Rift Valley Fever Virus Nucleoprotein Triggers Autophagy to Dampen Antiviral Innate Immune Responses

ABSTRACT

Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus that causes severe and potentially fatal hemorrhagic fever in humans. Autophagy is a self-degradative process that can restrict viral replication at multiple infection steps. In this study, we evaluated the effects of RVFV-triggered autophagy on viral replication and immune responses. Our results showed that RVFV infection triggered autophagosome formation and induced complete autophagy. Impairing autophagy flux by depleting autophagy-related gene 5 (ATG5), ATG7, or sequestosome 1 (SQSTM1) or treatment with autophagy inhibitors markedly reduced viral RNA synthesis and progeny virus production. Mechanistically, our findings demonstrated that the RVFV nucleoprotein (NP) C-terminal domain interacts with the autophagy receptor SQSTM1 and promotes the SQSTM1–microtubule-associated protein 1 light chain 3 B (LC3B) interaction and autophagy. Deletion of the NP C-terminal domain impaired the interaction between NP and SQSTM1 and its ability to trigger autophagy. Notably, RVFV-triggered autophagy promoted viral infection in macrophages but not in other tested cell types, including Huh7 hepatocytes and human umbilical vein endothelial cells, suggesting cell type specificity of this mechanism. It was further revealed that RVFV NP-triggered autophagy dampens antiviral innate immune responses in infected macrophages to promote viral replication. These results provide novel insights into the mechanisms of RVFV-triggered autophagy and indicate the potential of targeting the autophagy pathway to develop antivirals against RVFV.

IMPORTANCE We showed that RVFV infection induced the complete autophagy process. Depletion of the core autophagy genes ATG5, ATG7, or SQSTM1 or pharmacologic inhibition of autophagy in macrophages strongly suppressed RVFV replication. We further revealed that the RVFV NP C-terminal domain interacted with SQSTM1 and enhanced the SQSTM1/LC3B interaction to promote autophagy. RVFV NP-triggered autophagy strongly inhibited virus-induced expression of interferon-stimulated genes in infected macrophages but not in other tested cell types. Our study provides novel insights into the mechanisms of RVFV-triggered autophagy and highlights the potential of targeting autophagy flux to develop antivirals against this virus.

INTRODUCTION

Rift Valley fever virus (RVFV) is a highly pathogenic mosquito-borne virus that causes zoonotic diseases. Although RVFV is traditionally endemic in African countries, recent evidence has revealed the spread of the virus to the Arabian Peninsula and Middle East (1–3). Disease symptoms caused by RVFV infection in animals include fever, listlessness, abortion in pregnant animals, and acute mortality among newborns. The mortality rate in infected adult sheep is 20 to 30% (2), whereas the case fatality rate in adult cattle is 0 to 5% (4). However, mortality rates in livestock can be as high as 95% in newborns, and abortion rates can reach 100% (5, 6). In humans, symptoms of RVFV infection include hemorrhagic fever, hepatitis, encephalitis, and retinal vasculitis, and the death rate can reach 50% in cases of severe infection (7). Currently, there are no approved antiviral drugs or vaccines against RVFV for human use. Understanding the mechanisms through which RVFV establishes efficient viral infection could provide important insights to facilitate the development of antivirals targeting this virus.

