ABSTRACT
Prototype foamy virus (PFV), a complex retrovirus belonging to Spumaretrovirinae, maintains lifelong latent infection. The maintenance of lifelong latent infection by viruses relies on the repression of the type I interferon (IFN) response. However, the mechanism involving PFV latency, especially regarding the suppression of the IFN response, is poorly understood. Our previous study showed that PFV promotes autophagic flux. However, the underlying mechanism and the role of PFV-induced autophagy in latent infection have not been clarified. Here, we report that the PFV viral structural protein Gag induced amphisome formation and triggered autophagic clearance of stress granules (SGs) to attenuate type I IFN production. Moreover, the late domain (L-domain) of Gag played a central role in Alix recruitment, which promoted endosomal sorting complex required for transport I (ESCRT-I) formation and amphisome accumulation by facilitating late endosome formation. Our data suggest that PFV Gag represses the host IFN response through autophagic clearance of SGs by activating the endosome-autophagy pathway. More importantly, we found a novel mechanism by which a retrovirus inhibits the SG response to repress the type I IFN response.
IMPORTANCE Maintenance of lifelong latent infection for viruses relies on repression of the type I IFN response. Autophagy plays a double-edged sword in antiviral immunity. However, the role of autophagy in the regulation of the type I IFN response and the mechanism involving virus-promoted autophagy have not been fully elucidated. SGs are an immune complex associated with the antiviral immune response and are critical for type I IFN production. Autophagic clearance of SGs is one means of degradation of SGs and is associated with regulation of immunity, but the detailed mechanism remains unclear. In this article, we demonstrate that PFV Gag recruits ESCRT-I to facilitate amphisome formation. Our data also suggest that amphisome formation is a critical event for autophagic clearance of SGs and repression of the type I IFN response. More importantly, we found a novel mechanism by which a retrovirus inhibits the SG response to repress the type I IFN response.
INTRODUCTION
Foamy viruses (FVs), also known as spumaviruses, make up the only genus (Spumavirus) of the Spumaretrovirinae subfamily in the family Retroviridae (1). Prototype foamy virus (PFV) was isolated in the 1970s from a human nasopharyngeal carcinoma (2). The isolate was initially named human FV (HFV) but was later renamed PFV due to significant large homologies to simian FV in chimpanzees (3, 4). PFV is a member of the FVs, which can maintain a lifelong infection in the host and seem to be nonpathogenic in either naturally or accidentally infected hosts, thus differing from human immunodeficiency virus (HIV) and human T-cell leukemia virus (HTLV) (5, 6). PFVs are complex retroviruses having reading frames encoding structural proteins Gag, Pol, and Env and other regulatory factors (1). Many of the biological features of PFVs can be attributed to their Gag proteins (7). Gag protein plays critical roles in viral genes integration, viral budding, and intracellular trafficking during the PFV life cycle (7). However, its role in type I interferon (IFN) response regulation is little known.
Retroviral Gag proteins play important roles in both the early stage and the late phase of infection. These proteins are involved in the trafficking of incoming viruses and nuclear import in the early stage of infection (7). They coordinate the assembly of viral particles, selecting the viral genome for encapsidation and directing the incorporation of the envelope glycoproteins and viral budding (7). The late domain (L-domain) of PFV Gag is located in the central region precursor containing the sequence Pro-Ser-Ala-Pro (PSAP motif) at amino acids (aa) 284 to 287 and plays a role in particle egress (8, 9). The L-domain engages the endosomal sorting complex required for transport (ESCRT, also termed VPS [vacuolar protein sorting]) membrane fission machinery to promote membrane fission to detach the viral lipid envelope from the plasma membrane during the last step of viral replication (10). The PFV L-domain interacts with TSG101, which is a component of the cellular export machinery that mediates viral particle release from the plasma membrane (11). Also, the PFV L-domain is critical for the recruitment of tumor susceptibility gene 101 (TSG101) and apoptosis-linked gene 2 protein (ALG-2)-interacting protein X (Alix), the key factors of the ESCRT pathway, and promotes viral budding (12). Previously, we found that PFV induces autophagy, which is a membrane-associated biological process, as is viral budding (13). However, whether PFV Gag is a key factor in PFV-induced autophagy remains unknown. Furthermore, whether the recruitment of ESCRT facilitates autophagy by acting on the plasma membrane as it promotes viral budding is also unclear.
Amphisome, a transient organelle that results from the fusion of an autophagosome with an endosome, is a close link between the maturation of late endosomes and the regulation of autophagy flux. This hybrid organelle is efficient at viral replication and antiviral immune regulation. However, the mechanism involving the formation of virus-induced amphisomes remains unclear. Moreover, the roles of amphisomes in viral replication and the antiviral response are also controversial, since autophagy is considered to be a double-edged sword during viral infection. In addition, ESCRTs serve vital functions, such as phagophore membrane sealing and vesicle tethering, during autophagy. ESCRT machinery deficiency interferes with autophagosome maturation (14). The ESCRT machinery affects autophagosomal maturation mainly through the control of amphisome formation (15). However, whether ESCRTs might be potential targets regulated by viruses for the regulation of amphisome formation is still unknown, and their roles in antiviral immunity and viral replication are also not yet fully understood.
Stress granules (SGs) are large conserved granules that are formed from pools of untranslated messenger ribonucleoproteins (16). SGs are cleared by autophagy in a process termed granulophagy (17, 18). With the exception of autophagy-related genes (Atgs) and autophagic receptors, only valosin-containing protein (VCP) has been identified as a critical regulator of granulophagy. However, whether there are other critical regulators in granulophagy is not well known (17, 19). SGs facilitate the establishment of an antiviral state by limiting viral protein accumulation and regulating signaling cascades that affect virus replication and immune responses. SG formation would lead a robust translation arrest, which can repress viral gene expression (20, 21), but the genes encoding antiviral factors, such as in interferon-stimulated genes (ISGs), are not limited to the arrest of bulk translation and recruited to SGs (22). Given the biological roles of SGs in the antiviral response, a crucial goal is to understand the mechanisms that control autophagic clearance of SGs. This raises the interesting question of whether ESCRT can participate in the regulation of granulophagy.
