Amino acid profiling of the BmNPV budded virus (BV)

To understand the amino acid composition of BmNPV, we purified and assayed BmNPV-eGFP virions according to the flowchart in Fig 1A. When BmN cells were infected with BmNPV-eGFP, intense green fluorescence was observed after 72 h (Fig 1B), indicating successful viral propagation and protein expression. The culture medium of infected BmN cells was used for BmNPV purification by ultracentrifugation using a discontinuous sucrose gradient. It was observed that the BmNPV virus particles in the samples were intact and that the background was relatively free of impurities (Fig 1C). The size of the purified BmNPV virions was consistent with the literature (typically 40 nm–60 nm in diameter and 240 nm–390 nm in length [22] (Fig 1C). Targeted amino acid metabolism analysis of BmNPV-eGFP virions showed that the 10 most abundant amino acids in BmNPV included arginine, lysine, proline, isoleucine, histidine, leucine, valine, aspartic acid, glutamic acid, and phenylalanine which were all essential amino acids for silkworms (Fig 1D). Most notably, arginine is the most abundant amino acid in BmNPV BV virus particles (Fig 1D).

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Fig 1. Amino acid composition content of BmNPV.

(A) Flow chart of BmNPV purification and amino acid detection. (B) Green fluorescence was observed 72 h after BmN cells were infected with BmNPV-eGFP. Scale bar: 100 μm. (C) Examination of purified BmNPV using transmission electron microscopy (TEM). Red arrows: Purified virus particles (BV) (D) Amino acid composition content of BmNPV.

https://doi.org/10.1371/journal.ppat.1013331.g001

BmNPV reprograms amino acid metabolism in the silkworm and arginine promotes BmNPV proliferation

To understand the dynamic changes of amino acid abundance in the hemolymph of BmNPV-infected silkworms, BmNPV-eGFP was injected into silkworm larvae and hemolymph samples were collected at 24 and 72 hpi. Subsequently, the expression level of viral capsid gene vp39 in the hemocyte samples was detected by RT-PCR successfully (Fig 2A). Heatmap visualization from targeted amino acid metabolomics was used to analyze amino acid changes in hemolymph samples, which showed that most of them exhibited a decreasing trend at 24 hpi and an increasing trend at 72 hpi compared to the control (Fig 2B).

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Fig 2. BmNPV reprograms amino acid metabolism in the silkworm and arginine promotes BmNPV replication in BmN cells.

(A) Hemocytes were collected at 24 h and 72 h after BmNPV injection into the silkworm and the viral gene vp39 was detected using RT-PCR using TIF4A as a reference gene. 1-3: Hemocytes infected with BmNPV for 24 h; 4-6: Hemocytes uninfected with BmNPV for 24 h; 7-9: Hemocytes infected with BmNPV for 72 h; 10-12: Hemocytes uninfected with BmNPV for 72 h. (B) Targeted amino acid metabolomics was used to analyze changes in amino acid abundance in the hemolymph of the silkworm at 24 h and 72 h after BmNPV infection. (C-G) Changes of arginine (C), methionine (D), phenylalanine (E), valine (F), and tryptophan (G) in the hemolymph of BmNPV-infected silkworms at 24 and 72 h. (H) Changes of arginine content in the hemolymph of BmNPV-infected silkworms, at 24 and 72 hpi. (I, J) The concentration of arginine in BmN cells (I) and BmN cell supernatants (J) was measured at 24 h and 72 h after BmNPV infection of BmN cells. (K, L) After pretreatment of BmN cells with 3 mM arginine for 12 h and incubation of cells with 1 MOI of BmNPV, cell and supernatant samples were collected at 24, 48, and 72 hpi. The mRNA level of the viral gene vp39 was detected using qPCR (K) and the viral TCID₅₀ was determined (L). Each bar represents the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, not significant. NC, negative control.

https://doi.org/10.1371/journal.ppat.1013331.g002

The levels of essential and non-essential amino acids during BmNPV infection were analyzed by targeted metabolomics. The levels of arginine (Fig 2C), methionine (Fig 2D), phenylalanine (Fig 2E), valine (Fig 2F), and tryptophan (Fig 2G) increased significantly in the hemolymph at 72 hpi after BmNPV infection. However, methionine was not detected in the amino acid targeted metabolome analysis of BV amino acid content (Fig 1D). In addition, the contents of most of the nonessential amino acids such as alanine, tyrosine, glycine, serine and 3-phospho-serine also increased significantly at 72 hpi, with sarcosine, N-acetyl-L-alanine and hydroxyisocaproic acid as exceptions (S1 Fig). These results suggested that BmNPV infection could induce reprogramming of host amino acid metabolism to facilitate its own proliferation.

