Molecular insights into species-specific ACE2 recognition of coronavirus HKU5

The authors claim that "fatty acid binding plays a role in stabilizing the closed conformation of the S protein, but is not essential for ACE2-mediated entry." While the structural data clearly show the presence of oleic and palmitic acid in the identified pockets, the functional data (Supplementary Fig. 5f, g) show no significant difference in entry efficiency between the wild-type and the fatty acid-binding mutants (SDM1 and SDM2). The authors should clarify whether this suggests that the closed state is not the dominant conformation under the specific conditions of the pseudovirus entry assay, or if the stabilization provided by these fatty acids is redundant in the presence of other stabilizing factors. A more nuanced discussion of the physiological relevance of these fatty acid pockets in the context of viral transmission would strengthen the paper.

Regarding the cross-species transmission risk, the authors state that "these findings provide functional evidence that avian ACE2 orthologs are not only structurally compatible but also operationally competent to mediate HKU5 infection." This is a strong claim. While the pseudovirus and cell-cell fusion assays are well-executed, they are performed in heterologous cell lines (Lenti-X 293T and Caco-2). The authors should explicitly acknowledge the limitations of these models in predicting actual zoonotic risk, particularly the absence of relevant host-specific factors (e.g., proteases like TMPRSS2, which are often required for efficient viral entry in vivo). The discussion should be tempered to emphasize that this is a receptor-level observation rather than a definitive assessment of the virus's ability to infect avian hosts.

The authors state that 'HKU5 can exploit ACE2 orthologs across species, providing molecular insights into its host adaptation and underscoring the importance of surveillance for this virus and its zoonotic risk.' While the structural and biochemical data clearly establish that HKU5 RBD can bind to Pitta sordida ACE2, the leap to 'zoonotic risk' and 'conduits for cross-class transmission' is significant. The authors should temper these claims by acknowledging that receptor binding is only one of many barriers to cross-species transmission, and that the absence of evidence for natural infection in these avian species remains a critical gap. A more nuanced discussion of the barriers (e.g., TMPRSS2 usage, intracellular replication factors) would strengthen the manuscript.

Regarding the fatty acid binding sites, the authors note that 'disruption of these interactions in HKU5 induces a structural shift toward a more relaxed conformation' but also report that 'pseudoviruses carrying either WT S or the fatty acid binding site mutants... revealed no significant differences in entry efficiency.' This discrepancy suggests that while the closed state is stabilized by these lipids, it is not a bottleneck for entry in the cell culture models used. The authors should explicitly discuss why this might be the case—for example, whether the experimental conditions (e.g., high expression levels, use of TPCK-trypsin) bypass the regulatory role of these fatty acids, or if the 'relaxed' conformation is still competent for receptor engagement.

The authors state that 'fatty acid binding plays a role in stabilizing the closed conformation of the S protein, but is not essential for ACE2-mediated entry.' While the structural evidence for the fatty acid binding is convincing, the functional data (Supplementary Fig. 5f, g) show no significant difference in entry efficiency for the SDM1 and SDM2 mutants. It would be beneficial for the authors to discuss whether these fatty acids might play a more significant role in vivo, perhaps in the context of immune evasion or stability in the environment, rather than just receptor binding, to better justify the biological relevance of these pockets.

Regarding the cross-species analysis, the authors claim that 'these findings provide functional evidence that avian ACE2 orthologs are not only structurally compatible but also operationally competent to mediate HKU5 infection.' This is a strong claim. Given that the study uses an overexpression system in Lenti-X 293T and Caco-2 cells, it would be appropriate to include a caveat in the discussion that receptor compatibility is only one of many barriers to cross-species transmission, and that the 'potential for cross-species transmission' should be interpreted with caution until further ecological or live-virus studies are conducted.