Dual roles and evolutionary implications of P26/poxin in antagonizing intracellular cGAS-STING and extracellular melanization immunity

Yin, Mengyi; Kuang, Wenhua; Wang, Qianran; Wang, Xi; Yuan, Chuanfei; Lin, Zhe; Zhang, Huanyu; Deng, Fei; Jiang, Haobo; Gong, Peng; Zou, Zhen; Hu, Zhihong; Wang, Manli

doi:10.1038/s41467-022-34761-0

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Published: 14 November 2022

Dual roles and evolutionary implications of P26/poxin in antagonizing intracellular cGAS-STING and extracellular melanization immunity

Nature Communications volume 13, Article number: 6934 (2022) Cite this article

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Abstract

P26, a homolog of the viral-encoded nuclease poxin that neutralizes the cGAS-STING innate immunity, is widely distributed in various invertebrate viruses, lepidopteran insects, and parasitoid wasps. P26/poxin from certain insect viruses also retains protease activity, though its biological role remains unknown. Given that many P26s contain a signal peptide, it is surmised that P26 may possess certain extracellular functions. Here, we report that a secretory baculoviral P26 suppresses melanization, a prominent insect innate immunity against pathogen invasion. P26 targets the cofactor of a prophenoloxidase-activating protease, and its inhibitory function is independent of nuclease activity. The analysis of P26/poxin homologs from different origins suggests that the ability to inhibit the extracellular melanization pathway is limited to P26s with a signal peptide and not shared by the homologs without it. These findings highlight the independent evolution of a single viral suppressor to perform dual roles in modulating immunity during virus-host adaptation.

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Introduction

The cytosolic cGAS-STING pathway is part of an important innate immune response for antagonizing DNA virus infection. Recently, an immune nuclease poxin was identified in poxviruses to inhibit this cytosolic DNA-sensing pathway by selectively degrading 2′3′-cyclic GMP-AMP (cGAMP)¹. Poxin homologs widely exist in the genome of invertebrate DNA and RNA viruses, as well as in hymenopteran parasitoid wasps and lepidopteran insects¹, suggesting horizontal gene transfer events between insect hosts and their viruses/parasitoids, and further radiation to mammalian viruses. Structural and functional analyses suggest that poxins may have evolved from viral proteases through a gain of secondary nuclease activity²; however, the biological role of these viral proteases remains unknown. In addition, many poxin homologs contain a signal peptide, implying an extracellular function in addition to counteracting the cytosolic cGAS-STING pathway.

The homolog of poxin was initially identified in insect viruses of Baculoviridae and called P26, because it encodes a viral protein with a molecular mass of 26 kDa³. The detailed role of P26 during baculovirus infection remains unclear^4,5,6. In the parasitoid wasps Microplitis mediator and Microplitis demolitor, P26 is secreted and highly abundant in venom, which is introduced into lepidopteran hosts during wasp oviposition to suppress host immune response^7,8. Prediction of the signal peptide and phylogenetic analysis suggest that secreted P26/poxin homologs are extensively present in parasitoid wasps, alphabaculoviruses, and lepidopteran insects but not in others (Fig. 1a). Some baculoviruses harbor two p26 copies: copy-1 with and copy-2 without signal peptide. Due to the low sequence similarity between these two copies, it was hypothesized that they might have been acquired in independent events during evolution⁹.

**Fig. 1: Comparison of 2′3′-cGAMP nuclease activities and the active sites of different sources.**

Melanization is a unique and prominent innate immune defense mechanism in the hemolymph of insects and crustaceans. Sequential activation of an extracellular serine protease cascade converts prophenoloxidase (PPO) to active phenoloxidase (PO) to catalyze the formation of melanin^10,11,12. This immune response is crucial for insects to combat viruses and other invading pathogens and, in turn, viruses have evolved versatile strategies to inhibit host melanization response to ensure their survival^13,14. For example, we previously demonstrated that the baculovirus HearNPV successfully attenuates the melanization response of the cotton bollworm¹⁵. However, identity of the baculoviral gene(s) responsible for suppressing this extracellular innate immune response largely remains unknown.

Here, we provided first evidence to show that the baculovirus HearNPV P26 inhibits host PPO activation by targeting a key cofactor, serine protease homologs (SPHs), of PPO activating protease¹¹. We then determined the crystal structure of HearNPV P26 and performed structure-based mutagenesis and functional analysis to identify sites crucial for its functions. Finally, we confirmed a broad-spectrum melanization-inhibitory effect of the secreted P26s from different baculovirus and wasp origins.

