The BmCPV-derived viral small peptide vsp1S4(-) suppresses viral replication by triggering apoptosis via the ROS-JNK signalling pathway

Abstract

Viral antisense RNAs are generally considered noncoding. Here, we show that segment 4 of Bombyx mori cypovirus (BmCPV) encodes a 78-aa antisense-microprotein, vsp1S4(-), that triggers a host-restrictive reactive oxygen species (ROS)-c-Jun N-terminal kinase (JNK)-apoptosis axis. vsp1S4(-) localizes to mitochondria, induces superoxide release, and activates JNK signalling. Consequently, cells undergo caspase-3-dependent apoptosis and S-phase arrest, while the levels of the viral structural protein (VP7) and progeny virions are strongly suppressed. Pharmacologic interruption of either ROS with N-acetylcysteine (NAC) or JNK signalling with SP600125, a dominant-negative JNK effectively rescues VP7 expression and restores viral replication, confirming that vsp1S4(-)-elicited signalling shows antiviral activity. Thus, BmCPV autonomously limits its own propagation through an antisense-encoded peptide that weaponizes host mitochondrial ROS and JNK, representing a paradigm of programmed self-attenuation operating via antisense translation.

Introduction

The evolutionary arms race between viruses and their hosts has forged exquisitely tuned immune circuits that determine infection outcomes. Metazoans deploy germline-encoded pattern recognition receptors (PRRs) to detect viral nonself, triggering interferon (IFN) cascades, autophagy and programmed cell death (PCD) that collectively curtail replication [1–3]. Insects, which lack adaptive immunity, rely heavily on these intrinsic defences; consequently, they represent powerful, genetically tractable models for decoding conserved antiviral logic [4, 5].

The silkworm Bombyx mori is acutely susceptible to Bombyx mori cypovirus (BmCPV), a double-stranded RNA (dsRNA) virus of the family Reoviridae that infects midgut columnar cells and causes cytoplasmic polyhedrosis, a major source of economic loss in sericulture [6, 7]. BmCPV virions package ten genomic dsRNA segments; structural proteins encoded by the plus strands, particularly VP1-VP7, are essential for entry, transcription and particle assembly and are therefore prime targets for host restriction [8].

Apoptosis is a front-line antiviral response that eliminates infected cells before progeny virions are released [9, 10]. Mitochondrial (intrinsic) apoptosis is executed through the loss of the mitochondrial membrane potential (ΔΨm), the release of cytochrome c and second mitochondria-derived activator of caspases (SMAC), and the formation of the apoptosome, which is nucleated by APAF-1 and activates initiator and effector caspases [11–16]. Additional pro-apoptotic regulators, including SUR1 and SUR2, amplify this cascade [17]. Viruses, in turn, encode inhibitors or modulate signalling thresholds to delay PCD until replication is complete. Reactive oxygen species (ROS), crucial second messengers that regulate immunity and apoptosis [18, 19], are predominantly produced by mitochondria. As the primary ROS source, mitochondria release cytochrome c following the loss of membrane potential (ΔΨm), which activates caspase cascades [20].

ROS act as pleiotropic second messengers that bridge innate sensing and effector responses. Mitochondria are the dominant source of inducible ROS; excessive superoxide and hydrogen peroxide oxidize mitochondrial membranes, potentiating ΔΨm loss and apoptosome assembly [21]. ROS also feedback on signalling kinases, most notably mitogen-activated protein kinases (MAPKs) [22]. Among these, c-Jun N-terminal kinase (JNK) is a central node that phosphorylates c-Jun to drive the transcriptional programmes governing apoptosis, inflammation and autophagy [23]. Although JNK activation can reinforce antiviral immunity [24], hepatitis B virus (HBV) hijack JNK to suppress IFN responses or promote survival [25]. Whether and how JNK signalling contributes to antiviral immunity in Lepidoptera remains largely unexplored.

