Cyclophilin A stabilizes the capsid protein p72 to facilitate African swine fever virus replication

Domain: chemistry
Published: 2026-03-07
Editorial outcome: minor_revision

Abstract
African swine fever virus (ASFV) is a large DNA virus that poses a major threat to the global swine industry. Its virion is encapsulated by an icosahedral capsid predominantly composed of the structural protein p72, which constitutes approximately one-third of the total virion mass. Despite its abundance, the mechanisms regulating p72 stability remain poorly understood. Here, we demonstrate that host-mediated stabilization of p72 is essential for efficient ASFV replication. Mass spectrometry of p72 co-precipitates identified host cyclophilin A (CypA, also known as PPIA) as a key binding partner of p72. CypA interacts with p72 both in vitro and in vivo, specifically engaging exposed regions of p72 via its hydrophobic cavity. CypA interaction stabilizes p72 by reducing its ubiquitination and preventing proteasomal degradation, whereas cyclic CypA inhibitors destabilize p72 by disrupting this interaction and promoting its ubiquitination. Importantly, genetic or pharmacological inhibition of CypA markedly impairs ASFV replication. Mechanistically, CypA inhibition disrupts viral factory formation and virion assembly by decreasing p72 protein accumulation without affecting its transcription. Together, our findings uncover a host-dependent mechanism regulating capsid protein stability and highlight host CypA as a promising target for antiviral strategies against ASFV.

Introduction
African swine fever (ASF) is currently the most devastating infectious disease affecting swine, causing substantial economic losses worldwide1, 2. It is characterized by high morbidity and mortality rates (reaching up to 100%) in both domestic and wild pigs, and is designated as a notifiable disease by the World Organization for Animal Health (WOAH)3. First reported in Kenya in 1921, ASF has since spread globally over the past century, affecting numerous countries and regions4. Despite its severe impact, there are currently no safe and effective vaccines or antiviral therapeutics commercially available for its prevention or treatment, posing significant challenges to ASF control.

ASF virus (ASFV), the causative agent of ASF, is a large double-stranded DNA virus with a complex icosahedral architecture5. ASFV particles, approximately 200 nm in diameter, consist of multilayered structures, including a genome-containing nucleoid, core shell, inner envelope, icosahedral capsid, and an external lipid envelope6. The most abundant component of the ASFV virion is the major capsid protein p72, which constitutes about 33% of the total mass of the virion7. p72 has been well-demonstrated to play a critical role in capsid formation and viral assembly8-11, suggesting that viral or cellular factors regulating p72 may affect ASFV infection. Previous studies have shown that the ASFV-encoded protein pB602L functions as a chaperone facilitating p72 folding12, 13. Repression of pB602L expression results in the decreased p72 protein levels and the formation of aberrant zipper-like structures instead of icosahedral ASFV particles14. In contrast, p72 degradation is essential during the early phase of ASFV replication, driving capsid disassembly and enabling the release of core particles containing the viral genome into the cytoplasm following fusion of the viral inner envelope with host endosomal membranes, thereby facilitating viral DNA delivery into host cells15, 16. These findings suggest that p72 must be precisely and temporally regulated throughout the ASFV life cycle, likely through host factors that stabilize p72 during later stages of infection. Despite its essential role in virion assembly, the mechanisms governing p72 stability remain largely unknown.

CypA is abundantly expressed and possesses chaperone-like and prolyl isomerase activities that modulate protein folding and function, thereby influencing diverse biological processes, including MAPK signaling, RIG-I pathway, and cytokine production17-19. It has also been implicated in pathogen infections and a wide range of disease processes18. Whether CypA plays a role in ASFV infection remains unclear.

In this work, we identify host CypA as a critical regulator of p72 stability and ASFV infection. Co-immunoprecipitation (co-IP) combined with mass spectrometry reveals a specific interaction between CypA and p72, which is further characterized by AlphaFold3 structural modeling, mutagenesis, and binding assays. CypA interaction protects p72 from K63-linked ubiquitination-mediated degradation, whereas inhibition or depletion of CypA accelerates p72 turnover, impairing viral replication. Furthermore, CypA inhibition disrupts viral factory formation and impedes virion assembly by reducing p72 protein accumulation during ASFV infection. These findings uncover a host-dependent mechanism controlling p72 stability and suggest that targeting the CypA-p72 interaction could represent a potential antiviral strategy.

Results
Host CypA is a potential binding partner of the ASFV p72 protein
As the major component of the ASFV capsid, the viral p72 protein — encoded by the B646L gene — plays a critical role in ASFV virion assembly8, 11. It is well-established that B646L transcription initiates at 5 ~ 6 h post-infection (hpi) and is progressively upregulated during the late stages of ASFV infection, displaying a distinct expression pattern compared to the early genes CP312R and CP204L (encoding p30)20. In this study, porcine alveolar macrophages (PAMs) were infected with the genotype II ASFV HLJ/18 strain (referred to as ASFV unless otherwise noted), and samples were harvested at various time points post-infection for Western blot analysis to examine the expression kinetics of these viral proteins. Consistent with the transcriptional data20, p72 protein levels markedly increased at the later stages, in contrast to the early expression of CP312R and p30 (Fig. 1A). Given that protein abundance is controlled by multiple interconnected processes, including mRNA transcription, post-translational modifications, and protein degradation21, it is plausible that the accumulation of p72 at the late-stage of ASFV replication is also regulated by certain host factors after its synthesis. However, the mechanisms governing the stability of p72 protein in host cells remain largely unknown.

To explore potential mechanisms regulating p72 stability, we first sought to identify host factors that interact with p72. ASFV-infected PAMs were subjected to a co-IP with an anti-p72 antibody. As expected, viral p72 was specifically enriched by the anti-p72 antibody but not by the control IgG (Fig. 1B). The resulting co-precipitates were then analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), which identified a porcine cyclophilin-derived peptide, “FDDENFILK” (Fig. 1C). This peptide showed strong signal intensity in the p72-enriched sample but was absent in the IgG control (Fig. S1A). Sequence alignment of porcine cyclophilin family members revealed that both CypA (PPIA) and PPIE contain this peptide (Fig. 1D), suggesting p72 may associate with either or both proteins. Because cyclophilin family members share high sequence similarity22, we conducted GST pulldown assays using recombinant GST-tagged porcine cyclophilins and lysates from HEK293T cells expressing p72 to preliminarily assess their interactions. While most cyclophilins showed weak p72 binding, CypA and PPIE exhibited the strongest and comparable ability to recruit p72 (Fig. S1B). To determine the predominant binding partner, we examined the subcellular distributions of p72, CypA, and PPIE. Whereas p72 was mainly cytoplasmic and PPIE was strictly nuclear, CypA was present in both cytoplasm and nucleus (Fig. 1E), indicating that CypA, but not PPIE, has the spatial basis to interact with p72 in the cytoplasm. Together, these results suggest that host CypA may serve as a major binding partner of p72.

CypA is highly conserved across humans and animal species, including Ornithodoros, exhibiting approximately 100% identity between Sus scrofa and Phacochoerus africanus (warthog) (Fig. 1F). Emerging evidence suggests that CypA modulates the stability of different viral proteins, such as the HIV capsid23, the poxviruses C6 protein24, and the SARS-CoV-2 spike25. Therefore, it is reasonable to speculate that host CypA may likewise regulate p72 stability, warranting further investigation.

CypA interacts with viral p72 protein both in vitro and in vivo
To confirm the interaction between CypA and p72, we performed a series of binding experiments. Recombinant Strep-tagged trimeric p72 protein (referred to as recombinant Strep-p72) (Fig. S2) purified from yeast was subjected to Strep pulldown assays with GST or GST-CypA obtained from E. coli. We found that GST-CypA, but not GST alone, was efficiently recruited by Strep-Tactin resin with the recombinant Strep-p72 (Fig. 2A). In contrast, in the absence of recombinant Strep-p72, Strep-Tactin resin did not capture GST-CypA (Fig. 2B), which indicates that it is p72 that directly interacts with CypA in vitro. Moreover, co-IP in HEK293T cells co-expressing Strep-p72 and CypA-HA revealed that p72 was specifically detected in the CypA-enriched precipitates (Fig. 2C), confirming the formation of CypA-p72 complex in cells. Thus, these results demonstrate that CypA binds to ASFV p72 in vitro and in vivo.

Since intracellular co-localization is often a prerequisite for protein-protein interactions, we further examined subcellular co-localization of CypA and p72. Consistent with Fig. 1E, in plasmid-transfected HEK293T cells, Strep-p72 predominantly localized in the cytoplasm, whereas CypA-HA was present in both the cytoplasmic and nuclear regions (Fig. 2D). Importantly, co-expression of Strep-p72 and CypA-HA in HEK293T cells revealed strong cytoplasmic co-localization of these two proteins (Fig. 2D, E). We further assessed the dynamics of this co-localization in PAMs during the course of ASFV infection. At 6 hpi, only a small fraction of PAMs expressed low levels of p72, which localized to a restricted cytoplasmic region without detectable co-localization with CypA (Fig. 2F). However, as the infection progressed, p72 accumulated and showed pronounced cytoplasmic co-localization with endogenous CypA, particularly within viral factories (Fig. 2F). This co-localization was initially observed at 12 hpi, persisting to 24 hpi. A weaker p72 signal in factories was observed in a small fraction (~10%) of infected cells at late stages, potentially reflecting extensive p72 accumulation and redistribution. Importantly, the production of infectious progeny virions was initiated at 12 hpi and increased over time (Fig. 2G), coincided temporally with CypA-p72 co-localization, suggesting a potential role of CypA in promoting efficient virion assembly. Collectively, these findings demonstrate that host CypA co-localizes with ASFV p72 in host cells, likely contributing to viral replication.

CypA binds to the exposed regions (ERs) of p72 through its hydrophobic cavity
Prompted by the above interaction data, we sought to explore the binding mode between CypA and p72. While the structure of human CypA has been extensively characterized26, 27, the porcine CypA structure remains unresolved. Using the AlphaFold3 server, we predicted the structure of porcine CypA, which displayed a high similarity to its human counterpart (Fig. 3A), consistent with their high sequence identity (Fig. 1F). Given the demonstrated accuracy of AlphaFold3 in modeling biomolecular interactions28, we employed it to predict the CypA-p72 binding complex. Considering that p72 exists as a large trimer (~219.5 kDa) and CypA is a relatively small protein (~17.8 kDa), we hypothesized that the p72 trimer may bind CypA at a molar ratio of 1:3. The AlphaFold3-predicted model of a p72 trimer complexed with three CypA molecules exhibited high confidence (pTM = 0.74), with each CypA molecule binding to p72 in an identical fashion (Fig. 3B). Further detailed inspection of the binding interface using PyMOL29 revealed that CypA likely interacts with the apical exposed regions (ERs) of p72 via its 33 amino acid residues (spanning positions 54 ~ 153) (Fig. 3C, D). Notably, the interaction interface includes multiple residues (R55, F60, M61, Q63, A101, F113, W121, L122, and H126) of the hydrophobic cavity of CypA (Fig. 3D), suggesting that this hydrophobic pocket may be essential for p72 binding.

To experimentally verify this prediction, we generated a series of CypA truncation mutants with sequential deletions of 20, 40, 60, or 80 amino acids from either the N- or C-terminus (Fig. 3E). These mutants were expressed in E. coli, purified, and subjected to in vitro binding assays with the recombinant Strep-tagged p72 trimers. Deletion of 20 or 40 residues from either terminus had no significant effect on the binding between CypA and p72. However, removal of 60 or 80 residues from either end nearly abolished the interaction (Fig. 3F). As shown in Fig. 3E, deletions of 60 or more residues from either terminus disrupted key elements within the hydrophobic cavity (spanning residues 54 ~ 126), thereby impairing its structural integrity. To directly examine the role of the hydrophobic cavity in p72 binding, we generated a CypA(8A) mutant in which all key residues within the cavity were substituted with alanine (Fig. 3G). This mutant showed a marked loss of p72-binding capacity (Fig. 3H). These results support our structural prediction, confirming that the hydrophobic cavity of CypA serves as the primary binding region for p72.

We next sought to validate the corresponding CypA-binding site on p72. Previous structural studies have shown that p72 monomer adopts a double jelly-roll fold with four exposed regions (ERs) or insertions8, 9, 11. In our earlier study, we generated five porcine monoclonal antibodies (B1, F11, C9, H3, and G6) targeting p72 and determined their complex structures with p7213. Comparative analysis revealed that the binding epitopes of four of these antibodies (B1, F11, C9, and H3) significantly overlapped with the predicted CypA-binding region, all of which are located in the ERs of p72; Notably, each p72 trimer is capable of simultaneously binding three antibody molecules (Fig. 3I). In vitro binding assays confirmed that these four p72 antibodies effectively competed with CypA for p72 binding, supporting the idea that p72 recruits CypA via its ERs (Fig. 3J, K). In contrast, antibody G6, which binds the central apical region of the p72 trimer at a 1:1 ratio, failed to compete with CypA for p72 binding, similar to the negative control anti-p30 antibody (Fig. 3J, K). Given that the four competing antibodies (B1, F11, C9, and H3) all targeted ER1 on the outer apex of p72, while the non-competing G6 did not bind ER1 (Fig. 3I), we propose that the critical amino acid residues mediating p72-CypA interaction are primarily located within ER1. Collectively, our findings indicate that the CypA-p72 interaction is mediated through the hydrophobic cavity of CypA and the ER1 region of p72, laying a foundation for further elucidating how CypA may modulate p72 stability.

CypA stabilizes viral p72 by reducing its K63-linked ubiquitination
ASFV p72 has been reported to undergo degradation in host cells15, 30. To elucidate the major pathway responsible for p72 degradation, we treated p72-expressing cells with different inhibitors. While the lysosomal inhibitor bafilomycin A1 (BafA1) induced LC3-II accumulation, it had minimal impact on p72 protein levels (Fig. 4A). In contrast, treatment with MG132, a classical proteasomal inhibitor, not only enhanced Ub conjugation but also markedly increased p72 accumulation (Fig. 4A), indicating that p72 degradation is predominantly mediated by the proteasome. Furthermore, the stability of p72 was also significantly promoted by TAK-243 (MLN7243) (Fig. 4A), a first-in-class UBE1 inhibitor31 that depletes cellular polyubiquitin chains. Thus, the ubiquitin-proteasome system is primarily responsible for p72 degradation in host cells. Notably, the ASFV pB602L protein is essential for the correct folding and trimer assembly of p72, and the expression of p72 alone leads to the formation of misfolded p72 aggregates11, 12, 14. To further investigate the regulation of pB602L on p72 stability, we co-expressed p72 and pB602L in cells and conducted inhibitor treatment assays. Consistent with the results above, we observed similar effects of the inhibitors on p72 stability when the experiments were performed in the presence of pB602L (Fig. 4A). These findings indicate that the ubiquitin-proteasome system serves as the primary pathway for p72 degradation, irrespective of the presence or absence of pB602L.

It is noteworthy that co-expression of pB602L upregulated p72 protein levels (Fig. 4B), in agreement with a previous study14. Given that CypA has been reported to regulate the stability of several viral proteins24, 25, 32, we examined its potential role in modulating p72 levels. Similar to pB602L, CypA expression increased p72 protein levels (Fig. 4B and Fig. S3A). To ensure physiological relevance, p72 was co-expressed with pB602L in all subsequent experiments, unless otherwise specified. Notably, CypA exhibited a dose-dependent effect in enhancing p72 accumulation in cells (Fig. 4C), highlighting its critical role in regulating p72 expression or protein stability. However, CypA co-expression did not change the mRNA level of p72 (Fig. 4D), which indicates that CypA probably regulates the protein folding or stability of p72. Notably, while pB602L promoted p72 trimer formation regardless of CypA expression, CypA alone failed to induce trimerization (Fig. S3B), indicating that CypA does not exert a chaperone-like function in p72 folding. Deletion of endogenous CypA in cells resulted in a decreased p72 protein level, whereas re-expression of CypA restored p72 accumulation (Fig. 4E), further supporting its regulatory role in maintaining p72 stability. To assess p72 degradation rate, we used cycloheximide (CHX) to block protein synthesis and found that CypA expression suppressed p72 degradation (Fig. 4F). These results suggest that CypA enhances p72 protein stability in cells.

Since p72 was reported to undergo ubiquitination-mediated degradation30, we investigated whether CypA affects p72 ubiquitination. To this end, p72 was enriched from cells expressing p72 alone or co-expressing p72 and CypA under denaturing conditions, and its ubiquitination levels were analyzed. While strong ubiquitination of p72 was detected in the absence of CypA, co-expression of CypA markedly reduced its ubiquitination (Fig. 4G, H and Fig. S3C). To further characterize the ubiquitin (Ub) linkage type, cells were co-transfected with p72 and HA-tagged wild-type Ub or a series of Ub mutants, each retaining a single lysine residue at the indicated position. This analysis revealed that p72 is predominantly modified by K63-linked polyubiquitin chains in cells (Fig. 4I, J). Notably, overexpression of CypA resulted in a pronounced reduction of K63-linked polyubiquitination on p72 (Fig. 4K). Although K63-linked ubiquitination is typically associated with signal transduction33, 34, several studies have demonstrated that it can also target substrates for degradation35-37, and a recent study specifically implicated K63-linked ubiquitination in promoting p72 degradation30. Together, these findings suggest that CypA enhances protein stability of p72 by suppressing its K63-linked ubiquitination.