Results

Nuclease activity of P26 homologs is not correlated with protein secretion

Since not all P26 homologs contain predicted nuclease active-site residues (H-Y-K) and signal peptide (Supplementary Fig. 1), we first asked whether the cGAMP nuclease activity is a universal feature of P26s and whether the activity is associated with secretion. Several representative baculoviral P26 homologs along with M. mediator P26 and vaccinia virus (VACV) poxin were expressed in bacteria and purified for the 2′3′-cGAMP nuclease activity assay (Table 1 and Supplementary Fig. 2a). The results showed that VACV poxin (no signal peptide), AcMNPV P26 (no signal peptide), and wasp M. mediator P26 (with signal peptide) had the highest 2′3′-cGAMP nuclease activity; the three signal peptide-containing baculoviral P26 proteins showed moderate activities of the nuclease, while the two baculoviral copy-2 P26s (no signal peptide) and the negative control VACV poxin^H17A mutant lacked nuclease activity (Fig. 1b). Therefore, the nuclease activity is neither conserved in all P26 homologs nor associated with protein secretion, which is consistent with results from a recent study².

Table 1 Information of baculovirus and wasp P26 homologs

Full size table

The crystal structure of baculovirus HearNPV P26

To provide structural insights into the activity differences in 2'3'-cGAMP degradation across P26 homologs, we determined the crystal structure of P26 from HearNPV, a representative baculovirus with a single copy of secreted P26, at a resolution of 1.9 Å (Supplementary Table 1). Similar to other reported poxin/P26 structures¹, HearNPV P26 contains an N-terminal β-barrel domain and a C-terminal β-sandwich domain, and assembles into a V-shaped dimeric complex in the crystal lattice, with a putative serine protease-like active-site (S56-H110-D58) located in the N-terminal domain of each monomer and the putative nuclease active sites (H66-Y205-K215) at the interface of the two monomers (Fig. 1c, d). Similar to previously determined VACV poxin and AcMNPV P26 structures, a conserved H-Y-K catalytic triad for 2′3′-cGAMP hydrolysis is located in a deep cleft of the HearNPV P26 structure at the dimer interface between the N-terminal domain from one monomer and the C-terminal domain from the other. A loop substructure is present between Y205 and K215 in the HearNPV P26 structure, while these two residues are located in a single helix in the structures of VACV poxin and AcMNPV P26 (Fig. 1e). Structural modeling indicated that both SeMNPV P26-1 and SfMNPV P26-1 with moderate nuclease activity also have an inserted loop between the Y and K residues, whilst the M. mediator P26 with high nuclease activity does not, implying that the poxin/P26 nuclease activity could be modulated by this insertion. The two baculoviral copy-2 P26s lack the nuclease activity, likely due to the absence of H-Y-K catalytic center (Fig. 1b and Supplementary Fig. 3).

HearNPV P26 inhibits host melanization response

Led by these pieces of evidence, we examined whether P26 is involved in the extracellular melanization pathway, using HearNPV and cotton bollworm as a testing system. We first detected the presence of HearNPV P26 in hemolymph from the virus-infected insect via liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis (Fig. 2a). P26 was one of the most abundant viral proteins detected in the infected hemolymph at 48 and 72 h post infection (h p.i.) (Fig. 2b and Supplementary Table 2), implying a role in the host hemolymph. We detected significantly decreased activity of hemolymph PO, a key enzyme of the melanization pathway, at 48 and 72 h post HearNPV infection. Consistently, the P26 protein reached peak levels at these times (Fig. 2c). Therefore, we suggest that P26 accumulation in the hemolymph might be involved in viral counteraction against the melanization response. To test this premise, we expressed HearNPV P26 proteins using a Drosophila Expression System (Invitrogen) (Supplementary Fig. 2b) and found that purified P26 proteins reduced hemolymph PO activity in vitro in a dose-dependent manner, with PO activity decreasing by up to 64% at a protein concentration of 12 mg/mL (Fig. 2d, P < 0.0001). To determine the impact of P26 on PPO activation and viral infectivity in vivo, a p26-deleted (Δp26) and a repaired (REp26) HearNPV were constructed. Helicoverpa armigera larvae were infected with Δp26, REp26, and wild type (WT) viruses, and the PO activity and viral loads in the hemolymph were measured at 72 h p.i.. The results showed that significantly higher PO activity was detected in the infected hemolymph of Δp26 viruses than that of the hemolymph infected with WT and REp26 viruses (Fig. 2e). The DNA copy numbers of HearNPV in Δp26 infected hemolymph decreased to approximately 20% of the WT levels (Fig. 2f). Moreover, deletion of p26 significantly extended the median survival time (ST₅₀) of the virus in infected larvae (Supplementary Table 3). The in vitro and in vivo data above suggest that HearNPV P26 functions as an inhibitor of the host melanization response, which is crucial for viral infection in vivo.