Autophagy is an evolutionarily conserved process responsible for maintaining cellular homeostasis by eliminating protein aggregates and damaged organelles (8). Autophagy occurs via the formation of a cup-shaped double-membrane structure called the phagophore, which originates from various cellular membrane compartments, such as the endoplasmic reticulum (ER), mitochondria, Golgi complexes, endosomes, and plasma membranes (9). The phagophore further expands and closes to form large double-membrane vesicles (DMVs), known as autophagosomes, with diameters of 500 to 1,500 nm in mammalian cells (9, 10). Host cells can harness autophagy to exert antiviral effects through diverse mechanisms (8). For example, the cellular autophagy receptor p62 (also called sequestosome 1 [SQSTM1]) binds to the Sindbis virus capsid protein and targets it to autophagosomes for protein clearance (11). The ER transmembrane protein SCOTIN recruits hepatitis C virus (HCV) nonstructural protein NS5A to autophagosomes for degradation, thereby inhibiting HCV replication (12). Other studies have demonstrated that autophagy can also be subverted or hijacked by viruses to promote viral replication. For example, bunyavirus severe fever with thrombocytopenia syndrome virus (SFTSV) nucleoprotein (NP) triggers Beclin1-dependent autophagy, and the virus utilizes autophagic vesicles for virus assembly and egress (13). Both M2 and NP of influenza A virus interact with LC3B and induce autophagy to promote viral ribonucleoprotein (vRNP) export and viral replication (14).

Type I interferon (IFN-I) responses act as the first line of host defense against viral infection through the upregulation of numerous IFN-stimulated genes (ISGs), which can inhibit viral infection through diverse mechanisms (15, 16). Recently, an increasing number of studies have demonstrated that autophagy is involved in regulating innate immune responses during viral infection. For example, the matrix protein M of human parainfluenza virus 3 (HPIV3) interacts with the mitochondrial translation elongation factor Tu (TUFM), inducing TUFM-dependent mitophagy to inhibit IFN-I responses and promote viral replication (17). Inhibition of autophagy by knocking down the autophagy-related proteins Beclin1 and ATG7 increases the expression of IFN-α and IFN-β, thereby inhibiting HCV replication, indicating that HCV-induced autophagy negatively impairs the IFN-I signaling pathway to facilitate viral replication (18). Whether RVFV can trigger autophagy to regulate innate immune responses and viral replication remains unclear.

In this study, we investigated the underlying mechanisms of RVFV-triggered autophagy and regulation of antiviral immune responses. Our results provide novel insights into the mechanisms of RVFV-triggered autophagy and indicate the potential of targeting autophagic flux for developing antivirals against RVFV.

RESULTS

RVFV infection induced complete autophagy.

To investigate whether RVFV triggered autophagy during infection, we performed immunofluorescence assays with antibodies against LC3B and found that RVFV infection led to the formation of punctuate LC3B in phorbol 12-myristate 13-acetate (PMA)-treated THP-1 (THP-1^PMA) macrophages, similar to rapamycin-treated cells, which were included as the positive control (Fig. 1A). RVFV infection also induced punctuate LC3B formation in human Huh7 hepatocytes (Fig. 1B), primary mouse bone marrow-derived macrophages (mBMDMs) (see Fig. S1A in the supplemental material), and mouse embryonic fibroblasts (MEFs) (Fig. S1B). Transmission electron microscopy (TEM) analysis showed that similar to rapamycin-treated cells, RVFV infection led to the formation of DMVs (Fig. 1C). Previous studies have shown that the autophagy receptor SQSTM1 binds to LC3B and that both are degraded during autophagy (19). Depletion of the phosphatidylethanolamine-conjugated LC3B form (known as LC3B-II) and degradation of SQSTM1 are regarded as an indication of complete autophagy (20). In infected THP-1^PMA cells (Fig. 1D), mBMDMs (Fig. 1E) and Huh7 cells (Fig. 1F), RVFV induced a reduction in both SQSTM1 and LC3B-II during the course of infection. These results suggested that RVFV infection triggered complete autophagy in these cell types.

FIG 1.