CypA inhibitors destabilize p72 by disrupting CypA binding and enhancing p72 ubiquitination
Cyclosporine A (CsA), a cyclic undecapeptide with the highest known binding affinity for CypA, is a well-characterized CypA inhibitor17, 38. CsA is an immunosuppressive agent derived from fungi, whereas its non-immunosuppressive analogs, such as NIM811 and Alisporivir (Debio-025), have been developed as therapeutic alternatives39. Structural studies have resolved the complexes formed between CypA and these cyclic undecapeptide inhibitors40-42, revealing that their binding sites overlap with the p72-binding surface of CypA (Fig. 5A). This structural overlap suggests that CsA and its analogs may disrupt the interaction between CypA and p72. To test this, we performed an in vitro binding assay using the recombinant Strep-p72 and GST-CypA proteins in the presence or absence of CypA inhibitors. As shown in Fig. 5B, the addition of CsA to the in vitro binding system effectively blocked the binding of CypA to p72. Similarly, NIM811 and Alisporivir also inhibited CypA-p72 binding (Fig. 5B), confirming that these inhibitors can competitively interfere with this interaction. Since these cyclic undecapeptide inhibitors occupy the hydrophobic cavity of CypA43, 44, these findings further support that p72 engages this same hydrophobic pocket, reinforcing the conclusion that CypA binds p72 through its hydrophobic interface.

Given that CypA enhances p72 stability, we hypothesized that inhibitors interfering with this interaction could destabilize p72. To investigate this, p72-expressing cells were treated with CypA inhibitors for 24 h and p72 protein levels were analyzed by Western blot. CsA treatment induced a dose-dependent reduction in p72 protein levels (Fig. 5C, E), with noticeable effects starting from 5 μM and a maximal reduction at 20 μM. NIM811 similarly decreased p72 levels in a dose-dependent manner (Fig. 5C, D). A CHX chase assay further demonstrated that NIM811 accelerated p72 degradation, shortening the time for the protein intensity to reach 50% of its initial value from 6 h to 2.95 h (Fig. 5F, G). Moreover, analysis of p72 ubiquitination in the cells treated with CypA inhibitors revealed that CsA or NIM811 exhibited a robust ability to increase the ubiquitination of p72 (Fig. 5H, I and Fig. S4), in contrast to the reduction induced by CypA overexpression (Fig. 4K). Together, these findings establish that the cyclic peptide inhibitors of CypA promote p72 degradation by enhancing its ubiquitination.

Inhibition of CypA impairs ASFV infection
The data described above suggest that CypA may play a critical role in ASFV infection. To investigate this, we performed viral infection assays using BK2258 cells, a wild boar-derived cell line known to support ASFV replication45. BK2258 cells were engineered to stably express either a control sgRNA (Ctrl-sg) or CypA-targeting sgRNAs (CypA-sg) via the CRISPR/Cas9 system. Western blot analysis confirmed that CypA protein expression was efficiently ablated in CypA-sg cells (Fig. S5A). Upon ASFV infection, CypA-sg cells exhibited a marked reduction of viral p30 expression compared to control cells (Fig. S5A). Quantitative PCR (qPCR) further revealed that loss of CypA significantly impaired the production of extracellular ASFV virions in the supernatants (Fig. S5B). To validate these findings in a primary cell context, PAMs were transfected with either CypA-targeting siRNAs (siCypA-1/2/3) or a negative control siRNA (NC) prior to ASFV infection. Similarly, CypA knockdown led to substantial reduction in both viral protein expression and ASFV replication in PAMs (Fig. 6A, B). These results indicate that CypA is essential for the efficient ASFV infection.

Since cyclic peptide inhibitors of CypA destabilize p72, we next examined whether these inhibitors could impact ASFV infection. PAMs were infected with ASFV in the presence of CypA inhibitors, including CsA, NIM811, and Alisporivir. All three inhibitors significantly decreased virions production in the supernatants of infected cells (Fig. 6C). Western blot analysis revealed that treatment with these compounds decreased the intracellular accumulation of viral proteins (Fig. 6D), and immunofluorescence staining showed a marked decrease in the number of p72-positive cells in the inhibitor-treated groups (Fig. 6E). To further quantify the infection ratio, we employed an artificially attenuated ASFV strain, HLJ/18-7GD, which expresses the fluorescent markers EGFP and mCherry46. CsA and NIM811 caused reduction in fluorescent signals in the infected PAMs (Fig. 6F), in a dose-dependent manner (Fig. S5C, D). Flow cytometry analysis corroborated these findings, showing that treatment with CypA inhibitors decreased the proportion of the infected cells by approximately tenfold or more (Fig. 6G, H and Fig. S5E). Consistently, Western blot analysis revealed a dose-dependent suppression of p72 and p30 expression in the infected PAMs by CypA inhibitors (Fig. 6I, J and Fig. S5F), and qPCR quantification of extracellular virions in the cell supernatants confirmed a dose-dependent inhibitory effect of CypA inhibitors on ASFV infection (Fig. 6K, L and Fig. S5G). Notably, these CypA inhibitors had minimal effects on cell viability at the indicated concentrations (Fig. S6A, B). Collectively, these results demonstrate that CypA inhibition, either through genetic decrease or pharmacological intervention, effectively suppresses ASFV infection.

Cyclic undecapeptides targeting CypA suppress ASFV replication kinetics
To evaluate the impact of CypA inhibition on ASFV replication kinetics, PAMs were treated with CypA inhibitors and infected with ASFV HLJ/18-7GD strain. The infected cells, which express fluorescent signals, were monitored and collected at different time-points for analysis. At 12 and 24 hpi, fluorescence intensities remained comparably low in both the DMSO-treated control and inhibitor-treated groups (Fig. 7A, B). However, as ASFV infection progressed (48 hpi late), fluorescence intensity in the control group increased substantially, indicating robust viral replication, whereas only a modest increase was observed in the inhibitor-treated groups (Fig. 7A, B). Consistently, multistep growth curve analyses showed that CypA inhibitors markedly reduced infectious virion replication, especially during the later stages of infection (Fig. 7C, D). Together, these findings demonstrate that cyclic peptide inhibitors of CypA effectively impair ASFV replication dynamics.

We next assessed the expression of viral proteins p72 and p30 during ASFV infection. Treatment with CsA or NIM811 markedly inhibited the time-dependent accumulation of both viral proteins (Fig. S7A, B), suggesting that CypA inhibition may finally interfere with the expression of viral genes. To investigate the effect of CypA inhibitors on viral gene transcription further, RNA-seq analysis was performed on NIM811-treated or untreated PAMs, with ASFV infection at 72 hpi (Fig. S7C, D). In line with previous studies20, 47, more than 160 viral genes were highly expressed in ASFV-infected cells (Fig. 7E). However, NIM811 treatment obviously reduced the levels of the viral gene transcription (Fig. 7E and Fig. S7E, Supplementary Data 1). qRT-PCR analysis of CP204L (p30) and B646L (p72) mRNA levels further confirmed that although viral gene transcription was elevated at 48 hpi and much higher at 72 hpi, this transcriptional activity was significantly suppressed by CypA inhibitors (Fig. 7F, G). These results demonstrate that CypA inhibitors can impair the transcription of viral genes during multi-cycle replication.

ASFV infection is known to induce hemorrhagic fever, accompanied by exacerbated inflammatory responses in multiple organs, ultimately causing pig mortality48. To determine whether CypA inhibitors can mitigate ASFV-induced host inflammation, we conducted host transcriptomic analyses of the infected PAMs. ASFV infection upregulated numerous host genes (Fig. S8A, Supplementary Data 2), many of which are associated with inflammatory pathways, including cytokine receptor signaling and the PI3K signaling pathway (Fig. S8B, Supplementary Data 3). Gene Ontology (GO) enrichment analysis revealed significant activation of inflammation-related processes and notable alterations in plasma membrane components following ASFV infection (Fig. S8C, Supplementary Data 4). Notably, volcano plot analysis showed that NIM811 treatment effectively suppressed the ASFV-induced upregulation of host gene expression (Fig. S8A), a finding further supported by heatmap analysis (Fig. S8D, Supplementary Data 5). Collectively, these data suggest that CypA inhibition not only disrupts ASFV replication but also attenuates virus-induced inflammatory responses, highlighting the therapeutic potential of cyclic peptide inhibitors of CypA as antiviral agents against ASFV replication.

Inhibition of CypA impairs viral factory formation and virion assembly by reducing p72 protein levels
Because viral adsorption, internalization, uncoating, biosynthesis, assembly, and budding are essential steps in the ASFV life cycle49, 50, we next investigated which of these processes are regulated by CypA. To this end, BK2258 cells expressing either Ctrl-sg or CypA-sg were used to evaluate the effect of CypA on ASFV adsorption and internalization. Comparable levels of adsorption and internalization were observed in both cell lines, independent of CypA expression (Fig. 8A). Similarly, treatment of PAMs with the CypA inhibitors CsA or NIM811 did not significantly affect viral adsorption or internalization (Fig. 8B), indicating that CypA primarily modulates post-entry steps during ASFV infection.

To define the specific stages controlled by CypA, we conducted single-cycle ASFV replication assays in PAMs and collected samples over time to monitor viral DNA replication, as well as the mRNA and protein levels of B646L (p72). As shown, viral DNA replication in a single-cycle infection was not significantly affected by CsA or NIM811 (Fig. S9A). Transcription of B646L began to increase significantly at 8 hpi, peaked at 12 hpi, and remained at high levels thereafter, with no significant differences between inhibitor-treated and control groups (Fig. 8C). In contrast, Western blotting revealed that p72 protein was markedly reduced upon treatment with CsA or NIM811, whereas in control group, p72 became detectable at 8 hpi and accumulated progressively over time (Fig. 8D, E and Fig. S9B). Notably, the levels of another viral protein, p30, were unaffected by CypA inhibition (Fig. 8D, E). These results demonstrate that CypA primarily regulates p72 protein levels, rather than broadly influencing viral genome replication or early viral gene expression.

Given that certain viral proteins, including p72, help retain structural proteins within the assembly area and thus contribute to ASFV virus factory organization14, 51, we next asked whether CypA inhibition disrupts this process. BK2258 cells were subjected to immunofluorescence staining for p72 and p54, the latter serving as a marker for viral factory localization52. Treatment with CypA inhibitors markedly reduced p72 signals and disrupted the formation of p54-positive puncta (Fig. 8F). Confocal microscopy further revealed that large cytoplasmic viral factories co-stained with p54 and DAPI were present in DMSO-treated cells, whereas inhibitor-treated cells exhibited substantially smaller factories (Fig. 8G, H), indicating compromised viral factory formation and impaired virion assembly. To directly assess virion assembly, we examined ASFV-infected PAMs treated with DMSO or CypA inhibitors by transmission electron microscopy (TEM). Numerous mature icosahedral virions were readily observed within viral factories in DMSO-treated control cells (Fig. 8I, J). In contrast, CypA inhibitor-treated cells contained markedly fewer mature virions and exhibited obvious aberrant tubular structures in viral factories (Fig. 8I), partially resembling previously reported zipper-like assemblies induced by disruption of p72 or pB602L14, 53. Consistently, CypA deficiency also promoted the formation of aberrant structures in viral factories of ASFV-infected cells (Fig. S9C). Together, these findings demonstrate that CypA inhibition disrupts ASFV viral factory organization and virion assembly by destabilizing p72 protein, thereby impairing ASFV infection.

Methods
Facility and Ethics Statements
All experiments involving live ASFVs were conducted in the enhanced biosafety level 3 (P3+) facility at the Harbin Veterinary Research Institute (HVRI), Chinese Academy of Agricultural Sciences (CAAS), with approval from the Ministry of Agriculture and Rural Affairs of China. All animal experiments were approved by the Committee on the Ethics of Animal Experiments of the HVRI, CAAS (Approval number: 250417-01-GR), and were performed in accordance with institutional guidelines.

Cell and viruses
Primary porcine alveolar macrophages (PAMs) were isolated from 5-week-old healthy specific-pathogen-free (SPF) Landrace piglets, regardless of sex, as previously described79, 80, and were maintained in RPMI-1640 medium (Gibco, #C11875500BT) supplemented with 20% porcine serum (HyClone, #SH30908.04), 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco, #15140122), and 2 mmol/mL glutamine at 37 °C with 5% CO₂ in a humidified incubator. The wild boar kidney cell line (BK2258), human embryonic kidney 293T (HEK293T, CRL-3216, ATCC), and human cervix epithelial HeLa cells (CCL-2, ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, #C1 1995500BT) containing with 10% Fetal bovine serum (FBS; Sigma, #F8687-500mL) and 1% penicillin/streptomycin at 37 °C with 5% CO₂. For transfection experiments, cells were transfected using Lipo293 (Beyotime, #C0521), lipofectamine 2000 (Life Technologies, #11668019), or polyethylenimine (PEI; Polysciences, #23966-100), according to the manufacturer's instructions. The wild-type, virulent genotype II ASFV strain HLJ/18, previously isolated from domestic pigs, and the artificially attenuated strain HLJ/18-7GD were maintained in our laboratory46, 80.

Plasmid construction
The gene encoding porcine CypA (Uniprot: P62936; NM_214353.1) was amplified from PAM cDNA and cloned into pCDNA3.0 for mammalian expression and into pGEX-4T-1 for bacterial expression. The ASFV B646L gene encoding p72 (GenBank: QBH90570.1) and the B602L gene encoding pB602L (GenBank: QBH90568.1) were also cloned into the pCDNA3.0 for mammalian expression. Truncated variants of CypA and other porcine cyclophilins were generated by PCR and inserted into pGEX-4T-1 for recombinant expression and purification in E. coli. BL21 (DE3).

Antibodies and reagents
The anti-p72 rabbit polyclonal antibody, anti-p72 mouse monoclonal antibody, anti-p30 mouse antibody, anti-p54 antibody, and anti-CP312R mouse antibody were generated in-house, which are available from the corresponding author upon reasonable request. Mouse monoclonal antibodies against Flag (#L-MAb03), HA (#L-MAb01), and GST (#L-MAb04) were obtained from Biolinkedin (Shanghai). Rabbit monoclonal anti-Flag antibody (#F7425) and DAPI (#D9542) were purchased from Sigma. Rabbit anti-HA antibody (#AE036) was obtained from ABclonal. Anti-β-actin antibody (#A00702) and mouse lgG isotype control (#A01007) were purchased from GenScript. Monoclonal anti-CypA antibody (#67880-1-Ig), polyclonal anti-CypA rabbit antibody (#10720-1-AP), and polyclonal anti-LC3 rabbit antibody (#14600-1-AP) were from Proteintech. Anti-Strep antibody (#K200012M) was purchased from Solarbio. Rabbit monoclonal anti-Ub antibody (E4I2J) was provided by Cell Signaling Technology. K63-linked Ub antibody (#F0528) was obtained from Selleck. Fluorescent secondary antibodies were obtained from Thermo Fisher Scientific, including Goat anti-Mouse IgG (H+L) Alexa Fluor™ 555 (#A-21422), Goat anti-Rabbit IgG (H+L) Alexa Fluor™ 555 (#A-21429), Goat anti-Mouse IgG (H+L) Alexa Fluor™ 488 (#A-11001), and Goat anti-Rabbit IgG (H+L) Alexa Fluor™ 488 (#A-11034). Normal rabbit IgG control (#CR1) was purchased from Sino Biological. Goat anti-mouse IgG (H+L) peroxidase AffiniPure® secondary antibody (#115-035-003) and goat anti-rabbit IgG (H+L) peroxidase AffiniPure® secondary antibody (#111-035-003) were ordered from Jackson immunoresearch.

Affinity resins included Strep-Tactin resin (#L2302), anti-Flag agarose affinity gel (L-1109S), and anti-HA agarose affinity gel (L-1107S) from Biolinkedin (Shanghai). Glutathione Sepharose 4B resin for GST-tagged protein purification was sourced from Cytiva (#10250335) or BBI (#C600031-0100). PureProteome Protein A/G magnetic beads (#LSKMAGAG10) were obtained from Merck-Millipore. Chemical inhibitors used in this study included the proteasome inhibitor MG132 (#S2619) from Selleck, autophagy inhibitor bafilomycin A1 (BafA1; #HY-100558) and the ubiquitin-activating enzyme (E1) inhibitor TAK-243 (#HY-100487) from MCE. Cycloheximide (CHX; #66-81-9) was ordered from Merck. Cyclosporin A (CsA; #HY-B0579), Alisporivir (#HY-12559), and NIM811 (#HY-P0025) were obtained from MCE.