Here, we report the discovery of vsp1S4(-), a 78-aa antisense microprotein from BmCPV segment 4 that potently restricts viral replication. To elucidate its functional mechanism, we employed a combination of ectopic expression, pharmacological modulation, and transcriptomic profiling. We demonstrate that vsp1S4(-) localizes to mitochondria, triggers superoxide release, and thereby activates a ROS-dependent JNK cascade. This signalling axis drives caspase-3-mediated apoptosis and S-phase cell cycle arrest, extinguishing viral progeny production. Pharmacological or genetic interruption of either ROS (N-acetyl-cysteine, a mitochondrial superoxide scavenger) or JNK (SP600125, a dominant-negative JNK) restores the viral structural protein VP7 and viral replication. Thus, BmCPV autonomously attenuates its own propagation through an antisense-encoded peptide that weaponizes host mitochondrial ROS and JNK signalling. These findings reveal a previously unrecognized mode of viral self-limitation and position vsp1S4(-) as a prototype for host-targeted antiviral design.

Materials and methods

Cell culture

BmN cells (provided by the Molecular Biology Laboratory, School Of Life Sciences, Soochow University, China) were maintained at 26 °C in TC-100 medium (PanReac AppliChem, Barcelona, Spain) supplemented with 10% (v/v) fetal bovine serum (FBS). Cells were passaged every 3–4 days when reaching 80–90% confluence. For all experiments, cells were seeded at appropriate densities and cultured for 24 h prior to transfection or treatment to ensure proper attachment and growth.

Plasmid construction

The pUC57 backbone (Thermo Scientific) was converted to the insect-cell expression vector pUC57-OpIE2 by inserting the Orgyia pseudotsugata nucleopolyhedrovirus OpIE2 promoter immediately upstream of the MCS; all the derivatives were sequence-verified (Sangon Biotech, Shanghai) and comprised the following: (i) control reporters EGFPwt and EGFPmut (ATG→ATT); (ii) vsp1S4(-) expression plasmid pUC57-OpIE2-rORF1; (iii) translational fusion rORF1-EGFPmut (rORF1 in-frame with start-codon-deficient EGFPmut); and (iv) corresponding start-codon mutant controls rORF1mut and rORF1mut-EGFPmut.

Cell cycle analysis

BmN cells were harvested 48 h post-transfection, washed twice with ice-cold PBS, and fixed dropwise in 70% ethanol at 4 °C overnight. After rehydration in PBS, the cells were pelleted and resuspended in 500 µl of propidium iodide (PI) staining solution (Beyotime, Shanghai, China) containing 50 µg/mL PI and 100 µg/mL RNase A (Thermo Scientific) and then incubated at 37 °C for 30 min in the dark. The DNA content was acquired on a BD Accuri C6 flow cytometer (≥ 10,000 events per sample) and analyzed with FlowJo v10.8.1 using the Watson pragmatic model.

Apoptosis assay

BmN cells were harvested 48 h post-transfection, washed twice with ice-cold PBS, and resuspended in 100 µl of Annexin-V-FITC binding buffer containing 2.5 µl of Annexin-V-FITC and 1 µl of propidium iodide (Beyotime, Shanghai, China). After 15 min of incubation at 25 °C in the dark, 400 µl of binding buffer was added, and the samples were immediately analyzed on a BD Accuri C6 flow cytometer. Fluorescence compensation was set with single-stained controls; 10,000 events were recorded per sample. Annexin-V⁺/PI⁻ and Annexin-V⁺/PI⁺ populations were gated as early apoptotic and late apoptotic/necrotic cells, respectively, and quantified with FlowJo v10.8.1.

Mitochondrial membrane potential (ΔΨm)

The cells were harvested 48 h post-transfection, washed once with warm PBS, and resuspended at 1 × 10⁶ cells ml⁻¹ in prewarmed JC-1 working solution (Beyotime, Shanghai, China). After 20 min of incubation at 37 °C in the dark, the cells were washed twice with JC-1 staining buffer, resuspended in 500 µl of assay buffer, and analyzed immediately. Fluorescence was captured on a Nikon Ti-E microscope (green: 490/530 nm; red: 525/590 nm); the red/green fluorescence ratio was quantified with ImageJ and is expressed as the fold change relative to that of the untreated controls. A ratio decrease of ≥ 20% was scored as mitochondrial depolarization.

