1. Author Response:

    Reviewer #2 (Public Review):

    Dieterle et al set out to determine the receptors needed by hantaviruses to infect human endothelial cells. Prior to this publication, the authors identified protocadherin-1 (PCDH1) as a putative viral receptor for New-World Hantaviruses, but not Old-World hantaviruses. Additionally, both Integrins and DAF have been reported as receptor candidates for hantaviruses. However, whether these molecules function alone, or in combination to promote hantavirus entry and infection remains unclear. Dieterle et al generate and validate single and combinatorial knockouts of these 4 genes (PCDH1, DAF, ITGB1, ITGB3) and test the ability of the resulting cells to support viral replication in two independent assays. Dieterle et al confirm that New World hantaviruses require PCDH1 for infection. Furthermore, Dieterle et al fail to find a functional role for Integrins (Beta 3/Beta1) or DAF in hantavirus infection, even when knocked out in combination. Overall, the data is clearly presented and well controlled. The findings help clarify entry mechanisms used by hantaviruses and provide a foundation to identify receptor candidates for old-world hantaviruses. A few minor points are worth mentioning.

    1. The authors clearly demonstrate a lack of genetic requirement for (DAF, ITGB1, ITGB3). However, a second orthogonal approach to block access to Integrins or DAF would strengthen the conclusion and alleviate any minor concerns of incomplete genetic knockout.

    We agree that additional approaches would be helpful to rule out some caveats to our study and that they are warranted. For this short report, we chose to focus on a loss-of-function genetic approach. Our genetic and biochemical evidence indicate that incomplete genetic knockout is unlikely to explain our findings. However, we cannot rule out more complex scenarios, including those we articulate in the manuscript. We concur that more work will be required to fully rule out (or ‘rule in’) the involvement of the various entry factors that have been proposed.

    1. The authors are commended for a nuanced conclusion. In particular lines 181-185 the authors state "We note that our results do not rule out that one or more of these proteins is involved in hantavirus entry into other cell types not examined herein, or that they are involved in endothelial cell subversion post-viral entry, as shown previously (Gavrilovskaya et al. 1998; Gavrilovskaya et al. 1999; Krautkrämer and Zeier 2008)." It should be noted that studies demonstrating a requirement for DAF used polarized cells. This would suggest in addition to cell type, growth conditions, may play an important distinction in receptor utilization studies. None the less, under the conditions tested the authors clearly demonstrate that DAF is not absolutely required for hantavirus infection in human endothelial cells.

    We have added language to the “Results and DIscussion” indicating as an additional caveat that some of the previously proposed receptors could play roles in polarized cell layers, as indeed has been proposed for DAF (Krautkramer and Zeier, 2008).

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  2. Evaluation Summary:

    This is a well-performed study that defines the role of purported host cell receptors in the entry of hantaviruses into human endothelial cells. This is an important study as it applies CRISPR-mediated deletion studies for candidate receptors in the same endothelial cell line (TIME).

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

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  3. Reviewer #1 (Public Review):

    In this article, the authors perform CRISPR-mediated gene deletion studies to define the role of host cell factors previously reported to serve as entry receptors for hantaviruses. The authors focus their work on beta1/3 intergrin subunits, decay accelerating factor (DAF/CD55), and protocaderhin-1, which have all been previously described as hantavirus primary receptors. The authors generate a panel of human endothelial cells lines (TIME) that are depleted of the above-desribed candidate receptors and show conclusively that only depletion of protocadherin-1 reduces infection. The authors thus conclude that PCDH1 is the primary receptor for virulent hantaviruses and that other factors previously described are unlikely to play major roles in infection of endothelial cells.

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  4. Reviewer #2 (Public Review):

    Dieterle et al set out to determine the receptors needed by hantaviruses to infect human endothelial cells. Prior to this publication, the authors identified protocadherin-1 (PCDH1) as a putative viral receptor for New-World Hantaviruses, but not Old-World hantaviruses. Additionally, both Integrins and DAF have been reported as receptor candidates for hantaviruses. However, whether these molecules function alone, or in combination to promote hantavirus entry and infection remains unclear. Dieterle et al generate and validate single and combinatorial knockouts of these 4 genes (PCDH1, DAF, ITGB1, ITGB3) and test the ability of the resulting cells to support viral replication in two independent assays. Dieterle et al confirm that New World hantaviruses require PCDH1 for infection. Furthermore, Dieterle et al fail to find a functional role for Integrins (Beta 3/Beta1) or DAF in hantavirus infection, even when knocked out in combination. Overall, the data is clearly presented and well controlled. The findings help clarify entry mechanisms used by hantaviruses and provide a foundation to identify receptor candidates for old-world hantaviruses. A few minor points are worth mentioning.

    1. The authors clearly demonstrate a lack of genetic requirement for (DAF, ITGB1, ITGB3). However, a second orthogonal approach to block access to Integrins or DAF would strengthen the conclusion and alleviate any minor concerns of incomplete genetic knockout.

    2. The authors are commended for a nuanced conclusion. In particular lines 181-185 the authors state "We note that our results do not rule out that one or more of these proteins is involved in hantavirus entry into other cell types not examined herein, or that they are involved in endothelial cell subversion post-viral entry, as shown previously (Gavrilovskaya et al. 1998; Gavrilovskaya et al. 1999; Krautkrämer and Zeier 2008)." It should be noted that studies demonstrating a requirement for DAF used polarized cells. This would suggest in addition to cell type, growth conditions, may play an important distinction in receptor utilization studies. None the less, under the conditions tested the authors clearly demonstrate that DAF is not absolutely required for hantavirus infection in human endothelial cells.

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