Arginine had the highest concentration in BmNPV virions (Fig 1D), and there was a significant change in the arginine content in the hemolymph after viral infection (Fig 2C). Therefore, arginine was selected for further analysis of its regulation during infection. The detection of arginine in the hemolymph of BmNPV-infected and uninfected silkworms by biochemical assay showed that the arginine content in the hemolymph of BmNPV-infected silkworms was significantly reduced at 24 hpi, but significantly increased at 72 hpi (Fig 2H). This result was consistent with the results of amino acid targeted metabolomics analysis in the hemolymph (Fig 2C).

Similarly, in BmNPV-infected BmN cells, the arginine content decreased significantly at 24 hpi and increased significantly at 72 hpi (Fig 2I). By contrast, the arginine content in the cellular medium was significantly decreased at 72 hpi (Fig 2J). Next, to explore the effect of arginine on BmNPV replication, arginine was added to the culture medium and the cells and supernatants were collected at 24, 48 and 72 h, respectively, to detect the replication of BmNPV. The results showed that the addition of arginine significantly increased the mRNA level of vp39 (Fig 2K) as well as the viral titer of BmNPV, as measured by the TCID₅₀ assay (Fig 2L). The same experiment also confirmed that another constituent amino acid (top 12) of BV, serine (Fig 1D), could promote BV proliferation in BmN cells at 48 and 72 hpi (S2A and S2B Fig). These results suggested that the constituent amino acids of virions could indeed promote the proliferation of the virus.

Arginine transporter Slc7a6 increases intracellular arginine intake and promotes BmNPV proliferation

As an essential amino acid for silkworms, arginine cannot be synthesized at sufficient quantities and therefore needs to be assimilated from the food. We therefore analyzed the amino acid transporters of Bombyx mori using the previously determined hemocyte transcriptome [19] and found that the amino acid transporter gene Slc7a6 was significantly up-regulated in BmNPV-infected hemocytes. Slc7a6 is capable to function as a transporter of arginine (Fig 3A) [23]. To further explore the response of Slc7a6 to virus infection, the expression level of Slc7a6 was determined in BmNPV-infected BmN cells, which showed a significant up-regulation at 48 and 72 hpi (Fig 3B). In fat body, the expression of Slc7a6 was significantly induced by BmNPV infection at 72 hpi (Fig 3C). In addition, we found that Slc7a6 expression was significantly up-regulated in BmNPV-infected hemocytes at 24, 48 and 72 hpi (Fig 3D). Obviously, the response of the Slc7a6 to viral infection implied its potential role in the process of BmNPV replication through arginine transport (Fig 3A-D). Therefore, the effect of Slc7a6 on arginine content inside and outside the cell, and BmNPV replication was further verified after overexpression of Slc7a6 in BmN cells.

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Fig 3. BmNPV infection induces significant up-regulation of Slc7a6 to promote self-replication.

(A) Schematic diagram of amino acid transport by Slc7a6. Slc7a6 participates in the transport of cationic and large neutral amino acids. (B-D) BmN cells (B), fat body tissue (C), and hemocytes (D) were collected at 24, 48, and 72 h after infection with BmNPV, and the expression levels of the Slc7a6 gene were detected by qPCR. (E) BmN cells were seeded on a 24-well plate, and when the cell density reached 70-80%, 500 ng of pIEX-Slc7a6-V5 was transfected into BmN cells, and the same concentration of pIEX served as a control. Cell samples were collected at 24, 48, 72 and 96 h after transfection. The expression level of Slc7a6 was detected by Western blot using V5 antibody. (F, G) After pIEX-Slc7a6-V5 was transfected into BmN cells, cells (F) and supernatants (G) were collected at 24, 48, 72, and 96 h and assayed for the concentration of arginine. (H, I) BmN cells were transfected with pIEX-Slc7a6-V5 for 24 h. After 24 h, BmN cells were incubated with 1 MOI of virus for 1 h and then washed and cultured with fresh Grace’s medium, and cell samples were collected at 24, 48, and 72 hpi. The mRNA level of the viral gene vp39 (H) and the viral DNA load (I) were detected respectively. (J) Detection of viral titers using the TCID₅₀ assay. Each bar represents the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, not significant.

https://doi.org/10.1371/journal.ppat.1013331.g003

Western blot analysis showed that Slc7a6-V5 was successfully expressed in BmN cells (Fig 3E), which was accompanied by a significant increase in the intracellular arginine content at 72 and 96 h post-transfection (Fig 3F). Conversely, the arginine content in the cellular medium was significantly reduced at 72 and 96 h post-transfection (Fig 3G). When BmN cells were infected with BmNPV at 24 h after transfection of Slc7a6, it was observed that the mRNA expression level of the viral gene vp39 (Fig 3H), the viral DNA load (Fig 3I), and the titer of the virus (Fig 3J) were both significantly up-regulated at 72 hpi. These results suggested that overexpression of Slc7a6 could enhance the cellular uptake of arginine to benefit BmNPV proliferation.