ROS detection

BmN cells (1 × 10⁵ per well in 6-well plates) were transfected with pUC57-rORF1; 24 h post-transfection, the medium was removed, and the cells were washed with warm PBS and loaded with 10 µM DCFH-DA (Beyotime, Shanghai, China) in serum-free TC-100 for 20 min at 37 °C in the dark. After two additional washes with serum-free medium, ROS-driven fluorescence (Ex/Em 488/525 nm) was quantified immediately by flow cytometry (BD Accuri C6; ≥ 10,000 events) or imaged on a Nikon Ti-E microscope. The mean fluorescence intensity (MFI) was calculated with FlowJo v10.8.1 and normalized to that of untransfected controls.

CCK-8 viability assay

BmN cells were seeded in 96-well plates at 8 × 10³ cells/well, allowed to attach overnight (16–20 h), and exposed to serial dilutions of the test compounds for 48 h. Subsequently, 10 µl of Cell Counting Kit-8 reagent (Beyotime, Shanghai, China) was added to each well, and the plates were incubated for 4 h at 26 °C. The absorbance was read at 450 nm (reference 630 nm) on a SpectraMax i3x microplate reader; background-corrected values are expressed as percentage viability relative to vehicle-treated controls.

RNA-seq

BmN cells were transfected with different vectors, and 48 h post-transfection, total RNA was extracted from three biological replicates per condition and shipped on dry ice to Shanghai OE Biotech Co., Ltd. Ribosomal RNA was depleted with the NEBNext rRNA Depletion Kit, and libraries were prepared via NEBNext Ultra II Directional RNA Library Prep and sequenced on an Illumina NovaSeq 6000 (2 × 150 bp, ~ 30 M clean reads per sample). The raw reads were deposited in the NCBI SRA under BioProject PRJNA1308017.

Western blotting

The cells were harvested 48 h post-transfection, lysed on ice for 30 min in RIPA buffer containing 1× protease/phosphatase inhibitor cocktail (Beyotime, Shanghai). Protein concentrations were determined with a BCA kit (Beyotime); 30 µg per lane was resolved on 10–12% SDS‒-PAGE gels (Vazyme, Nanjing) and electrotransferred to 0.22 μm PVDF membranes (Millipore). The membranes were blocked with 5% (w/v) BSA in TBST for 1 h at RT and then incubated overnight at 4 °C with the following primary antibodies (all from Huabio unless otherwise stated): anti-JNK (1:1000), anti-p-JNK (1:1000), anti-c-JNK (1:1000), anti-cleaved caspase-3 (1:1000), anti-VP7 (1:10000) and anti-α-tubulin (Proteintech, 1:2000). After three TBST washes, HRP-conjugated goat anti-rabbit or anti-mouse IgG (Proteintech, 1:5 000) was applied for 1 h at RT. Immunoreactive bands were visualized with NCM ECL Ultra (NCM Biotech, Suzhou) on a ChemiDoc MP imager and quantified with ImageJ; all signals were normalized to the corresponding tubulin loading control.

Statistical analysis

All the data are presented as the means ± SEMs from ≥ 3 independent experiments. Statistical analyses were performed in GraphPad Prism 9.0 via two-tailed unpaired Student’s t test or one-way ANOVA followed by Tukey’s multiple-comparison test as appropriate; *, **, *** and **** denote P ≤ 0.05, 0.01, 0.001 and 0.0001, respectively.

Results

vsp1S4(-) triggers caspase-dependent apoptosis and S-phase arrest in BmN cells

To determine how the viral antisense microprotein vsp1S4(-) suppresses BmCPV replication, we transfected BmN cells with pIZ-OpIE2-rORF1 (expressing vsp1S4(-) under the immediate-early OpIE2 promoter) or an empty vector (pIZ-OpIE2). Forty-eight hours post-transfection, real-time PCR revealed robust upregulation of pro-apoptotic transcripts (SUR1, SUR2, APAF-1 and the effector caspase BmCaspase-1) (Fig. 1A, P < 0.0001). Similarly, Western blot analysis revealed a significant increase in cleaved caspase-3 (C-Cas3) relative to that of the vector control (Fig. 1B, P < 0.001). Annexin-V/PI flow cytometry confirmed a dose-dependent increase in apoptosis (Fig. 1C; P < 0.01).