Alignment of amino acid sequences
Gene sequences of porcine cyclophilins, including CypA/PPIA (NM_214353.1), PPIB (XM_021093880), PPIC (XM_003129041), PPID (NM_001244386), PPIE (XM_013999195), PPIF (XM_001929483), PPIH (XM_003128098), and PPIL1 (XM_003128368), were retrieved from NCBI. Gene or protein sequences of CypA orthologs from human (NM_021130.5), monkey (NM_001032809.1), cattle (NM_178320.3), dog (XM_038689274.1), cat (NM_001009370.1), chicken (NM_001166326.2), mouse (NM_008907.2), rabbit (NM_001082057.1), Phacochoerus africanus (warthog, XP_047619276.1), and Ornithodoros turicata (XP_064487992.1) were also obtained from NCBI. Amino acid sequences were translated from coding sequences (CDS) of genes using SnapGene software (Dotmatics, available at https://www.snapgene.com). Multiple sequence alignment was performed with MUSCLE algorithm81.

Co-immunoprecipitation (co-IP)
Co-IP assays were conducted using ASFV-infected PAMs or HEK293T cells co-transfected with Strep-tagged p72 and HA-tagged CypA for 36 hours. Cells were collected by centrifugation at 500 g for 5 minutes and lysed in Co-IP buffer consisting of 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, and 1× protease inhibitor cocktail. Lysates were subjected to ultrasonication using a non-contact automated ultrasonic disruptor (5 seconds on, 8 seconds off, for 60 cycles). After centrifugation at 12,000 g for 20 minutes at 4 °C, the clarified supernatants were incubated overnight at 4 °C with either anti-p72 or anti-HA antibodies for immunoprecipitation. Immune complexes were washed five times with Co-IP buffer, resuspended in 1× SDS loading buffer, and boiled at 100 °C for 30 minutes prior to analysis by Western blotting.

Mass spectrometry analysis
Mass spectrometry analysis was performed on control IgG- and anti-p72-enriched samples (one biological replicate each) from PAM lysates, following previously described procedures with minor modifications82, 83. Protein bands were excised from SDS-PAGE gels and subjected to in-gel digestion. Gel pieces were destained with 30% acetonitrile in 50 mM NH₄HCO₃, reduced with 10 mM dithiothreitol at 56 °C for 30 min, and alkylated with 60 mM iodoacetamide in the dark for 30 min. After dehydration, samples were rehydrated with sequencing-grade trypsin (Promega) in 50 mM NH₄HCO₃ and incubated overnight at 37 °C. Peptides were sequentially extracted with 60-90% acetonitrile containing 0.1% trifluoroacetic acid, dried, and desalted on C18 columns. The resulting peptide samples were redissolved with 0.1% formic acid and analyzed by liquid chromatography (LC) using an UltiMate3000 Rapid Separation system (Thermo Fisher Scientific) coupled to a Q Exactive HF-X Orbitrap (Thermo Fisher Scientific) operated in data-dependent acquisition (DDA) mode. Raw MS data were processed with MaxQuant (v2.7.5.0)84 using default and specified parameters, including trypsin digestion allowing up to two missed cleavages, variable modifications of methionine oxidation and protein N-terminal acetylation, and the use of both unique and razor peptides for protein quantification. MS tolerances were set to 20 ppm for FTMS precursors and 0.5 Da for ITMS fragments. Peptide-level false discovery rate (FDR) was set at 1%, with a minimum peptide length of seven amino acids. Database searches were performed against both the Sus scrofa reference proteome (UPE000008227) and the ASFV HLJ/18 strain proteome (GenBank: MK333180.1).

Expression and purification of recombinant proteins
Recombinant Strep-tagged p72 trimer protein was expressed and purified in yeast cells as previously described85, 86. For bacterial expression of GST-tagged proteins, pGEX-4T1 plasmids containing full-length CypA or its truncation constructs were transformed into E. coli BL21 cells. Transformed bacteria were first grown on LB agar plates containing 100 µg/mL ampicillin at 37 °C. A single colony was then picked and cultured in 5 mL LB medium with shaking at 220 rpm overnight. In the next day, the culture was inoculated into 500 mL fresh LB medium and grown at 37 °C for 2~3 h, until the OD600 reached 0.6~0.8. Protein expression was induced by adding IPTG to a final concentration of 0.5 mM, followed by incubation at 16 °C overnight. Cells were harvested by centrifugation at 5,000 g for 10 min, washed once with PBS, and resuspended in NETN buffer containing 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.5% TritonX-100, 1 mM EDTA, 2 mM PMSF, and 1 mM DTT. Cell lysis was performed using a high-pressure cell disruptor. After centrifugation at 12,000 g for 30 min at 4 °C, the supernatant was collected and incubated with Glutathione Sepharose 4B beads for 2 h at 4°C with gentle rotation. After washing five times with NETN buffer, GST-tagged proteins were eluted using freshly prepared elution buffer containing 200 mM Tirs-HCl (PH 8.0), 150 mM NaCl, and 50 mM reduced glutathione. Eluted proteins were dialyzed overnight at 4°C in 2 L of dialysis buffer containing 20 mM Tris-HCl (pH 7.6), 500 mM NaCl, 2 mM DTT, and 20% glycerol. After dialysis, proteins were aliquoted and stored in -80 °C refrigerator. Protein purity and concentration were assessed by SDS-PAGE followed by Coomassie Brilliant Blue staining.

Strep pull-down assay
Recombinant GST or GST-tagged proteins, Strep-tagged p72 trimer, Strep-Tactin resin, and a protease inhibitor cocktail (Roche) were combined into 1.5 mL microcentrifuge tubes. The mixtures were supplemented with co-IP buffer (50 mM Tris, pH 7.6; 150 mM NaCl; 1% TritonX-100; 1× protease inhibitor cocktail; 0.5% Micrococcal Nuclease (M0247S); 1mM CaCl2; and 2 mM DTT) to a final volume of 800 µL. For competitive binding assay, 5 µM cyclic undecapeptide CypA inhibitors or 10 µg p72-specific antibodies were added to the reaction. The samples were incubated at 4 °C for 4 h or overnight with gentle rotation. After incubation, the Strep-Tactin resin was collected by centrifugation at 500 g for 1 min and washed five times with pre-chilled co-IP buffer. The bound proteins were eluted by mixing the resin with 1× SDS loading buffer and boiling at 100 °C for 30 min. Samples were subsequently analyzed by Western blotting.

Immunofluorescence and confocal microscopy
Immunofluorescence was performed to analyze the subcellular localization of the indicated proteins, as previously described87. PAMs or HeLa cells were seeded in 12-well plates containing sterile glass coverslips and infected with ASFV or transfected with the indicated plasmids for 24 h. Cells were fixed with 4% paraformaldehyde (PFA) for 10 min and permeabilized with 0.1% Triton X-100 for 20 min at room temperature. After three washes with PBS (10 min each), cells were blocked in 1 % BSA in PBST (PBS containing 0.1% Tween-20) for 1 h at room temperature. Primary antibodies diluted in the blocking buffer were applied to the cells and incubated overnight at 4 °C. The following day, cells were washed three times with PBST and incubated with the appropriate fluorescently labeled secondary antibodies (1:1000 dilution in blocking buffer) for 1 h at room temperature. After three additional washes with PBST, nuclei were counterstained with DAPI (0.2 μg/mL) for 20 min. Coverslips were then washed, mounted onto glass slides using fluorescence mounting medium (Dako Omnis, #NS3023), and allowed to air-dry overnight in the dark. Fluorescence images were acquired using a standard fluorescent microscope or a Zeiss LSM 800 confocal microscope.

Cycloheximide (CHX) chase assay
Transfected or inhibitor-treated cells were exposed to CHX (100 μM) for the indicated time periods, followed by Western blot analysis of p72. Band intensities were quantified using Adobe Photoshop and normalized to the corresponding loading control (if needed) and to the initial time point (t = 0) of each group. As the normalized intensity-time data displayed an approximately linear decline, the degradation rates were determined by performing a simple linear regression in GraphPad Prism (version 9.5.0). The rate of linear decrease was obtained from the slope of the fitted linear equation Y = slope × X + b, where b represents the initial intensity at t = 0. For clarity, we report the estimated time required for the intensity to decrease to 50% of the initial value, calculated as t50% = b/ (2 × |slope|).

In vivo ubiquitylation assay
In vivo ubiquitination was performed as previously described88, with minor modifications. Cells expressing Strep-p72, with or without CypA co-expression or CypA inhibitor treatment, were harvested by centrifugation at 500 g for 5 min, washed once with PBS, and resuspended in 100 µL TSD buffer (50 mM Tris-HCl; pH 7.6; 2 % SDS; and 5 mM DTT). Samples were boiled at 100 °C for 30 min, then centrifuged at 12,000 g for 10 min. The supernatants were collected and diluted 10-fold with co-IP buffer to reduce the SDS concentration from 2% to 0.2%. Diluted lysates were incubated with 20 µL Strep-Tactin resin at 4 °C overnight with gentle rotation. The resin was washed five times with cold co-IP buffer and boiled in 1× SDS loading buffer. Precipitated proteins were analyzed for Western blot analysis using anti-ubiquitin or other indicated antibodies.

Gene knockdown by siRNA transfection
Three siRNAs targeting Sus scrofa CypA (GenBank: XM_021078519.1) and a non-targeting control siRNA (NC) were synthesized by GENCEFE Biotech (Wuxi, China):
(1) siCypA-1: Forward: GGUUCCUGCUUUCACAGAATT;
Reverse: UUCUGUGAAAGCAGGAACCTT;
(2) siCypA-2: Forward: GGGCAUGAAUAUUGUGGAATT;
Reverse: UUCCACAAUAUUCAUGCCCTT;
(3) siCypA-3: Forward: CUGACUGUGGACAAAUCUATT;
Reverse: UAGAUUUGUCCACAGUCAGTT;
(4) NC: Forward: UUCUCCGAACGUGUCACGUTT;
Reverse: ACGUGACACGUUCGGAGAATT.
PAMs were seeded in 12-well plates and incubated for 6 h prior to transfection. Cells were transfected with 80 pmol of siRNA per well using the Gencefe Super Trans transfection reagent (GENCEFE Biotech) and incubated for 12h. Following transfection, the medium was replaced with fresh complete growth medium, and cells were cultured for an additional 36 ~ 48 h. Subsequently, cells were infected by ASFV at a MOI of 0.2 and incubated for 72 h. After infection, culture supernatant was collected for viral DNA quantification, and cells lysates were prepared for Western blot analysis.

Construction of CypA-deficient cell lines by CRISPR/Cas9 system
CypA-deficient HEK293T cell line was generated using the CRISPR/Cas9 technology, as described previously25. Briefly, three sgRNA sequences targeting the human CypA gene (NM_021130.5) were designed using the CRISPOR online tool (http://crispor.tefor.net/) and cloned into the lentiCRISPRv2 vector (Addgene, #52961) following the standard procedures89. HEK293T cells were seeded into 6-well plates and cultured overnight. Cells were then co-transfected with packaging plasmid psPAX2 (Addgene, #12260), envelope plasmid pMD2.G (Addgene, #12259), and the lentiCRISPRv2-sgRNA plasmids at a ratio of 2.5:1:3.5. After 48~72 h, lentivirus-containing supernatant was collected and filtrated through 0.45 μm filters. For transduction, 2 mL of viral supernatant was mixed with 2 mL of fresh complete growth medium containing 10 μg/mL polybrene and added to target cells in 6-well plates. After 24 h, the medium was replaced with fresh growth medium, and cells were cultured for an additional 24 h. Cells were then passaged into 10-cm dishes and cultured in medium containing the appropriate antibiotic for at least five passages to enrich for transduced populations. To isolate knockout clones, cells were sorted by flow cytometry into 96-well plates (one cell per well). Clones were screened by Western blot using anti-CypA antibody, and successful knockout clones were further confirmed by gene sequencing.

A similar strategy was used to construct CypA-deficient BK2258 cells. Three sgRNA sequences targeting the Sus scrofa CypA gene (XM_021078519.1) were designed using CRISPOR and cloned into the lentiCRISPRv2 vector. Lentiviral particles were produced as described above and used to transduce BK2258 cells. After 48 hours, transduced cells were selected in antibiotic-containing medium for at least five passages and assessed for CypA knockout by Western blotting.

The sgRNA sequences used in the study were as follows:
(1) Human CypA sgRNAs25: 5’- CTTGGGCCGCGTCTCCTTTG -3’;
5’- TTCTTCGACATTGCCGTCGA -3’;
5’- ATCCTAAAGCATACGGGTCC -3’.
(2) Porcine CypA sgRNAs: 5’- GCGATGTCGAAGAAGACGGT -3’;
5’- TCGAAGGAGACGCGGCCCAA -3’;
5’- AACTTCCGTGCTCTGAGCAC -3’.
(3) Non-targeting control sgRNA: 5’- ACGGAGGCTAAGCGTCGCAA -3’.

ASFV infection assay
The ASFV infection procedure was performed as described before45, 90, 91, with minor modifications. PAMs or BK2258 cells were seeded in 12 well-plates and cultured for 6 h or 12 h prior to ASFV infection. Cells were then incubated with ASFV at the indicated multiplicity of infection (MOI) at 37 °C for 2 h. Cells were washed three times with PBS and maintained in culture medium containing 20 % porcine serum (for PAMs) or 3% FBS (for BK2258) in a 5% CO2 incubator at 37 °C. At the indicated time points, both supernatants and cells were collected for further analysis. ASFV genomic DNA was extracted from supernatants using QIAamp® DNA Mini Kits (Qiagen, Germany) and quantified by quantitative PCR (qPCR) using an ABI QuantStudio5 system (Applied Biosystems, USA) with primers targeting the B646L gene. Infected cells were processed as followed: fixed in 4% PFA for immunofluorescence staining; harvested in 1× SDS loading buffer for Western blot analysis; or lysed in TRIzol reagent for total RNA extraction and subsequent gene expression analysis.

Virus adsorption and internalization assays
ASFV binding and internalization assays were performed as previously described92. For the adsorption assay, BK2258 stable cell lines or PAMs treated with CypA inhibitors were incubated with ASFV (MOI = 5) at 4 °C for 2 h to allow viral binding. Unbound virions were removed by washing the cells five times with ice-cold PBS. Cells were then harvested for total DNA extraction, followed by qPCR quantification of ASFV genomic DNA. For the internalization assay, BK2258 cells or PAMs were first incubated with ASFV (MOI = 5) at 4 °C for 2 h to enable viral attachment. After five washes with ice-cold PBS, cells were shifted to 37 °C and incubated in growth medium for an additional 2 h to facilitate viral internalization, in the presence or absence of CypA inhibitors. The cells were then incubated with trypsin-EDTA for 2 min at 37 °C to remove noninternalized particles. Cells were then collected for total DNA extraction and qPCR analysis of internalized ASFV genomes.

Flow cytometry analysis
PAMs were seeded in 12-well plates and cultured for 6 h prior to infection with ASFV HLJ/18-7GD (MOI = 0.1) for 72 h, in the presence or absence of CypA inhibitor treatment. Infected cells were either examined by fluorescent microscopy or analyzed by flow cytometry. For flow cytometry analysis, cells were harvested by trypsinization, neutralized with complete medium, washed with PBS, and fixed with 4% paraformaldehyde (PFA). Single-cell suspensions were prepared by passing the cells through a 70-μm cell strainer. Cells were first gated on forward scatter (FSC-A) versus side scatter (SSC-A) to exclude debris and cell aggregates. mCherry-positive infected cells were subsequently identified by gating on mCherry fluorescence versus SSC-A, using uninfected cells as the negative control. Data were acquired on a flow cytometer and analyzed using FlowJo software (v10.8.1).

RNA sequencing and bioinformatic analysis
PAMs were infected with or without ASFV (MOI = 0.1) for 72 h in the presence or absence of NIM811. Total RNA was extracted using TRIzol reagent, and high-throughput RNA sequencing was performed by BMKgene. Raw FASTQ files from RNA sequencing were quality-filtered using fastp93, followed by alignment to the reference genome using HISAT294. Gene-level counts were quantified with featureCounts95, and differential expression analysis was conducted using DESeq2 package96, applying thresholds of |log2(Fold change)| ≥ 2 and adjusted p-value ≤ 0.01. Functional enrichment analysis of differentially expressed genes (DEGs) was performed using clusterProfiler R package97, with reference to the Gene Ontology98 (GO: https://www.geneontology.org) and Kyoto Encyclopedia of Genes and Genomes99 (KEGG: https://www.genome.jp) pathway databases.