RVFV infection induced autophagic signaling. (A and B) Autophagosomes were visualized by IFA using confocal microscopy. THP-1^PMA cells (A) and Huh7 cells (B) were treated with rapamycin, mock infected, or infected with RVFV at an MOI of 5 for 24 h. Cells were fixed and stained for RVFV with anti-RVFV NP (red) antibody, autophagosomes with anti-LC3B (green) antibody and nuclei with DAPI (blue). Rapamycin (5 μM) served as the positive control for autophagy, and cells were treated for 6 h before harvest. Bars, 10 μm. The LC3B puncta in each cell were counted, and at least 100 cells were included for each group (graphs). (C) Mock-infected, rapamycin (5 μM, 6 h)-treated, or RVFV (MOI = 10)-infected THP-1^PMA cells were processed and analyzed at 6 or 24 hpi by TEM for autophagosomes. Arrows indicate autophagic vacuoles. Bars, 500 nm. At least 50 cells were collected for statistical analysis in each group (bottom). (D to F) WB analysis of the indicated proteins in THP-1^PMA cells (D), mBMDMs (E), and Huh7 cells (F) infected with RVFV for the indicated times. The gray values of SQSTM1 and LC3B-II protein bands from three independent WB experiments were calculated with Image J and underwent statistical analysis. α-Tubulin was used as a control for normalization (right). Data are means and standard deviations (SD). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, no significant difference.

To verify whether RVFV infection induced complete autophagy, we employed a tandem reporter construct, pmCherry-eGFP-LC3B, which indicated whether the autophagosome fused with lysosomes based on the chemical properties of mCherry and enhanced green fluorescent protein (eGFP) fluorophores (21). The eGFP fluorescence signal of this tandem reporter construct is attenuated in a lysosomal acidic environment, whereas the mCherry fluorescence signal remains unaffected (21). Therefore, the fusion of autophagosomes with lysosomes resulted in the loss of yellow mCherry-eGFP fluorescence signals and the appearance of red mCherry fluorescence signals, indicating complete autophagic flux (22). To induce complete autophagy, Earle’s balanced salt solution (EBSS) was applied to cells as a positive control. We found that in both EBSS-starved cells and RVFV-infected cells, there were large numbers of mCherry-positive autophagic vacuoles and only a few eGFP-positive vacuoles compared with mock-infected cells (Fig. 2A). In contrast, in cells treated with chloroquine (CQ), an inhibitor of lysosomal acidification and autophagosome-lysosomal fusion (23), to block autophagy, most LC3B-positive autophagic vacuoles remained yellow, consistent with the inhibition of autophagosome-lysosome fusion by CQ treatment. Tracking autophagosomes with transiently overexpressed eGFP-LC3B and lysosomes with lysosome-associated membrane protein 2 (LAMP2) revealed that a large number of eGFP-LC3B molecules colocalized with LAMP2 in RVFV-infected cells, similar to EBSS-treated controls (Fig. 2B). To further confirm the induction of complete autophagy flux during RVFV infection, we used another autophagy inhibitor, bafilomycin A1 (BAFA1), which blocks the acidification of H⁺-ATPase and fusion between the autophagosome and lysosome (24, 25). RVFV-infected THP-1^PMA cells were treated with BAFA1 for 6 h before harvest (Fig. 2C). We found that, compared with the infected cells without BAFA1 treatment, BAFA1 treatment partially rescued the level of SQSTM1 and LC3B-II at 24 h postinfection (hpi) in the infected cells (Fig. 2D). Meanwhile, compared with the uninfected control, RVFV infection led to reduction of both SQSTM1 and LC3B-II with or without BAFA1 treatment. Treatment with BAFA1 resulted in pronounced accumulation of LC3B-II, masking the detection of LC3B-II in untreated cells to a certain extend. Nevertheless, RVFV infection led to a reduced LC3B-II level in BAFA1-treated cells, indicating occurrence of a complete autophagy flux. Similar results were observed in Huh7 cells (Fig. 2E). Taken together, these findings suggest that RVFV infection led to the fusion of autophagosomes with lysosomes and triggered complete autophagy.

FIG 2.

