Phosphatidylserine receptors are critical for lethal infection by Crimean-Congo hemorrhagic fever virus

J. Berrigan; S. R. Monticelli; M. D. Ramirez; P. T. Donovan; T. S. Powell; A. I. Kuehne; R. R. Bakken; S. B. Sulkosky; S. R. Coyne; X. Zeng; C. P. Stefan; A. S. Herbert; W. J. Maury; K. Chandran; E. Kasikci

doi:10.64898/2026.10.29.762781

AI summary · DOI: 10.64898/2026.10.29.762781 · bioRxiv: Microbiology

Phosphatidylserine receptors are critical for lethal infection by Crimean-Congo hemorrhagic fever virus

J. Berrigan, S. R. Monticelli, M. D. Ramirez, P. T. Donovan, T. S. Powell, A. I. Kuehne, R. R. Bakken, S. B. Sulkosky,

and 7 more authors

S. R. Coyne, X. Zeng, C. P. Stefan, A. S. Herbert, W. J. Maury, K. Chandran, E. Kasikci

Original paper Published on 30 Oct. 2026 bioRxiv preprint License: CC BY-NC-ND 4.0 Also in: fr

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The essentials

AXL, TIM-1, and TIM-4 support CCHFV entry and disease; deleting two receptors protected mice in a lethal-challenge model.

Question

Do phosphatidylserine receptors contribute to CCHFV entry in relevant cells and to infection and disease in vivo? Can blocking or removing these receptors reduce infection?

Method

The authors combined genome-wide CRISPR/Cas9 screens with receptor knockout, complementation, knockdown, overexpression, and inhibitor or antibody experiments in cell lines and liver-derived cells, using CCHFV, viral surrogates, and HAZV. They challenged Ifnar1-deficient mice carrying individual or paired receptor knockouts with 10 PFU of CCHFV-Turkey2004; groups had 15–16 mice, with five per group sampled on day 5 and the remaining animals monitored for 28 days.

Findings

Result AXL and TIM-1 loss reduced CCHFV entry or infection in immortalized cells and liver-derived cell models, while complementation or overexpression supported their roles.
See the passage in the paper
We conclude that the PSRs AXL and TIM-1 contribute to CCHFV entry in multiple liver cell types associated with CCHFV infection and pathogenesis in vivo.
Result PtdSer liposomes inhibited CCHFV entry, and a TIM-1 mutant deficient in PtdSer binding did not enhance entry.
Result Combined AXL and TIM-1 targeting inhibited cell infection more effectively than targeting either receptor alone.
Result Individual receptor knockouts provided partial protection in mice, whereas paired Axl/Timd4 loss prevented detectable infectious virus in serum and sampled organs and protected against disease.
See the passage in the paper
PSR dKO reduced viral infectivity to baseline in serum and all sampled solid organs (Fig. 7d) and afforded greater diminutions in viral genome loads than the PSR single-KOs (Fig. 7d).
Authors’ interpretation The authors interpret the overlapping receptor effects as evidence that these PSRs are determinants of CCHFV infection and pathogenesis, and potential antiviral targets.
See the passage in the paper
We conclude that PSRs are not just cell entry factors but bona fide determinants of viral infection, dissemination, and disease and legitimate targets for the development of new antiviral countermeasures against CCHFV.

Limitations

The animal findings come from an Ifnar1-deficient mouse challenge model, so their applicability to infection in immunocompetent animals or humans remains unestablished.
See the passage in the paper
These mice lacked the gene encoding interferon α/β receptor (Ifnar1; to render the mice susceptible to CCHFV) and an individual PSR-encoding gene: Axl, Havcr1, or Timd4 (Fig. S5; see Methods for details)[22, 50].
The study establishes genetic and cell-based effects but does not demonstrate therapeutic efficacy of receptor inhibitors in infected animals.
See the passage in the paper
Together with our findings in vivo, the potency of bemcentinib (Fig. 5c) and anti-PSR mAb cocktails (Fig. 6a) in cellulo raises the prospects for the development of PSR inhibitors as host-directed antivirals against CCHFV and other emerging orthonairoviruses.

Why it matters

The authors argue that PSRs contribute to CCHFV disease in vivo, not merely cell entry, and that overlapping receptor use may make simultaneous targeting more effective. Their results nominate PSR inhibition as a host-directed antiviral strategy, while noting challenges from the receptors’ roles in immune function.

Written by AI from the paper. Passages are checked word for word against the text we analysed; the rest may contain errors.