Quantitative RT-PCR analysis
Total RNAs were extracted from ASFV-infected PAMs using TRIzol reagent, and RNA concentrations were measured with a NanoDrop spectrophotometer. A total of 1 μg RNA was reverse-transcribed into cDNA using the PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (#RR047A, Takara). Quantitative PCR was performed in triplicate in a 96-well plate using 50 ng cDNAs per well, the SYBR® Green Realtime PCR Master Mix (TOYOBO, #QPK-201), and an ABI QuantStudio5 system. Relative gene expression levels were calculated using the 2(–ΔΔCt) method, with GAPDH as the internal reference. The PCR primers used were: ASFV B646L (forward: 5′-ACGGCGCCCTCTAAGGGT-3′; reverse: 5′-CATGGGCAGCTTCAAACGTTTC-3′) and pig GAPDH (forward: 5′-ACCCAGLAAGACTGTGGATGG-3′; reverse: 5′-ACGCCTGCTTCACCACCTTC-3′).

Transmission electron microscopy (TEM)
PAMs were infected with ASFV (MOI = 5) for 24 h in the presence or absence of CypA inhibitor treatment. Infected PAMs were harvested and fixed with 2.5% glutaraldehyde in PBS (PH 7.4) at 4°C overnight. Fixed samples were then dehydrated through a graded ethanol series, embedded in epoxy resin, and processed for ultrathin sectioning and staining according to standard protocols. Sections were examined using a Hitachi H-7650 transmission electron microscope (Hitachi, Tokyo, Japan) operated at 80 kV. Digital images were captured and analyzed using Adobe Photoshop.

Statistics and reproducibility
Data are presented as mean ± standard deviation (SD). Unless otherwise stated, all experiments were independently repeated at least three times with similar results, and representative images are shown. Statistical significance between groups was determined using two-tailed unpaired Student’s t-test or ordinary one-way ANOVA followed by Dunnett’s multiple-comparisons test, as indicated in the figure legends. All statistical analyses were performed using GraphPad Prism (version 9.5.0).