Knocking down of Slc7a6 reduces intracellular arginine content and inhibits BmNPV replication

After transfection of dsRNA-Slc7a6, BmN cell samples showed significant suppression of Slc7a6 mRNA compared with the control group (Fig 4A), together with a significant drop in intracellular arginine content and a significant increase in extracellular arginine in the cellular medium at 72 and 96 h after transfection of dsRNA-Slc7a6 (Fig 4B and 4C). Moreover, knockdown of Slc7a6 in BmN cells and subsequent infection with BmNPV revealed a significant decrease in virus vp39 expression (48 and 72 hpi), viral DNA load (72 hpi), and viral titer (72 hpi) in the later stages of infection (Fig 4D, 4E and 4F).

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Fig 4. Knockdown of Slc7a6 reduces intracellular arginine content and inhibits BmNPV replication.

(A) BmN cells were seeded on a 24-well culture plate, 5 μg of dsRNA-Slc7a6 was transfected into BmN cells, and cell samples were collected at 24, 48, 72, and 96 h to detect the knockdown efficiency of Slc7a6. (B, C) After transfection of BmN cells with the same concentration of dsRNA-Slc7a6, cell (B) and supernatant (C) fractions were harvested at 24, 48, 72, and 96 h post-transfection, respectively, to assay arginine concentration. (D, E) At 24 h after BmN cells were transfected with dsRNA-Slc7a6, BmN cells were incubated with 1 MOI of BmNPV for 1 h. Subsequently, the cells were washed with PBS and incubated with fresh Grace’s medium. At 24, 48 and 72 hpi, cell samples were collected and the mRNA level of viral gene vp39 (D) and the viral DNA load (E) were detected by qPCR. (F) Supernatants were collected 24, 48 and 72 h after BmNPV infection of cells and viral titers were assayed using the TCID₅₀ assay. (G) Sequence analysis of the targeted sequence in WT and Slc7a6^KO BmN cell lines. (H, I) Of the Slc7a6^KO and WT BmN celllines, cells (H) and the corresponding supernatants (I) were collected to assay the arginine content. (J, K) Slc7a6^KO cells were inoculated in 24-well cell culture plates and subsequently infected with 1 MOI of BmNPV virus. At 24, 48 and 72 hpi, cell samples were collected and tested for mRNA levels of the viral gene vp39 (J) and the viral DNA load (K) using qPCR. (L) Detection of supernatant titers 24, 48 and 72 h after BmNPV infection of Slc7a6^KO cells using the TCID₅₀ assay. Each bar represents the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, not significant.

https://doi.org/10.1371/journal.ppat.1013331.g004

To further validate the effect of Slc7a6 on arginine content as well as BmNPV proliferation, Slc7a6^KO BmN cells were generated by CRISPR/Cas9 technology (Figs 4G, S3A and S3B). After transfection of pSL1180-Cas9-U6-Slc7a6, surviving BmN cells were selected by Zeocin and further analyzed for expression levels of Slc7a6 (S3A and S3B Fig). Sequence analysis showed that the ORF of Slc7a6 was successfully deleted by 2–16 nucleotides (Fig 4G), presumably corresponding to different cellular subpopulations. Additionally, the cell viability showed no difference from that of normal cells (S3C Fig).

When analyzed for intracellular arginine content, its levels were found to be significantly reduced in the Slc7a6^KO cell line compared with the control group (Fig 4H), while the arginine content in the cellular medium of the Slc7a6^KO group was significantly up-regulated (Fig 4I). Obviously, the absence of Slc7a6 significantly affected the cellular uptake of arginine. As observed for silencing of Slc7a6 with dsRNA, deficiency in intracellular arginine inhibited BmNPV infection in Slc7a6^KO cells, illustrated by quantification of vp39 mRNA, viral DNA load assessment, and viral titer measurements (Fig 4J, 4K and 4L). The difference is that in Slc7a6^KO cells, each detection time point of 24, 48 and 72 hpi showed significant inhibition of virus proliferation, rather than 48 or 72 hpi in RNAi knockdown cells (Fig 4D,4E,4F,4J, 4K and 4L). These results suggest that inhibition of Slc7a6 expression attenuated cellular uptake of arginine and inhibited BmNPV proliferation.

