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To investigate whether individual ASFV proteins trigger autophagy, we transfected Flag-tagged ASFV E183L, E199L, F317L, MGF 505-4R, CP530R and B602L expression plasmids into Vero cells and used Western blot to measure the conversion of LC3-Ⅰ to LC3-Ⅱ and P62 degradation, which is a general method for evaluating autophagy process (Bjorkoy et al. 2005). As shown in Fig. 1A, transient transfection of E199L protein induced the conversion of LC3-Ⅰ to LC3-Ⅱ and P62 degradation. We carried out densitometry analysis of the density band and the statistical results also confirmed the induction of autophagy (Fig. 1B). In contrast, E183L, F317L, CP530R and B602L proteins did not induce autophagy.
Figure 1. ASFV E199L protein triggers autophagy in Vero and HEK-293T cells. A Vero cells were transfected with empty vectors or various plasmids expressing Flag-tagged ASFV E183L, E199L, F317L, MGF 505-4R, CP530R, B602L proteins and empty vector (EV). At 24 h post-transfection, cells were harvested and Western blotting was performed. Blots are representative of the 3 independent experiments. β-actin was used as sample-loading control. B Densitometric LC3-Ⅱ/LC3-Ⅰ and P62/β-actin ratios from at least 3 independent experiments were shown. Error bars show standard error of the mean (SEM). Significance was analyzed with two-tailed Student's test. ***P < 0.001. C and E Vero and HEK-293T cells were transfected with ASFV E199L protein expression plasmids (E199L) or empty vectors (EV). Cells were harvested at indicated time points (6, 12, 24 and 36 h) and detected with anti-LC3B antibody. Blots are representative of the 3 independent experiments. β-actin was used as sample-loading control. D and F Densitometric LC3-Ⅱ/LC3-Ⅰ ratios from at least 3 independent experiments were shown. Error bars show standard error of the mean (SEM). Significance was analyzed with two-tailed Student's test. *P < 0.05, **P < 0.01, ***P < 0.001. G and H Vero and HEK-293T cells were transfected with ASFV E199L protein expression plasmids or empty vectors. The fluorescent puncta of LC3B were observed by confocal microscopy with scale bars indicating 10 µm.
To confirm autophagy induction by ASFV E199L protein, we measured induction over time after transfection of ASFV E199L protein. The conversion of LC3-Ⅰ to LC3-Ⅱ was monitored at 6, 12, 24, and 36 h post-transfection with empty vector (EV) treatment as a negative control. As demonstrated in Fig. 1C, E199L-transfected Vero cells had significant conversion of LC3-Ⅰ to LC3-Ⅱ since 12 h. To quantify these results, we further used densitometry analysis to measure band intensity. The ratio of LC3-Ⅱ to LC3-Ⅰ was increased at 12, 24, and 36 h in ASFV E199L-transfected cells but not in untreated control cells at the corresponding time points (Fig. 1D). Accordingly, we detected similar phenomena in HEK-293T cells (Fig. 1E, 1F).
To further confirm that the conversion of LC3-Ⅰ to LC3-Ⅱ was indeed related to the induction of autophagy, we further observed the formation of LC3 fluorescent puncta from the morphological point of view. Vero and HEK-293T cells were transfected with pPRK5-Flag-E199L for 24 h. In ASFV E199L-transfected cells, the amount of LC3 fluorescent puncta noticeably increased while a largely dispersed fluorescence distribution was observed in EV-treated groups (Fig. 1G, 1H). Collectively, these data demonstrate that pPRK5-Flag-E199L transfection indeed induced an autophagic response in Vero and HEK-293T cells.
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The accumulation of autophagosomes may be due to autophagy induction or a block in autophagosome maturation (Klionsky et al. 2016). To further explore whether the autophagic response triggered by E199L was a complete process, the expression level of P62 was measured by western blot analysis. As an adaptor of LC3-Ⅱ, P62 was generally considered a marker to asses autophagic flux (Yasui et al. 2016). Compared to EV-treated group, P62 was not significantly degraded at early stages, but significantly decreased at later stages (24 and 36 h) (Fig. 2A, 2B), suggesting that the E199L protein induced complete autophagy. Furthermore, we observed the similar results in HEK-293T cells (Fig. 2C, 2D).
