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Summary and Assessment

This manuscript by Tervonen et al. investigates the mechanobiological remodeling of the host nucleus during herpes simplex virus type 1 (HSV-1) infection. The authors are conducting this study to understand how HSV-1 infection alters nuclear mechanics, a fundamental yet underexplored aspect of host–virus interactions. This is important because nuclear softening may facilitate viral replication, genome organization, and egress, providing new mechanobiological insight into infection. Using AFM, cryo-SXT, FIB-SEM, confocal microscopy, FLIM, GRO-Seq, and computational modeling, the authors show that the nucleus softens dramatically during infection. They attribute this to decreased outward forces, a combination of osmotic pressure and cytoskeletal pull, rather than lamina instability alone. This was convincingly demonstrated using AFM measurements (Fig. 1c, L88-L102), which is supported by the orthogonal imaging and modeling approaches mentioned above.  The study is important for viral cell biology and mechanobiology because it reconciles conflicting prior reports (e.g., stiffness increase in isolated nuclei, L391–L393) and provides mechanistic insight that nuclear softening during HSV-1 infection is driven primarily by reduced outward forces which are 1. loss of osmotic pressure and 2. diminished cytoskeletal pull, rather than by lamina disintegration or chromatin decondensation. The strength of evidence is high, supported by multimodal approaches and robust modeling.

This is a significant and rigorous study that substantially advances our understanding of nuclear mechanobiology during viral infection. I recommend that authors revise the manuscript, focusing on clarifying mechanistic interpretations, improving figure accessibility, and standardizing terminology. No additional experiments are strictly required, but greater transparency and emphasis on interpretational limits will strengthen the manuscript.

Major Points

• Direct Experimental Evidence for Osmotic Pressure Changes:

The central claim that reduced intranuclear osmotic pressure drives softening is (L334–L344, Fig. 6e–f), currently supported by modeling and density analysis (L125–L147, Fig. 2). Without direct measurements, this conclusion remains inferential. To improve reader understanding, the authors can explicitly acknowledge in the Discussion (L407–L411) that osmotic pressure changes are inferred, and suggest future methods (e.g., ion concentration or nuclear transport assays).

• Temporal Relationship Between SUN2 Downregulation and Early Softening:

AFM shows softening by four hours post-infection (L88–L102, Fig. 1c), while GRO-Seq reveals SUN2 downregulation only at eight hours post-infection (L249–L258, Fig. 5). This temporal mismatch weakens a direct causal interpretation. To address this, the authors can clarify in Results/Discussion (L447–L453) that early softening likely arises from additional mechanisms such as rapid cytoskeletal remodeling or chromatin decondensation. Rapid cytoskeletal remodeling can reduce mechanical tension transmitted to the nucleus, while chromatin decondensation lowers nuclear rigidity by decreasing chromatin compaction. Both processes occur quickly after infection onset and could therefore explain the observed softening before SUN2 downregulation becomes significant.

• Nuclear Lamina Observations and Interpretation:

The data show increased lamin A/C immunolabeling at 8–12 hpi (L265–L271, Fig. 5d–e) despite nuclear softening. Modeling (L295–L331) reconciles this by prioritizing outward force reduction; however, interpretation is complicated by potential changes in antibody accessibility (L269–L272). The authors can emphasize in Results/Discussion that increased labeling may reflect conformational exposure rather than actual protein abundance.

• Functional Implications of Increased Nuclear Membrane Tension:

The model and FLIM data show increased nuclear membrane tension at late infection (L217–L221, Fig. 4e–f; L355–L361, Fig. 6g–i). The functional significance is not fully developed. To improve reader understanding, the authors can expand Discussion (L435–L442) to consider how elevated membrane tension could facilitate capsid egress.

• Terminology for 'Outward Forces':

“Outward forces” (L334–L344) are variably described as osmotic pressure, cytoskeletal pull, or both. The authors can provide a consistent definition in Methods (L668–L674) and clarify in all figure legends (Figs. 1, 4, 6).

Minor Points

• To improve clarity, reiterate in Discussion (L417–L419) that although swelling and lamin upregulation often stiffen nuclei, here reduced outward forces dominate.

• To give readers more context, cite literature (e.g., L444–L453) where SUN2 suppression softens nuclei, to reinforce its proposed role. For example, Yue et al. (2003) showed that SUN2 suppression mediates nuclear softening and delays stress-induced senescence. Jiao et al. (2003) demonstrated that SUN1/2 deficiency decreases nuclear size and stiffness in microphages. Krshnan et al. (2022) reported that regulated degradation of SUN2 alters nuclear envelope mechanics and architecture.

• Figures 2, 4, and 6 contain complex 3D/modeled data. Adding intuitive legends, color-coding of force components, and scale references would enhance accessibility.

• In Discussion (L469–L477), add speculation on the evolutionary advantage of nuclear softening (e.g., enhanced capsid trafficking or egress). Speculation on the evolutionary advantage of nuclear softening would help readers connect the mechanistic findings to viral fitness and pathogenesis. By considering how softening might facilitate processes like capsid trafficking, nuclear egress, or chromatin remodeling, authors can frame their results within a broader biological context. This would make the study more impactful and appealing to a wider audience.

• To strengthen clarity of presentation, ensure uniform use of 'hours post-infection (hpi)' (appears variably, e.g., L120–L123) and consistent mechanical descriptors ('stiffness' vs. 'softness')

Competing interests

The authors declare that they have no competing interests.