**Fig. 2: HearNPV P26 inhibits the prophenoloxidase (PPO) activation.**

HearNPV P26 targets the cofactor of PPO activation reaction

The PPO activation pathway of H. armigera is mediated by a cascade of three serine proteases SP41-cSP1-cSP6, and a combined cofactor of cSPH11 and cSPH50 (Fig. 3a)¹⁶. Upon activation by pathogen invasion, these components are converted from zymogens to active forms and finally activate PPO to catalyze melanin formation. Note that cSPH11 and cSPH50 act synergistically as a cofactor in PPO activation, and cSPH11 or cSPH50 alone does not enhance the activation reaction¹⁶. To investigate the molecular mechanism of P26-mediated inhibition of PPO activation, we first examined a possible interaction between P26 and component(s) in the cascade. The pull-down experiments showed that none of the serine proteases (proSP41, procSP1, or procSP6) interacted with P26, whereas procSPH11 and procSPH50 of the cofactor showed strong interactions with P26 (Fig. 3b). We then used an established in vitro PO activity assay¹⁶ to further screen for P26 targets by pre-incubating it with different cascade component(s). The results showed that pre-incubation of P26 with proSP41, procSP1, or procSP6 did not affect the PPO activation; in contrast, when P26 was pre-incubated with procSPH11 and procSPH50, the PO activity was significantly decreased (Fig. 3c). Further, P26 could inhibit the cofactor-mediated PPO activation in a dose-dependent manner (Fig. 3d, P < 0.0001). As procSPH11 and procSPH50 are cleaved by the upstream cSP6 during activation¹⁶, we investigated whether interaction with P26 would affect their cleavage. Immunoblotting showed that pre-incubation of P26 with procSPH11, procSPH50, or procSPH11 and procSPH50 did not affect their cleavage by cSP6 (Fig. 4a–c).

**Fig. 3: Molecular mechanism for inhibiting PPO activation by HearNPV P26.**

**Fig. 4: The impact of HearNPV P26 on the proteolytic cleavages of the serine protease homologs.**

The important functional sites in HearNPV P26

To better understand the structure-function relationship of HearNPV P26, we focused on three characteristic regions identified by structural analysis: the putative serine protease-like catalytic triad (S56-H110-D58) (Fig. 1d), the nuclease active-site (H66-Y205-K215) (Fig. 1e), and the distinct electrostatic surface with negative or positive potentials (Fig. 5a). Three sets of mutations at the key residues within these regions were designed and expressed using the Drosophila S2 Cell Expression System to determine their functions in terms of suppressing PPO activation (Supplementary Fig. 2c). The point mutations that changed the surface charge of the protein (D241H/E243H, H66D/H67D, D241H/E243H/H66D/H67D) had no impact on the suppression of PPO activation (Fig. 5b). Notably, two mutants harboring a combination of mutations in surface charge residues also contained the key H66 residue for nuclease activity, suggesting that the nuclease activity of P26 is not correlated with its function of suppressing PPO activation. Mutation of the key nuclease activity residue H66 (H66A) led to complete abrogation of cGAMP hydrolysis activity (Fig. 5d). However, this mutant did not show a compromised ability to inhibit PPO activation (Fig. 5e), further confirming that the nuclease activity is independent of melanization inhibition. In contrast, simultaneous mutations of the three key residues (S56A/H110A/D58A) constituting the putative serine protease catalytic triad in P26 completely abolished the suppression of PPO activation (P < 0.0001); the H110A (P < 0.0001) and S56A (P < 0.05) point mutation also significantly impaired P26 function, while the D58A mutant had no significant influence (Fig. 5c). Notably, both S56A/H110A/D58A and H110A showed levels of cGAMP nuclease activity comparable to the WT P26 (Fig. 5d). The results also showed that the melanization-inhibitory function of the P26 is independent of its nuclease activity.

**Fig. 5: Mutational analysis of key residues in HearNPV P26.**

Broad-spectrum melanization-inhibitory function of signal peptide-containing P26s/poxins

To investigate whether the extracellular role of antagonizing host melanization is evolutionarily conserved among diverse P26/poxin homologs, the eight P26/poxin proteins tested earlier in Fig. 1b (except poxin H117A mutant) were expressed in the Drosophila S2 Cell Expression System and further purified (Supplementary Fig. 2b) to test their inhibitory activity on PPO activation. The results showed that all the signal peptide-containing P26s, including those from baculoviruses and a parasitoid wasp, could inhibit hemolymph PPO activation of H. armigera, with the highest inhibitory effect achieved by HearNPV P26. In contrast, the ones without signal peptide did not possess this ability (Fig. 6a). To further confirm this finding, the experiment was conducted using hemolymph from another lepidopteran insect Spodoptera frugiperda. As in H. armigera, only the signal peptide-containing P26s were able to inhibit the hemolymph melanization reaction of S. frugiperda, with the highest inhibitory effect achieved by SfMNPV P26 copy-1, a protein from the native virus to which the insect is susceptible (Fig. 6b). These findings suggest that the secreted P26 homologs of baculoviruses and parasitoids can suppress extracellular immune function and seem to have evolved adaptively with the host immune system.