Data availability
The mass spectrometry data generated in this study have been deposited in the ProteomeXchange Consortium via the iProX partner repository under accession code PXD073232 [https://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD073232]. The RNA-seq data generated in this study have been deposited in the Gene Expression Omnibus (GEO) under accession code GSE300823 [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE300823]. The previously published PDB structures referred to in this study include 3K0N [https://www.rcsb.org/structure/3K0N], 1CWA [https://www.rcsb.org/structure/1CWA], 1CWM [https://www.rcsb.org/structure/1CWM], and 5HSV [https://www.rcsb.org/structure/5HSV]. All data are available within the Article, Supplementary Information, or Source Data file. Source data are provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript the authors investigate the interaction of the African swine fever virus p72 major capsid protein with cyclophilin A (CypA). Although CypA has previously been shown to have important and diverse roles during replication of several other viruses. This is the first study to describe a role for CypA during replication of ASFV. CypA was one of the main proteins the authors detected by mass spectrometry in co-precipitates by an antibody against p72 from ASFV infected porcine alveolar macrophages. Subsequently this interaction was confirmed by direct binding between the tagged recombinant proteins and by coprecipitation following transient expression in transfected cells. The interaction of CypA with p72 was mapped to exposed regions on p72 and a sequence of CypA spanning a cavity region. Interaction of CypA was shown to reduce K63 linked ubiquitination of p72 and proteasomal degradation. Inhibitors of CypA reduced the size of virus factories and resulted in aberrant assembly of virions thus reducing virus replication kinetics. The authors indicate this interaction as a promising target for antiviral drug development against ASFV. The manuscript provides interesting novel data on an ASFV host interaction to stabilise the p72 capsid protein hence enhancing virus replication. The experiments are generally comprehensively carried out and clearly described and results support the conclusions drawn.\nSpecific Points:\n1. As described by the authors, the p72 protein assembly into trimers which are incorporated into the virus capsid requires the virus encoded B602L chaperone although this protein isn’t incorporated into virus particles. Without co-expression of the B602L p72 is not expressed in the native trimeric form. To obtain recombinant p72 protein the authors express p72 with or without B602L in yeast. Although this co-expression with B602L has previously been shown to produce the native trimeric form of p72 in this manuscript the data to confirm this is achieved in the current study is not presented. The authors should present this data to confirm that the recombinant p72 used in experiments is assembled into trimers and that the monomeric form of p72 is absent. This data is important for the interpretation of experiments shown in Fig 1, and S1.\n2. One important question, which is not fully resolved in this study, is the stage of replication when p72 is associated with CypA. The authors data suggest this interaction is important during virus assembly most likely after the assembly of the trimeric form of p72. In Figure 2 F immunofluorescence images from ASFV infected porcine alveolar macrophages fixed at 24 hpi infection at high multiplicity of infection are shown. Staining with anti-p72 and anti-CypA shows co-localization across the cytoplasm although not in factory structures which at this stage seem largely devoid of either DNA or p72 protein. At this late stage of infection it would be expected that progeny infectious virions have been produced some and would be moving to the cell surface or already been released. This data seems to suggest that CypA colocalises with virions as they move to the cell surface and even in projections from the cell, suggesting a possible role for the interaction of CypA with virions after virus assembly. To better understand the role of the CypA interaction with p72 the authors should repeat this experiment at different time points after infection. A parallel time course of infectious virus titre would establish when progeny virions are produced. This analysis may also help to establish if the CypA remains associated with p72 in the virus factories, no CypA is detected in the factories in the image shown in Fig 2F at this time point (24 hpi).\n3. In Figure 6 the authors use Western blotting to measure accumulation of protein p30, a protein expressed from early times, during ASFV infection rather than p72 which is expressed from later times and is incorporated into virions. The authors should explain the reason why Western blotting with anti-p72 isn’t used in these experiments as Western blotting against both p30 and p72 is used in Fig 7 C. If both were used consistency between experiments would be better.\n4. In Fig 7 ASFV replication kinetics are compared in CypA inhibitor treated and non-treated cells by several assays including measurement of numbers of fluorescent cells, expression of proteins by Western blotting, global transcription of ASFV genes, genome copy numbers. The impact of the treatment on the infectious virus titres are not shown. These data should be included to support the claim by the authors that treatment with drugs targeting CypA may be an effective antiviral treatment.\n5. Figure 9 shows a cartoon to summarise the results from the study. This is helpful although some points haven’t been established or are confusing. The diagram shows the interaction between p72 and CypA in the cytoplasm but not in the virus factory whereas as discussed in point 2 above this hasn’t been established in the current study. Indeed the data shown in Fig 2F suggest CypA may be associated with migrating virions. In addition transcription of the mRNA for B646L appears to be shown outside the factory whereas the late transcription is assumed to be inside the factory where the replicating genomes are accumulated.\nMinor Points:\n1. In the text and Figure 1 legend the numbers of replicates of the co-precipitation and mass spectrometry analysis is not given.\n2. Legend Figure 4A states that p72 was expressed in the presence or absence of B602L. On the figure the label shows B602L was included in all of these experiments.", "reviewer_2": "Zhao and colleagues present data to characterize the interaction between ASFV p72 with cyclophilin A which was discovered by AP-MS. Truncation experiments and use of inhibitors allowed to narrow down the interacting site. Function of CYPA with respect to ASFV replication are tested with infection experiments using PAM and genetically engineered knock-out BK2258 porcine cells. The main findings are that CYPA interacts with p72, stabilizes it against proteasomal degradation and enhances ASFV infection. These findings are important in the ASFV field however interactions of CYPA with matrix proteins of other viruses have been described before.\nThe subject of the study is of great importance as ASFV threatens the pig industries almost worldwide. The manuscript is well written. However, some of the presented data needs more explanation and experimental confirmation, for details please see the section with the ‘Minor points’. Also, some figures seem overcrowded and could be rearranged and greater care should be taken to give appropriate references for all tools that were used, some are mentioned in this section.\nThe presented data is interesting but also raises questions. In a number of experiments the authors describe the negative effect of CYPA loss or inhibition on the expression of ASFV proteins, which can be explained by the decreased number of cells infected in the inoculated cell batch (see Figure 6). Protein expression data on single cell basis are not presented, but Figure 8 shows an obvious reduction of ASFV particles within an infected cell in the absence of CYPA suggesting lower expression of at least structural proteins. It remains unclear why successful internalisation of ASFV leads to low level infection in only very few cells in the absence of CYPA while viral entry is not affected (Fig 8). Unfortunately, this crucial experiment (adsorption / internalisation) is not described precisely, no reference is given, and no controls are used (if available at all). The manuscript would also improve if the authors added some information from the literature about the impact of CYPA on a number of cellular processes that may, in addition to the described interaction with p72, influence the outcome of an ASFV infection (MAPK signalling, RIG-I IFN, regulation of cytokines) in the Introduction section.\nMinor points\nLine 101: the peptide mentioned here and in Fig 1 is also present in porcine PPIE, please explain why PPIA was chosen for further experiments.\nLine 137: Fig 2 shows localisation of both proteins in the cytoplasm which explains the overlap but does not prove the interaction, pls correct. The magnification of Fig 2D,F is too low to draw this conclusion.\nLine 150: pls add a reference for PyMOL\nLine 442, Methods:\nAs MS data is shown in Figure 1, pls add a detailed description of the of MS analysis. The authors should consider to make the MS data available to the public in a repository. The same accounts for RNA sequencing.\nLine 634: the adsorption assay is not described sufficiently, no reference is given. Pls specify ‘cold acidic PBS’ and if that was used or the trypsin treatment, specify all conditions.\nLine 663: pls add citations for GO and KEGG repositories\nFigure 1: pls add sequences from other species that are relevant for ASFV infection (e.g. from genera Potamochoerus, Ornithodoros), add reference for MUSCLE algorithm.\nFigure 1C: looks like Maxquant software has been used. If this is so, please add a citation.\nFigure 1D: what is the coloring based on?\nFigure 4F: the band intensities for WT and KO cells are very different, I find it hard to judge if the degradation is really ‘accelerated’ based on the very different starting levels.\nFig 4G: these diffuse bands are hard to interpret, pls add a quantitation of the experiment.\nFig 4H: the conclusion is difficult to follow without quantitation of the bands including a correction for the input of Ub. Pls avoid ‘above’ for references within the manuscript but specify in detail how the blots were done. The lower HA panel has 9 tracks, all others have 10, pls correct.\nFig 4I: the upper panel is hard to interpret, pls replace\nFig 5E: this blot needs quantitation and statistical evaluation to demonstrate the claimed acceleration of degradation.\nFig 5F: pls add a detailed description of the procedure to calculate the half-lives to the Method section. The shown intensity-time relation seems linear, is this a logarithmic scale?\nFig 6: there is some redundancy here which makes the figure confusing. Consider re-arranging\nfor instance by combining panel A with D, and panel B with C. Panels G, H demonstrate that there is a lower number of infected cells in the absence of CYPA and explains A,B,C, and D. Pls explain ‘BF’ in panel B and Fig 7.\nFig 6B,K: only extracellular virions were assessed, pls correct\nFig 7B: which MOI was chosen?\nFig 7C: considering results from panels A,B, panel C is dispensable\nFig 7D: hard to interpret. How can the 2 separate ‘tracks' of points in the ASFV vs mock panel be explained? What does ‘left’ and ‘right’ in the legend refer to? The RNA-seq data is not publicly available, nor are details for data evaluation. The volcano representation can be replaced by a dot-plot (DMSO vs NIM811) for clarity. There is redundance with panel E. Pls consider rearranging the Figure or putting parts of it in a supplement.\nFig 8A: what do the bars represent, how is ‘relative’ adsorption and internalisation defined, pls explain.", "reviewer_3": "In this study, Kong et al identify cyclophilin A (CypA) as a host interacting partner of the African swine fever virus (ASFV) major capsid protein p72. Their findings suggest that p72-CypA interaction prevents proteosome-mediated degradation of p72, thereby facilitating viral replication. Conversely, suppression of CypA expression or treatment with CypA inhibitors significantly impairs viral replication, highlighting a novel potential therapeutic target against ASFV.\nWhile the work presents novel and intriguing data, the effect of CypA suppression and CypA inhibitors on viral assembly contradicts previous evidence on the consequences of p72 knockdown and needs clarification. Furthermore, key aspects of CypA's mechanism of action on p72 stability remain unclear and would require further investigation.\nMajor points:\n- Inconsistencies in ASFV assembly defects. The reported effects of CypA inhibitors on viral assembly do not align with established phenotypes resulting from p72 abrogation. Previous studies (e.g., Ref. 9) have shown that suppression of p72 expression leads to the massive formation of aberrant viral structures known as “zippers” in viral factories, resulting from the co-assembly of unprocessed polyproteins pp220 and pp62 with inner envelope precursors. Similar structures are observed when the viral chaperone pB602L is abrogated, which blocks p72 trimerization (Refs. 11 and 13). Surprisingly, these hallmark features are absent in the presence of CypA inhibitors, and this discrepancy is not addressed in the manuscript. Instead, CypA inhibitors reduce viral factory size—an effect more suggestive of compromised formation of ER-derived viral membrane rather than decreased p72 levels.\nThese discrepancies raise questions about whether CypA inhibitors specifically act only through modulation of p72 degradation, or whether they impact additional viral components or replication stages. To clarify this, it would be highly informative to examine the ultrastructure of viral factories following CypA knockdown or knockout. If similar effects to those of the inhibitors are observed, off-target or broader effects should be considered to account of the effects of CypA abrogation and inhibitors.\nDoes CypA solely regulate p72 stability? The data do not conclusively demonstrate that CypA’s only role is in stabilizing p72, since most infection experiments were conducted at low MOI (0.1–1 PFU) and analyzed at late post-infection times (72–96 hpi), allowing for multiple replication cycles. To unravel primary from secondary effects, it would be beneficial to re-analyse the impact of CypA silencing and CypA inhibitors on viral DNA replication and early (e.g., p30) and late viral expression (p72 and other late proteins) in single-cycle assays.\n- Mechanism of Cyp A action on p72 stability. Beyond p72 stabilization, it remains unclear whether CypA acts as a chaperone for proper p72 folding and trimerization. Can the authors determine if p72 trimers are formed in CypA knockout cells, with or without co-expression of pB602L?\n- K63-linked ubiquitination. The claim in the Abstract that ´interaction with CypA stabilizes p72 by reducing K63-linked ubiquitination and preventing proteasomal degradation` appears overstated. This conclusion is based solely on overexpression assays in transfected cells lacking pB602, which is essential for proper p72 folding and trimerization (indeed, ´expression of p72 alone leads to the formation of misfolded p72 aggregates`; refs. 11, 13, 16). Moreover, K63-linked ubiquitination is typically associated with signaling rather than degradation. The authors should temper this conclusion and contextualize the limitations of their system, especially as it differs markedly from the infection setting.\n- p72 immunofluorescence patterns. The intracellular distribution of p72 in infected cells (Figs. 2F and 8F) appears diffuse and, surprisingly, largely excluded from assembly sites, which is inconsistent with that expected for the major capsid protein. This contrasts with established reports showing p72 localization to viral factories and cytoplasmic puncta corresponding to scattered virions. This discrepancy undermines claims about colocalization with CypA (Fig. 2F) and the late virion protein p54 (Fig. 8F), and should be addressed.\nOther Points\n- In Fig. 6, CypA inhibitors appear to suppress ASFV replication more strongly than CypA knockdown. What accounts for this difference? Would similar results be observed in CypA knockout cells?\n- The work does not analyse if p72-Cyp A interaction persists upon ASFV assembly. This would be relevant, as retention of p72-Cyp A complexes in virions could influence early replication events after infection.\n- In Fig. 8, the low viral production in the control factories (DMSO) is striking. Is this representative of PAM infections with ASFV strain HLJ/18? The reduction in factory size in the presence of CypA inhibitors also does not appear as significant as in the graphs.\n- Viral genome quantification alone does not distinguish between infectious and non-infectious particles. A viral titration assay should be performed to assess the actual impact on infectivity.\n- Fig. 2A shows an unexplained band between GST and GST– CypA. Is this a degradation product or a contaminant? Clarification is needed.\n- The Discussion lacks depth and does not contextualize the findings against prior literature, particularly regarding the phenotypic consequences of p72 and pB602 abrogation. It also misses the opportunity to compare and discuss the broader roles of CypA in the life cycles of other viruses (e.g., HIV, SARS-CoV-2, IBV, etc).\n- References: Literature citations are sometimes not accurate enough, and self-citations are sometimes overused. For instance:\n· Line 56–57 cites one relevant reference (own) on p72’s role in capsid formation and viral assembly (Wang et al. 2019. Science), omitting two other relevant cryo-EM studies on ASFV structure (Liu et al. 2019. Cell Host & Microbe, and Andres et al. 2019. J Biol Chem), and a significant structural analysis of p72 capsomers (Liu et al., Cell Res, 2019). It would be fair to acknowledge the work of other researchers as well.\n·Line 54–56 attributes p72 abundance data to Ref. 8, when it was actually reported in Andrés et al. (J Virol, 1997, 71:2331). Please correct\n· The use of self-reference 5 on ´promising p15 immunogen` to support general statements about the lack of antiviral therapies (lines 47–49) and self-reference 17 to support late p72 expression (already well-established) appears unnecessary."}, "author_response": "Response to Referees Letter\nDear Editor & Reviewers,\nWe sincerely thank you for the opportunity to revise and improve our manuscript (Manuscript ID:\nNCOMMS-25-33627). We are pleased to resubmit the revised version (New manuscript ID: NCOMMS-25-\n33627A), entitled “Cyclophilin A Stabilizes the Capsid Protein p72 to Facilitate African Swine Fever\nVirus Replication”, for your kind consideration for publication in Nature Communications.\nWe greatly appreciate the reviewers for their insightful and constructive comments, which have been\ninstrumental in improving the quality of our study. In response, we have performed the suggested\nexperiments, thoroughly addressed all concerns, and substantially revised the manuscript. We believe that\nthese efforts have significantly strengthened the work.\nBelow, we provide a detailed, point-by-point response to each reviewer comment. Reviewer comments are\nshown in italic magenta, followed by our responses in regular font. We hope that our revisions and\nclarifications adequately meet your expectations. Thank you very much again for your time, thoughtful\nevaluation, and continued support!\n---------------------------------------------------------------------------------------------------------------------------------\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nIn this manuscript the authors investigate the interaction of the African swine fever virus p72 major capsid\nprotein with cyclophilin A (CypA). Although CypA has previously been shown to have important and\ndiverse roles during replication of several other viruses. This is the first study to describe a role for CypA\nduring replication of ASFV. CypA was one of the main proteins the authors detected by mass spectrometry\nin co-precipitates by an antibody against p72 from ASFV infected porcine alveolar macrophages.\nSubsequently this interaction was confirmed by direct binding between the tagged recombinant proteins\nand by coprecipitation following transient expression in transfected cells. The interaction of CypA with p72\nwas mapped to exposed regions on p72 and a sequence of CypA spanning a cavity region. Interaction of\nCypA was shown to reduce K63 linked ubiquitination of p72 and proteasomal degradation. Inhibitors of\nCypA reduced the size of virus factories and resulted in aberrant assembly of virions thus reducing virus\nreplication kinetics. The authors indicate this interaction as a promising target for antiviral drug\ndevelopment against ASFV. The manuscript provides interesting novel data on an ASFV host interaction to\nstabilise the p72 capsid protein hence enhancing virus replication. The experiments are generally\ncomprehensively carried out and clearly described and results support the conclusions drawn.\nResponse #1: We sincerely thank the reviewer for the encouraging comments and the positive evaluation of\nour work. We truly appreciate your recognition of our experimental design, the clarity of our presentation,\nand the overall quality of the data. Your supportive feedback is highly motivating and inspires us to further\nrefine and improve our study.\nSpecific Points:\n1. As described by the authors, the p72 protein assembly into trimers which are incorporated into the virus\n1 / 24\ncapsid requires the virus encoded B602L chaperone although this protein isn’t incorporated into virus\nparticles. Without co-expression of the B602L p72 is not expressed in the native trimeric form. To obtain\nrecombinant p72 protein the authors express p72 with or without B602L in yeast. Although this co-\nexpression with B602L has previously been shown to produce the native trimeric form of p72, the data to\nconfirm this in the current study is not presented. The authors should present this data to confirm that the\nrecombinant p72 used in experiments is assembled into trimers and that the monomeric form of p72 is\nabsent. This data is important for the interpretation of experiments shown in Fig 1, and S1.\nResponse #2: We greatly appreciate the reviewer’s insightful comment. In accordance with your\nsuggestion, we examined the oligomeric state of the purified recombinant p72 used in our experiments by\nnative PAGE, followed by Coomassie Blue staining and anti-p72 Western blotting. The recombinant p72\nexhibited a mobility corresponding to ~200 kDa, consistent with the trimeric form, and was specifically\nrecognized by the anti-p72 antibody. Importantly, no signal was detected at ~70 kDa, confirming the\nabsence of monomeric p72 in the preparations and thereby ensuring the validity of our experimental\ninterpretations. These results are now presented in Fig. S2 of the revised manuscript.\nFig. S2. Confirmation of trimeric status of the recombinant p72 protein. Increasing amounts of recombinant\np72 protein was analyzed by native PAGE, followed by anti-p72 WB or Coomassie Blue staining.\n2. One important question, which is not fully resolved in this study, is the stage of replication when p72 is\nassociated with CypA. The authors data suggest this interaction is important during virus assembly most\nlikely after the assembly of the trimeric form of p72. In Figure 2F immunofluorescence images from ASFV\ninfected porcine alveolar macrophages fixed at 24 hpi infection at high multiplicity of infection are shown.