Fig. 1.

vsp1S4(-) induces caspase-dependent apoptosis in BmN cells. (A) Real-time PCR analysis of pro-apoptotic genes (SUR1, SUR2, APAF, and Caspase) in BmN cells transfected with pUC57-OpIE2-rORF1 [vsp1S4(-)] or the empty vector control pUC57-OpIE2 (48 h posttransfection). The data were normalized to actin and are presented as the mean fold change ± SD (n = 3). ****P < 0.0001. (B) Western blotting analysis of cleaved caspase-3 (C-Cas3) in transfected cells (representative blot on the left, quantification on the right). The data are presented as the means ± SDs (n = 3). **P < 0.01, ****P < 0.0001). (C) Flow cytometry analysis of apoptosis via Annexin V/PI staining (representative plots in D, quantification in E). The data are presented as the means ± SDs (n = 3). *P < 0.5. (D) Effects of vsp1S4(-) on the mitochondrial membrane potential. The data are presented as the means ± SDs (n = 3). ***P < 0.001, ****P < 0.0001. (E) Cell cycle distribution analyzed by flow cytometry (PI staining) in BmN cells transfected with vsp1S4(-) or the control vector (48 h posttransfection). Representative histograms (left) and quantification of cell cycle phases (right) are shown. The data are presented as the means ± SDs (n = 3). **P < 0.01, ***P < 0.001 (S-phase and G2/M comparisons, respectively)

Because mitochondrial outer-membrane permeabilization (MOMP) is rate-limiting for Lepidopteran apoptosis, we quantified the ΔΨm with JC-1. vsp1S4(-) expression caused a significant decrease in the red/green fluorescence ratio within 12 h (Fig. 1D, P < 0.001), indicating severe mitochondrial dysfunction. Cell cycle profiling by EdU/PI click chemistry revealed the selective accumulation of cells in S phase with a reciprocal decline in G2/M (Fig. 1E, P < 0.01). Collectively, these data demonstrate that vsp1S4(-) simultaneously initiates MOMP-driven apoptosis and S-phase stalling, thereby depriving the virus of replication-competent host cells.

vsp1S4(-) triggers mitochondrial ROS- to execute apoptosis in BmN cells

Because mitochondrial outer-membrane permeabilization is frequently preceded by an oxidative burst, we asked whether vsp1S4(-) perturbs redox homeostasis. The transfection of BmN cells with the vsp1S4(-) construct increased DCFH-DA fluorescence within 6 h (Fig. 2A, P < 0.0001).

Fig. 2.

vsp1S4(-) triggers mitochondrial ROS-dependent apoptosis in BmN cells. (A) Intracellular ROS levels measured by DCFH-DA fluorescence in BmN cells transfected with vsp1S4(-) or the control vector (48 h posttransfection). The data are presented as the mean fluorescence intensity (MFI) ± SD (n = 3). ****P < 0.0001. (B) Cell viability (CCK-8 assay) of BmN cells treated with H₂O₂ (0-0.5 mM) for 24 h. Data are presented as the means ± SDs (n = 3). ****P < 0.0001. (C) Viability of BmN cells treated with NAC (0–8 mM) for 24 h. Data are presented as the means ± SDs (n = 3). ****P < 0.0001. (D) Flow cytometry analysis of the effects of H₂O₂ and NAC on apoptosis induced by vsp1S4(-). The data are presented as the means ± SDs (n = 3). ***P < 0.001, ****P < 0.0001. (E) ROS levels (DCFH-DA fluorescence) in vsp1S4(-) BmN cells treated with 0.2 mM H₂O₂ or 2 mM NAC for 6 h. Representative histograms (left) and quantification of the MFI (right). The data are presented as the means ± SDs (n = 3). ***P < 0.001, ****P < 0.0001. (F) Western blotting analysis of cleaved caspase-3 (C-Cas3) in the same treatment groups

To establish a pharmacological window for modulation, we titrated the oxidative stressor H₂O₂ and the thiol antioxidant N-acetyl-cysteine (NAC). No apparent cytotoxicity was observed at H₂O₂ ≥ 0.2 mM (Fig. 2B), whereas NAC ≥ 2 mM was detected with overt toxicity (Fig. 2C). We therefore used 0.2 mM H₂O₂ and 2 mM NAC for all subsequent experiments.