**Fig. 6: Secreted P26 homologs from different sources evolved the function of suppressing PPO activation.**

Discussion

In summary, we have unraveled a novel extracellular function of P26/poxin, thereby demonstrating a single protein capable of suppressing both intracellular and extracellular immune responses (Fig. 7). The viral P26 is an antagonist of host melanization, targeting the cofactor downstream of the PPO activation pathway, at least in H. armigera. The ability to inhibit the extracellular response is uniquely associated with the signal peptide-containing P26s. The wide distribution of secreted P26/poxins suggests that the suppression of PPO activation is an ancient mechanism recruited by parasitoids and insect viruses. The ability to inhibit melanization and the nuclease function are structurally and functionally independent. Among the P26/poxin proteins tested, the signal peptide-containing ones appear to contain both extracellular and intracellular immune suppression functions, whilst the ones without signal peptide either only harbor intracellular cGAMP degrading activity or lack both extracellular and intracellular activities, suggesting the occurrence of gain- and loss-of-function events during P26 evolution.

**Fig. 7: A model depicting the extracellular and intracellular roles of immunosuppression of poxin/P26.**

The results of this study raised some new questions. Here, we have not investigated the function of P26/poxin homologs in lepidopteran insects, which include proteins both with and without signal peptide. Some of the lepidopteran P26/poxin showed nuclease function^1,2, thus it would be interesting to investigate their function as PPO activation inhibitors. Previous proteomic and transcriptomic data showed that H. armigera P26 (signal peptide-containing) is one of the differentially expressed genes after inoculation of a wide variety of pathogens, including fungi, baculovirus and wasp (NCBI SRA: PRJNA349107, https://www.ncbi.nlm.nih.gov/bioproject/PRJNA349107/)^17,18, indicating it is an immune responsive gene. Its possible role in regulating the insect immune system deserves further investigation, as it would elucidate how P26/poxin genes evolved during long-term adaption between insect hosts and pathogens.

In mammals, the intracellular cGAS-STING pathway plays a pivotal role in antiviral defense. cGAS produces 2′3′-cGAMP, which can be degraded by virus-encoded poxins¹. In lepidopteran insect Bombyx mori, baculovirus BmNPV (a dsDNA virus) induces host cellular 2′3′-cGAMP production at an early stage of infection, which activates STING-mediated antiviral signaling pathway, although its upstream receptor is still unclear¹⁹. Additionally, BmNPV-encoded P26 can efficiently degrade 2′3′-cGAMP¹. Interestingly, two cGAS-like receptors (cGLRs), cGLR1 and cGLR2, were recently identified in Drosophila^20,21. Like cGAS in mammals, they synthesize cyclicdinucleotides to activate STING. However, Drosophila cGLR1 senses dsRNA ligand instead of DNA ligand to produce a novel cGAMP isomer 3′2′-cGAMP, which shows higher potency against RNA viruses than 2′3′-cGAMP. And 3′2′-cGAMP has been shown to be resistant to poxin cleavage²⁰. Therefore, it will be interesting to investigate if insect RNA viruses encode any nuclease against this signaling pathway (Fig. 7).

Besides its intracellular role, 2′3′-cGAMP also exists as a soluble, extracellular immunotransmitter^22,23,24. Therefore, it is possible that apart from being the melanization inhibitor in insects, the secretory P26/poxin homologs might also be involved in degrading extracellular 2′3′-cGAMP in mammals and insects (Fig. 7). These hypotheses, of course, need further study.

The detailed mechanism of how P26 suppresses PPO activation is unclear. Although HearNPV P26 contains a serine protease-like triad, but the residues do not occur in the typical order of H-D-S in the primary structure. And the serine protease-like triad is present in only some P26 homologs (Supplementary Fig. 4). We tried to overlay the structure of HearNPV P26 with that of Zika virus NS3 protease, as well as with that of lepidopteran Trichoplusia ni poxin. The results showed that the protease-like triad of HearNPV P26 does not overlap with the other two (Supplementary Fig. 5). In addition, the mutation of the putative catalytic serine residue S56 showed only minor impact on suppression of PO activity (Fig. 5c). Most importantly, we failed to show proteolytic cleavage activity (Fig. 4). Thus, it is unlikely that HearNPV P26 acts through a functional protease domain. Moreover, the detailed mechanism of the cofactor in the PPO activation reaction remains unclear. The cofactor is composed of two noncatalytic SPHs which contain a clip domain at their amino terminus (cSPHs)²⁵. PPO activation occurs efficiently in the presence of the cofactor, and the cSPHs are covalently associated with PO to form a high M_r melanization complex²⁵. According to the most thoroughly studied Manduca sexta model, two components of the cofactor form scaffold structures to orient PPO activating protease and PPO for optimal cleavage²⁶. Therefore, P26 may bind to cSPHs and physically block the scaffolds and consequently inhibit the activation of PPO. Since the PPO activation pathway usually contains more than one upstream branch, targeting downstream components appears to be an effective strategy and has been reported in other invertebrate viruses, such as polydnaviruses (carried by many parasitoid wasps) and white spot syndrome virus^27,28,29.