\nStaining with anti-p72 and anti-CypA shows co-localization across the cytoplasm although not in factory\nstructures which at this stage seem largely devoid of either DNA or p72 protein. At this late stage of\ninfection, it would be expected that progeny infectious virions have been produced some and would be\nmoving to the cell surface or already been released. This data seems to suggest that CypA colocalizes with\nvirions as they move to the cell surface and even in projections from the cell, suggesting a possible role for\n2 / 24\nthe interaction of CypA with virions after virus assembly. To better understand the role of the CypA\ninteraction with p72 the authors should repeat this experiment at different time points after infection. A\nparallel time course of infectious virus titer would establish when progeny virions are produced. This\nanalysis may also help to establish if the CypA remains associated with p72 in the virus factories, no CypA\nis detected in the factories in the image shown in Fig 2F at this time point (24 hpi).\nResponse #3: We are sincerely grateful to the reviewer for this important and professional comment. We\nfully agree that clarifying the stage of infection at which p72 associates with CypA is critical for\nunderstanding the biological significance of this interaction. As suggested, we have performed ASFV\ninfection assays in PAMs (MOI = 5) for multiple time points (0, 6, 12, 18, and 24 hpi), followed by\nimmunofluorescence confocal macroscopy and TCID assays.\n50\n(1) Re-analysis of p72 co-localization with endogenous CypA. As shown in Fig. 2F, at 6 hpi, only a few\ncells expressed low levels of p72, which localized to restricted cytoplasmic regions and showed no overlap\nwith CypA. From 12 hpi, p72 accumulated in the cytoplasm and displayed pronounced co-localization with\nendogenous CypA, particularly within viral factories. This co-localization persisted to 24 hpi, suggesting\nthat CypA likely interacts with viral p72 during or after virions assembly, but not at the onset of p72\nexpression after infection.\n(2) Parallel measurement of infectious virus production. In the supernatant, no infectious progeny virion\nproduction was found at 6 hpi, whereas robust increases in viral titers were observed from 12 hpi onward\n(Fig. 2G). The temporal consistency between CypA-p72 co-localization and progeny virus production\nsupports the notion that CypA interaction may contribute to efficient virion assembly.\n(3) Explanation for unusual p72 staining in a few cells. Notably, at later stages (18-24 hpi), in a subset of\ninfected cells, p72 staining appeared weaker within viral factories compared with surrounding cytoplasm,\nsimilar to the observation in the previous Fig. 2F of the initial version. Although viral factories seemed\ndevoid of p72 in these cases, closer examination revealed that p72 signals were still present, albeit weaker\nthan in the surrounding cytoplasm. This phenomenon may result from excessive accumulation of p72\noutside the factories at late stages, thereby reducing the relative staining intensity within factories. As this\nphenomenon occurred only in a small fraction of cells and lacks generality, we have decided not to include\nthese images in the revised manuscript.\n3 / 24\nFig. 2. (F) Co-localization of viral p72 and endogenous CypA in ASFV-infected PAMs. (G) Quantitative\nanalysis of infectious virus production over time.\n3. In Figure 6 the authors use Western blotting to measure accumulation of protein p30, a protein expressed\nfrom early times, during ASFV infection rather than p72 which is expressed from later times and is\nincorporated into virions. The authors should explain the reason why Western blotting with anti-p72 isn’t\nused in these experiments as Western blotting against both p30 and p72 is used in Fig 7 C. If both were\nused consistency between experiments would be better.\nResponse #4: We sincerely thank the reviewer for this valuable comment. In the original experiments for\nFig. 6, Western blotting of p72 was attempted in parallel with p30, but at that time the anti-p72 antibody did\nnot yield clear signals, so only p30 was shown. We considered that the reduction of p30 expression was\nsufficient to demonstrate the inhibitory effects of CypA inhibitors on ASFV infection.\nWe fully agree, however, that simultaneous analysis of both early (p30) and late (p72) viral proteins would\nimprove data consistency and strengthen our conclusions. Accordingly, we have repeated the experiments\nand successfully obtained high-quality Western blot data for p72. These results have now been incorporated\ninto the revised manuscript (Please see Fig. 6D, 6I-J).\n4. In Fig 7 ASFV replication kinetics are compared in CypA inhibitor treated and non-treated cells by\nseveral assays including measurement of numbers of fluorescent cells, expression of proteins by Western\nblotting, global transcription of ASFV genes, genome copy numbers. The impact of the treatment on the\ninfectious virus titers are not shown. These data should be included to support the claim by the authors that\ntreatment with drugs targeting CypA may be an effective antiviral treatment.\nResponse #5: We appreciate the reviewer for this insightful suggestion. In the revised manuscript, we have\nexamined the effect of CypA inhibitor treatment on the production of infectious virus using TCID assays.\nsupernatant. These new data further support our conclusion that drugs targeting CypA could serve as an\neffective antiviral strategy against ASFV (Please see Fig. 7D).\n4 / 24\nDMSO CsA NIM811\n12 h 1.500 1.500 1.400 1.500 1.250 1.400 1.429 1.357 1.417\n24 h 2.155 2.250 2.400 1.500 1.500 2.000 2.500 2.500 2.400\n36 h 3.500 3.500 3.600 3.000 3.400 3.600 3.000 3.250 3.400\n48 h 4.000 3.750 3.875 2.875 3.250 2.600 3.600 3.500 2.750\n60 h 4.400 4.250 4.500 4.000 3.400 3.500 3.600 3.400 3.000\n72 h 5.250 5.250 4.600 3.750 4.000 4.000 3.600 4.000 3.600\n84 h 5.364 5.000 5.250 4.250 4.000 4.000 3.600 3.600 3.875\n96 h 6.000 6.500 5.500 4.600 4.750 4.600 4.000 4.400 4.250\nFig. 7. (D) Treatment with CypA inhibitors decreases infectious virion production in a multistep\ngrowth assay. (Note: Outlier values observed at 36 h in the CsA group were excluded from the analysis.)\n5. Figure 9 shows a cartoon to summarise the results from the study. This is helpful although some points\nhaven’t been established or are confusing. The diagram shows the interaction between p72 and CypA in the\ncytoplasm but not in the virus factory whereas as discussed in point 2 above this hasn’t been established in\nthe current study. Indeed the data shown in Fig 2F suggest CypA may be associated with migrating virions.\nIn addition, transcription of the mRNA for B646L appears to be shown outside the factory whereas the late\ntranscription is assumed to be inside the factory where the replicating genomes are accumulated.\nResponse #6: We sincerely thank the reviewer for the constructive and professional concern regarding the\nproposed model cartoon of our study.\n(1) As discussed in Response #3, we examined the dynamics of p72-CypA co-localization during ASFV\ninfection in PAMs and found that p72 strongly co-localized with endogenous CypA in the cytoplasm,\nparticularly within viral factories at later stages, but not at the onset of p72 expression (Please see also Fig.\n2F). To better illustrate this observation, the schematic has been revised to depict the CypA-p72 interaction\nprimarily occurring within viral factories.\n(2) We also agree that late gene B646L transcription primarily occurs inside viral factories. Accordingly, we\nhave relocated this process into the factory in the revised model. We believe that these modifications make\nthe schematic more accurate and easier to interpret (Please see Fig. 9).\n5 / 24\nFig. 9. Proposed model of CypA-mediated regulation of p72 stability and ASFV replication.\nMinor Points:\n1. In the text and Figure 1 legend the numbers of replicates of the co-precipitation and mass spectrometry\nanalysis is not given.\nResponse #7: We sincerely thank the reviewer for this helpful reminder. The co-precipitation and mass\nspectrometry analyses presented in the manuscript are representative of two independent experiments. As\nthe mass spectrometry data shown included only a single replicate per group, to ensure the rigor and\nreliability of data presentation, we have moved these data (previously Fig. 1D) to the Supplementary\nFigures (now Fig. S1A) and indicated them as preliminary results.\n2. Legend Figure 4A states that p72 was expressed in the presence or absence of B602L. On the figure the\nlabel shows B602L was included in all of these experiments.\nResponse #8: We sincerely thank the reviewer for this helpful reminder and apologize for the unclear\nlabeling in the figure. We have revised Fig. 4A to clearly indicate the groups, labeling “Ctrl” for the\nabsence of pB602L and “pB602L” for its presence.\n6 / 24\nFig. 4A. p72 degradation is primarily mediated by the ubiquitin-proteosome pathway.\nReviewer #2 (Remarks to the Author):\nZhao and colleagues present data to characterize the interaction between ASFV p72 with cyclophilin A\nwhich was discovered by AP-MS. Truncation experiments and use of inhibitors allowed to narrow down the\ninteracting site. Function of CYPA with respect to ASFV replication are tested with infection experiments\nusing PAM and genetically engineered knock-out BK2258 porcine cells. The main findings are that CYPA\ninteracts with p72, stabilizes it against proteasomal degradation and enhances ASFV infection. These\nfindings are important in the ASFV field however interactions of CYPA with matrix proteins of other viruses\nhave been described before.\nResponse #9: We sincerely thank the reviewer for the constructive summary of our study. We greatly\nappreciate the recognition of our main findings, including the interaction between CypA and ASFV p72,\nits role in stabilizing p72 against proteasomal degradation, and its enhancement of ASFV infection. While\nCypA interactions with other viral proteins have been reported previously, our study is the first to\ndemonstrate the functional relevance of CypA during ASFV replication, providing novel insights into host-\nvirus interactions in this field.\nThe subject of the study is of great importance as ASFV threatens the pig industries almost worldwide. The\nmanuscript is well written. However, some of the presented data needs more explanation and experimental\nconfirmation, for details please see the section with the ‘Minor points’. Also, some figures seem\novercrowded and could be rearranged and greater care should be taken to give appropriate references for\nall tools that were used, some are mentioned in this section.\n7 / 24\nResponse #10: We sincerely thank the reviewer for the positive evaluation of our study and for the\nconstructive suggestions. In the revised manuscript, we have provided additional explanations and\nexperimental confirmation for the presented data. We have also rearranged certain figures, particularly Fig.\n7, to reduce overcrowding, and ensured that all tools used in the study are appropriately cited or their\nsources clearly stated.\nThe presented data is interesting but also raises questions. In a number of experiments the authors describe\nthe negative effect of CYPA loss or inhibition on the expression of ASFV proteins, which can be explained\nby the decreased number of cells infected in the inoculated cell batch (see Figure 6). Protein expression\ndata on single cell basis are not presented, but Figure 8 shows an obvious reduction of ASFV particles\nwithin an infected cell in the absence of CYPA suggesting lower expression of at least structural proteins. It\nremains unclear why successful internalisation of ASFV leads to low level infection in only very few cells in\nthe absence of CYPA while viral entry is not affected (Fig 8). Unfortunately, this crucial experiment\n(adsorption / internalisation) is not described precisely, no reference is given, and no controls are used (if\navailable at all).\nResponse #11: We highly appreciate the reviewer’s interest and the important concerns.\n(1) Inhibitory effect of CypA inhibition on ASFV protein expression. As noted, we demonstrated the\ninhibitory effect of CypA loss or inhibition on ASFV protein expression using different approaches. In Fig.\n6, multi-step infection assays (48 ~ 72 h post- infection) showed that the reduced viral protein expression\ncould be attributed to the decreased number of ASFV-infected cells. In Fig. 8, one-step infection assays\nallowed us to examine the specific ASFV stage affected by CypA inhibition, confirming that accumulation\nof the major structural protein p72, but not the early viral protein p30, was impaired by the inhibitors. The\nreduced p72 levels at the late stages disrupt ASFV virion assembly and production, leading to a marked\ndecrease in infected cells after prolonged infection. Thus, these results indicate an overall consistency\nwithin the data of this study.\n(2) Virus adsorption and internalization assays. In the revision, these crucial experiments were\ncarefully re-performed and precisely described in the Methods section (please see lines 756-767 in the\nrevised manuscript), according to a previous study (PMID: 32788374, Ref. 92). Quantitative PCR was\nused to measure specific genome copy numbers from bound or internalized ASFV virions; therefore,\nuninfected controls are not required. Our new data demonstrated that CypA depletion or inhibition\ndoes not affect ASFV attachment and internalization (please see Fig. 8A, B).\n(3) Mechanism analysis. Although ASFV internalization occurs successfully, CypA inhibition-induced\ndegradation impairs p72 accumulation. Given that certain viral proteins, including p72, help retain\nstructural proteins within the assembly area and thereby contribute to ASFV virus factory organization\n(PMID: 17035321, Ref. 14; PMID: 15047843, Ref. 51), the decrease in p72 likely disrupts the\nlocalization and function of other structural proteins, impairing viral factory formation. This\nmechanism may explain why only low levels of infection in a few cells were observed upon CypA\ninhibition. Notably, while CypA inhibitors could potentially affect additional viral proteins, the levels of\np30 were not impacted (please see Fig. 8D, E), highlighting a specific role of CypA in stabilizing p72.\n8 / 24\nThese findings provide a mechanistic explanation for the observed decrease in factory size and reduction in\nvirion assembly. Other potential mechanisms are further discussed in the Discussion section (Please see\nlines 470-482) and warrant further investigation.\nThe manuscript would also improve if the authors added some information from the literature about the\nimpact of CYPA on a number of cellular processes that may, in addition to the described interaction with\np72, influence the outcome of an ASFV infection (MAPK signaling, RIG-I IFN, regulation of cytokines) in\nthe Introduction section.\nResponse #12: We really thank the reviewer for this insightful suggestion. We fully agree that, as an\nabundantly expressed protein, CypA participates in multiple cellular processes that could potentially affect\nASFV infection beyond its interaction with p72. In line with this recommendation, we have added\ninformation in the Introduction section to highlight CypA’s involvement in MAPK signaling, the RIG-I\npathway, and cytokine regulation (Refs. 17-19) (please see lines 68-71). Furthermore, we have expanded\nthe Discussion to address how CypA-mediated regulation of these processes may influence the outcome of\nASFV infection in addition to its role in stabilizing p72 (please see lines 470-482).\nMinor points:\nLine 101: the peptide mentioned here and in Fig 1 is also present in porcine PPIE, please explain why\nPPIA was chosen for further experiments.\nResponse #13: We thank the reviewer for this critical comment. The identified peptide “FDDENFILK” is\nindeed present in both CypA/PPIA and PPIE (Fig. 1D). Moreover, CypA and PPIE both showed strong\nassociation with p72 compared with other cyclophilins in a GST pulldown assay (Fig. S1B). However,\nimmunofluorescence confocal microscopy revealed distinct subcellular localizations: p72 is mainly\ncytoplasmic, whereas PPIE is strictly nuclear, suggesting no spatial overlap between PPIE and p72 (Fig.\n1E). In contrast, CypA is distributed in both the cytoplasm and nucleus, providing the spatial basis for\ninteraction with cytoplasmic p72. For this reason, CypA, but not PPIE, was selected for further experiments\n(please see lines 107-117).\nFig. 1. (D) Alignment of the identified peptide sequences within pig cyclophilins. Residues differing from\n9 / 24\nthe CypA are highlighted in yellow. (E) Cellular localization of overexpressed p72 and CypA or PPIE.\nLine 137: Fig 2 shows localisation of both proteins in the cytoplasm which explains the overlap but does\nnot prove the interaction, pls correct. The magnification of Fig 2D, F is too low to draw this conclusion.\nResponse #14: We thank the reviewer for raising this important point. We agree that the cytoplasmic\nlocalization of p72 and CypA indicates spatial overlap but does not itself demonstrate interaction. We have\ncorrected the relevant description of Fig. 2 accordingly (please see lines 152-153). Moreover, we have not\nonly replaced the previous Fig. 2D, F with new confocal images, but also provided higher magnification to\nmore clearly display the overlap (Fig. 2D, F).\nLine 150: pls add a reference for PyMOL\nResponse #15: We thank the reviewer for this suggestion. We have added the appropriate citation for\nPyMOL in the revised manuscript (please see line 166).\nLine 442, Methods:\nAs MS data is shown in Figure 1, pls add a detailed description of the of MS analysis. The authors should\nconsider to make the MS data available to the public in a repository. The same accounts for RNA\nsequencing.\nResponse #16: We thank the reviewer for this valuable suggestion. We have now added a detailed\ndescription of the mass spectrometry (MS) analysis in the Methods section (please see lines 605-617),\nincluding sample preparation, data acquisition, and analysis parameters. Furthermore, we have included the\nMS intensity data in Excel format in the Source Data of this study. In addition, the RNA sequencing data\nhave also been deposited in the Gene Expression Omnibus (GEO) with the accession number GSE300823.\nThe relevant accession numbers have been added to the Data Availability statement. To review GEO\naccession GSE300823, please visit https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE300823, and\nenter token mjsvmymojvohlsd into the box to access the RNA-seq data.\nLine 634: the adsorption assay is not described sufficiently; no reference is given. Pls specify ‘cold acidic\nPBS’ and if that was used or the trypsin treatment, specify all conditions.\nResponse #17: We thank the reviewer for raising this concern. As detailed in Response #11, we have now\nprovided a full description of the adsorption and internalization assays and included an appropriate\nreference (Ref. 92). In particular, we clarified the washing conditions for the internalization assay: “The\ncells were then incubated with trypsin-EDTA for 2 min at 37 °C to remove noninternalized particles”.\n(please see lines 756-767)\nLine 663: pls add citations for GO and KEGG repositories\nResponse #18: We thank the reviewer for this suggestion. We have now added appropriate citations for the\nGene Ontology (GO) and KEGG databases in the revised manuscript (Refs. 98-99; please see lines 786-\n787).\n10 / 24\nFigure 1: pls add sequences from other species that are relevant for ASFV infection (e.g. from genera\nPotamochoerus, Ornithodoros), add reference for MUSCLE algorithm.\nResponse #19: We thank the reviewer for this valuable suggestion. We agree that including CypA\nsequences from other species relevant to ASFV infection improves the alignment. Accordingly, CypA\northologs from Phacochoerus africanus (warthog, XP_047619276.1) and Ornithodoros turicata\n(XP_064487992.1) were retrieved from NCBI and included in the amino acid alignment, showing high\nsimilarity to porcine CypA (see Fig. 1F). In addition, we have provided detailed information on the\nsequence alignment in the Methods section, including NCBI accession numbers, the software used, and the\nMUSCLE algorithm with an appropriate reference. (please see lines 581-591)\nFigure 1C: looks like Maxquant software has been used. If this is so, please add a citation.\nResponse #20: We thank the reviewer for this suggestion. Indeed, MaxQuant software was used to analyze\nthe mass spectrometry data in Fig. 1C. As described in Response #16, we have now provided a detailed\ndescription of the MS analysis in the Methods section, including the appropriate citation for MaxQuant\n(please see line 616).\nFigure 1D: what is the coloring based on?\nResponse #21: We thank the reviewer for this question. In the original Fig. 1D, the coloring represented the\nintensities of the identified peptides in the mass spectrometry analysis comparing anti-p72 and control IgG\ngroups. In the revised manuscript, the MS intensity data are provided in Excel format in the Source Data.\nNotably, as described in Response #7, the original Fig. 1D has been moved to Supplementary Figures (Fig.\nS1A).\nFigure 4F: the band intensities for WT and KO cells are very different, I find it hard to judge if the\ndegradation is really ‘accelerated’ based on the very different starting levels.\nResponse #22: We sincerely thank the reviewer for raising this concern. We agree that the different band\nintensities at time zero for WT and KO cells limited the interpretation of accelerated degradation. To\naddress this, we re-performed the CHX chase assays in CypA-KO cells with or without CypA rescue. As\nshown in Fig. 4F, the initial bands at time zero for WT and KO cells now exhibit comparable intensities,\nand the presence of CypA clearly suppresses the rate of p72 degradation.\nFig. 4. (F) CypA inhibits p72 degradation in cells. CypA-KO HEK293T cells expressing Strep-p72 and\n11 / 24\npB602L were transfected with or without CypA for 36 h, then treated with cycloheximide (CHX, 100 μM)\nfor indicated times prior to WB analysis.\nFig 4G: these diffuse bands are hard to interpret, pls add a quantitation of the experiment.\nResponse #23: We thank the reviewer for this helpful suggestion. In the revised manuscript, we have\nincluded quantitative analysis of the results shown in Fig. 4G to enhance clarity and interpretation. The\nquantification of band intensities is now provided in Fig. 4H. In addition, for consistency, we have also\nprovided quantification (shown as Fig. 5I) of the relevant data of Fig. 5H.\nFig 4H: the conclusion is difficult to follow without quantitation of the bands including a correction for the\ninput of Ub. Pls avoid ‘above’ for references within the manuscript but specify in detail how the blots were\ndone. The lower HA panel has 9 tracks, all others have 10, pls correct.\nResponse #24: We thank the reviewer for this valuable comment.\n(1) In the revised manuscript, we have added quantitative analysis of the results in Fig. 4I to clearly support\nthe conclusion. The quantification of Ub intensities in Strep-p72 precipitates is now presented in Fig. 4J.\n(2) As suggested, we have revised the corresponding figure legend to avoid using the term “above” and\nnow provide a detailed description of how the blots were performed: “Cells were lysed and denatured in\nTSD buffer at 100 ℃, followed by a 10-fold dilution with binding buffer. The lysates were then subjected to\nIP with Strep-Tactin resin and analyzed by anti-Ub Western blotting.”