Autophagy rescues intracellular amino acid content and promotes BmNPV proliferation in Slc7a6^KO cells

To investigate the role of autophagy in amino acid metabolism during BmNPV infection, Slc7a6^KO cells were used to minimize the influence of exogenous arginine uptake. We first confirmed that BmNPV infection could also induce autophagy in Slc7a6^KO cells (Fig 5A and 5D). That is, 48 and 72 h after BmNPV infection, the conversion of ATG8 to ATG8-PE was enhanced in Slc7a6^KO cells (Fig 5A and 5D). Next, the autophagy inhibitor TSA and autophagy inducer C646 were used to treat the Slc7a6^KO cells. Using toxicity assays, the optimal concentrations of TSA (S4A Fig) and the C646 (S4B Fig) were determined as 4 nM and 100 nM, respectively. Subsequently, BmNPV-infected Slc7a6^KO cells were treated with TSA or C646, and the occurrence of autophagy in the cells was examined. The results showed that TSA was able to inhibit BmNPV-induced conversion of ATG8 to ATG8-PE in Slc7a6^KO cells (Fig 5B and 5E), indicating that TSA suppressed BmNPV - induced autophagy in Slc7a6^KO cells. Conversely, the use of C646 enhanced autophagy (Fig 5C and 5F). Next, the concentration of arginine in the cells was assayed following treatment with TSA (Fig 5G) or C646 (Fig 5K). Treatment of uninfected Slc7a6^KO cells with TSA or C646 had no effect on intracellular arginine content compared to untreated controls (Fig 5G and 5K). On the other hand, after 24 h of infection with BmNPV in Slc7a6^KO cells, the intracellular arginine was significantly decreased (Figs 5G, 5K and S3D), similar to wild-type BmN cells (Fig 2I). After infection with Slc7a6^KO cells for 48 and 72 h, the levels of arginine in the Slc7a6^KO cells were replenished, especially at 72 hpi, which was basically the same as the control group (Figs 5G, 5K and S3D). However, unlike the significant upregulation of intracellular arginine in wild-type BmN cells at 72 hpi (Fig 2I), there was no upregulation of intracellular arginine in Slc7a6^KO cells after 72 h of BmNPV infection, which may be due to the inability of Slc7a6^KO cells to take up arginine from the culture medium (Figs 5G,5K and S3D). These results indicated that during infection of Slc7a6^KO with BmNPV at 48 and 72 hpi, certain mechanisms within the cells restored amino acid balance, which is likely due to virus induced autophagy. Treatment with autophagy inducers and inhibitors confirmed this assumption. After treating BmNPV - infected cells with the autophagy inhibitor TSA, the intracellular arginine content decreased significantly (Fig 5G). Conversely, treating Slc7a6^KO cells with the autophagy inducer C646 led to a significant increase in intracellular arginine levels at 24–72 h post - BmNPV infection, compared to untreated controls (Fig 5K). Moreover, the autophagy inhibitor TSA was found to inhibit BmNPV proliferation in Slc7a6^KO  cells (Fig 5H, 5I and 5J) while autophagy inducer C646 could promote of BmNPV proliferation (Fig 5L, 5M and 5N). These results suggested that BmNPV infection induced cells to initiate autophagy, which in turn replenishes the intracellular arginine required to maintain their proliferation.

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Fig 5. Effect of autophagy on amino acid content and BmNPV replication in Slc7a6^KO cells.

(A) Western blotting was used to detect ATG8-PE in Slc7a6^KO cells after BmNPV infection. (B, C) After pretreatment of Slc7a6^KO cells with 4 nM TSA (B) or 100 nM C646 (C) for 12 h, the cells were incubated with 1 MOI of BmNPV, followed by incubation of the cells with fresh medium containing 4 nM TSA (B) or 100 nM C646 (C). Cells were collected at 24, 48 and 72 hpi, and Western blotting was used to detect ATG8-PE in Slc7a6^KO cells. (D) Quantification of ATG8-PE in Panel A. (E) Quantification of ATG8–PE in Panel B. (F) Quantification of ATG8-PE in Panel C. (G, K) Uninfected and BmNPV-infected Slc7a6^KO BmN cells were treated with 4 nM TSA (G) or 100 nM C646 (K). Cell samples collected at 24, 48 and 72 hpi were assayed for the concentration of arginine. (H, I) After pretreating Slc7a6^KO cells with 4 nM TSA for 12 h (DMSO was used as a control), the cells were incubated with 1 MOI of BmNPV, followed by incubation with fresh medium containing 4 nM TSA. Cells were collected at 24, 48 and 72 hpi to detect changes in mRNA expression levels of the viral gene vp39 (H) and the viral DNA load (I). (J) Detection of viral titer by TCID₅₀. (L, M) Slc7a6^KO BmN cells were pretreated with 100 nM C646 for 12 h (DMSO was used as a control). At the end of pretreatment, cells were infected with 1 MOI of BmNPV virus and continued in fresh medium containing 100 nM C646. At 24, 48 and 72 hpi, cell samples were collected and examined for changes in mRNA expression levels of the viral gene vp39 (L) and the viral DNA load (M). (N) Detection of viral titer by TCID₅₀. The Western blotting results are representative of one of three independently performed. Each bar represents the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, not significant.

https://doi.org/10.1371/journal.ppat.1013331.g005

Effect of BmNPV-induced mitochondrial autophagy on intracellular arginine levels

Our previous study found that mitochondrial morphology was altered and mitochondrial function was impaired after BmNPV infection of BmN cells [24]. Furthermore, we used the Mito-QC system to detect whether BmNPV infection leads to mitochondrial autophagy. The results showed that the mitochondrial green fluorescent signal was significantly reduced in BmNPV-infected wild-type BmN cells compared with normal BmN cells (S5A and S5B Fig), indicative of quenching of green fluorescence that occurs during delivery to lysosomes by mitophagy [25]. Furthermore, the Mito-QC system confirmed that BmNPV infection of Slc7a6^KO-non-eGFP cells also induces mitophagy (Fig 6A and 6B).