It remains unclear how ancestral P26/poxin evolved to perform dual functions. Eaglesham et al. showed that poxins were descended from self-cleaving RNA-virus proteases². The distinct amarilloviral proteases they identified, however, lack the signal peptide for secretion, which is a prerequisite for melanization-inhibitory activity. Studies dedicated to addressing the above questions will further shed light on the function-evolution relationships and precise working mechanism of this crucial viral immune inhibitor.

Methods

Insect cells, insects, and viruses

The Helicoverpa zea cell line HzAM1 was maintained at 28 °C in Grace’s insect medium (Sigma-Aldrich, G8142) supplemented with 10% fetal bovine serum (FBS) (Gibco, 10091148). The S2 cell line (Invitrogen, R69007) was maintained at 27 °C in ESF921 medium (Expression Systems, 96-001-01). H. armigera larvae were reared on an artificial diet at 27 °C. The control virus vHaBac-egfp-ph was previously constructed in our laboratory³⁰.

The p26 gene from HearNPV bacmid HaBacHZ8 was knocked out and repaired using the method of homologous recombination, as previously described³¹. HzAm1 cells were transfected with the recombinant bacmids, using Lipofectin (Invitrogen, 18292037) according to the manufacturer’s protocol. Five days post-transfection, the culture supernatant containing infectious viruses was collected and used to infect HzAm1 cells to amplify the recombinant viruses vHacBacΔp26-egfp-ph and vHacBacRep26-egfp-ph.

Prokaryotic expression of recombinant proteins

The open reading frames of p26/poxin homologs including HearNPV p26, VACV poxin, VACV poxin^H17A, AcMNPV p26, SfMNPV p26−1, SfMNPV p26-2, SeMNPV p26−1, SeMNPV p26-2, and M. mediaror p26 with signal peptide sequences deleted were amplified using viral genomic DNA as templates. PCR products were cloned into the pET-28a(+) (Novagen) or pET-32a(+) (Novagen) vector containing an N-terminal histidine tag (His-tag) and His-tag plus a thioredoxin tag (Trx-tag), respectively. Recombinant plasmids were transformed into E. coli strain BL21 (DE3) for protein expression. The proteins were purified by HIS-Select Nickel Affinity Gel (Millipore, P6611) and Superdex-200 column (GE, 17-5175-01). For the HearNPV P26 samples used in crystallization, both the N- terminal His-tag and Trx-tag of the proteins were removed by thrombin digestion. The cleaved and purified proteins were concentrated to 10 to 20 mg/mL, then flash frozen by liquid nitrogen as single-use aliquots and stored at −80 °C.

Selenomethionine-labeled HearNPV P26 proteins were expressed in BL21 (DE3) cells using M9 minimal medium (47.5 mM Na₂HPO₄, 22 mM K₂HPO₄, 8.5 mM NaCl, 18.7 mM NH₄Cl, 0.4% w/v glucose, 2 mM MgSO₄, 0.1 mM CaCl₂) supplemented with 100 mg/L lysine, phenylalanine, and threonine; 50 mg/L isoleucine, leucine, and valine; and 60 mg/L selenomethionine (SeMet, Sigma, 1611955). The protein was purified using the procedure described above.

2′3′-cGAMP nuclease activity assays

To determine 2′3′-cGAMP hydrolysis activity of recombinant proteins, 1 μL of each recombinant protein (2 μM) and 1 μL of 2′3′-cGAMP (21 μM) were added to 8 μL of buffer (50 mM HEPES-KOH pH 7.5, 40 mM KCl, 1 mM DTT). Reactions were carried out at 37 °C for 1 h. Then, each mixture was diluted to 3 mL and transferred to a 10 kDa ultrafiltration tube (Millipore, UFC8010), followed by centrifugation at 4000 g for 5 min at 4 °C. After centrifugation, 50 μL of each supernatant was used to assay the amount of 2′3′-cGAMP with the 2′3′-cGAMP ELISA Kit (Cayman, 501700), according to the manufacturer’s instructions. Each test was performed three times. Results were analyzed statistically via two-tailed Student’s t test.

Protein crystallization, X-ray diffraction data collection, and structure determination

Crystallization screening was performed at 16 °C by vapor diffusion in sitting drops containing 0.4 μL of protein (10 mg/mL) and 0.4 μL of precipitant/reservoir solution. Native and SeMet-labeled HearNPV P26 crystals were obtained with a reservoir solution of 0.2 M ammonium sulfate, 0.1 M MES monohydrate (pH 6.7), and 28% (w/v) polyethylene glycol monomethyl ether 5000. For collection of X-ray diffraction data, the crystals were harvested and transferred into the reservoir solution, followed by incremental replacement of a cryosolution (reservoir solution supplemented with 20% [vol/vol] glycerol) before flash frozen by liquid nitrogen.