Figure 2. ASFV E199L protein-induced autophagy is a complete process. A and C Vero and HEK-293T cells were transfected with ASFV E199L protein expression plasmids or empty vectors. Cells were harvested at indicated time points (6, 12, 24 and 36 h) and detected with anti-P62 antibody. Blots are representative of the 3 independent experiments. β-actin was used as sample-loading control. B and D Densitometric P62/β-actin ratios from at least 3 independent experiments were shown. Error bars show standard error of the mean (SEM). Significance was analyzed with two-tailed Student's test. ***P < 0.001. E and F Vero and HEK-293T cells were transfected with ASFV E199L protein expression plasmids or empty vectors for 24 h and then treated with Lyso-Tracker for 2 h. The fusion between the autophagosomes and lysosomes were observed by confocal microscopy with scale bars indicating 10 µm.
Subsequently, to investigate the accumulation of autophagosomes, cells were labeled with Lyso-Tracker Red to label acidic compartments or organelles in living cells (Gu et al. 2019). Autophagosomes and Lyso-Tracker Red were co-localized in cells following E199L-transfected. By contrast, in EV-treated group, almost no co-localization was observed between Autophagosomes and Lyso-Tracker Red (Fig. 2E, 2F). These results suggested that autophagosomes could fuse with acidic compartments or organelles following the transfection of E199L. Taken together, these data indicate that E199L protein-induced autophagy is a complete process in Vero and HEK-293T cells.
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The study of protein-protein interactions is an essential process to understand the biological functions of proteins and the underlying mechanisms. Co-immunoprecipitation coupled with mass spectrometry (CoIP-MS) is one of the most extensively used high-throughput techniques to discover novel protein-protein interactions (Ngounou et al. 2014). To explore this issue, we preformed mass spectrometry analysis of E199L protein and screened 119 potential interacting proteins (Supplementary Table S1). To identify putative functional processes associated with E199L-interacting proteins, we used GO cluster analysis to provide relevant information about biological processes (BP), cellular components (CC) and molecular functions (MF) and KEGG pathway analysis (Supplementary Fig. S1). We have noted that the GO term intracellular and membrane-bounded organelle were enriched in the CC category, while the binding-associated GO terms were enriched in the MF category, and various metabolic processes were enriched in the BP category. The top 10 KEGG pathways enriched by the target genes included ribosome, tight junction and endoplasmic reticulum pathways.
Furthermore, we have chosen 5 possible interaction proteins (ARL8B, LAMP2, PYCR2, BAG5 and FLII) which are related to cell autophagy and constructed eukaryotic expression plasmid respectively (Table 1). Co-immunoprecipitation assays and Western blot analysis showed that PYCR2 can interact with E199L (Fig. 3A) whereas others do not (Supplementary Fig. S2). In addition, confocal immunofluorescence suggested that host protein PYCR2 (mCherry) co-localized with ASFV-E199L (EGFP) in the cytoplasm (Fig. 3B). This further illustrates the interaction between PYCR2 and E199L. Interestingly enough, we found that the expression levels of PYCR2 seem to be regulated by E199L. After ASFV E199L-transfected, the fluorescence puncta of PYCR2 significantly decreased as shown in Fig. 3B.
Gene name Protein name Fold change LFQ intensity E199L-IP LFQ intensity E199L-IgG Functional description References ARL8B ADP-ribosylation factor-like protein 8B 3.428346353 44, 963, 000 0 A critical regulator of cargo delivery to lysosomes (Garg et al. 2011) LAMP2 lysosomal associated membrane protein 2 3.365582298 36, 729, 000 0 An important role in autophagosome formation (Fukushima et al. 2020) (Nguyen et al. 2018) PYCR2 Pyrroline-5-carboxylate reductase 2 58.15290957 351, 840, 000 0 Downregulation of PYCR2 induces the autophagy (Ou et al. 2016) BAG5 BAG family molecular chaperone regulator 5 32.82806482 101, 330, 000 0 Bcl-2-associated BAG5 regulates autophagy (De snoo et al. 2019) FLII Protein flightless 1 homolog 3.152638472 16, 234, 000 0 FLII interacts with p62 to block its recognition of LC3 (He et al. 2018) Table 1. Potential E199L-interacting proteins which are related to cell autophagy.
Figure 3. ASFV E199L protein interacts with host protein PYCR2. A Vero cells were co-transfected with pEGFP-C1, pEGFP-E199L, pmCherry-N1 and pmCherry-PYCR2 plasmid in pairs and immunoprecipitation was performed with anti-EGFP antibody. Immunoblotting analysis was performed with anti-EGFP antibody and anti-mCherry antibody. Blots are representative of the 3 independent experiments. B Vero cells were co-transfected with pEGFP-C1, pEGFP-E199L, pmCherry-N1 and pmCherry-PYCR2 plasmid in pairs. The fluorescence signals were visualized by confocal immunofluorescence microscopy with scale bars indicating 10 µm. C Vero cells were transfected with ASFV E199L protein expression plasmids or empty vectors. Quantitative real-time PCR of the mRNA expression levels of PYCR2 were detected. GAPDH, as an inner reference, was used as sample-loading control. D Vero cells were transfected with ASFV E199L protein expression plasmids or empty vectors. At 24 h post-transfection, cells were harvested and western blotting was performed with anti-PYCR2 antibody. Blots are representative of the 3 independent experiments. β-actin was used as sample-loading control. Densitometric PYCR2/β-actin ratios from at least 3 independent experiments are shown. Error bars show standard error of the mean (SEM). Significance was analyzed with two-tailed Student's test. ***P < 0.001.