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Fig 6. Supplementation of intracellular arginine by mitochondrial autophagy benefits BmNPV replication.

(A) The Mito-QC reporting system detects mitochondrial autophagy in BmNPV-infected Slc7a6^KO-non-eGFP cells. Scale bar: 100 μm and 10 μm. (B) Calculation of green fluorescence intensity of mitochondria in Slc7a6^KO-non-eGFP cells using FiJi software. (C) Slc7a6^KO cells were pretreated with 30 nM of CSA for 12 h (DMSO was used as a control) followed by infection with 1 MOI of BmNPV. Cell samples were collected at 24, 48, and 72 hpi to assay for arginine content. (D, E) Slc7a6^KO cells were pretreated with CSA for 12 h and Slc7a6^KO cells followed by infection with 1 MOI of BmNPV. Cell samples were collected at 24, 48 and 72 hpi to detect the mRNA level of the viral gene vp39 (D) and the viral DNA load (E), respectively. (F) The supernatant of CSA-treated BmNPV-infected cells at 24, 48, and 72 h was collected for viral titer detemination using the TCID₅₀ method. Each bar represents the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant. (G) Overview of the mechanisms by which BmNPV maintains intracellular arginine levels to benefit self-replication. Panel a display the processes in silkworm larvae: both hemocyte and fat body cell disruption because of BmNPV infection and nutrient transport from the gut epithelium after feeding contribute to the increase of amino acid levels in the hemolymph. Panel b shows the regulation of the levels of the amino acids in individual cells: induction of mitochondrial autophagy and transmembrane transport of by Slc7a6 result in an increase of cytosolic arginine levels.

https://doi.org/10.1371/journal.ppat.1013331.g006

To investigate whether mitochondrial autophagy affects intracellular arginine content, we used the mitochondrial autophagy inhibitor, Cyclosporine A (CSA) [26]. At the optimal CSA concentration (30 nM; S4C Fig), the content of arginine was significantly reduced in the Slc7a6^KO cells at 48 and 72 hpi (Fig 6C). More importantly, after treating  Slc7a6^KO cells with mitochondrial autophagy inhibitor CSA, the proliferation of BmNPV showed a significant decrease in the middle and late stages of infection at the level of vp39 expression (Fig 6D), the viral DNA load (Fig 6E), and the viral titer (Fig 6F).

These results suggested that BmNPV induces cellular mitochondrial autophagy to replenish intracellular arginine, thereby favoring its replication.

Cell line, silkworm strain, expression plasmid and recombinant BmNPV

The silkworm BmN cell line was cultured in Grace’s medium, supplemented with 10% Fetal Bovine Serum (FBS, Gibco, USA), at 28°C in a humidified incubator. Silkworm larvae (Bombyx mori, Dazao P50 strain) were reared under conditions of 28°C and 70–80% relative humidity. The over-expression plasmid pIEX-his was obtained from Wuhan Miaoling Biotech Co., Ltd. The recombinant BmNPV-eGFP virus was stored in the Key Laboratory of Agricultural Genomics and Molecular Breeding in Guangdong Province.

Targeted metabolomics: sample preparation and detection

Silkworms were injected with 10 μL of BmNPV-eGFP (TCID₅₀ = 10^5.8/0.1 mL) at day 1 of the 5th instar, while the control group was injected with an equivalent volume of sterile water. At 24 and 72 hours post-infection (hpi), hemolymph samples from both the treatment and control groups were collected and processed following the method described by Feng et al. (2021a), with each sample comprising hemolymph from six silkworms and each group having three replicates [19]. Subsequently, the supernatant samples (after removal of hemocytes) were rapidly frozen in liquid nitrogen and stored at -80°C. After the successful infection of the samples with BmNPV was confirmed by RT-PCR, targeted metabolomics analysis was subsequently performed on the hemocytes. Specifically, 100 μL of hemolymph was aliquoted into 2 mL centrifuge tubes, followed by the addition of 800 μL of 80% methanol containing an internal standard at a concentration of 100 ng/mL. The mixture was vortexed for 1 min, homogenized for 5 min, and sonicated at 4°C for 30 min. Thereafter, the samples were incubated at 60°C for 10 min and centrifuged at 12,000 rpm for 10 min. The supernatant obtained post-centrifugation was used for subsequent instrumental analysis. Targeted metabolomics sequencing analysis was conducted by Gene Denovo Biotechnology Co. (Guangzhou, China).