Diffraction data were collected at BL18U1 and BL17U1 beamline at the Shanghai Synchrotron Radiation Facility (SSRF). For native HearNPV P26 crystals, 180 degrees of data was collected in 1° oscillation steps at 0.97780 Å. For SeMet-labeled crystals, 720 degrees of single-wavelength anomalous diffraction (SAD) data was collected in 1° oscillation steps at the Se-K edge (0.97915 Å). Diffraction data were integrated, merged, and scaled using the HKL2000 program³². The SeMet P26 structure (2.7 Å) was solved using the AutoSol³³ program in PHENIX Suite³⁴ by Se atom site search, phase calculations, density modification, and automated model-building. The native P26 structure (1.9 Å) was obtained via molecular replacement (MR) program PHASER³⁵ using the SeMet P26 structure as the initial search model. After several cycles of manual model rebuilding in Coot³⁶ and automated refinement in PHENIX³⁴, the final model was refined to have R_work and R_free values of 0.183 and 0.211, respectively. Structural superimpositions of HearNPV P26 with other correlated proteins were performed using the maximum likelihood-based structure super-positioning program THESEUS³⁷.

Sample preparation for mass spectrometry

Cell-free hemolymph (100 μL) was collected from mock and recombinant virus-infected H. armigera larvae at 48 and 72 h p.i. with the addition of 1 mM N-phenylthiourea (Sigma, P7629) and 1 mM p-aminobenzamidine (Sigma, 857661). All samples were precipitated by adding 25 μL 100% w/v trichloroacetic acid (Sigma, T6399) and incubated on ice for 30 min. The protein pellet was collected by centrifugation at 140,000 × g for 10 min at 4 °C, and the supernatant was removed. Cold acetone at −20 °C was used to wash the protein pellet, followed by centrifugation at 140,000 × g for 10 min at 4 °C, after which acetone was removed. This step was repeated twice, and the remaining solvent was allowed to evaporate. Two technical replicates were performed for each sample. The dissolved protein pellet from each sample was reduced, alkylated, followed by digestion with trypsin. The purified peptides were subjected to LC-MS/MS and analyzed on a Q Exactive HF mass spectrometer coupled with an UltiMate 3000 RSLCnano system (Thermo Fisher Scientific)³⁸. Raw MS data were analyzed by MaxQuant V1.6.6 software (Max Planck Institute of Biochemistry) using the Andromeda database search algorithm³⁹. The spectra were searched against protein sequences downloaded from NCBI database (NC_002654.2, https://www.ncbi.nlm.nih.gov/nuccore/NC_002654.2/) for HearNPV protein identification. The iBAQ value, which represent the protein abundance normalized to the number of theoretically observable peptides, was used to estimate the absolute amounts of protein⁴⁰.

Viral infection of H. armigera larvae

To collect infected hemolymph for mass spectrometry, PO activity assay, and quantitative PCR analysis, H. armigera larvae were infected with HearNPV by the droplet feeding method⁴¹. Occlusion bodies (OBs) of vHacBac-egfp-ph, vHacBacΔp26-egfp-ph, and vHacBacRep26-egfp-ph were harvested and purified from diseased larvae. Third instar H. armigera larvae were starved for 16 h, and then, OBs of each recombinant virus were fed to the larvae at a final concentration of 10⁷ per mL. Infected larvae were reared separately in 24-well plates and monitored daily.

The oral infectivity assay of the recombinant viruses was conducted by the droplet feeding method as described above. Larvae were fed eight concentrations (3 × 10⁶, 1 × 10⁶, 3 × 10⁵, 1 × 10⁵, 3 × 10⁴, 1 × 10⁴, 3 × 10³, and 1 × 10³ OBs/mL) of each recombinant virus. Median lethal concentration (LC₅₀) values were calculated by POLO probit analysis and were compared via the lethal dose ratio method. ST₅₀ of the recombinant viruses was measured in third instar larvae exposed to 3 × 10⁶ OBs/mL of each virus. Forty-eight insects were used, and the mortality was checked every 6 h. The ST₅₀ values were calculated using the Kaplan-Meier estimator and further compared using Log-rank test. All experiments were performed in duplicate.

Expression of recombinant proteins in the Drosophila S2 cell system

For PO activity analysis, p26/poxin homologs including HearNPV p26, HearNPV p26^S56A, HearNPV p26^D58A, HearNPV p26^H110A, HearNPV p26^{S56A/H110A/D58A}, HearNPV p26^H66A, HearNPV p26^H66D/H67D, HearNPV p26^D241H/E243H, HearNPV p26^{H66D/H67D/D241H/E243H}, VACV poxin, AcMNPV p26, SfMNPV p26−1, SfMNPV p26-2, SeMNPV p26−1, SeMNPV p26-2, and M. mediaror p26 without a signal peptide sequence were amplified from viral genomes. The PCR products were cloned into pMT-BiP/V5-HisA vector. The plasmids were transfected into Drosophila S2 cells along with pCoHygro hygromycin selection vector, and stable cell lines were screened according to the manufacturer’s instruction of DES Inducible/ Secreted Kit (Invitrogen, K413001)¹⁶. The cell supernatants containing recombinant proteins were harvested and loaded onto a nickel-charged resin and eluted with imidazole. The eluted samples were concentrated for further purification using a Superdex-200 column. The purified proteins were concentrated to 10 to 20 mg/mL and stored at −80 °C before use.