Combined vsp1S4(-) expression with 0.2 mM H₂O₂ synergistically elevated Annexin-V positivity (Fig. 2D, P < 0.001). Conversely, NAC pretreatment reduced apoptosis to baseline levels, confirming a causal role for ROS. Quantitative ROS imaging corroborated these findings: H₂O₂ amplified vsp1S4(-)-induced DCF fluorescence, whereas NAC quenched DCF fluorescence (Fig. 2E, P < 0.0001). Immunoblot analysis revealed that H₂O₂ further increased cleaved caspase-3 (C-Cas3) in vsp1S4(-)-expressing cells, an effect completely abrogated by NAC (Fig. 2F). Together, these data demonstrate that vsp1S4(-) triggers a mitochondrial ROS burst that is both necessary and sufficient for downstream caspase activation and apoptosis.

vsp1S4(-) couples mitochondrial ROS to apoptotic JNK signalling

RNA-seq of vsp1S4(-)-expressing BmN cells (48 h) revealed MAPK signalling as the top-enriched pathway (FDR < 0.001, Fig. 3A). Western blotting confirmed a significant increase in phosphorylated JNK (p-JNK) without a change in total JNK (Fig. 3B, P < 0.01), indicating posttranslational activation.

Fig. 3.

vsp1S4(-) activates JNK signalling. (A) Transcriptomic analysis (RNA-seq) of MAPK signalling pathway enrichment in vsp1S4(-) -transfected vs. control cells (48 h). (B) Western blot analysis of phosphorylated JNK (p-JNK) and total JNK in transfected cells. The data are presented as the mean p-JNK/JNK ratio ± SD (n = 3). **P < 0.01. (C, D) Cell viability (CCK-8 assay) of vsp1S4(-) -transfected BmN cells treated with anisomycin (JNK agonist, 0–50 µM) or SP600125 (JNK inhibitor, 0–50 µM) for 24 h. Data are presented as the means ± SDs (n = 3). ****P < 0.0001. (E) Western blotting analysis of p-JNK, c-JNK, and C-Cas3 in vsp1S4(-) -transfected cells treated with 5 µM anisomycin or 5 µM SP600125 for 12 h. (F) Flow cytometry analysis of the effects of anisomycin and SP600125 on the degree of apoptosis induced by vsp1S4(-). The data are presented as the means ± SDs (n = 3). ****P < 0.0001

To position JNK relative to ROS, we exploited the pharmacological agents anisomycin (agonist) and SP600125 (inhibitor). CCK-8 titration revealed that 5 µM anisomycin and 5 µM SP600125 were the highest nontoxic concentrations that maximize or abolish JNK phosphorylation, respectively (Fig. 3C and D). Anisomycin (5 µM) superinduced p-JNK and elevated cleaved caspase-3 (C-Cas3) in vsp1S4(-)-positive cells, whereas SP600125 (5 µM) completely blocked both p-JNK and C-Cas3 accumulation (Fig. 3E). Annexin-V flow cytometry mirrored these changes: anisomycin increased apoptosis, whereas SP600125 reduced it (Fig. 3F, P < 0.001). Importantly, SP600125 also reversed the mitochondrial ROS burst, placing JNK downstream of ROS but upstream of MOMP. Collectively, these data establish that vsp1S4(-)-elicited mitochondrial ROS obligatorily signals through JNK to execute caspase-dependent apoptosis.

ROS-dependent JNK activation is both necessary and sufficient for vsp1S4(-)-induced apoptosis

To establish a causal sequence linking ROS generation, JNK signaling and apoptosis execution in cells expressing vsp1S4(-), we used an H₂O₂-driven ROS boost combined with acute pharmacological modulation of JNK. BmN cells stably overexpressing vsp1S4(-) were treated with 0.2 mM H₂O₂ and immediately coexposed to either the JNK agonist anisomycin (5 µM) or the selective JNK inhibitor SP600125 (5 µM) for 2 h. After compound washout and overnight culture, apoptosis was quantified via Annexin-V/propidium iodide flow cytometry (Fig. 4A). Anisomycin synergistically amplified H₂O₂-triggered apoptosis, whereas SP600125 reduced apoptosis to baseline levels (Fig. 4B), indicating that JNK activity is necessary and sufficient downstream of ROS.

Fig. 4.