AI-generated keywords: Crimean-Congo hemorrhagic fever; phosphatidylserine receptors; viral entry; AXL; host-directed therapy

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Comprehensive Summary
Layman's Summary
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Original abstract

Crimean-Congo hemorrhagic fever virus (CCHFV) can cause severe and sometimes fatal disease, and no virus-specific treatments have been approved. This bioRxiv preprint investigates whether three phosphatidylserine receptors—AXL, TIM-1, and TIM-4—help the virus enter cells and contribute to infection and disease in animals. These receptors recognize phosphatidylserine, a lipid exposed on the surface of enveloped viruses, and may facilitate viral attachment and uptake. The researchers conducted genome-wide CRISPR knockout screens in human U2OS and SW-13 cells. They selected cells that survived exposure to either a recombinant vesicular stomatitis virus bearing CCHFV glycoproteins or the related Hazara virus. AXL emerged as a prominent cell-surface candidate in both screens. To assess receptor function, the researchers performed gene-knockout and receptor-restoration experiments, overexpressed receptors, and used short-hairpin RNA to reduce receptor expression. Their assays included recombinant virus, CCHFV transcription- and entry-competent virus-like particles, and authentic CCHFV. They also tested primary human endothelial cells, liver-derived endothelial cells, and liver-like HepaRG cells. To evaluate receptor dependence in vivo, the team challenged interferon-receptor-deficient mice—which are susceptible to CCHFV—with 10 plaque-forming units of the Turkey2004 strain. The mice lacked one or two of the receptor genes. The researchers measured survival, clinical signs, infectious virus, viral RNA, and tissue pathology. In cultured cells, loss of either AXL or TIM-1 reduced CCHFV entry or infection, while restoring the missing receptor largely restored infection. These effects were observed across several CCHFV strains and in liver-associated cell models. Increasing AXL or TIM-1 expression enhanced infection, but a TIM-1 mutant unable to bind phosphatidylserine did not. Phosphatidylserine-containing liposomes inhibited infection in a dose-dependent manner, whereas control phosphatidylcholine liposomes had little effect. Together, these results support a role for viral-envelope phosphatidylserine in receptor-mediated entry. Targeting both receptor pathways often had a stronger effect than targeting either alone. Combined anti-AXL and anti-TIM-1 antibodies blocked entry more effectively than either antibody individually. AXL/TIM-1 double-knockout cells showed more than a 90% reduction in infection, compared with reductions of roughly 50–75% in cells lacking one receptor. The AXL inhibitor bemcentinib had only a modest effect in cells expressing both receptors but reduced infection by about 90% in TIM-1-knockout cells. This finding is consistent with overlapping entry routes and a contribution from AXL kinase activity. In mice, deleting any one of the receptor genes improved survival, with the strongest single-gene effects reported for TIM-1 and TIM-4. Single knockouts also reduced viral burdens and tissue damage, although the effects varied by organ and measure. Deleting AXL together with TIM-1 or TIM-4 provided substantially greater protection. In both double-knockout groups, infectious virus fell to baseline levels in serum and sampled organs. AXL/TIM-4 double-knockout mice were reported to be completely protected from challenge, with no weight loss or clinical signs. Viral RNA, liver and spleen inflammation, and tissue pathology were also reduced. The authors conclude that AXL, TIM-1, and TIM-4 collectively support CCHFV infection and disease, and that blocking multiple receptors could be a promising host-directed treatment strategy. However, the study is a preprint that has not been certified by peer review.

How CCHFV Uses Cell-Surface Receptors to Drive Infection

Crimean-Congo hemorrhagic fever virus (CCHFV) can cause severe, sometimes fatal illness. Yet no treatment specifically targeting the virus has been approved. A new preprint, “Phosphatidylserine receptors are critical for lethal infection by Crimean-Congo hemorrhagic fever virus,” investigates how the virus enters cells—and whether blocking that process could reduce disease.

Led by J. Berrigan, S. R. Monticelli, M. D. Ramirez, P. T. Donovan, T. S. Powell, A. I. Kuehne and colleagues, the study focuses on three cell-surface proteins: AXL, TIM-1 and TIM-4. These proteins can recognize phosphatidylserine, a lipid found on the outer surface of enveloped viruses. The researchers asked whether this interaction helps CCHFV attach to and enter cells, and whether the receptors also influence infection in animals.