Ultra-high performance liquid chromatography mass spectrometry (UPLC-MS)

UPLC-MS analysis was conducted using a Waters UPLC BEH Amide chromatographic column (1.7 µm, 2.1 mm × 100 mm) interfaced with a mass spectrometry system. The mobile phase was composed of water (solvent A) and acetonitrile (solvent B). The elution gradient was as follows: from 0 to 1 minute, 85% B; from 1 to 9 min, a linear gradient from 85% to 55% B; from 9 to 10 min, isocratic at 55% B; from 10 to 11 min, a linear gradient from 55% to 85% B; and from 11 to 12 min, isocratic at 85% B. The column temperature was maintained at 40°C, and the sample injection volume was set to 6 μL.

For the mass spectrometry conditions, electrospray ionization (ESI) was employed. The curtain gas flow rate was set at 35 arbitrary units (arb), the collision gas flow rate at 7 arb, the ion spray voltage at 4500 V, and the ion source temperature at 450°C. The flow rates for both ion source gas 1 and ion source gas 2 were maintained at 55 arb. Then, the amino acids in the samples were identified according to the chromatographic and mass spectrometric conditions of each amino acid standard (Shanghai yuanye Bio-Technology, China), and the content of the amino acids was calculated.

Determination of the amino acid content in BmNPV

BmN cells were evenly seeded onto 10 cm² cell culture dishes and infected with BmNPV-eGFP at a multiplicity of infection (MOI) of 1. At 72 hpi, after successful observation of cell fluorescence, 300 mL of cell supernatant was collected for virus purification. The purification of BmNPV was achieved through discontinuous sucrose gradient ultracentrifugation at 45,000 g for 6 hours at 4°C. The purified BmNPV samples were examined by transmission electron microscopy to confirm their quality before proceeding to amino acid composition analysis. Briefly, the samples were ground into a powder under liquid nitrogen and then lyophilized. To the lyophilized samples, 1.5 mL of 6 M HCl was added, and the samples were hydrolyzed at 110°C for 24 h. After cooling to room temperature, the pH was adjusted to neutrality with a sodium hydroxide solution, and the volume was made up to 1 mL. The resulting solution was filtered through a 0.22 μm aqueous filter membrane and stored at -20°C until analysis on the instrument.

The analysis was conducted using a high-performance liquid chromatography (HPLC) system (Agilent 1260) interfaced with a triple quadrupole mass spectrometer (Agilent 6420A). The mobile phase consisted of water adjusted to pH 3 (solvent A) and 90% acetonitrile (solvent B). The chromatographic separation was achieved with the following gradient elution program: 95% B from 0 to 0.01 min; a linear gradient from 95% to 80% B from 0.01 to 4 min; isocratic at 80% B from 4 to 6 min; a linear gradient from 80% to 55% B from 6 to 12 min; a linear gradient from 55% to 95% B from 12 to 12.1 min; and isocratic at 95% B from 12.1 to 17 min. The sample injection volume was 1 μL, and the detection wavelength was set at 254 nm. For the mass spectrometric analysis, electrospray ionization (ESI) in positive ion mode with multiple reaction monitoring (MRM) was employed. The source parameters included a gas temperature of 350°C, a gas flow rate of 10 L/min, a nebulizer pressure of 45 pounds per square inch (psi) and capillary voltages of 4000 V for positive ions and 3500 V for negative ions. Then, the amino acids in the samples were identified according to the chromatographic and mass spectrometric conditions of each amino acid standard (biovina biotech, China), and the content of the amino acids was calculated.

Effect of exogenous amino acids on BmNPV replication

L-Arginine and L-Serine were procured from Sigma-Aldrich, USA. These amino acids were individually dissolved in insect Grace medium. BmN cells at a density of 1.25 × 10⁵ cells were seeded in 24-well plates and left to attach. BmN cells were infected with 1 MOI of BmNPV for 1 h at 28°C. The supernatant was then removed and replaced with fresh insect Grace medium containing 10% FBS, supplemented with either serine (5 mM) or arginine (3 mM). The time at which the supernatant was replaced was designated as 0 hpi. Cells and supernatants were collected at 24, 48, and 72 hpi. Targeting the viral capsid gene vp39, the intracellular viral mRNA level was quantified using qPCR, while 50% endpoint viral titers in the supernatant were determined using the Reed-Muench method.