RVFV infection induced a complete autophagy process. (A) The effect of RVFV infection on autophagic flux in HeLa cells was observed by confocal microscopy. HeLa cells were transfected with pmCherry-eGFP-LC3B plasmid expressing mCherry-LC3B and eGFP-LC3B for 18 h and then starved in EBSS (6 h), treated with CQ (10 μM, 6 h), and mock infected or infected with RVFV (MOI = 5). At 12 hpi, cells were fixed and stained for RVFV with anti-RVFV NP (violet) antibody. Bars, 10 μm. The quantification of mCherry⁺ eGFP⁺ or mCherry⁺ eGFP⁻ fluorescence is representative of at least 20 cells (graph). (B) The effect of RVFV infection on autophagic flux in HeLa cells was observed by confocal microscopy. eGFP-LC3B was expressed in HeLa cells for 18 h, and then cells were treated with EBSS (6 h), mock infected, or infected with RVFV (MOI = 5). At 12 hpi, cells were fixed and stained for RVFV with anti-RVFV NP (violet) antibody and for lysosomes with anti-LAMP2 (red) antibody. Bars, 10 μm. The number of autolysosomes (eGFP and LAMP2 positive) in 40 cells was quantified (right). (C to E) Effect of RVFV infection on autophagic flux in THP-1^PMA and Huh7 cells treated with BAFA1. The experimental design for THP-1^PMA cells (D) and Huh7 cells (E) is displayed in panel C. THP-1^PMA and Huh7 cells were infected with RVFV for 18 h and then treated with BAFA1 for 6 h before WB analysis. The gray values of SQSTM1, LC3B-II, and NP bands were measured (bottom). The data are representative of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, no significant difference.

RVFV NP promoted autophagy through interaction with SQSTM1.

To identify the RVFV protein that triggers autophagy, we transiently coexpressed eGFP-LC3B with viral RNA-dependent RNA polymerase (RdRp), glycoprotein Gn, glycoprotein Gc, nonstructural protein NSm, nonstructural protein NSs, and nucleoprotein NP in HEK293T cells and analyzed eGFP-LC3B punctum formation. The results showed that the expression of NP, but not the other viral proteins, resulted in a significant increase in eGFP-LC3B punctum formation (Fig. 3A). Additionally, we found that the expression of RVFV NP in HeLa cells increased the abundance of LC3B-II in a concentration-dependent manner (Fig. 3B). Next, we investigated whether RVFV NP triggered autophagy by interacting with autophagy-related proteins. By screening a series of proteins functioning in autophagy flux (i.e., ATG5, ATG7, ATG12, Beclin1, SQSTM1, and LC3B), we found that NP interacted specifically with SQSTM1 (Fig. 3C). SQSTM1 also reciprocally precipitated RVFV NP (Fig. S2A). Interaction between SQSTM1 and NP was also observed in Huh7 cells (Fig. S2B). Confocal microscopy analysis further revealed the colocalization of NP and SQSTM1 in RVFV-infected HeLa cells (Fig. 3D). Transient expression of NP and SQSTM1 in HeLa cells also demonstrated the distinct colocalization of these two proteins (Fig. 3E).

FIG 3.