Searching for Cellular Entry Factors

To identify potential entry factors, the team carried out genome-wide CRISPR knockout screens in human U2OS and SW-13 cells. They exposed the cells to engineered viruses bearing CCHFV glycoproteins or to the related Hazara virus, then identified gene disruptions associated with cell survival. AXL stood out as a prominent candidate in both screens.

The researchers then tested the roles of AXL, TIM-1 and TIM-4 through complementary approaches. These included deleting receptor genes, restoring them, increasing receptor expression, and reducing expression with short-hairpin RNA. They tested the effects using recombinant viruses, virus-like particles that model CCHFV entry, and authentic CCHFV. Their experiments also included several types of human cells, among them endothelial cells, liver-associated endothelial cells and liver-like HepaRG cells.

In cultured cells, loss of either AXL or TIM-1 reduced viral entry or infection, while restoring the missing receptor largely restored infection. Increasing AXL or TIM-1 expression boosted infection. The effects appeared across multiple CCHFV strains and in liver-associated cell models, suggesting that the findings were not confined to a single cell line or viral strain.

Evidence for Phosphatidylserine-Mediated Entry

Several experiments linked receptor activity to phosphatidylserine. A TIM-1 mutant that could not bind the lipid failed to promote infection as effectively as the normal receptor. Meanwhile, liposomes containing phosphatidylserine inhibited infection in a dose-dependent manner. Control liposomes made with phosphatidylcholine had little effect.

Together, these results support a model in which phosphatidylserine on the virus surface helps it engage cellular receptors and enter cells. The receptors may provide overlapping routes: disrupting one can reduce infection, but the remaining receptors may still help the virus gain access.

That overlap was evident when the team targeted receptors in combination. Antibodies against both AXL and TIM-1 blocked entry more effectively than either antibody alone. Cells lacking both receptors showed a reduction in infection of more than 90%, compared with reductions of roughly 50–75% when only one receptor was missing.

The AXL-blocking drug bemcentinib had only a modest effect in cells that still expressed TIM-1. But in TIM-1-knockout cells, it reduced infection by about 90%. This pattern is consistent with AXL and TIM-1 providing partially redundant entry routes, while also suggesting that AXL kinase activity contributes to infection.

Testing Receptor Deletions in Mice

To examine the receptors’ role in disease, researchers challenged interferon-receptor-deficient mice with a low dose of the Turkey2004 CCHFV strain. These mice are susceptible to infection. The animals lacked one or two of the receptor genes, allowing the team to compare survival, symptoms, infectious virus, viral RNA and tissue damage.

Deleting any one receptor improved survival, though protection varied. The strongest single-gene survival effects were reported for TIM-1 and TIM-4. Single knockouts also reduced viral burden and tissue damage, with the changes differing across organs and measurements.

Removing two receptors provided substantially greater protection. In both AXL/TIM-1 and AXL/TIM-4 double-knockout mice, infectious virus fell to baseline levels in serum and sampled organs. AXL/TIM-4 double-knockout mice were completely protected from the challenge in this experiment: they showed no weight loss or clinical signs. These mice also had less viral RNA, inflammation in the liver and spleen, and tissue pathology.

What the Findings Could Mean

The results point to AXL, TIM-1 and TIM-4 as host factors that collectively support CCHFV infection and disease. Because these receptors belong to the host rather than the virus, targeting them could offer a host-directed treatment strategy. The strong effects of combined receptor disruption suggest that blocking several routes may be more effective than targeting one alone.

There are important limits to keep in mind. The mouse experiments used animals with impaired interferon signaling, and genetic deletion is not the same as treating an established infection with a drug. The findings therefore do not show that receptor-blocking treatments will work in people. The paper is also a preprint and has not been certified by peer review.

Even with those caveats, the study offers a useful account of how CCHFV may exploit phosphatidylserine-recognizing receptors. Further research will be needed to establish whether these pathways can be safely and effectively targeted—and whether the approach can translate into treatment for a dangerous disease with few specific therapeutic options.

Created on 02 Nov. 2026

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Weekly letter Presented in "Biology and medicine this week" of 2 November 2026. Biology and medicine this week: Hidden partners, adjustable gates and overlooked disease

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Phosphatidylserine receptors are critical for lethal infection by Crimean-Congo hemorrhagic fever virus

The essentials

Question

Method

Findings

Limitations

Why it matters

Read further

Definitions

How CCHFV Uses Cell-Surface Receptors to Drive Infection

Searching for Cellular Entry Factors

Evidence for Phosphatidylserine-Mediated Entry

Testing Receptor Deletions in Mice

What the Findings Could Mean

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