PO activity assay

PO activity of infected hemolymph was determined as previously described¹⁵. Briefly, 1 μL cell-free hemolymph was added to a 200-μL substrate solution (2 mM dopamine in 50 mM sodium phosphate buffer, pH 6.5) in 96-well plates. PO activity was determined by measuring the absorbance at 470 nm with a microplate reader (Synergy H1, BioTek). The values of PO activity were obtained via Gen5 software (BioTek). One unit of PO activity was defined as ΔA470 of 0.001 in one minute.

To analyze the inhibitory effect of PO activation by P26/poxin homologs against naive hemolymph, cell-free hemolymph from healthy third instar H. armigera or S. frugiperda larvae was collected. Hemolymph (1 μL) was added to 9 μL of recombinant proteins (HearNPV P26 containing twin-strep tag, HearNPV P26^S56A, HearNPV P26^D58A, HearNPV P26^H110A, HearNPV P26^{S56A/H110A/D58A}, HearNPV P26^H66A, HearNPV P26^H66D/H67D, HearNPV P26^D241H/E243H, HearNPV P26^{H66D/H67D/D241H/E243H}, VACV poxin, AcMNPV P26, SfMNPV P26-1, SfMNPV P26-2, SeMNPV P26-1, SeMNPV P26-2, or M. mediaror P26) expressed in Drosophila S2 cells at final protein concentrations of 0, 1.5, 3, 4.5, and 6 mg/mL (for H. armigera) or 0, 3, 6, 9, and 12 mg/mL (for S. frugiperda). The reaction mixtures were incubated on ice for ~40 min. Then, the 10-μL reaction mixtures were subjected to the PO activity assay. BSA was used as the negative control. Each test was performed three times. Statistical analysis was performed using two-way analysis of variance (ANOVA).

Time-course analysis of HearNPV P26

Cell-free hemolymph was collected from H. armigera larvae infected with vHaBac-egfp-ph at 0, 12, 24, 36, 48 and 72 h p.i., treated with SDS-PAGE loading buffer (50 mM Tris-HCl, 2% SDS, 0.1% bromophenol blue, 10% glycerol, 5% β-mercaptoethanol) and heated at 95 °C. Proteins were separated on a 12% SDS-PAGE and western blotting was carried out using primary polyclonal-antibodies against HearNPV P26, H. armigera HSP27.2¹⁵, and HRP-conjugated secondary antibody (Affinity, S0001). All the antibody dilutions are 1:5000, and the scans were obtained using Gel Capture Microchem (DNR Bio Imaging System).

To generate antisera against HearNPV P26, the prokaryotically expressed recombinant HearNPV P26 proteins were used for rabbit immunization. The animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Wuhan Institute of Virology, Chinese Academy of Sciences (ethics number: WIVA01201601).

Quantitative PCR (qPCR) analysis of genomic DNA copy numbers in hemolymph

The cell-free hemolymph from third instar larvae (n = 5) infected with vHacBac-egfp-ph, vHacBacΔp26-egfp-ph, and vHacBacRep26-egfp-ph were collected, and viral genomic DNA was isolated. Cell-free hemolymph (10 μL) was mixed with 90 μL H₂O and incubated with 100 μL 20% PEG8000 (Sigma, V900156) in 1 M NaCl for 30 min. The virions were then spun down at 12,000 × g, 4 °C for 15 min and resuspended in 20 μL H₂O. Virions were lysed by adding 80 μL virus disruption buffer (10 mM Tris-HCl pH 7.5, 10 mM EDTA, 0.25% SDS) and 5 μL proteinase K (20 mg/mL; Sigma, P2380). Virions were lysed at 50 °C for 1 h. The viral DNA was extracted with phenol, precipitated with ethanol, and dissolved in 10 μL H₂O. The virus loads in hemolymph were determined via qPCR analysis of viral DNA using primers of ha39 (ha39-F: 5′-GAAATGCGAATCAGACAGATTACTCG-3′ and ha39-R: 5′-CGCAACCTAACATTTGAGAACACAC-3′), as described previously⁴². Each test was performed twice. Data was collected using CFX manager (Bio-Rad). Results were analyzed statistically via two-tailed Student’s t test.

Pull-down assay

Purified HearNPV P26 with twin-strep tag expressed by S2 cells was incubated with Strep-Tactin XT IBA beads at 4 °C for 4 h. Then, the beads were washed five times, incubated with proSP41, procSP1, procSP6, procSPH11, or procSPH50 at 4 °C overnight. After extensive washes, the beads were resuspended in SDS-PAGE loading buffer. The samples were heated at 95 °C and then centrifuged at 12,000 × g for 10 min. The supernatant was subjected to western blotting as described above, using primary antibodies against HearNPV P26, V5 tag (Abcam, ab15828) and HRP-conjugated secondary antibody. All the antibody dilutions are 1:5000. The scans were obtained using Gel Capture Microchem (DNR Bio Imaging System).

