PPATHOGENS-D-26-00573

SV40 exploits the Nesprin-2-SUN1-KPNA4 axis for stepwise targeting and entry into the host nucleus to promote infection

PLOS Pathogens

Dear Dr. Tsai,

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Walter J. Atwood

Academic Editor

PLOS Pathogens

Alison McBride

Section Editor

PLOS Pathogens Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

Journal Requirements:

1) Please ensure that the CRediT author contributions listed for every co-author are completed accurately and in full.

At this stage, the following Authors/Authors require contributions: Luke Gohmann, and Billy Tsai. Please ensure that the full contributions of each author are acknowledged in the "Add/Edit/Remove Authors" section of our submission form.

The list of CRediT author contributions may be found here: https://journals.plos.org/plospathogens/s/authorship#loc-author-contributions

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Reviewers' Comments:

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: The stepwise penetration of SV40 particles through ER membranes, partial disassembly in the cytoplasm, and trafficking to nuclear pores provides an elegant entry mechanism that remains only partially understood. The Tsai laboratory has long been a leader in this field, advancing understanding of this complex entry mechanism. Here, they provide new mechanistic insights, implicating the SUN-KASH complex proteins Nesprin2 and SUN1 in mediating transport of SV40 particles from the cytosolic face of the ER to the nuclear periphery, where KPNA4 is involved in targeting particles to nuclear pores to complete the uncoating process.

This manuscript is exceptionally well written and the data is very clearly and logically presented. Methods are well described.

The authors’ claims are very well supported by the dataset. These findings represent a significant advance in our understanding of the process of polyomavirus entry into host cells. Undoubtedly, some of these findings may be relevant to understanding how other viruses traffic to the nucleus as well.

The data analysis and presentation is of exceptionally high quality.

Reviewer #2: This is strong well designed set of experiments demonstrating step-by-step how Nesprin-2-SUN1-KPNA4 targets and assists the nuclear entry of SV40 into the nucleus. I really cannot not think of anything that needs to changed or added.

Reviewer #3: This manuscript investigates how SV40 exploits components of the LINC complex and nuclear import machinery to achieve nuclear entry. The authors identify a stepwise mechanism in which the Nesprin-2–SUN1 complex mediates targeting of cytosolic virus to the nuclear envelope, followed by KPNA4-dependent translocation through the nuclear pore. The study addresses an important question in DNA virus biology and presents several interesting observations, particularly the specificity for SUN1 over SUN2 and for KPNA4 among importin-α isoforms.

However, while the data generally supports the requirement of Nesperin-2 /Sun1 for SV40 nuclear targeting, several key mechanistic aspects remain insufficiently resolved. In particular, the relationship between SUN1 (inner nuclear membrane), Nesprin-2 (outer nuclear membrane), cytosolic trafficking, and NPC engagement are not fully clarified. A careful examination of these would further strengthen the manuscript and at this stage requires major revision.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: (No Response)

Reviewer #2: N/A

Reviewer #3: 1) A central conceptual issue is how SUN1, an inner nuclear membrane protein, influences the capture of cytosolic SV40 via Nesprin-2 on the outer nuclear membrane. The data in Fig. 3G suggest that SUN1 is required for the Nesprin-2–SV40 interaction, yet the mechanistic basis for this is not resolved. Given that SV40 accumulates at ER foci in SUN1-depleted cells, it is critical to determine whether microtubule-dependent transport toward the nucleus is impaired. Specifically does SUN1 depletion disrupt BICD2–dynein–Nesprin-2 interactions? Is directional movement of SV40 toward the nuclear envelope affected?

2) The finding that SUN1 is required for Nesprin-2 binding to SV40 is interesting and intriguing and raises an important question. Does SUN1 directly participate in viral recruitment, or does it regulate the conformation/organization of Nesprin-2 at the NPC? Although the authors did not observe any visual difference in Nesperin -2 localization in Sun1 depleted cells in Fig 1, it is important to determine if the observed effects are indirect via motor recruitment.

3) Whether SV40 is preferentially targeted to specific NPC-associated regions, or whether KPNA4 recruitment occurs at defined nuclear envelope subdomains is unclear from the proposed models and the data. At minimum, this limitation should be more clearly discussed.

4) The specificity of KPNA4 (but not KPNA1/2) in promoting SV40 infection is a key finding. However, the mechanistic basis for this specificity is not explored for a article that needs to be published in PLOS Pathogens. For instance the NLS in VP2/3, does that play any role in this Nesperin-2/Sun1/KPNA4 mediated nuclear targeting. There are different classes of NLS in proteins and each NLS can perform distinct function and associate with distinct at nuclear import factors. The authors need to carefully examine the role of VP NLS in this context or at the least discuss this.

5) The quantification of disassembled versus assembled virus in Fig. 2E is not sufficiently clear. It would be important to show VP1 levels across individual gradient fractions rather than only the combined quantification presented. From the western blots, the distribution of VP1 across fractions does not appear to differ substantially between control and SUN1 KD conditions, raising questions about how disassembly is being assessed. In addition, given that BICD2 is implicated in SV40 disassembly and transport, it is unclear whether SUN1 depletion affects BICD2 function or engagement with the virus. If disassembly is indeed unchanged, this would suggest that SUN1 acts downstream of BICD2-mediated disassembly.

6) Preventing SV40 from accessing the nuclear interior during infection and halting them to stay exposed in the cytosol, does that trigger an innate response?

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: The only minor critique is that some of the representative microscopy data, as in Fig. 1F, was quite pixelated, and greater care should be taken to export high quality images in the appropriate format for review.

Reviewer #2: N/A

Reviewer #3: 1) Line 80 is a bit confusing, what is extracted out, is it SV40 or the genetic material? Or are the authors talking about naked virus getting into the cytoplasm.

2) Typo error in line 115

3) The authors may have to look carefully at Fig 1F, the cells shown seem to be Myco positive (from DAPI stain).

4) The orientation in which figures are oriented is a bit confusing in this article. Examples are Figure 3D which is not in alignment with others in the same panel and another if Fig 5A. In Fig 5A, it might be better to show Kd data of siRNA first and then T-Ag levels.

5) In fig 6D, consider putting the graph next to the western (Fig 6C) or changing the Y axis to include Sun1 Flag IP

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Dear Prof. Tsai,

We are pleased to inform you that your manuscript 'SV40 exploits the Nesprin-2-SUN1-KPNA4 axis for stepwise targeting and entry into the host nucleus to promote infection' has been provisionally accepted for publication in PLOS Pathogens.

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Best regards,

Walter J. Atwood

Academic Editor

PLOS Pathogens

Alison McBride

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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Reviewer Comments (if any, and for reference):