Our earlier study showed that PFV induces complete autophagy and is associated with endoplasmic reticulum (ER) stress (13). We report here that PFV Gag recruits ESCRT to promote amphisome formation and facilitate endosomal autophagic clearance of SGs. PFV Gag-induced granulophagy inhibits the type I IFN response, and amphisome formation is a key event in this process. This study reveals a novel mechanism of virus-induced autophagy and repression of antiviral immunity.
RESULTS
PFV induces autophagy to negatively regulate the type I IFN response.Our previous study suggested that PFV induces autophagy (13), but the mechanism of how the virus triggers autophagy is still unknown. To confirm that PFV induces autophagy, we subjected HT1080 cells, which are a PFV-permissive cell line, to PFV infection and found that PFV infection significantly promoted LC3 lipidation at 24 and 48 h postinfection and p62 degradation compared to mock infection (Fig. 1A). Also, PFV infection seemed to promote LC3 lipidation and p62 degradation in a time-dependent manner (Fig. 1A to C). We also investigated whether PFV formation increased autophagosome formation. HT1080 cells were transfected with GFP-LC3-expressing plasmid for 24 h and then subjected to PFV infection for 24 h. We found that PFV infection obviously increased green fluorescent protein (GFP) punctum formation relative to mock infection (Fig. 1D), suggesting that PFV promoted autophagosome formation.
PFV induces autophagy to restrain the type I IFN response. (A) HT1080 cells were subjected to PFV infection at an MOI of 0.5 for various times as indicated or mock infection. Cell lysates were subjected to immunoblotting assay for LC3 and p62. The data are representative of the results of three independent experiments. (B and C) Quantitation of the data in panel A. (D) EGFP-LC3-expressing HT1080 cells were infected with PFV at an MOI of 0.5 for 24 h. GFP-LC3 dots were visualized via confocal microscopy. Scale bars, 5 μm. (E) HT1080 cells were subjected to PFV infection at an MOI of 0.5 for various times as indicated or mock infection and then treated with saline or 2 μg/ml poly(I·C). The cells were subjected to luciferase assays. Graphs show the means ± the SD (n = 3). ***, P ≤ 0.001. (F) HT1080 cells were transfected with Atg5-targeted shRNA or control shRNA for the times indicated. Cell lysates were subjected to immunoblotting assay for Atg5. The data are representative of the results of three independent experiments. (G and H) Atg5-knockdown HT1080 cells (shAtg5) or control HT1080 cells (shCtrl) were infected with PFV or mock treated at an MOI of 0.5 for 24 h. (I and J) HT1080 cells were infected with PFV at an MOI of 0.5 for 24 h or subjected to mock treatment in the presence of 50 nM bafilomycin A1 or dimethyl sulfoxide (DMSO). (G and I) The cells were subjected to luciferase assays. Graphs show the means ± the SD (n = 3). ***, P ≤ 0.001. (H and J) The relative levels of ISG56 mRNA were determined by RT-PCR. The data are representative of the results of three independent experiments.
Interestingly, we further explored whether PFV infection-induced autophagy affects the type I IFN response, which mediates strong antiviral immunity. First, we found that PFV infection obviously promoted the promoter activity of IFN-β (Fig. 1E). Subsequently, we pretreated HT1080 cells with poly(I·C), a synthetic viral RNA mimic which can cause a type I IFN response, and then infected the cells with PFV. We found that PFV infection led to reduction of the poly(I·C)-induced increase in IFN-β promoter activity (Fig. 1E), suggesting that PFV may suppress the type I IFN response, although it could induce a moderated type I IFN response. Second, we blocked autophagy by Atg5 knockdown (Fig. 1F) upon infection with PFV. Atg5 knockdown obviously promoted a PFV-induced increase in the promoter activity of IFN-β (Fig. 1G) and the transcription of interferon-stimulated gene 56 (ISG56), which is an IFN-β-specific target gene (Fig. 1H), suggesting that autophagy played a negative role in the PFV-induced type I IFN response. Third, we inhibited the fusion of autophagosome and lysosome by bafilomycin A1 and found that the bafilomycin treatment also promoted a PFV-induced increase in the promoter activity of IFN-β (Fig. 1I) and the transcription of ISG56 (Fig. 1J) accordingly. These data suggest that PFV induces autophagy to suppress the type I IFN response.
PFV Gag induces endosomal autophagy.Since PFV induces autophagy (13), we wanted to explore the detailed mechanism of how the virus triggers autophagy. Given that the structural protein Gag of retroviruses is capable of interacting with the plasma membrane (7, 23) and that autophagy is a plasma membrane-related biological process initiated at the isolation membrane (also termed the phagophore), which forms specifically from the mitochondrion-associated ER membrane and other membranes (24–26), we reasoned that the Gag protein participates in the regulation of PFV-induced autophagy.
First, we determined the physiological dose of Gag of PFV-induced autophagy. Our previous study (13) showed that cells infected with PFV at a multiplicity of infection (MOI) of 0.5 for 24 h undergo complete autophagy. To reproduce a physiological dose of Gag with PFV infection at an MOI of 0.5, we determined the Gag level for PFV infection at an MOI of 0.5 for 24 and 48 h and determined the Gag level after transfection of Gag-expressing plasmid at various gradient doses by Western blotting. As shown in Fig. 2A to C, transfection of the Gag-expressing plasmid at a minimum of 400 ng per well in a 6-well plate (200 ng/ml) for 24 h could nearly reproduce the PFV infection at an MOI of 0.5 for 24 h.
