be beneficial.

Response
Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work.The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.• The dosing schedule of the mAB 5A3 to abolish the IFN response does not follow the published schedule for CCHFV.Thus, the authors need to carry out an endpoint animal study to prove their timing schedule works.

Response: Thx for the comments. The protocol used in this study has been documented in a publication (Hawman D.W. et al, eBioMedicine, 2022). It is now referenced line 685.
In the subsequent mouse study we conducted, we maintained a group of knockout (KO) mice for a survival analysis.The corresponding data are illustrated in Figure 5d.
• Most importantly, the authors cannot make the statement that the mice were protected against the disease if the animals were euthanized on day four, as it is done in line 81.Experiments must be conducted to extend the time to evaluate potential sickness and survival for at least 21 days.Studies have shown that the onset of disease in mice infected with CCHF can be delayed.There was just as much viral genome in some of the LDLR-/-mouse samples as the wild type, which makes me suspect they would have caught up if given a few more days.

Response:
We appreciate the reviewer's comments and would like to express our gratitude.In the second mouse study, we conducted additional experiments, including the retention of a group of knockout (KO) mice for a survival study.The corresponding data are presented in Figure 5d.The manuscript text has been adjusted to incorporate these new findings.These supplementary data have influenced our conclusions, as emphasized in the revised manuscript.
To ensure the infection of all susceptible cells before their rapid growth (haploid cells exhibit high growth rates), a high Multiplicity of Infection (MOI 10) is employed to enhance the likelihood of infecting all susceptible cells.Conversely, for validation purposes with the goal of investigating the impact of knockout (KO) on infection, and to avoid forcing the virus into cells, a lower MOI (here MOI 0.1) was utilized.This has been mentioned in revised MS line 506 361: If the actual stocks of the CCHFV virus were grown and amplified in SW-13 cells, why then were A549 and Vero's cells chosen as the haploid knockouts?Please justify the switch from a human adrenal carcinoma cell line to a monkey kidney cell line.Why were not liver cells are chosen, such as Huh-7 or HepG2 cells?Both of these liver cell lines play a role in hemorrhagic fever virus tropisms, LDL/LDLR, and support the growth of CCHFV to high titers.Please justify why liver cells were not used in these experiments.

Response:
Regarding diploid cells, SW13 was not employed as a knockout (KO) due to the challenges associated with knocking them out, and the same difficulty applied to Huh7.The decision not to use hepatoma cells does not diminish the significance of the findings-as our data were confirmed in human blood vessels.In the revised manuscript, we have also included HepG2 cells for a different purpose (figure 7).
Figure 3: The normalization of BRET scoring seems arbitrary.1.00-1.02changes do not seem profound at all, and this reviewer questions if any of this is noise without explaining how this readout is supposed to be interpreted in results.Because in subsequent panels, the changes are on the orders of 1.2-1.4,yet both are supposed to be significant changes?Please justify what these results mean in this context.
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Response: BRET is the ratio of acceptor to donor counts.Over the years, different donor and acceptor pairs have been engineered to decrease noise and increase signal.As a BRET donor, Nluc usually produces smaller increases in BRET with a cleaner signal-to-noise, whereas Rluc typically has larger responses that can however be more sensitive to substrate, BRET acceptor and dipole-dipole orientations.As such, it is not meaningful to compared the raw BRET ratios or the normalized responses between assays with different donor-acceptor pairs (and different biological questions), specifically the binding assay which requires an N-terminally tagged LDLR (using Nluc) and the internalization assay that used Rluc.Notably, we have presented all data with the appropriate positive and negative controls and applied statistical analysis throughout (Figure 3).
Figure 5D: the axis in the serum is off compared to the others in this panel.Why include 10^-5 as the x-axis in that panel, but 10^3 in the others, please address this or correct it.
Response: Now modified Figure 5c 5.In the Introduction section, line 66, it would be preferable to include the specific name of the tick species that serves as a CCHFV reservoir.
Response: Agree, Now added line 96 6. Line 183: This sentence should be moved or modified (e.g., "These data indicate that soluble LDLR can prevent CCHFV infections").

Response: now modified line 273
7. Line 202-203: it is not a title.
Response: Thanks for highlighting this mistake.It is now modified line 295 8. Line 410: The tubes were then incubated at 37°C with shaking for 30 minutes.