ROS-induced apoptosis requires JNK activation. (A) vsp1S4(-) BmN cells were treated with 0.2 mM H₂O₂ alone or in combination with 5 µM anisomycin (a JNK agonist) or 5 µM SP600125 (a JNK inhibitor) for 2 h, followed by overnight culture. Apoptosis quantification by flow cytometry (annexin V/PI). The data are presented as the means ± SDs (n = 3). ****P < 0.0001. (C, D) Western blot analysis of p-JNK and C-Cas3 in the same treatment groups is shown in Fig. 4A

Western blotting confirmed that H₂O₂/anisomycin combination markedly elevated phospho-JNK (p-JNK) and that the active executioner fragment cleaved caspase-3 (C-Casp-3) (Fig. 4C). Conversely, SP600125 inhibited both p-JNK and C-Casp-3 induction (Fig. 4C). Importantly, H₂O₂ alone was sufficient to increase total JNK and p-JNK in vsp1S4(-)-expressing cells, and this response was completely abrogated by pretreatment with the ROS scavenger N-acetyl-cysteine (NAC, 5 mM), confirming that JNK activation is ROS dependent (Fig. 4D).

Collectively, these data establish a linear pathway in which vsp1S4(-) expression evokes an ROS burst that activates JNK and then drives the apoptotic programme through caspase-3 cleavage and subsequent cell death.

ROS-JNK-dependent repression of viral structural protein expression

To determine whether the vsp1S4(-)-triggered ROS-JNK cascade feeds back to viral gene expression, we monitored the levels of the major structural protein VP7 after selective manipulation of the pathway. BmN cells stably expressing vsp1S4(-) were exposed to 200 µM H₂O₂ (a ROS boost), 5 µM anisomycin (a JNK agonist), 5 mM N-acetyl-cysteine (a ROS scavenger), or 10 µM SP600125 (a JNK inhibitor). Whole-cell lysates harvested 8 h posttreatment were probed for VP7 by quantitative Western blotting.

H₂O₂ and anisomycin each reduced VP7 abundance compared to the control, respectively (Fig. 5A). This repression was fully reversed by cotreatment with NAC or SP600125, and the expression level of VP7 was restored compared with that of the control, confirming that suppression is mediated specifically through the ROS-JNK axis (Fig. 5B).

Fig. 5.

vsp1S4(-) suppresses viral replication by triggering apoptosis via the ROS-JNK signaling pathway. (A, B) Western blotting analysis of the viral structural protein VP7 in vsp1S4(-) -transfected BmN cells treated with H₂O₂ (0.2 mM), anisomycin (5 µM), NAC (5 mM), or SP600125 (10 µM) for 24 h

Thus, vsp1S4(-) initiates a signalling cascade in which ROS-activated JNK not only drives apoptosis but also simultaneously affects the synthesis of the viral structural protein VP7, coupling oxidative stress to the posttranscriptional regulation of viral gene expression.

Discussion

This study reveals a sophisticated self-limiting strategy in BmCPV, where the viral peptide vsp1S4(-) paradoxically restricts viral propagation by co-opting the host ROS-JNK-apoptosis axis. We demonstrated that vsp1S4(-) triggers a mitochondrial ROS burst, which serves as the primary activator of JNK signalling. Activated JNK then drives caspase-3-dependent apoptosis and S-phase arrest, creating a hostile environment for BmCPV replication. Crucially, this pathway directly suppresses viral structural protein synthesis: JNK activation (via anisomycin) or ROS induction (via H₂O₂) downregulates VP7 expression, whereas JNK inhibition (SP600125) or ROS scavenging (NAC) rescues it. Unlike canonical viral antagonists of host defence, vsp1S4(-) exploits oxidative stress signalling to eliminate infected cells and suppress viral gene expression, revealing a novel mechanism for intrinsic viral replication control.

The JNK pathway plays a dual role in viral infections: while it restricts dissemination by triggering infected apoptosis, it can simultaneously modulate viral replication efficiency through direct effects on viral gene expression [26]. Our findings establish that vsp1S4(-) exploits this dichotomy, activating JNK signalling primarily through mitochondrial ROS generation. This cascade culminates in JNK phosphorylation (p-JNK) and the downstream upregulation of apoptotic executors such as caspase-3. Strikingly, this pro-apoptotic strategy directly opposes the canonical antagonism employed by baculovirus inhibitor of apoptosis (IAP) proteins [17, 27]. For example, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) IAP sustains host cell viability by directly suppressing caspase activity to maximize viral yield [28]. This fundamental divergence underscores how distinct viral families evolve opposed strategies to manipulate host cell fate—either accelerating death to limit spread (vsp1S4(-)) or prolonging survival to increase replication.