\n(3) We apologize for the error in the original figure, where the lower HA panel showed only 9 tracks due to\nomission of the first blank lane. This has been corrected in the revised Fig. 4I to include all 10 tracks.\nFig 4I: the upper panel is hard to interpret, pls replace\nResponse #25: We thank the reviewer for pointing out this concern. In the revised manuscript, the original\nFig. 4I has been renumbered as Fig. 4K, and the unclear upper K63-Ub panel has been replaced with newly\ngenerated data.\nFig 5E: this blot needs quantitation and statistical evaluation to demonstrate the claimed acceleration of\ndegradation.\nResponse #26: We thank the reviewer for this valuable suggestion. In the revised manuscript, the original\nFig. 5E has been renumbered as Fig. 5F. We have now included relative quantitation of the p72 band\nintensities to support the conclusion. In addition, statistical evaluation of the degradation kinetics is\nprovided in the new Fig. 5G, as further detailed in the following response (Response #27).\nFig 5F: pls add a detailed description of the procedure to calculate the half-lives to the Method section.\nThe shown intensity-time relation seems linear, is this a logarithmic scale?\nResponse #27: We thank the reviewer for this important comment. In the revised manuscript, we have\nadded a detailed description of the half-life calculation procedure to the Methods section. Briefly, band\nintensities of p72 were quantified and normalized to the corresponding loading control (if needed) and to\n12 / 24\nthe initial time point (t = 0). The normalized intensities were then plotted against time. Since the intensity-\ntime relationship showed an approximately linear decline, half-lives were estimated using simple linear\nregression (GraphPad Prism). Notably, the data were plotted on a linear, not logarithmic, scale. The detailed\nprocedure is provided in the Methods section (please see lines 668-678).\nFig. 5. (G) Quantification of p72 half-life. Relative p72 band intensities were measured by Adobe\nPhotoshop. The half-life (t ) was calculated by fitting the normalized intensities to a simple linear\n1/2\nregression, shown as dashed lines.\nFig 6: there is some redundancy here which makes the figure confusing. Consider re-arranging for instance\nby combining panel A with D, and panel B with C. Panels G, H demonstrate that there is a lower number of\ninfected cells in the absence of CYPA and explains A,B,C, and D. Pls explain ‘BF’ in panel H and Fig 7.\nResponse #28: Thank you for this helpful suggestion.\n(1) As suggested, we have re-arranged the data in Fig. 6. Given that the original Fig. 6A and 6B were\nobtained from sgRNA-expressing BK2258 cells and the original Fig. 6C and 6D were from siRNA-\ntransfected PAMs, it would be difficult to combine 6A with 6D and 6B with 6C. To avoid redundancy, we\ndecide to move the original Fig. 6A and 6B to the supplementary figures, as Fig. S5A and S5B.\n(2) The “BF” in panel H and Fig. 7A is the abbreviation of “bright-field”. To avoid confuse, we have\npresented “bright-field” rather than “BF” in the revised figures.\nFig 6B,K: only extracellular virions were assessed, pls correct\nResponse #29: We thank the reviewer for this reminder. In the revised manuscript, we have corrected the\nfigure legend and relevant text to specify that only extracellular virions were assessed (Please see lines 292\nand 312).\nFig 7B: which MOI was chosen?\nResponse #30: We thank the reviewer for the question. In the revised manuscript, the original Fig. 7B has\nbeen renumbered as Fig. 7C. The experiment was performed at an MOI of 0.1, and we have added “(MOI\n= 0.1)” to the corresponding figure legend of the new Fig. 7C.\n13 / 24\nFig 7C: considering results from panels A,B, panel C is dispensable.\nResponse #31: We thank the reviewer for this helpful comment. We agree that, given the results shown in\npanels A (fluorescent intensities) and B (genome copy numbers) of the original Fig. 7, panel C (viral\nprotein analysis by WB) is largely redundant. In the revised manuscript, the panel C has been removed\nfrom Fig. 7 to simplify the figure and improve clarity, and the original panel C has been relocated to the\nSupplementary Figures as Fig. S7A.\nFig 7D: hard to interpret. How can the 2 separate ‘tracks' of points in the ASFV vs mock panel be\nexplained? What does ‘left’ and ‘right’ in the legend refer to? The RNA-seq data is not publicly available,\nnor are details for data evaluation. The volcano representation can be replaced by a dot-plot (DMSO vs\nNIM811) for clarity. There is redundance with panel E. Pls consider rearranging the Figure or putting\nparts of it in a supplement.\nResponse #32: We thank the reviewer for raising these critical concerns regarding the original Fig. 7D, 7E.\n(1) In the original Fig. 7D, the two “tracks” of points in the ASFV vs. mock panels (volcano plot)\nrepresented the expression levels of viral genes in the absence (DMSO) and presence (NIM811) of the\ninhibitor. The terms “right” and “left” in the legend were incorrect and should be “top” and “bottom.” We\nagree that this volcano representation in the previous Fig. 7D was confusing. As suggested, we have\nreplaced it with a clearer dot plot (DMSO vs. NIM811), now shown as Fig. 7E in the revised manuscript.\n(2) We apologize for not making the RNA-seq data available in the initial submission. The dataset has now\nbeen deposited in the Gene Expression Omnibus (GEO) under accession number GSE300823, and this\ninformation has been added to the Data Availability statement. To review the dataset, please visit\nhttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE300823 and enter the token mjsvmymojvohlsd.\nPlease also refer to Response #16.\n(3) We agree that the heat map in the original panel E was redundant. In the revised manuscript, this panel\nhas been moved to the Supplementary Figures (now Fig. S7E), and Fig. 7 has been reorganized to\nminimize redundancy and improve clarity.\nFig. 7. (E) NIM811 alters the ASFV transcriptome. Dot plots show differential expression of ASFV\n14 / 24\ngenes between groups (three replicates per group).\nFig 8A: what do the bars represent, how is ‘relative’ adsorption and internalisation defined, pls explain.\nResponse #33: We thank the reviewer for this concern. The original Fig. 8A and 8B showed the relative\nlevels of viral adsorption and internalization. These relative values were calculated based on viral DNA\ncopy numbers determined by qPCR and then normalized to the control group (Ctrl-sg or DMSO).\nIn the revision, we have re-performed the viral adsorption and internalization assays and replaced the\noriginal Fig. 8A and 8B with new data. In the revised Fig. 8A, 8B, the absolute viral genome copy numbers\nfrom bound or internalized ASFV virions are now presented to improve clarity. Further details of the\nadsorption and internalization assays can be found in the Methods section (see lines 756-767). Please also\nrefer to Response #11.\nFig. 8. (A) CypA depletion in BK2258 cells does not affect ASFV attachment and\ninternalization. (B) CypA inhibitor treatment does not alter ASFV attachment and internalization in PAMs.\nReviewer #3 (Remarks to the Author):\nIn this study, Kong et al identify cyclophilin A (CypA) as a host interacting partner of the African swine\nfever virus (ASFV) major capsid protein p72. Their findings suggest that p72-CypA interaction prevents\nproteosome-mediated degradation of p72, thereby facilitating viral replication. Conversely, suppression of\nCypA expression or treatment with CypA inhibitors significantly impairs viral replication, highlighting a\nnovel potential therapeutic target against ASFV.\nResponse #34: We sincerely thank the reviewer for the thorough and professional evaluation of our study.\nWhile the work presents novel and intriguing data, the effect of CypA suppression and CypA inhibitors on\nviral assembly contradicts previous evidence on the consequences of p72 knockdown and needs\nclarification. Furthermore, key aspects of CypA's mechanism of action on p72 stability remain unclear and\nwould require further investigation.\nResponse #35: We sincerely thank the reviewer for recognizing the novelty and significance of our study.\nWe also appreciate the insightful comments pointing out these critical issues. In the revised manuscript, we\n15 / 24\nhave included additional data and a more detailed analysis to clarify the effects of CypA suppression versus\np72 knockdown, as well as to further elaborate on the mechanisms by which CypA stabilizes p72.\nThese revisions and clarifications are addressed in detail in the following responses (particularly Responses #36-\n#39).\nMajor points:\n- Inconsistencies in ASFV assembly defects. The reported effects of CypA inhibitors on viral assembly do\nnot align with established phenotypes resulting from p72 abrogation. Previous studies (e.g., Ref. 9) have\nshown that suppression of p72 expression leads to the massive formation of aberrant viral structures known\nas “zippers” in viral factories, resulting from the co-assembly of unprocessed polyproteins pp220 and pp62\nwith inner envelope precursors. Similar structures are observed when the viral chaperone pB602L is\nabrogated, which blocks p72 trimerization (Refs. 11 and 13). Surprisingly, these hallmark features are\nabsent in the presence of CypA inhibitors, and this discrepancy is not addressed in the manuscript. Instead,\nCypA inhibitors reduce viral factory size—an effect more suggestive of compromised formation of ER-\nderived viral membrane rather than decreased p72 levels. These discrepancies raise questions about\nwhether CypA inhibitors specifically act only through modulation of p72 degradation, or whether they\nimpact additional viral components or replication stages.\nResponse #36: We are sincerely grateful to the reviewer for these professional and constructive comments.\nWe apologize for not providing sufficient analysis of the potential occurrence of zipper-like aberrant\nstructures in viral factories.\n(1) Aberrant structures were observed. Upon careful re-examination of our TEM data, we did observe\naberrant zipper-like structures in viral factories of CypA inhibitor-treated cells, although these features were\nnot highlighted in the initial version. In the revised manuscript, we have replaced the original Fig. 8H with\nrepresentative TEM images showing such zipper-like intermediates (new Fig. 8I), with aberrant structures\nindicated by blue arrows. The relevant text in the manuscript has been updated to describe these aberrant\nstructures (lines 395-399).\n(2) Functional p72 trimers were partially impaired. Notably, the frequency and extent of these aberrant\nstructures under CypA inhibition were lower, compared with the classical phenotypes reported upon\ncomplete abrogation of p72 or pB602L (PMID: 17035321, Ref. 14; PMID: 9580160, Ref. 53). This\nsuggests that CypA inhibition partially impairs p72 stability and capsid assembly, but does not completely\neliminate functional p72 trimers, resulting in a mixed phenotype — limited zipper formation together with\nmature icosahedral virions (please see lines 444-449).\n(3) Decrease of p72 affects viral factory organization. Certain viral proteins, including p72, help retain\nstructural proteins within the assembly area, thereby contributing to ASFV virus factory organization\n(PMID: 17035321, Ref. 14; PMID: 15047843, Ref. 51). For instance, suppression of p72 can lead to the\nrelease and translocation of protein pE120R from viral factories (PMID: 17035321, Ref. 14). Therefore,\nreduced p72 levels likely disrupt the localization and function of other viral structural proteins, impair\nviral factory formation, and results in smaller factories.\n16 / 24\nTaken together, while we cannot exclude the possibility that CypA inhibitors affect additional viral\ncomponents, the degradation of p72 induced by CypA inhibition appear to be a major mechanism\nunderlying both increased occurrence of aberrant structures and the reduction in viral factory size.\nFig. 8. (I) Transmission electron microscopy (TEM) analysis reveals that CypA inhibition disrupts\nvirion assembly within viral factories. Viral factories are outlined in black boxes; mature virions are\nindicated by magenta arrows, while aberrant structures are indicated by blue arrows.\nTo clarify this, it would be highly informative to examine the ultrastructure of viral factories following\n17 / 24\nCypA knockdown or knockout. If similar effects to those of the inhibitors are observed, off-target or\nbroader effects should be considered to account of the effects of CypA abrogation and inhibitors.\nResponse #37: We thank the reviewer for the helpful suggestion.\n(1) Ultrastructural analysis following CypA knockout. As suggested, we examined viral factories in\nCypA-knockout cells. Consistent with CypA inhibitor treatment, we observed reduced numbers of mature\nvirions and increased aberrant viral structures in CypA-sg-expressing cells, compared with Ctrl-sg cells\n(Fig. S9C). These results demonstrate functional consistency between CypA inhibitors and genetic\nablation, reinforcing the conclusions of our study.\n(2) Mechanistic interpretation. As discussed in Response #36, although we cannot entirely exclude the\npossibility that CypA inhibitors affect additional viral components, p72 degradation induced by CypA\ninhibition remains the major mechanism accounting for both the increased formation of aberrant structures\nand the reduction in viral factory size.\nFig. S9. (C) CypA deficiency induces aberrant structures in viral factories.\nViral factories are outlined (black boxes); mature virions (magenta arrows) and aberrant structures (blue\narrow) are indicated. Scale bar, 1 μm.\nDoes CypA solely regulate p72 stability? The data do not conclusively demonstrate that CypA’s only role is\nin stabilizing p72, since most infection experiments were conducted at low MOI (0.1–1 PFU) and analyzed\nat late post-infection times (72–96 hpi), allowing for multiple replication cycles. To unravel primary from\nsecondary effects, it would be beneficial to re-analyse the impact of CypA silencing and CypA inhibitors on\nviral DNA replication and early (e.g., p30) and late viral expression (p72 and other late proteins) in single-\ncycle assays.\nResponse #38: We thank the reviewer for this important and insightful comment. We fully agree that\n18 / 24\ninfection experiments at low MOI and late time points (72-96 hpi) could reflect cumulative effects from\nmultiple replication cycles, thereby obscuring primary versus secondary consequences of CypA inhibition.\nTo address this concern, we performed single-cycle infection assays at an MOI of 5 and analyzed viral\nDNA replication as well as early (p30) and late (p72) protein expression within one replication cycle (24\nhpi).\n(1) CypA inhibition reduces p72 but not p30 or viral DNA replication. Viral DNA replication was not\nsignificantly affected by CypA inhibitors (Fig. S9A). In contrast, Western blotting revealed a marked\nreduction of p72 protein upon treatment with CsA or NIM811 (Fig. 8D, E), while levels of the early protein\np30 remained unchanged. These results indicate that CypA primarily regulates p72 stability, rather than\nbroadly influencing viral genome replication or early viral gene expression.\n(2) CypA may also exert additional effects. While our single-cycle assays underscore a specific role of\nCypA in stabilizing p72, we recognize that CypA’s diverse cellular functions may allow it to influence\nASFV replication through additional mechanisms. These possibilities are further discussed in the revised\nmanuscript (lines 470-482).\nFig. 8. (D, E) CypA inhibitors markedly decrease p72 protein levels during ASFV single-cycle replication.\n- Mechanism of CypA action on p72 stability. Beyond p72 stabilization, it remains unclear whether CypA\nacts as a chaperone for proper p72 folding and trimerization. Can the authors determine if p72 trimers are\nformed in CypA knockout cells, with or without co-expression of pB602L?\nResponse #39: We thank the reviewer for this excellent comment regarding whether CypA also functions\nas a chaperone in p72 folding and trimerization. To address this, we performed experiments in WT and\nCypA-KO HEK293T cells. Cells were transfected with p72 alone, and WT cells co-transfected with p72\nand pB602L served as a positive control. As shown in Fig. S3B, while pB602L co-expression strongly\npromoted p72 trimer formation, the presence or absence of CypA alone did not induce detectable\ntrimerization of p72. These results indicate that CypA does not act as a chaperone to directly facilitate\np72 folding or trimerization.\n19 / 24\nFig. S3. (B) CypA does not promote trimer assembly of p72.\n- K63-linked ubiquitination. The claim in the Abstract that ´interaction with CypA stabilizes p72 by\nreducing K63-linked ubiquitination and preventing proteasomal degradation` appears overstated. This\nconclusion is based solely on overexpression assays in transfected cells lacking pB602, which is essential\nfor proper p72 folding and trimerization (indeed, ´expression of p72 alone leads to the formation of\nmisfolded p72 aggregates; refs. 11, 13, 16). Moreover, K63-linked ubiquitination is typically associated\nwith signaling rather than degradation. The authors should temper this conclusion and contextualize the\nlimitations of their system, especially as it differs markedly from the infection setting.\nResponse #40: We thank the reviewer for this important comment.\n(1) The overexpression assays examining the role of CypA in reducing p72 ubiquitination and degradation\nwere performed in cells co-expressing pB602L. As noted in the figure legend of Fig. 4C, “(Note: unless\notherwise specified, p72 plasmid was co-transfected with pB602L in subsequent experiments.)”. To\nimprove clarity, we have now added a corresponding statement in the main text (lines 226-228).\n(2) We agree that K63-linked ubiquitination is most commonly associated with signaling rather than\ndegradation. Nevertheless, recent studies have shown that K63-linked ubiquitination can also target\nsubstrates for degradation (Refs. 35-37), and a recent report specifically implicated K63-linked\nubiquitination in promoting p72 degradation (PMID: 37815352; Ref. 30). These findings support the\npossibility that CypA suppresses p72 degradation by reducing its K63-linked ubiquitination. Please see\nlines 249-252.\n(3) Nevertheless, according to the reviewer’s suggestion, to avoid overstating our conclusions and to reflect\nthe potential limitations of our system compared with real infection conditions, we have tempered this\nstatement in the Abstract by deleting the specification of “K63-linked.” The revised text now reads: “CypA\ninteraction stabilizes p72 by reducing its ubiquitination and preventing proteasomal degradation.”\n- p72 immunofluorescence patterns. The intracellular distribution of p72 in infected cells (Figs. 2F and 8F)\nappears diffuse and, surprisingly, largely excluded from assembly sites, which is inconsistent with that\n20 / 24\nexpected for the major capsid protein. This contrasts with established reports showing p72 localization to\nviral factories and cytoplasmic puncta corresponding to scattered virions. This discrepancy undermines\nclaims about colocalization with CypA (Fig. 2F) and the late virion protein p54 (Fig. 8F), and should be\naddressed.\nResponse #41: We appreciate the reviewer’s insightful comment regarding p72 immunofluorescence\npatterns. This prompted us to re-examine our confocal data and re-perform localization assays.\n(1) As described in Response #3, we have performed ASFV infection assays in PAMs (MOI = 5) for\nmultiple time points (0, 6, 12, 18, and 24 hpi), followed by immunofluorescence confocal macroscopy.\nFrom 12 hpi, p72 accumulated in the cytoplasm and displayed pronounced co-localization with endogenous\nCypA, particularly within viral factories (Fig. 2F).\n(2) Re-exanimating of confocal data related to original Fig. 8F confirmed that, in most infected cells, p72\npredominantly localized within viral factories, overlapping with CypA. Representative images have been\nupdated in the revised Fig. 8G.\n(3) Explanation for apparent diffuse staining. Notably, at later stages (18-24 hpi), a subset of infected\ncells showed relatively weaker p72 staining within viral factories compared with the surrounding\ncytoplasm, consistent with the previous Fig. 2F and Fig. 8F. Closer inspection revealed that p72 signals\nwere still present in factories but appeared reduced relative to cytoplasmic accumulation, likely due to\nextensive p72 synthesis and redistribution. As this phenotype was rare and not generalizable, these images\nwere not included in the revised manuscript.\nOverall, these revisions clarify that p72 largely localizes to viral factories and co-localizes with CypA under\nphysiologically relevant conditions, while the diffuse staining observed in a small subset of cells represents\na minor late-stage phenomenon.\nOther Points\n- In Fig. 6, CypA inhibitors appear to suppress ASFV replication more strongly than CypA knockdown.\nWhat accounts for this difference? Would similar results be observed in CypA knockout cells?\nResponse #42: We thank the reviewer for the careful observation and insightful question. Indeed, ASFV\nreplication was suppressed more strongly by CypA inhibitors than by CypA knockdown, and similar trends\nwere observed in CypA knockout cells (Fig. S5A, B). This difference likely reflects the broader activity of\nCypA inhibitors, which could target multiple cyclophilin family proteins in addition to CypA, whereas\nknockdown or knockout specifically affects CypA.\nWe have provided a relevant discussion in the revised manuscript (lines 484-496). Briefly, the\ncyclophilin family comprises multiple members with highly conserved hydrophobic cavities (Ref. 19),\nsuggesting that other cyclophilins may also interact with p72. Our data in Fig. S1B support this possibility,\nalthough CypA appears to be the predominant regulator. Importantly, although these cyclic undecapeptide\ninhibitors primarily target CypA, they also display partial activity against other cyclophilin family members\n(Ref. 22). Therefore, it is reasonable to speculate that the stronger suppression of ASFV replication by\nCypA inhibitors reflects combined inhibition of CypA and additional cyclophilins that contribute to p72\n21 / 24\nstabilization and viral replication.\n- The work does not analyse if p72-CypA interaction persists upon ASFV assembly. This would be relevant,\nas retention of p72-CypA complexes in virions could influence early replication events after infection.\nResponse #43: We are grateful to the reviewer for this important comment.\n(1) This concern is similar to another reviewer’s comment (see Response #3). Briefly, we performed ASFV\ninfection assays in PAMs (MOI = 5) at multiple time points (0, 6, 12, 18, and 24 hpi), followed by\nimmunofluorescence confocal microscopy. As shown in Fig. 2F, at 6 hpi, only a few cells expressed low\nlevels of p72, which localized to restricted cytoplasmic regions and did not overlap with CypA, indicating\nno co-localization at the onset of p72 expression. From 12 hpi onwards, p72 accumulated in the cytoplasm\nand showed pronounced co-localization with endogenous CypA, particularly within viral factories. This\nco-localization persisted to 24 hpi, suggesting that CypA interacts with viral p72 during or after\nvirion assembly.