PFV Gag induces endosomal autophagy. (A) HT1080 cells were subjected to PFV infection at an MOI of 0.5 for various times as indicated. (B) HT1080 cells were transfected with pcDNA6.0 as a negative control (NC) or His-tag plasmid expressing His-Gag (Gag) (dose as indicated). (A and B) Cell lysates were subjected to immunoblotting assay for PFV Gag. The data are representative of the results of three independent experiments. (C) Quantitation of the data in panels A and B. (D) HEK293T cells were transfected with pcDNA6.0 as a negative control (NC) or His-tag plasmid expressing His-Gag (Gag) (dose as indicated). An immunoblotting assay for LC3 and p62 was performed. The data are representative of the results of three independent experiments. (E) EGFP-LC3-expressing HEK293T cells were transfected with pcDNA6.0 as a negative control (NC) or His-tag plasmid expressing His-Gag (Gag) (200 ng/ml). Confocal microscopy analysis of EGFP-LC3 puncta was performed. Representative images of EGFP-LC3 puncta are shown. Scale bars, 10 μm. (F) HEK293T cells were transfected with plasmid expressing His-Gag (Gag) (dose as indicated). The intracellular distribution of CD63 was examined by confocal microscopy. Scale bars, 5 μm. (G) HEK293T cells were transfected with pcDNA6.0 as a negative control (NC) or His-tag plasmid expressing His-Gag (Gag) (dose as indicated). An immunoblotting assay for EEA1 and CD63 was performed. The data are representative of the results of three independent experiments. HEK293T cells were transfected with pcDNA6.0 (NC), His-tag plasmid expressing His-Gag (FL), or His-Gag with an L-domain deletion mutation (ΔLD). (H) Immunoblotting assay for EEA1 and CD63. (I) Immunoblotting assay for LC3 and p62. The data are representative of the results of three independent experiments. (J and K) Quantitation of the data in panel I. (L) HEK293T cells were transfected with pcDNA6.0 as a negative control (NC) or His-tag plasmid expressing His-Gag (Gag) with or without pretreatment with 2 μg/ml U18666A to inhibit late endosome formation. An immunoblotting assay for LC3 and p62 was performed. The data are representative of the results of three independent experiments. (M and N) Quantitation of the data in panel L.
Second, we tested whether Gag could affect autophagy. We cloned PFV Gag and investigated the change in autophagic flux caused by PFV Gag. Remarkably, PFV Gag could promote the lipidation of LC3 and the degradation of p62 (Fig. 2C), which has been confirmed as the receptor of PFV-induced autophagy (13). LC3 punctum accumulation was also observed in His-Gag-transfected cells, suggesting that PFV Gag induced autophagosome formation (Fig. 2E).
Third, we determined whether Gag induced autophagy by affecting endosomal biogenesis (endosomal autophagy), since the Gag protein of retroviruses can be directly associated with the ESCRT complex (12) and the ESCRT machinery positively regulates late endosome biogenesis (27). To ascertain whether PFV Gag promotes late endosome biogenesis, we detected the cellular distribution of the late endosome marker CD63, the early endosome marker early endosome antigen 1 (EEA1), and their levels in PFV Gag-transfected cells. Our results showed a striking difference in late endosome distribution and a marked change in EEA1 and CD63 levels in PFV Gag-transfected cells (Fig. 2F and G). Zhadina and Bieniasz reported that the L-domain of the retrovirus Gag protein plays a central role in Gag-induced late endosome biogenesis (12). To test whether this was the case with PFV Gag, EEA1 and CD63 levels were determined in cells transfected with PFV-Gag with an L-domain deletion or full-length PFV-Gag. Deletion of the L-domain led to a decrease in EEA1 and CD63 levels (Fig. 2H). Interestingly, the L-domain deletion of PFV Gag also attenuated p62 degradation but had no effect on LC3 lipidation (Fig. 2I to K). To ascertain that PFV Gag-induced autophagy is endosomal autophagy, LC3 lipidation and p62 degradation were detected following PFV Gag transfection in the presence of a late endosome inhibitor, U18666A. The pharmacological inhibition of late endosome formation also repressed PFV Gag-induced p62 degradation but without any defects in LC3 lipidation (Fig. 2L to N). These data suggest that the L-domain of PFV Gag promotes late endosome formation to facilitate PFV Gag-induced endosomal autophagy.
The L-domain plays a pivotal role in PFV Gag-triggered ESCRT-I recruitment.It has been reported that the L-domain of retrovirus protein Gag recruits the ESCRT-I complex in an Alix-dependent manner (12). To ascertain whether the PFV Gag L-domain mediates the recruitment of the ESCRT-I complex, the interaction between Alix and L-domain-deleted/full-length PFV Gag was tested. Remarkably, PFV Gag could interact with Alix and TSG101, an essential subunit of ESCRT-I, but the L-domain deletion led to a noninteracting state of Gag/Alix and Gag/TSG101 (Fig. 3A). The deletion of the PFV L-domain also weakened the interaction between Alix and TSG101 (Fig. 3A). Zhadina and Bieniasz reported that Alix is critical for ESCRT-I recruitment, which is induced by retroviral Gag during viral budding (12). To confirm whether Alix is essential for PFV Gag-induced ESCRT-I recruitment, the interaction between Gag and TSG101 was tested under Alix knockdown. Our results showed that Alix silencing abolished the interaction between PFV Gag and TSG101 (Fig. 3B). These results suggest that PFV Gag recruits the ESCRT-I complex via the interaction between its L-domain and Alix.