Overexpression and RNA interference (RNAi)

To assess the impact of the amino acid transporter Slc7a6 (BMSK0000671/ XM_004924899.4) on the replication of BmNPV, we modulated its expression by overexpression and RNAi experiments. Initially, primers for double-stranded RNA (dsRNA) production targeted at Slc7a6 were designed and synthesized (S1 Data) and dsRNA was produced using the T7 RiboMAX Express RNAi System. Subsequently, BmN cells were seeded in 24 well cell culture plates and 5 μg of dsRNA-Slc7a6 was transfected into BmN cells using FuGENE HD Transfection Reagent (Promega, USA) according to the manufacturer’s protocol, while dsRNA-dsRed was used as a control. Cell samples were collected at 24, 48, 72, and 96 h post-transfection to detect the mRNA expression levels of Slc7a6. Finally, after transfection of BmN cells with the same concentration of dsRNA-Slc7a6 for 24 h, the cells were infected with 1 MOI of BmNPV. After collecting cell samples at 24, 48, and 72 hpi, both the relative mRNA expression levels of BmNPV, the viral DNA load and the viral titer were assessed.

To overexpress Slc7a6 in BmN cells, we constructed the overexpression vector pIEX-Slc7a6-V5 (Primers in S1 Data). BmN cells were seeded in 24-well plates, and then 500 ng of the overexpression vector pIEX-Slc7a6-V5 or the empty vector pIEX were transfected into the BmN cells using FuGENE HD reagent. The cells were collected at 24, 48, 72, and 96 h post-transfection, and the protein expression level of external Slc7a6-V5 was analyzed by western. Subsequently, BmN cells were infected with 1 MOI of BmNPV 24 h after transfection with the same concentration of pIEX-Slc7a6-V5 (empty vector pIEX was used as control). Cell samples were collected at 24, 48 and 72 hpi and the relative mRNA expression level of BmNPV and viral DNA load were determined using qPCR. In addition, 50% endpoint viral titers in the supernatant were determined.

Detection of viral proliferation

To detect the expression level of mRNAs, cell samples were collected from different treatments. Total RNA was then extracted using the Fast2000 kit (Fastagen, China) according to the manufacturer’s instructions. Genomic DNA was removed using the PrimeScript RT kit (TaKaRa, Japan) with gDNA Eraser and RNA was reverse transcribed to obtain cDNA. In order to explore the fold change in gene expression, qPCR was performed with 2 × SYBR qPCR premix (Yeasen Biotechnology, China). The primers for qPCR were listed in S1 Data. All experimental data were obtained using the Bio-Rad real-time fluorescence quantitative PCR system and analyzed using the 2^-ΔΔCt method. BmTIF4A was used as the reference gene and detection of the viral gene vp39 was used to evaluate BmNPV replication.

For the determination of the viral DNA load, BmN cell samples from different treatments were collected and total DNA from cell samples was extracted using the SteadyPure Universal Genomic DNA Extraction Kit (Accurate Biology, China). The computational analysis of the viral DNA load was carried out according to the method of Chen et al. (2018). Briefly, qPCR was performed using primers specific for gp41 and 5 ng of DNA as template. The absolute amount of the target gene to assess the BmNPV viral DNA load was calculated by intrapolation into a standard curve obtained by dilutions of an external standard [64].

To measure the viral titer, supernatants from BmNPV -infected cells with different treatments were collected at 24, 48, and 72 hpi. Cells were then seeded into 96 -well plates. After the cells adhered to the plates, the collected supernatant samples were serially diluted by 10-fold at each step. Subsequently, 10 μL of each serially the diluted supernatant was added to wells containing 90 μL of BmN cells for incubation. At 72 hpi, green fluorescence in the wells was observed, and TCID₅₀ was calculated using the Reed-Muench method [65].

Western blot

The cell samples were lysed at 4°C for 30 min in a mixture containing the protease inhibitor phenylmethylsulfonyl fluoride (PMSF, 0.5mM). The lysates were then sonicated and the supernatant was collected by centrifugation. The supernatant was mixed with 5 × SDS loading buffer and incubated at room temperature for 30 min to allow denaturation. The samples were separated by SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking for 1 h, the membrane was incubated with V5 mouse monoclonal antibody (Invitrogen, USA), α-Tubulin Rabbit Monoclonal Antibody (Beyotime Biotechnology, China) or ATG8 rabbit polyclonal antibody (AtaGenix Biologicals Ltd, China) at 4°C overnight. Subsequently, the membrane was washed with 1 × TBST buffer (Tris buffered saline with Tween-20) and incubated with horse radish peroxidase (HRP)-labeled Goat anti-Mouse IgG (H + L) or Goat anti-Rabbit IgG (H + L) (Beyotime Biotechnology, China) at room temperature for 1 h. Finally, the antibody-antigen complexes were visualized using enhanced chemiluminescence (Bio-Rad, USA) and Gel XR gel imager (Bio-Rad, USA).