\n(2) We fully agree that the persistence of p72-CypA interaction upon ASFV assembly is biologically\nrelevant. While the current study did not directly test whether retention of p72-CypA complexes in virions\ninfluences early replication events, this represents an important question for future investigation.\n- In Fig. 8, the low viral production in the control factories (DMSO) is striking. Is this representative of\nPAM infections with ASFV strain HLJ/18? The reduction in factory size in the presence of CypA inhibitors\nalso does not appear as significant as in the graphs.\nResponse #44: We thank the reviewer for raising this important concern. Upon careful re-examination of\nour TEM datasets, we noted that viral factories in the DMSO control group generally displayed higher\nvirion production than was reflected in the image originally shown in Fig. 8H. To better represent the\noverall dataset, we have updated this panel with a new representative image showing more robust virion\nproduction in control factories.\nIn addition, to ensure consistency across conditions, we also replaced the images of inhibitor-treated cells\nfrom the original Fig. 8H with new representative images that more clearly display the significant reduction\nin factory size. The revised TEM data are now presented in Fig. 8I of the manuscript. Please also refer to\nthe Response #36 for additional details.\n- Viral genome quantification alone does not distinguish between infectious and non-infectious particles. A\nviral titration assay should be performed to assess the actual impact on infectivity.\nResponse #45: We thank the reviewer for this important suggestion. We fully agree that viral genome\nquantification alone cannot distinguish between infectious and non-infectious particles, and that viral\ntitration assays are essential to accurately assess the true impact on infectivity.\nDuring the revision, we collected culture supernatants from PAMs infected with ASFV over time, in the\npresence or absence of CypA inhibitors, and determined viral titers using TCID assays. As shown in Fig.\n50\n7D, the titration results were consistent with the genome quantification data, confirming that CypA\n22 / 24\ninhibition significantly reduces ASFV infectivity. Please also refer to the Response #5 for additional details.\n- Fig. 2A shows an unexplained band between GST and GST– CypA. Is this a degradation product or a\ncontaminant? Clarification is needed.\nResponse #46: We thank the reviewer for carefully noting the additional band in Fig. 2A, B. This band\nmost likely represents a degradation product of GST-CypA rather than a contaminant, as it was consistently\ndetected with anti-GST antibody in GST-CypA preparations but not in GST alone. To clarify, we have\nrevised the figure legend to note this observation.\n- The Discussion lacks depth and does not contextualize the findings against prior literature, particularly\nregarding the phenotypic consequences of p72 and pB602 abrogation. It also misses the opportunity to\ncompare and discuss the broader roles of CypA in the life cycles of other viruses (e.g., HIV, SARS-CoV-2,\nIBV, etc).\nResponse #47: We thank the reviewer for this constructive comment regarding our Discussion. As\nsuggested, we have substantially expanded the Discussion section in two key directions:\n(1) We now provide a comparison of the aberrant viral structures observed upon CypA inhibition with those\nreported following p72 or pB602L abrogation. Our analysis emphasizes that, while the underlying\nmechanisms appear similar, both leading to defective p72 function and aberrant assembly, the severity of\nthe phenotypes differs. Specifically, CypA inhibition results in fewer zipper-like intermediates and allows\nthe formation of some mature icosahedral virions, in contrast to the massive zipper accumulation seen upon\ncomplete loss of p72 or pB602L. These observations suggest that CypA inhibition partially destabilizes\np72, thereby impairing but not abolishing capsid assembly (see lines 441-451 of the revised Discussion).\n(2) We have also expanded the discussion to place our findings in the broader context of CypA biology. In\nparticular, we have compared the role of CypA in ASFV infection with its well-established functions in the\nlife cycles of other viruses, including RNA viruses (HIV, IBV, SARS-CoV-2, and HCV) and a DNA virus\n(poxvirus), highlighting both common principles and virus-specific mechanisms of CypA (see lines 453-\n468 of the revised Discussion).\n- References: Literature citations are sometimes not accurate enough, and self-citations are sometimes\noverused.\nResponse #48: We thank the reviewer for pointing out this. In the revision, all references have been\ncarefully re-checked and revised for accuracy. We have also reduced self-citations and added more relevant\nindependent studies to ensure a balanced citation of the literature.\nFor instance:\n• Line 56–57 cites one relevant reference (own) on p72’s role in capsid formation and viral assembly\n(Wang et al. 2019. Science), omitting two other relevant cryo-EM studies on ASFV structure (Liu et al.\n2019. Cell Host & Microbe, and Andres et al. 2019. J Biol Chem), and a significant structural analysis of\np72 capsomers (Liu et al., Cell Res, 2019). It would be fair to acknowledge the work of other researchers\n23 / 24\nas well.\nResponse #49: We sincerely apologize for omitting these relevant references. In the revised manuscript, we\nhave now cited Liu et al. (2019, Cell Host & Microbe), Andres et al. (2019, J Biol Chem), and Liu et al.\n(2019, Cell Research) alongside our previous reference to provide a more comprehensive and balanced\nacknowledgment of work on p72’s role in capsid formation and viral assembly (lines 56-57).\n• Line 54–56 attributes p72 abundance data to Ref. 8, when it was actually reported in Andrés et al. (J\nVirol, 1997, 71:2331). Please correct\nResponse #50: We thank the reviewer for pointing out this error. The reference has been corrected in the\nrevised manuscript, as Ref. 7 (Andrés et al. J Virol, 1997) in the revised manuscript.\n• The use of self-reference 5 on ´promising p15 immunogen` to support general statements about the lack\nof antiviral therapies (lines 47–49) and self-reference 17 to support late p72 expression (already well-\nestablished) appears unnecessary.\nResponse #51: We thank the reviewer for this comment. In the revised manuscript, we have removed the\nunnecessary self-citations (the original Refs. 5 and 17).\n--------------------------------------------------------------------------------------------------------------------------------\nFinally, we sincerely believe that the revised manuscript has been significantly improved through the\nincorporation of the reviewers’ insightful comments and suggestions. We truly hope that our efforts will be\nsatisfactory to all the reviewers.\nWe are deeply grateful for your time and dedication in reviewing our work!\nKindest regards,\nDongming Zhao, PhD\nXiangpeng Sheng, PhD\n24 / 24\nResponse to Referees Letter\nDear Editor & Reviewers,\nWe sincerely thank you for the opportunity to further improve our manuscript (Manuscript ID: NCOMMS-\n25-33627A). We are pleased to resubmit the second revised version (New manuscript ID: NCOMMS-25-\n33627B), entitled “Cyclophilin A Stabilizes the Capsid Protein p72 to Facilitate African Swine Fever\nVirus Replication”, for your kind consideration for publication in Nature Communications.\nWe would like to express our sincere gratitude to Reviewer #1 and Reviewer #2 for their thoughtful\nevaluation of our revised manuscript and for their assistance in assessing our responses to the comments\noriginally raised by Reviewer #3. We greatly appreciate the reviewers’ additional time and constructive\ninput, which have substantially contributed to improving the overall quality of our work.\nBelow, we provide a detailed, point-by-point response to each reviewer’s comment. Reviewer comments\nare presented in italic magenta, followed by our responses in regular font. We hope that our revisions\nand clarifications meet your expectations. Thank you again for your time, thoughtful evaluation, and\ncontinued support.\n---------------------------------------------------------------------------------------------------------------------------------\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors have addressed the points raised in my previous review satisfactorily.\nResponse #1: We greatly appreciate the reviewer’s time and efforts in evaluating our revised manuscript.\nThe reviewer’s supportive feedback is highly encouraging and motivates us to continue improving the\nquality of our work. Thank you very much for your positive assessment.\n....................................\nResponses to Reviewer 3\nResponse 36: The authors have modified the manuscript satisfactorily to take account of this point raised\nby the reviewer as summarised below.\n(1) The authors have re-examined TEMs and observe aberrant zipper-like structures in viral factories of\nCypA inhibitor-treated cells, although these features were not highlighted in the initial version. A revised\npanel is included illustrating the presence of zipper like structures in virus factories in Cyp- inhibitor\ntreated cells (Fig 8I replacing 8H and Fig S9C) and description included in the text (lines 395- 399).\n(2) The authors explain that they observed reduced appearance of aberrant zipper structures in factories\ncompared to observations from previously published experiments in which p72 or B602L expression was\nrepressed (ref 53). They suggest that CypA inhibition partially impairs p72 stability and capsid assembly,\nbut does not completely eliminate functional p72 trimers, resulting in a mixed phenotype — limited zipper\nformation together with mature icosahedral virions (revised text lines 444-449).\n(3) The authors also observed smaller factory sizes compared with previous publications in which p72 or\n1 / 6\nB602L expression was ablated. They suggest that reduced p72 levels may disrupt the localization and\nfunction of other viral structural proteins, impair viral factory formation, and results in smaller factories.\nThe authors state they cannot exclude the possibility that CypA inhibitors affect additional viral\ncomponents. However, the increased degradation of p72 induced by CypA inhibition is likely to represent\nan important mechanism underlying both occurrence of aberrant structures and the reduction in viral\nfactory size.\nResponse #2: We sincerely thank the reviewer for the additional evaluation of our responses to the\ncomments originally raised by Reviewer #3. We greatly appreciate your supportive assessment.\nResponse 37: This point has also been addressed satisfactorily by modification of the text (Lines 395-399\nand 444-449) and including Fig S9C.\n(1) As suggested by the reviewer the authors compared the effect of Cyp A knockout with the effect of drug\ntreatment on ASFV factory formation (Fig S9C). This confirmed a similar effect consistent with the\ninterpretation of the proposed role of Cyp A stabilisation of p72 trimers.\n(2) A response given in point 36 is reiterated to explain that the possibility that CypA inhibitors affect\nadditional viral components cannot be excluded, p72 degradation induced by CypA inhibition remains the\nmajor mechanism accounting for both the increased formation of aberrant structures and the reduction in\nviral factory size.\nResponse #3: We are thankful to the reviewer for their positive and encouraging evaluation.\nPoint 38: The authors have responded satisfactorily to the point raised.\nAs requested by the reviewer the authors performed single-cycle infection assays at an MOI of 5 and\nanalyzed viral DNA replication as well as early (p30) and late (p72) protein expression within one\nreplication cycle (24 hpi). The results are shown in Figures S9A, 8D, 8E and explained in the text. These\nresults indicate that CypA primarily regulates p72 stability, rather than broadly influencing viral genome\nreplication or early viral gene expression.\nTo address the valid concern that CypA’s diverse cellular functions may allow it to influence ASFV\nreplication through additional mechanisms further discussion is included (lines 470-482).\nResponse #4: We sincerely thank the reviewer for the supportive assessment.\nResponse 39: The authors have responded satisfactorily.\nThe authors included an additional experiment to demonstrate that Cyp A does not act as a chaperone to\nenhance p72 folding or trimerization (Fig S3B).\nResponse #5: We are grateful to the reviewer for the positive evaluation.\nResponse 40: The authors have responded satisfactorily to this point.\n(1) The authors clarify that a plasmid expressing B602L was added in overexpression experiments unless\notherwise stated. A corresponding statement was included in the main text (lines 226-228).\n(2, 3) The authors discuss the roles of K63 ubiquitination (Lines 249-252) and have included references\n(35-37) showing that K63 ubiquitination can target proteins for degradation including a previous report on\n2 / 6\np72 targeting for degradation (Ref 30). To avoid overstating conclusions a sentence in the abstract has\nbeen altered to “CypA interaction stabilizes p72 by reducing its ubiquitination and preventing proteasomal\ndegradation.”\nResponse #6: We are highly grateful to the reviewer for the encouraging evaluation.\nResponse 41: I agree that the authors have addressed the points (1, 2) by performing a time course\nexperiment over a single cycle of infection. It seems justified to replace the images shown in the previous\n2F and 8G, demonstrating weaker p72 staining within factories at later times, if these represent a minor\npopulation of the observed localisation for p72. However, within the relevant revised section (Lines 144-\n153) they should mention that this weaker localisation in factories was observed in a minority of cells\n(ideally giving an indication of the proportion of cells).\nResponse #7: We sincerely thank the reviewer for the supportive evaluation and the constructive\nsuggestion. We fully agree that the weaker localization of p72 in viral factories should be explicitly noted\nas occurring in a minority of cells (about 10%). Accordingly, we have revised the corresponding text to\ninclude this clarification. The revised sentence now reads (lines 149-151): “A weaker p72 signal in\nfactories was observed in a small fraction (~10%) of infected cells at late stages, potentially reflecting\nextensive p72 accumulation and redistribution.”\n(1, 2) Concerns about p72 immunofluorescence patterns were also raised by reviewer 1. The authors\ncarried out an additional time course experiment at high moi to observe p72, Cyp A and virus factory\nlocalisation (Fig 2F). Revised Figure 8G shows representative images of p72 localisation in virus factories.\n(3) In response to comments that, “at later stages (18-24 hpi), a subset of infected cells showed relatively\nweaker p72 staining within viral factories compared with the surrounding cytoplasm, consistent with the\nprevious Fig. 2F and Fig. 8F”.\nThe authors re-examined the localisation of p72 in images at these later times and conclude that the diffuse\nstaining observed in a small subset of cells represents a minor late-stage phenomenon possibly due to\nextensive p72 synthesis and redistribution. As this phenotype was rare and not generalizable, these images\nwere not included in the revised manuscript.\nResponse #8: We are grateful to the reviewer for the encouraging comments.\nAdditional points raised by reviewer 3, response 42 to 51:\nI agree that the authors have answered these points satisfactorily.\nResponse #9: We sincerely appreciate your positive feedback.\nIn response to point 44 the authors have re-examined TEM datasets and replaced the original images\nshown in Fig 8 to better represent factories in DMSO and Cyp A inhibitor treated cells.\nResponse #10: We are thankful to the reviewer for the positive evaluation.\nIn addition, I suggest that the authors clarify a line in the introduction.\nLine 61: The text states, “ In contrast, p72 degradation is necessary during early stages of viral\n3 / 6\nreplication to release viral DNA into host cells”. This should be clarified to explain that core particles\ncontaining the virus genome are released into the cell cytoplasm following capsid disassembly and fusion\nof the virus inner envelope with host endosomal membranes. This explanation is included in the discussion\nbut it would help the reader to clarify the entry process in the Introduction. Relevant references are cited:\n76. African Swine Fever Virus Undergoes Outer Envelope Disruption, Capsid Disassembly and Inner\nEnvelope Fusion before Core Release from Multivesicular Endosomes. Hernáez B, Guerra M, Salas ML,\nAndrés G. PLoS Pathog. 2016 doi: 10.1371/journal.ppat.1005595.\n15. Autophagy promotes p72 degradation and capsid disassembly during the early phase of African swine\nfever virus infection. Song J, Li J, Li S, Zhao G, Li T, Chen X, Hu B, Liu J, Lai X, Liu S, Zhou Q, Huang L,\nWeng C.J Virol. 2025 doi: 10.1128/jvi.01701-24.\nResponse #11: We sincerely thank the reviewer for the constructive and professional comments. We fully\nagree that clarifying the role of p72 degradation during the entry process in the Introduction will help\nreaders better understand ASFV entry. Accordingly, we have revised the text to explain that p72\ndegradation and capsid disassembly facilitate the release of core particles into the cytoplasm following\nfusion of the virus inner envelope with host endosomal membranes, as supported by the recommended\nreferences. Please refer to lines 61-64: “In contrast, p72 degradation is essential during the early phase of\nASFV replication, driving capsid disassembly and enabling the release of core particles containing the\nviral genome into the cytoplasm following fusion of the viral inner envelope with host endosomal\nmembranes, thereby facilitating viral DNA delivery into host cells15,16.”\nReviewer #2 (Remarks to the Author):\nThe authors have addressed the concerns i have raised in the review of the original manuscript\nappropriately.\nResponse #12: We sincerely thank the reviewer for taking the time to carefully evaluate our revised\nmanuscript. We greatly appreciate the positive and encouraging feedback, which is highly motivating for\nour work.\nOne correction still has to made before publication. The term 'half-life' has to be replaced throughout the\ntext. Half-life refers to exponential decays while the authors observed a linear decay in Fig 5G. The last\nsentence of the paragraph beginning in line 668 is wrong and has to be removed (line 677 'For cases more\nrapid decay...'). The new paragraph 'Cycloheximide (CHX) chase assay', line 668 in the merged .pdf\nreferring to Response #27 has to be changed accordingly, the given formulas do not describe a half-life\ncalculation in a non-logarithmic scale.\nResponse #13: We sincerely thank the reviewer for this helpful and insightful suggestion.\n(1) We fully agree that the term “half-life” is inappropriate for describing the linear decay observed in Fig.\n5G. Accordingly, all instances of “half-life” in the manuscript have been replaced with “degradation rate”\nto accurately reflect linear decay. The relevant sentence has also been revised to avoid using “half-life”:\n“shortening the time for the protein intensity to reach 50% of its initial value from 6 h to 2.95 h” (lines 280-\n281).\n4 / 6\n(2) As suggested, the last sentence of the “Cycloheximide (CHX) chase assay” paragraph in the Methods\nsection (“For cases more rapid decay…”) has been removed.\n(3) We acknowledge that the previously provided formulas only describe the linear degradation. The\nparagraph has now been revised to correctly describe linear decay. Please refer to lines 677-680 in the\nrevised manuscript.\n...........................................................\nResponses to Reviewer 3\nZhao and colleagues have responded appropriately to most issues raised by reviewer 3. However, some\nminor points remain to be resolved.\nResponse #14: We sincerely thank the reviewer for taking the time to further evaluate our responses to\nReviewer #3’s comments. We greatly appreciate your positive assessment and constructive feedback.\nResponse #38, Fig 8: in their rebuttal, the authors claim that lower amounts of p72 after single-cycle\ninfection show that CypA regulates stability of p72, however, downregulation of p72 synthesis can result in\nthe same effect. Thus, the statement in sentence beginning in line 379 seems an over-interpretation of the\nwestern blot.\nResponse #15: We sincerely appreciate the reviewer’s important comment. We acknowledge that decreased\np72 levels after single-cycle infection could result from both reduced protein stability and downregulated\nsynthesis. Although B646L transcription was not affected by CypA inhibitors in single-cycle infection, to\navoid potential over-interpretation, we have revised the sentence to replace “protein stability” with “protein\nlevels”. Please refer to lines 383-384: “These results demonstrate that CypA primarily regulates p72\nprotein levels”.\nResponse #38, (2): in the discussion of the possible impact of CypA expression on MAPK pathways and the\nconsequences for virus replication (beginning in line 473), references should be added that show the\ninteraction of ASFV with MAPK pathways, e.g. Wöhnke et al., 2022, https://doi.org/10.3390/v14102140\ndescribing the impact of ASFV infection on MAPK14/p38 expression and phosphorylation.\nResponse #16: We sincerely thank the reviewer for this helpful reminder. In the revised manuscript, we\nhave added a citation to highlight the interaction of ASFV with MAKP14/p38, as recommended. Please\nrefer to lines 476-479: “Given that many viruses exploit MAPK signaling to facilitate replication64, 65, and\nthat ASFV infection has been reported to modulate MAPK14/p38 expression and phosphorylation66, it is\nplausible that CypA-mediated MAPK activation contributes to ASFV proliferation.”\nResponse #39 does not seem to fully answer the reviewer’s question, as the positive control in the CypA-KO\ncells with pB602L is missing. The blot does not show if trimers are formed in the presence of pB602L and\nthe absence of CypA.\nResponse #17: We sincerely thank the reviewer for raising this important concern. We acknowledge that\nthe previous revision lacked the group showing p72 trimer formation in CypA-KO cells expressing\npB602L. To fully address this, we have updated Fig. S3B by including two groups showing p72 trimer\n5 / 6\nformation: CypA-KO cells expressing pB602L and CypA-KO cells expressing CypA-HA. As shown, in\nCypA-KO cells, expression of pB602L, but not CypA, is sufficient to promote p72 trimer formation. These\nupdated data further confirm that CypA does not function as a chaperone to directly facilitate p72 folding or\ntrimerization. Accordingly, we have revised the sentence in lines 233-235 to: “Notably, while pB602L\npromoted p72 trimer formation regardless of CypA expression, CypA alone failed to induce trimerization”.\nFig. S3. (B) CypA does not promote trimer assembly of p72.\n(Note: the two rightmost panels are newly provided.)\n--------------------------------------------------------------------------------------------------------------------------------\nFinally, we sincerely believe that the revised manuscript has been further strengthened by the insightful\ncomments and suggestions from the reviewers. We hope that our revisions satisfactorily address all\nconcerns.\nWe are deeply grateful for your time and dedication in reviewing our manuscript. Thank you once again for\nyour thoughtful and encouraging evaluation.\nKindest regards,\nDongming Zhao, PhD\nXiangpeng Sheng, PhD\n6 / 6"}]

Decision letter