The L-domain and Alix mediate PFV Gag-induced ESCRT-I recruitment. (A) HEK293T cells were transfected with His-tag plasmid expressing His-Gag (Gag [full length]) or His-Gag with an L-domain deletion mutation (Gag [ΔL-domain]). Lysates were immunoprecipitated with anti-His, anti-Alix, or anti-TSG101 antibody. Immune complexes were resolved by SDS-PAGE and immunoblotted with anti-His, anti-Alix, or anti-TSG101 antibody. (B) Alix-knockdown HEK293T cells (shAlix) or control HEK293T cells (shCtrl) were transfected with His-tag plasmid expressing His-Gag (Gag) at 200 ng/ml. Lysates were immunoprecipitated with anti-His or anti-TSG101 antibody. Immune complexes were resolved by SDS-PAGE and immunoblotted with anti-His or anti-TSG101 antibody. The data are representative of the results of three independent experiments.
PFV Gag induces amphisome formation and maturation in an ESCRT-I-dependent manner.Fusion of the autophagosome and late endosome to generate an amphisome is a key event during endosomal autophagy (15). Since PFV Gag could recruit ESCRT-I to promote the formation of the late endosome and induce endosomal autophagy, as shown above, we speculated that PFV Gag might regulate amphisome formation via the ESCRT-I machinery. First, to examine this hypothesis, we looked for the presence of amphisomes in PFV Gag-transfected cells. By detecting the cellular distribution of LC3 and CD63, we found colocalization of LC3 and CD63 in PFV Gag-transfected cells (Fig. 4A). Moreover, we observed that L-domain deletion repressed LC3 and CD63 colocalization (Fig. 3A). These data suggested that PFV Gag can induce amphisome formation and that the L-domain is critical for this process. Second, given that the recruitment of ESCRT-I to PFV Gag is mediated by Alix (12), we further tested whether ESCRT-I mediates amphisome formation and maturation by detecting the cellular distribution of CD63 and LAMP1 under Alix knockdown conditions. We found that transfection of PFV Gag induced CD63 and LAMP1 colocalized dots (Fig. 4B), but Alix knockdown (Fig. 4D) reduced the number of colocalized dots (Fig. 4B and C), suggesting that Alix is important for PFV Gag-induced maturation of amphisomes. Subsequently, to confirm that ESCRT-I mediates amphisome formation and maturation, the cellular distribution of CD63 and LAMP1 was detected in cells transfected with PFV Gag with an L-domain deletion. There were more CD63 and LAMP1 colocalized dots in the cells transfected with full-length PFV Gag than in those with the L-domain deletion (Fig. 4E and F), suggesting that the L-domain is essential for PFV Gag to promote amphisome formation. To further confirm that PFV Gag induced ESCRT-I-dependent amphisome effects, autophagic flux was detected in Alix-knockdown cells transfected with PFV Gag. Alix knockdown repressed PFV Gag-induced p62 degradation but without any change in LC3 lipidation (Fig. 4G). Altogether, these data suggest that PFV Gag induces amphisome formation and maturation in an ESCRT-I-dependent manner.
PFV Gag promotes mature amphisome formation through recruitment of the ESCRT-I complex. (A) GFP-LC3-expressing HEK293T cells were transfected with pcDNA6.0 as a negative control (NC), His-tag plasmid expressing His-Gag (Gag [FL]), or His-Gag with an L-domain deletion mutation (Gag [ΔLD]). The intracellular distribution of CD63 was examined by confocal microscopy. Scale bars, 5 μm. (B) Alix-knockdown HEK293T cells (shAlix) or control HEK293T cells (shCtrl) were transfected with His-tag plasmid expressing His-Gag (Gag). (D) HEK293T cells were transfected with Alix-targeted shRNA or control shRNA for the times indicated. Cell lysates were subjected to immunoblotting assay for Alix. The data are representative of the results of three independent experiments. (E) HEK293T cells were transfected with His-tag plasmid expressing His-Gag (Gag [FL]) or His-Gag with an L-domain deletion mutation (Gag [ΔLD]). (B and E) Mature amphisomes were examined by fluorescence microscopy (CD63 and LAMP1 serve as mature amphisome markers). Representative images of mature amphisomes are shown. Scale bars, 5 μm. (C) Quantitation of the data in panel B. (F) Quantitation of the data in panel E. Graphs show the means ± the SEM; six random fields and 10 cells per field were examined by confocal microscopy. *, P < 0.05. (G) Alix-knockdown HEK293T cells (shAlix) or control HEK293T cells (shCtrl) were transfected with His-tag plasmid expressing His-Gag (Gag) or pcDNA6.0 as a negative control (NC). An immunoblotting assay for LC3 and p62 was performed. The data are representative of the results of three independent experiments.
PFV Gag induces autophagic clearance of SGs.Repressing the SG response is a strategy that viruses apply to facilitate their replication. Retroviruses, such as HIV-1, can disassociate SGs to allow the production of infectious viral particles (28). However, it is still unknown whether autophagy is a strategy that viruses apply to clear SGs. First, to test whether PFV Gag-induced autophagy targets SGs, the change in poly(I·C)-induced SG formation in the presence of PFV Gag was determined. We found that the poly(I·C)-induced SG amount decreased in the PFV Gag-transfected cells compared to control cells (Fig. 5A to C). Next, we investigated whether PFV Gag-induced amphisomes could target poly(I·C)-induced SGs. The number of G3BP1 granules was significantly reduced, and there was less CD63, LC3, and G3BP1 colocalization in PFV Gag-transfected cells (Fig. 5D). Third, to further confirm PFV Gag-induced granulophagy, the number of poly(I·C)-induced SGs was determined in Atg5-knockdown cells and control cells that were transfected with PFV Gag. Atg5 knockdown reversed the SG reduction caused by PFV Gag (Fig. 5E to G). These data suggest that SGs can be cleared by PFV Gag-induced autophagy.