Construction of knockout cell lines

To further understand the effect of amino acid transporters on BmNPV replication, we constructed a Slc7a6 knockout (KO) BmN cell line using the CRISPR/Cas9 method [66]. Initially, a guide RNA (gRNA) targeting a specific site on the Slc7a6 mRNA was designed utilizing the CRISPRdirect online tool (https://crispr.dbcls.jp/). The synthesized gRNA primers were then annealed to form oligo dimers, which were subsequently inserted into the pSL1180-Cas9-U6-sgRNA vector containing eGFP cassette, Bombyx U6 promoter and gRNA scaffold [67]. The constructed vector, harboring the gRNA targeting the Slc7a6 mRNA site, was transfected into BmN cells. After 72 h, green fluorescence was observed, and cells were selected with 350 mg/L Zeocin. Two weeks later, BmN cells were harvested for the extraction of RNA and genomic DNA. PCR was carried out using primers that flank the CRISPR/Cas9 target site using genomic DNA as template. PCR fragments were subsequently checked for mutations in the Slc7a6 gene. RNA was used for qPCR to determine the mRNA expression levels of Slc7a6. Finally, this cell line was designated as Slc7a6^KO Cell. The Slc7a6 knockout cells used in Mitochondrial autophagy assay were generated using the same method as the Slc7a6^KO cells, but with a knockout plasmid that does not contain the green fluorescent protein eGFP. Therefore, this cell line was designated as Slc7a6^KO-non-eGFP cells. (gRNA sequences and primers shown in S1 Data).

Measurement of arginine concentration in BmN wild-type and Slc7a6^KO cells

The arginine content of cell samples from different treatment groups was determined according to the instructions of the arginine content assay kit (Solarbio, China). Briefly, after counting of the number of cells, cell samples were centrifuged at 500 g. To the cell pellets, 1 mL of extraction reagent I were added, and then ultrasonication was performed at 4°C (power 300 W, ultrasonic 3 s, interval 7 s, 3 min totally). Supernatants of cellular extracts were obtained after centrifugation at 12000 g for 10 min at 4°C. To 800 μL of supernatant, 150 μL of the extraction reagent II was added and mixed slowly followed by re-centrifugation at 12000 g for 10 min at 4°C. Final supernatants were added to the analytical solution for the determination of arginine by absorbance at 525 nm (colorimetric assay).

Regulation of autophagy by treatment of BmN cells with inhibitors/inducers

To investigate whether BmNPV infection of host cells regulates arginine content in host cells through autophagy, we used the autophagy inhibitor Trichostatin A (TSA) (MedChem Express, USA) and the autophagy inducer C646 (MedChem Express, USA), which have been studied to regulate the onset of autophagy in B. mori [24,68]. First, cytotoxicity was assessed using the CCK-8 assay kit (Beyotime Biotechnology, China). Specifically, Slc7a6^KO cells were grown in 96-well plates, followed by treatment of the cells with different concentrations of C646 or TSA for 24 hours. Afterwards, cell culture medium containing 10% CCK-8 reagent was added to the BmN cells and incubated at 28°C for 2 h to determine cell viability. The optimal concentration was considered the highest concentration at which no toxic effects were observed in the cells. The corresponding cells were first pretreated with the drugs for 12 h, and then the cells were incubated with 1 MOI of BV for 1 h. Cells were collected at 24 hpi, 48 hpi and 72 hpi and the expression of ATG8-PE was detected by Western blotting.

To investigate the effect of autophagy changes on the changes of intracellular arginine content and the proliferation level of BmNPV, cells were treated with the optimal concentration of TSA or C646 as described above, and after infection with BmNPV, cells were collected to detect the changes of arginine in the cells and the changes of BmNPV proliferation.

Mitochondrial autophagy assay

Previous studies have shown that BmNPV infection leads to mitochondrial damage [24]. The occurrence of mitochondrial autophagy during BmNPV infection was monitored using the Mito-QC reporting system [25]. In brief, we constructed a recombinant overexpression plasmid, pIEX-mCherry-eGFP-Fis1, which includes the tandem mCherry-eGFP tag fused to the mitochondrial targeting sequence of the silkworm mitochondrial fission 1 protein, Fis1 (BMSK0002319). After transfection of 1 μg of the pIEX-mCherry-eGFP-Fis1 plasmid into cells, cells were infected with BmNPV at 1 MOI. At 72 hpi, green and red fluorescence signals were observed, photographed and compared using a laser confocal microscope (Leica SP8, Germany). Fiji software (version 2.3) was used for green fluorescence statistics. To understand the role of mitochondrial autophagy during BmNPV infection, we used the mitochondrial autophagy inhibitor Cyclosporin A (CSA) (MedChem Express, USA) at the determined optimal concentration (S4C Fig) to perform experiments using the same treatments as TSA and C646.

Statistical analysis

Three independent replications of all experiments were performed (virus titer assay experiments were performed at least twice) to ensure the reliability of the results. The results of the experiments are presented as mean ± standard deviation (Mean ± SD). For analyzing the differences between the different groups, we used GraphPad Prism software (version 9.0) to perform Two-Way ANOVA tests to determine the significance of the variances. If the P-value was less than 0.05, the results were considered statistically significant. For other types of experiments, we used an unpaired t-test to assess statistical significance, again using a P-value of less than 0.05 as the criterion.