Response: It is now corrected line 570
Referee #3 (Remarks to the Author): Monteil et al. report the identification of the low density lipoprotein related receptor (LDLR) as a cellular receptor for Crimean Congo hemorrhagic fever virus (CCHFV).The authors used a genetic screen to identify LDLR as a candidate receptor.They then used a series of assays, including work with recombinant proteins, VSV pseudotypes, infectious viruses (lab and clinical isolates), and LDLR knockout mice to support their claim for the role of LDLR as a cellular receptor for CCHFV.Given the importance of CCHFV as an emerging human pathogen and the virus' epidemic potential, the work is significant, and the findings are of broad interest.The work is also exciting because of the increasingly recognized role of LDLR-related proteins (alluded to in the manuscript) as receptors for many viruses for which cellular receptors had previously remained elusive.
Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work.The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
However, certain issues would need to be addressed for the work to meet the standard usually required to make a strong claim that a given protein is a bona fide cellular receptor that also contributes to pathogenesis.This includes showing that receptor expression allows for the attachment and internalization of viruses or virus-like particles, which is a critical piece of information that is currently missing from the manuscript.There are also concerns about the design and execution of the in vivo study, as noted below.
Major 1.In regards to the VSV pseudotype, the authors write in lines 96-98, "This virus lacks the region coding for any glycoproteins, and therefore produces non-infectious particles unless reconstituted with a novel surface glycoprotein, i.e. in our screen with the Gc glycoprotein of the CCHFV."This phrasing suggests that only Gc is on the pseudotype.It seems unlikely that VSV pseudotype that contains Gc only on the surface would produce infectious particles, as the Gn/Gc heterodimer would be required.Could the authors clarify?(e.g., Figure 1a shows Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work.The images or other third party material in file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.Thank you for your patience as we've prepared the guidelines for final submission of your Nature Microbiology manuscript, "LDLR as an important receptor for Crimean-Congo Hemorrhagic Fever virus" (NMICROBIOL-23071732A).I am very sorry for the delay, since we encountered issues with our processes.

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In recognition of the time and expertise our reviewers provide to Nature Microbiology's editorial process, we would like to formally acknowledge their contribution to the external peer review of your manuscript entitled "LDLR as an important receptor for Crimean- Introduction 65: I thought Dengue was the most widespread HFV.Response: This has been addressed in revised MS line 94 68: expending -> expanding Response: This has been addressed in revised MS line 98 68: The global warming stance is not justified by publications; please cite for this.
J. et al, Arch.Pathol., 1997; Haddock E et al, Nat.Microbiol., 2018; Andersson I, J.Med.Virol, 2006).It is now better highlighted line 280.The fact that there is less virus in Ldlr -/-mice also highlight the importance of LDLR in CCHFV infection in liver (figure 5c).
: The challenge with infecting blood-vessel organoids lies in their growth within a collagen/matrigel matrix, rendering the cells less accessible to the virus.To address this, organoids were initially seeded in a 2D format to maintain cellular diversity and enhance virus accessibility.Further clarification on this matter is provided in the text (line 280).Additionally, in response to suggestions, we have modified the data presentation to viral copy numbers (fig 2b-c; fig 3e-f; fig 4b and e; fig 5b-c; fig 6; fig 7b,c,f and g; fig 8).
Gn-Gc attachment but also ApoE.(Figure 8, results line 345 to 373 and discussion) Lastly, as mentioned above, confirming a finding generated with one technique with a second technique is imperative.Thus, an IFA should be performed to also look at what percent of cells (=infectivity rate) are infected express protein.Response: The IFA data are added as Extended data figure 4, in revised MS Mouse experiments The in vivo experiments have some major experimental design flaws.• First of all, no model characterization of the LDLR knockout mice was done.For example, they are not on the same genetic background (B6.129) as the C57Bl.Response: We agree, however, C57BL/6 mice are the control suggested by Jackson Laboratory and LDLR KO mouse are extensively characterized and used (Ishibashi S et al, J.Clin.Invest.,1993;Ishibashi S et al, PNAS, 1994; Ishibashi S et al, PNAS, 1994; J.Clin.Invest.,1993;Hertx J et al, PNAS, 1995; Truong TQ et al, Biochim Biophys Acta, 2000; Keren P et al, Diabetes, 2000; Huszar D et al, Arterioscler Thromb Vasc Biol, 2000 etc) : Agree.Now done Figure4 and 73.Additional relevant literature that could be cited (lines 260-264) is the identification of LDLRrelated proteins as receptors for Oropouche virus (PMID: 35939689) and certain alphaviruses (PMID: 35939689, 34929721)Response.Agree but we are limited in number of citations.