PFV Gag induces endosomal autophagic clearance of SGs. (A) HEK293T cells were transfected with pcDNA6.0 as a negative control (NC) or His-tag plasmid expressing His-Gag (Gag) and treated with saline or 2 μg/ml poly(I·C). SGs were examined by fluorescence microscopy (G3BP1 and TIA1 serve as SG markers). Representative images of stress granules are shown. Scale bars, 10 μm. (B and C) Quantitation of the data in panel A. Graphs show the means ± the SEM; six random fields and 10 cells per field were examined by confocal microscopy. **, P ≤ 0.01; ***, P ≤ 0.001. (D) GFP-LC3-expressing HEK293T cells were transfected with pcDNA6.0 as a negative control (NC) or His-tag plasmid expressing His-Gag (Gag). Intracellular distributions of CD63, LC3, and G3BP1 were examined by confocal microscopy. Scale bars, 10 μm. (E) Atg5-knockdown HEK293T cells (shAtg3) or control HEK293T cells (shCtrl) were transfected with His-tag plasmid expressing His-Gag (Gag) or pcDNA6.0 as a negative control (NC) and treated with 2 μg/ml poly(I·C). SGs were examined by fluorescence microscopy (G3BP1 and TIA1 serve as SG markers). Representative images of stress granules are shown. Scale bars, 10 μm. (F and G) Quantitation of the data in panel E. Graphs show the means ± the SEM; six random fields and 10 cells per field were examined by confocal microscopy. *, P < 0.05; **, P ≤ 0.01.
The recruitment of ESCRT-I to PFV Gag is important for the regulation of PFV Gag-induced granulophagy.Since the data above suggest that PFV Gag recruits ESCRT-I to promote amphisome formation and PFV Gag-induced autophagy targets SGs, we further wanted to know whether the recruitment of ESCRT-I to PFV Gag could promote autophagic clearance of SGs. Hence, we performed the following experiments. First, given that the L-domain of PFV Gag is critical for the recruitment of ESCRT-I (12), we deleted the L-domain of PFV Gag to inhibit the recruitment of ESCRT-I and investigated the effects on PFV Gag-induced SG degradation. We found that deletion of the L-domain reversed the SG reduction caused by PFV Gag (Fig. 6A to C). Second, since Alix is the key factor for the recruitment of ESCRT-I to PFV Gag as shown in Fig. 2, we inhibited expression of Alix by short hairpin RNA (shRNA) to suppress the recruitment of ESCRT-I caused by PFV Gag, and we found that Alix knockdown reversed the SG reduction caused by PFV Gag (Fig. 6D to F). Third, since TSG101 is a core component of ESCRT-I (29), we suppressed TSG101 expression by shRNA (Fig. 6J) to inhibit ESCRT-I assembly. Accordingly, TSG101 knockdown also reversed the SG reduction caused by PFV Gag (Fig. 6G to I). These data suggest that the recruitment of ESCRT-I is a critical event in the process of PFV Gag-induced granulophagy.
The recruitment of ESCRT-I mediates PFV Gag-induced granulophagy. (A) HEK293T cells were transfected with pcDNA6.0 as a negative control (NC), His-tag plasmid expressing His-Gag (Gag [full length]), or His-Gag with an L-domain deletion mutation (Gag [ΔLD]) and treated with 2 μg/ml poly(I·C). (D) Alix-knockdown HEK293T cells (shAlix) or control HEK293T cells (shCtrl) were transfected with His-tag plasmid expressing His-Gag (Gag) or pcDNA6.0 as a negative control (NC) and treated with 2 μg/ml poly(I·C). (G) TSG101-knockdown HEK293T cells (shTSG101) or control HEK293T cells (shCtrl) were transfected with His-tag plasmid expressing His-Gag (Gag) or pcDNA6.0 as a negative control (NC) and treated with 2 μg/ml poly(I·C). SGs were examined by fluorescence microscopy (G3BP1 and TIA1 serve as SG markers). Representative images of stress granules are shown. Scale bars, 10 μm. (B and C) Quantitation of the data in panel A. (E and F) Quantitation of the data in panel D. (H and I) Quantitation of the data in panel G. Graphs show the means ± the SEM; six random fields and 10 cells per field were examined by confocal microscopy. *, P < 0.05; **, P ≤ 0.01. (J) HEK293T cells were transfected with TSG101-targeted shRNA or control shRNA for the times indicated. Cell lysates were subjected to immunoblotting assay for TSG101. The data are representative of the results of three independent experiments.
PFV Gag-induced granulophagy represses the type I IFN response.Virus sensors and ISG activation are closely related to the formation of SGs (22). Thus, we reasoned that PFV Gag-induced granulophagy would repress the type I IFN response. First, to verify this hypothesis, we transfected PFV Gag into cells that were treated with poly(I·C) and investigated the type I IFN response. As expected, PFV Gag decreased the promoter activity of IFN-β caused by poly(I·C) (Fig. 7A). Accordingly, PFV Gag also repressed the transcription of ISG56 caused by poly(I·C), (Fig. 7B). Second, since the L-domain is critical for the recruitment of ESCRT-I, which promotes amphisome formation and endosomal autophagy as the data presented above suggest, we deleted the L-domain to suppress PFV Gag-induced autophagy and investigated the consequent effect on the type I IFN response. We found that deletion of the L-domain reversed the repression of IFN-β promoter activity caused by PFV Gag (Fig. 7C). Accordingly, deletion of the L-domain also reversed the repression of ISG56 transcription caused by PFV Gag (Fig. 7D). Alix is the key factor that mediates PFV Gag-induced endosomal autophagy, as suggested by the data presented above. We inhibited Alix expression by shRNA to investigate the effect on PFV Gag-induced suppression of the type I IFN response, and we found that knockdown of Alix reversed the repression of IFN-β promoter activity and ISG56 transcription caused by PFV Gag (Fig. 7E and F). To further confirm whether the PFV Gag-induced endosomal autophagic clearance of SGs represses the type I IFN response, the effect of a pharmacological inhibitor of late endosome formation on the type I IFN response in PFV Gag-transfected cells was tested. Remarkably, the late endosome inhibitor U18666A reversed the repression of IFN-β promoter activity and ISG56 transcription caused by PFV Gag (Fig. 7G and H).