In vitro determination of the inhibitory step in the melanization pathway

To identify the target of HearNPV P26, a biochemically reconstituted pathway was used¹⁶. Using procSPH11 and procSPH50 as an example, 3 µM procSPH11 and procSPH50 were mixed with 0.8 mg/mL HearNPV P26 and incubated overnight at 4 °C. Concurrently, the upstream protease procSP6 was sequentially activated by Factor Xa-proSP41xa-procSP1. Next, the activated SP6 was added to the mixture and incubated at 27 °C for 1 h. Then, 3 µM proPO was added to the mixture before determining PO activity. The rest of the groups, namely, proSP41_Xa, procSP1, and procSP6, were individually mixed with HearNPV P26 and similarly assayed for PO activity. Each test was performed three times. Results were analyzed statistically via two-tailed Student’s t test.

To detect the cleavage of procSPHs, 3 µM procSPH11 or 3 µM procSPH50 was incubated with HearNPV P26, overnight at 4 °C. Then, 3 µM of activated cSP6_Xa was added to the mixture, incubated at 27 °C for 1 h, and subjected to western blotting using primary polyclonal-antibodies against HearNPV P26, procSPH11, procSPH50 and HRP-conjugated secondary antibody. The antibody dilutions are 1:5000. The scans were obtained using Gel Capture Microchemi (DNR Bio Imaging System).

Statistics

All of the analyses and data plotting were performed using GraphPad Prism v8.0.2 software. A two-tailed Student’s t test or two-way ANOVA test was applied to to calculate the significance as indicated in each figure legend. The bioassay (Supplementary Table 3) was analyzed using SPSS Statistics v22 software. The LC₅₀ values were calculated using a POLO probit analysis and was compared via the lethal dose ratio method. The ST₅₀ values were calculated using a Kaplan-Meier estimator and further compared applying a Log-rank test. A P value <0.05 was considered statistically significant. Error bars of all figures represent as mean ± SEM of two or three independent experiments.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The data generated in this study are available from the corresponding authors upon request. Atomic coordinates and structure factors for the crystal structure generated in this study have been deposited in the Protein Data bank (https://www.rcsb.org) under PDB code 7WN7. Previously published crystal structures used in this study are available from the PDB codes 6EA9, 6XB3, 6XB5 and 5GPI. The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium with the dataset identifier PXD037784. Source data are provided with this paper.

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Acknowledgements

This research was supported by the grants from the National Natural Science Foundation of China (No. 32170156 to Z.H.), the Strategic Priority Research Program of the Chinese Academy of Sciences (grant XDB11030400 to Z.H. and XDPB16 to Z.Z.), Hubei Natural Science Foundation for Distinguished Young Scholar (2021CFA050 to M.W.) and the Young Top-notch Talent Cultivation Program of Hubei Province to M. W. We thank Mr. Xijia Liu for antibody preparation; Dr. Guoliang Lu and Dr. Bo Shu for their help during X-ray diffraction data collection; Dr. Guiqing Peng for kindly providing the SeMet reagent; Dr. Qiangqiang Han from SpecAlly Life Technology Co., Ltd. for his assistance with MS data analysis. We thank staff of BL17U1 and BL18U1, Shanghai Synchrotron Radiation Facility and the Center for Instrumental Analysis and Metrology of Wuhan Institute of Virology for their technical support.

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These authors contributed equally: Mengyi Yin, Wenhua Kuang.

Authors and Affiliations

State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China
Mengyi Yin, Wenhua Kuang, Qianran Wang, Xi Wang, Chuanfei Yuan, Huanyu Zhang, Fei Deng, Peng Gong, Zhihong Hu & Manli Wang
University of Chinese Academy of Sciences, Beijing, China
Mengyi Yin & Zhen Zou
State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
Zhe Lin & Zhen Zou
Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK, USA
Haobo Jiang

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Contributions

M.Y., M.W., Z.H., Z.Z., and P.G. designed the experiments and supervised the study; M.Y., and W.K. conducted most of experiments with the assistance from Q.W., X.W., C.Y., Z.L., H. Z., and F.D.; M.Y., W.K., and M.W. wrote the draft; Z.H., Z.Z. H.J., and P.G., revised the draft. All the authors read and approved with the final paper.

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Correspondence to Peng Gong, Zhen Zou, Zhihong Hu or Manli Wang.

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Yin, M., Kuang, W., Wang, Q. et al. Dual roles and evolutionary implications of P26/poxin in antagonizing intracellular cGAS-STING and extracellular melanization immunity. Nat Commun 13, 6934 (2022). https://doi.org/10.1038/s41467-022-34761-0

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Received: 20 April 2022
Accepted: 07 November 2022
Published: 14 November 2022
DOI: https://doi.org/10.1038/s41467-022-34761-0

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