At the signalling level, vsp1S4(-) may directly target mitogen-activated protein kinase kinase 4/7 (MKK4/7, also known as JNKK1/2). This mechanism resembles how the HIV Tat protein activates the JNK pathway: both appear to bypass canonical upstream MAP3K regulation, directly activating the MAP2K (MKK4/7) cascade in a nonclassical manner [29]. In this study, anisomycin treatment elevated p-JNK levels, concomitant with reduced expression of the viral proteins VP7 and NSP9. Critically, this inhibitory effect was completely reversed by the JNK inhibitor SP600125. Furthermore, exogenous H₂O₂ elevated ROS levels, activated the JNK pathway, and similarly affected viral protein expression, indicating that vsp1S4(-) operates through the classical ROS-JNK axis. Evidence also suggests that JNK activation can further amplify ROS generation, potentially establishing a self-reinforcing positive feedback loop. Specifically, vsp1S4(-)-induced JNK activation may promote mitochondrial membrane potential depolarization, increasing mitochondrial ROS production. Elevated ROS could then further activate the ASK1-MKK4/7-JNK signalling axis, amplifying the cascade. This proposed feedback mechanism warrants further experimental validation.

With respect to cell cycle regulation, this study demonstrated that the vsp1S4(-) protein inhibits viral replication by inducing S-phase arrest in host cells. Flow cytometry confirmed a significantly greater proportion of S-phase cells upon vsp1S4(-) overexpression than in the controls. This strategy presents an intriguing contrast to the mechanism employed by the influenza virus NS1 protein, which prolongs the S-phase to increase viral replication [30]. This dichotomy likely stems from fundamental differences in viral replication strategies: RNA viruses (influenza) rely heavily on host RNA polymerase II for transcription, whereas DNA viruses (BmNPV) depend on the host DNA replication machinery. More notably, the S-phase arrest induced by vsp1S4(-) is functionally complementary to the G2/M-phase blockade triggered by the herpes simplex virus (HSV-1) ICP0 protein [31]. Whereas ICP0 promotes viral particle release by degrading cyclin-dependent kinase 1 (CDK1) regulators to induce G2/M arrest, vsp1S4(-)-mediated S-phase arrest primarily targets the inhibition of viral template strand formation, underscoring the diversity of viral strategies for manipulating the host cell cycle.

Despite these significant advances, several key questions remain. First, direct structural evidence for the interaction between vsp1S4(-) and MKK4/7 kinases is lacking. Future studies should employ coimmunoprecipitation (co-IP) coupled with surface plasmon resonance (SPR) to analyze the binding kinetics of this interaction. Second, the evolutionary significance of the ROS-JNK pathway within the insect immune system needs further exploration. From an adaptive evolution perspective, this signalling mechanism may enable the host to limit early viral dissemination via a ”novel strategy (inducing apoptosis of infected cells), although sustained pathway activation and consequent oxidative damage could impair tissue repair capacity. Additionally, as the current findings are derived primarily from cellular models, the in vivo antiviral efficacy of vsp1S4(-) requires validation in a larval infection model, with a particular focus on dynamic changes in JNK pathway activity and the viral load within the primary target organ, midgut tissue. Overall, this study offers novel avenues for antiviral strategy development, providing crucial insights into the molecular dynamics of the ongoing virus-host arms race.

Authors’ contributions

Xialing Chen, Xiaoyan Du, Lixuan Li, Yao Yao, Qian Teng, and Haoni Xue performed the experiments and analyzed the data. Xing Zhang, Chengliang Gong, and Xiaolong Hu designed the study, analyzed and interpreted the data. Qian Teng, Haoni Xue, and Min Zhu analyzed the data. Xing Zhang, Chengliang Gong, and Xiaolong Hu wrote the paper.

Funding

This study was supported by the National Natural Science Foundation of China (grant no. 31972620, 31602007) and a project funded by the Priority Academic Program of Development of Jiangsu Higher Education Institutions. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data availability

The raw reads were deposited in the NCBI SRA under BioProject PRJNA1308017.

Declarations

Ethics approval and consent to participate

Not applicable.

Competing interests

The authors declare that they have no conflicts of interest.