PFV Gag-induced granulophagy represses the type I IFN response. (A and B) HEK293T cells were transfected with pcDNA6.0 as a negative control (NC) or His-tag plasmid expressing His-Gag (Gag) and treated with saline or 2 μg/ml poly(I·C). (C and D) HEK293T cells were transfected with pcDNA6.0 as a negative control (NC), His-tag plasmid expressing His-Gag (Gag [FL]), or His-Gag with an L-domain deletion mutation (Gag [ΔLD]) and treated with 2 μg/ml poly(I·C). (E and F) Alix-knockdown HEK293T cells (shAlix) or control HEK293T cells (shCtrl) were transfected with His-tag plasmid expressing His-Gag (Gag) or pcDNA6.0 as a negative control (NC) and treated with 2 μg/ml poly(I·C). (G and H) HEK293T cells were transfected with pcDNA6.0 as a negative control (NC) or His-tag plasmid expressing His-Gag (Gag) with or without pretreatment with 2 μg/ml U18666A in the presence of 2 μg/ml poly(I·C). For panels A, C, E, and G, the cells were subjected to luciferase assays. Graphs show the means ± the SD (n = 3). *, P < 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. For panels B, D, F, and H, the relative levels of ISG56 mRNA were determined by RT-PCR. The data are representative of the results of three independent experiments.
Inhibition of amphisome formation restricts PFV Gag-induced granulophagy.The amphisome is a transient organelle in the endosomal autophagy process. PFV Gag promoted amphisome formation and targeted SGs, as shown in Fig. 4. To further confirm that amphisome formation is an essential step in PFV Gag-induced granulophagy, we detected changes in PFV Gag-induced granulophagy during inhibition of amphisome formation. Rab7a is a key factor that mediates late endosome maturation and the fusion of the endosome and autophagosome, so it is essential for amphisome formation (30). Our data showed that Rab7a knockdown (Fig. 8D) reversed the repression of poly(I·C)-induced SG formation caused by PFV Gag (Fig. 8A to C). EPG5 is a Rab7 effector that determines the fusion specificity of autophagosomes with late endosomes/lysosomes (31). Thus, knockdown of EPG5 would inhibit amphisome maturation. As expected, we found that EPG5 knockdown (Fig. 8H) reversed the repression of poly(I·C)-induced SG formation caused by PFV Gag (Fig. 8E to G). These data suggest that amphisome formation is a critical event in PFV Gag-induced granulophagy.
Inhibition of amphisome formation restricts PFV Gag-induced granulophagy. (A) Rab7a-knockdown HEK293T cells (shRab7a) or control HEK293T cells (shCtrl) were transfected with His-tag plasmid expressing His-Gag (Gag) or pcDNA6.0 as a negative control (NC) and treated with 2 μg/ml poly(I·C). (D) HEK293T cells were transfected with Rab7a-targeted shRNA or control shRNA for the times indicated. Cell lysates were subjected to immunoblotting assay for Rab7a. The data are representative of the results of three independent experiments. (E) EPG5-knockdown HEK293T cells (shEPG5) or control HEK293T cells (shCtrl) were transfected with His-tag plasmid expressing His-Gag (Gag) or pcDNA6.0 as a negative control (NC) and treated with 2 μg/ml poly(I·C). (A and E) SGs were examined by fluorescence microscopy (G3BP1 and TIA1 serve as SG markers). Representative images of stress granules are shown. Scale bars, 10 μm. (B and C) Quantitation of the data in panel A. (F and G) Quantitation of the data in panel E. Graphs show the means ± the SEM; six random fields and 10 cells per field were examined by confocal microscopy. *, P < 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (H) HEK293T cells were transfected with EPG5-targeted shRNA or control shRNA for the times indicated. Cell lysates were subjected to immunoblotting assay for EPG5. The data are representative of the results of three independent experiments.
PFV Gag-induced repression of the type I IFN response is reduced by amphisome inhibition.Since PFV Gag-induced repression of the type I IFN response is closely related to amphisome formation, we wanted to further confirm whether the inhibition of amphisome maturation would reverse PFV Gag-induced repression of the type I IFN response. As expected, we found that Rab7a knockdown reversed the repression of IFN-β promoter activity (Fig. 9A) and the decrease in ISG56 transcription (Fig. 9B). Accordingly, knockdown of EPG5 also reversed the repression of IFN-β promoter activity (Fig. 9C) and the decrease in ISG56 transcription (Fig. 9D). These data suggested that amphisome impairment reversed the PFV Gag-induced repression of the type I IFN response.
Amphisome impairment reverses PFV Gag-induced repression of the type I IFN response. (A and B) Rab7a-knockdown HEK293T cells (shRab7a) or control HEK293T cells (shCtrl) were transfected with His-tag plasmid expressing His-Gag (Gag) or pcDNA6.0 as a negative control (NC) and treated with 2 μg/ml poly(I·C). (C and D) EPG5-knockdown HEK293T cells (shEPG5) or control HEK293T cells (shCtrl) were transfected with His-tag plasmid expressing His-Gag (Gag) or pcDNA6.0 as a negative control (NC) and treated with 2 μg/ml poly(I·C). (A and C) The cells were subjected to luciferase assays. Graphs show means ± the SD (n = 3). *, P < 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (B and D) The relative levels of ISG56 mRNA were determined by RT-PCR. The data are representative of the results of three independent experiments.