To further clarify the detailed interaction between E199L and PYCR2, the expression levels of PYCR2 were measured in E199L-transfected cells. As we expected, the overexpression of E199L down-regulated the expression of PYCR2 both in mRNA and protein levels (Fig. 3C, 3D). These data demonstrate that the ASFV E199L protein interacts with host protein PYCR2 and down-regulates the expression level of PYCR2.
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There is little known about the function of PYCR2 and only one literature suggests that the down-regulation of PYCR2 seems to be associated with autophagy (Ou et al. 2016). We therefore hypothesized that the E199L-induced autophagy was mediated by down-regulation of PYCR2. To put this hypothesis to the test, we knocked down the expression of PYCR2 and detected the changes of autophagy markers. First, we verified the interference efficiency of siRNA by Western blot analysis. As shown in Fig. 4A, 4B, siRNA-1 has the highest interference effect in 24 h. After that, we collected cellular proteins treated by siRNA-1 at different time points. In siRNA-treated cells, the conversion of LC3-Ⅰ to LC3-Ⅱ and the degradation of P62 were observed as against NC-treated cells. Densitometry analysis also supports the results (Fig. 4C, 4D). These data indicate that depletion of PYCR2 promotes autophagy and it is consistent with previous studies (Ou et al. 2016).
Figure 4. Host protein PYCR2 regulates autophagy activation. A and B Vero cells were transfected with si-PYCR2 and si-NC treatment was used as negative control. At 36 h post-transfection, cells were harvested and western blotting was performed with anti-PYCR2 antibody. Blots are representative of the 3 independent experiments. β-actin was used as sample-loading control. Densitometric PYCR2/β-actin ratios from at least 3 independent experiments were shown. Error bars show standard error of the mean (SEM). Significance was analyzed with two-tailed Student's test. ***P < 0.001. C and E Cells were harvested at indicated time points (24, 36 and 48 h) and detected with anti-P62 antibody and anti-LC3B antibody. Blots are representative of the 3 independent experiments. β-actin was used as sample-loading control. C: Vero cells were transfected with si-PYCR2 and si-NC. E: Vero cells were transfected with ASFV E199L protein expression plasmids or empty vectors. D and F Densitometric LC3-Ⅱ/LC3-Ⅰ and P62/β-actin ratios from at least 3 independent experiments are shown. Error bars show standard error of the mean (SEM). Significance was analyzed with two-tailed Student's test. **P < 0.01, ***P < 0.001. ns: not significant.
To further investigate the regulation of PYCR2 on autophagy, we made use of a PYCR2 overexpression vector (pPRK5-Flag-PYCR2). We checked the influence of PYCR2 overexpression on autophagy as assessed by monitoring the conversion of LC3-Ⅰ to LC3-Ⅱ and the degradation of P62. Out of our expectation, overexpression of PYCR2 had no impact on these processes, which indicated the existence of complicated mechanism in this regulation (Fig. 4E, 4F).
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In order to clarify the interaction between E199L and PYCR2 on autophagy, we transfected cells with multiple plasmids either individually or co-transfected. As shown in Fig. 5, overexpression of E199L or knockdown of PYCR2 promoted autophagy to varying degree, and when E199L was co-transfected with si-PYCR2, the results were the same. Nevertheless, when PYCR2 was overexpressed, the activity of P62 inhibited by E199L was completely recovered; and the level of LC3-Ⅱ/LC3-Ⅰ was decreased. These results further confirmed that E199L induce autophagy by interacting with PYCR2 and down-regulating the expression level of PYCR2.
Figure 5. ASFV E199L protein induces autophagy by down-regulating PYCR2. A and C Vero cells were transfected with various expression plasmids. At 24 h post-transfection, cells were harvested and western blotting was performed with anti-PYCR2 antibody. Blots are representative of the 3 independent experiments. β-actin was used as sample-loading control. B and D Densitometric LC3-Ⅱ/LC3-Ⅰ and P62/β-actin ratios from at least 3 independent experiments were shown. Error bars show standard error of the mean (SEM). Significance was analyzed with two-tailed Student’s test. **P <0.01, ***P <0.001.