DISCUSSION
Substantial evidence accumulated in recent years has highlighted the role of ESCRT not only as a regulator of late endosome biogenesis but also as a critical player in autophagy (14). However, no direct evidence that ESCRT is applied by viruses to regulate the type I IFN response, which is essential for the antiviral response, has been reported. In addition, viruses could affect autophagy to regulate the antiviral response or viral replication by promoting or inhibiting the formation and maturation of amphisomes generated from the fusion of autophagosomes and endosomes (32). However, whether ESCRT is a target that viruses use to regulate the amphisome has not been reported to date. Here, we report that PFV Gag recruits ESCRT-I to promote amphisome formation and to facilitate granulophagy and inhibit the type I IFN response. Furthermore, the L-domain is critical for the recruitment of ESCRT-I to PFV Gag (Fig. 10).
Schematic representation of our key findings. PFV Gag induces amphisome formation and triggers autophagic clearance of SGs to repress the type I IFN response. Moreover, the L-domain of the Gag protein plays a central role in Alix recruitment, which promotes ESCRT-I formation and amphisome accumulation by facilitating late endosome formation.
The ESCRT pathway catalyzes analogous membrane fission events that require a series of vesiculation events (27). Viruses mimic cellular recruiting signals to usurp the ESCRT pathway and facilitate viral replication (33). Enveloped viruses, such as HIV-1, bud from infected cells through ESCRT (34–36). In addition to the role of the ESCRT pathway in the viral life cycle, some recent studies have suggested that the ESCRT pathway might also regulate the antiviral response. The ESCRT-II subunit EAP30 promotes IRF3-dependent induction of type I and III IFNs, IFN-stimulated genes (ISGs), and chemokines by binding to double-stranded RNA or viruses (37). ESCRT-0, which is composed of hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) and the signal transducing adaptor molecule (STAM), was recently implicated in post-Golgi trafficking by sorting ubiquitinated TLR7 and TLR9 to endosomes and was shown to regulate the TLR3-mediated antiviral response in murine astrocytes (38, 39). However, there has not been direct evidence showing that the ESCRT pathway is applied by the virus to counteract the antiviral response. Here, we report that ESCRT-I is essential for PFV Gag-promoted autophagic clearance of SGs and leads to the restriction of the type I IFN response. These findings reveal that ESCRT is a promising tool for virus suppression of the type I IFN response, the main antiviral response.
Amphisomes have been proven to be organelles that viruses target to impair antiviral immune responses and facilitate viral replication. HIV-1 inhibition of immune-amphisomes in dendritic cells impairs early innate and adaptive immune responses (40). Coxsackievirus B3 (CVB3) and enterovirus D-68 (EV-D68) harness autophagic membranes for RNA replication complexes and require amphisomes for viral maturation (41, 42). However, the mechanism by which viruses regulate amphisome formation has remained unclear. Here, we show that PFV Gag promotes amphisome formation to facilitate granulophagy, which results in the suppression of the type I IFN response.
Selective autophagy plays a crucial role in antiviral host defenses. The role of autophagy in antiviral immunity is still controversial due to its opposing effects during different viral infections. Dengue virus and hepatitis C virus regulate lipophagy (cytosolic lipid droplet-targeted autophagy) for replication (43). Zika virus impairs reticulophagy (ER-targeted autophagy) by NS2B3-mediated cleavage of the reticulophagy receptor FAM134B, thereby inhibiting FAM134B-mediated ER degradation and protecting the sites of viral RNA replication (44). However, mitophagy targets the virions of Sindbis virus and herpes simplex virus 1 to restrict viral replication (45, 46). Whereas studies of selective autophagy are expanding sharply, not all types of selective autophagy during virus infection have been investigated. Granulophagy, an SG-targeted autophagy, is one such selective autophagy. We found that PFV Gag causes autophagic clearance of SGs, suggesting that virus protein-induced granulophagy is a new type of virus-induced selective autophagy.
MATERIALS AND METHODS
Cells, virus, and plasmids.HEK293T cells (ATCC, CRL-11268) were maintained in Dulbecco modified Eagle medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; Biological Industries, lot no. 1719426) and penicillin (100 U/ml)/streptomycin (100 μg/ml) (BioSharp, catalog no. BL505A) at 37°C in a humidified atmosphere containing 5% CO2. HT1080 cells (ATCC, CCL-121) were maintained in minimum Eagle medium supplemented with 10% heat-inactivated FBS and penicillin (100 U/ml)/streptomycin (100 μg/ml) at 37°C in a humidified atmosphere containing 5% CO2.
Wild-type and L-domain-deleted PFV Gag genes were cloned into pcDNA6.0. The human LC3 gene was cloned into pEGFP-N1. shDNAs encoding specific shRNAs targeting Alix, Atg5, TSG101, Rab7a, and EPG5 were cloned into pGPU6 (GenePharma). A negative-control vector was constructed with a nonsense shRNA sequence. All inserted sequences were verified by DNA sequencing. The sequences of the shRNAs are shown in Table 1.
Insert sequences for each target gene
Antibodies and reagents.Rabbit anti-LC3 (Proteintech, catalog no. 18725-1-AP), rabbit anti-p62 (Proteintech, catalog no. 18420-1-AP), rabbit anti-β-actin (Proteintech, catalog no. 20536-1-AP), mouse anti-EEA1 (Proteintech, catalog no. 66218-1-Ig), rabbit anti-CD63 (Proteintech, catalog no. 25682-1-AP), mouse anti-His (Proteintech, catalog no. 66005-1-Ig), rabbit anti-Alix (Proteintech, catalog no. 12422-1-AP), and rabbit anti-TSG101 (Proteintech, catalog no. 14497-1-AP) were used at a dilution of 1:1,000 for Western blotting. Mouse anti-G3BP1 (Proteintech, catalog no. 66486-1-Ig), rabbit anti-TIA1 (Proteintech, catalog no. 12133-2-AP), rabbit anti-G3BP1 (Proteintech, catalog no. 13057-2-AP), mouse anti-CD63 (MBL, catalog no. D263-3), and rabbit anti-LAMP1 (Proteintech, catalog no. 21997-1-AP) were used for indirect immunofluorescence microscopy at a dilution of 1:200. Horseradish peroxidase-conjugated secondary antibodies were purchased from Proteintech and used at 1:5,000. Cy3-conjugated goat anti-mouse secondary antibody (catalog no. SA00009-1), Cy3-conjugated goat anti-rabbit secondary antibody (catalog no. SA00009-2), fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse secondary antibody (catalog no. SA00003-1), and FITC-conjugated goat anti-rabbit secondary antibody (catalog no. SA00003-2) were purchased from Proteintech and used at 1:200. For immunoprecipitation of His, mouse anti-His (Proteintech, catalog no. 66005-1-Ig) was used. For immunoprecipitation of Alix, rabbit anti-Alix (Proteintech, catalog no. 12422-1-AP) was used. For immunoprecipitation of TSG101, rabbit anti-TSG101 (Proteintech, catalog no. 14497-1-AP) was used. Protein A/G-agarose beads (Abmart, catalog no. A10001M) were used. U18666A (catalog no. HY-107433) was purchased from MedChemExpress. Polyinosinic-poly(C) sodium salt [poly(I·C); catalog no. P0913] was purchased from Sigma-Aldrich. The transfection reagent was purchased from Biomiga (catalog no. GT2211).
Immunoblotting and immunoprecipitation.For immunoblotting, whole-cell lysates were prepared in radioimmunoprecipitation assay (RIPA) buffer (Beyotime Technology, catalog no. P0013B) supplemented with protease inhibitor (Beyotime Technology, catalog no. P1005) and phosphatase inhibitor cocktail (Beyotime Technology, catalog no. P1005P1096) supplemented with complete protease inhibitor cocktail (Roche, catalog no. 04693132001) for 30 min on ice. Cell lysates were separated by SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore) by electroblotting. Proteins were visualized by enhanced chemiluminescence according to the manufacturer’s instructions (Pierce).
For immunoprecipitation, cells were lysed in RIPA buffer (Beyotime Technology, catalog no. P0013B) supplemented with protease inhibitor (Beyotime Technology, catalog no. P0013D) on ice for 30 min. Primary antibodies were incubated with protein agarose A/G beads for 4 h at 4°C, followed by incubation with cell lysates for 5 h with rotation at 4°C. The beads were washed four times with lysis buffer and analyzed by immunoblotting.
Immunofluorescence confocal microscopy.Cells were fixed with 4% paraformaldehyde for 20 min at room temperature and permeabilized for 20 min with 0.1% Triton X-100 in phosphate-buffered saline (PBS). Samples were blocked with 2% premium-quality normal goat serum in PBS for 90 min at room temperature. Cells were incubated with the indicated primary antibodies at 4°C overnight, washed, and incubated for 1 h with DyLight405-, Cy3- or FITC-conjugated secondary antibodies. After washing, the slides were mounted with a reagent containing DAPI (4′,6′-diamidino-2-phenylindole). Images were captured by a confocal fluorescence microscope (Nikon A1R).
RNA extraction and RT-PCR.Total RNA was extracted using TRIzol reagent (Invitrogen). Reverse transcription was performed using 500 ng of the purified RNA samples as a template (Thermo Fisher Scientific). The obtained cDNA samples were subjected to PCR (Bio-Rad iQ5) by using a PCR kit (TsingKe, catalog no. TSE006). The products were examined by agarose gel electrophoresis and visualized by Goldview (Solarbio, catalog no. G8142). The primers used for reverse transcription-PCR (RT-PCR) are listed in Table 2.
Primer sequences for each target gene
Luciferase reporter assay.Cells were transfected with reporter plasmid containing the IFN-β promoter, and the indicated amounts of the expression plasmids were transfected. The pRL-TK Renilla luciferase reporter plasmid was added to each transfection reaction to normalize for transfection efficiency. The luciferase reporter assays were performed with a dual-luciferase reporter assay system (Promega, catalog no. E1980) according to the procedures recommended by the manufacturer. Firefly luciferase activity was normalized to Renilla luciferase activity. The relative luciferase activities were expressed as fold changes over the empty-plasmid-transfected or mock-treated controls.
Data analysis.All data are presented as the means ± the standard deviations (SD) or means ± the standard errors of the means (SEM) as indicated. Comparisons between two groups were performed using the Student t test, whereas comparisons among multiple groups were performed using one-way analysis of variance. For all tests, P values of <0.05 were considered statistically significant.
ACKNOWLEDGMENTS
We thank Ying Zhou (Medical Structural Biology Research Center of Wuhan University) for technical assistance with the confocal microscopy assay.
This study was supported by grants to W.L. from the National Natural Sciences Foundation of China (no. 81371790), the Fundamental Research Funds for Shenzhen Science and Technology Innovation Committee (no. JCYJ20170818143952175), the Central Universities of China and the Translational Medical Research Fund of Wuhan University School of Medicine (2042018gf0034, 2042017kf0240), and the Creative Research Groups of Hubei Natural Science Foundation (no. 2017CFA017) and to G.Z. from the National Natural Sciences Foundation of China (no. 81701571), the Natural Science Foundation of Hubei Province (no. 2016CFB306), and the Health and Family Planning Commission of Hubei Province (no. WJ2017H0041).
FOOTNOTES
- Received 7 October 2019.
- Accepted 7 January 2020.
- Accepted manuscript posted online 22 January 2020.
- Copyright © 2020 American Society for Microbiology.