RVFV NP promoted autophagy through interacting with SQSTM1. (A) IFA analysis of exogenously expressed RVFV proteins for autophagy activation in HEK293T cells by confocal microscopy. eGFP-LC3B and empty vector or Strep-tagged or Twin-Strep-tagged viral proteins of RVFV were coexpressed in HEK293T cells. Viral proteins of RVFV were labeled with anti-Strep (red) antibody. Arrows indicate NP-induced autophagosomes. Bars, 10 μm. The graph shows quantitation of autophagosomes by taking the average number of eGFP-LC3B dots in 50 cells. (B) WB analysis of RVFV NP for autophagy activation in HeLa cells. HeLa cells were transfected with increasing NP-expressing plasmids for 24 h and the cell lysates were harvested and detected by WB. LC3B and Strep-tagged NPs were detected by the anti-LC3B antibody and anti-Strep antibody, respectively. The gray values of LC3B-II bands were measured (right). (C) Screening of autophagy-related proteins for the interaction with NP by streptavidin pulldown experiments in HEK293T cells. Strep-tagged NP and Flag-tagged eGFP, ATG5, ATG7, ATG12, Beclin1, SQSTM1 and LC3B were coexpressed in HEK293T cells. After 24 h, cells were subjected to pulldown assays with streptavidin MagBeads, followed by WB with anti-Strep antibody for NP and anti-Flag antibody for autophagy-related proteins. (D) IFA analysis of colocalization of NP with endogenous SQSTM1 in HeLa cells. HeLa cells were mock infected or infected with RVFV (MOI = 5) for 12 h. RVFV and SQSTM1 were labeled with anti-RVFV NP (red) antibody and anti-SQSTM1 (green) antibody. Bars, 5 μm (main images) and 2.5 μm (magnified image). Colocalization between SQSTM1 and NP was calculated using Fiji software (graph). (E) IFA analysis of colocalization of exogenously expressed NP with SQSTM1 in HeLa cells. Flag-tagged SQSTM1 and Strep-tagged NP were coexpressed in HeLa cells. NP and SQSTM1 were labeled with anti-Strep (red) antibody and anti-Flag (green) antibody, respectively. Bars, 5 μm (main images) and 2 μm (magnified image). Colocalization between SQSTM1 and NP was calculated using Fiji software (graph). (F) The effect of RVFV NP on the interaction between SQSTM1 and LC3B was analyzed by pulldown assays in HEK293T cells. Strep-tagged eGFP or NP, eGFP-tagged LC3B, and Flag-tagged SQSTM1 were coexpressed in HEK293T cells. After 24 h, cell lysates were subjected to pulldown assays with Flag MagBeads and analyzed by WB. The gray values of eGFP-LC3B protein bands from three independent WB experiments were calculated (bottom). (G) The effect of RVFV NP on the colocalization between endogenous SQSTM1 and LC3B was analyzed by IFA in HeLa cells. Strep-tagged NP and eGFP-tagged LC3B were coexpressed in HeLa cells for 24 h. NP and SQSTM1 were labeled with anti-Strep (violet) antibody and anti-SQSTM1 (red) antibody. Nuclei were stained with DAPI (blue). Bars, 10 μm. Colocalization between eGFP-LC3B and SQSTM1 was calculated using Fiji software (graph). Statistical analysis was performed with Student’s t test; **, P < 0.01; ***, P < 0.001; ns, no significant difference.

There are also other selective autophagy receptors (19, 26) that recognize cargos to form autophagosomes through binding with LC3 (27). Therefore, we analyzed whether additional representative autophagy receptors (i.e., OPTN, NBRI, NIX, and TOLLIP) can also interact with RVFV NP. The results showed that SQSTM1, but not the other selective autophagy receptors, interacts with RVFV NP (Fig. S2C). Next, we analyzed whether NP-SQSTM1 interaction depends on ubiquitination modification. For this purpose, we expressed streptavidin (Strep)-tagged NP or mitochondrial antiviral signaling (MAVS) protein, which is known to be modified by ubiquitination and serves as a positive control, together with HA-tagged ubiquitin in HEK293T cells. Strep-tagged proteins were pulled down, and the ubiquitination was analyzed. The results showed that while MAVS was strongly ubiquitinated, NP did not show a specific ubiquitination signal (Fig. S2D), suggesting that SQSTM1 binds to NP in a ubiquitination-independent manner. Additionally, we evaluated whether NP affected the interaction between SQSTM1 and LC3B. Coexpression experiments showed that the presence of NP promoted the interaction between SQSTM1 and LC3B and was associated with the complex (Fig. 3F). Additionally, immunofluorescence assays showed that NP expression in transfected HeLa cells triggered distinct colocalization between SQSTM1 and eGFP-LC3B (Fig. 3G). Taken together, these results suggested that RVFV NP interacts with SQSTM1 to promote SQSTM1-LC3B interaction and autophagic flux.

To further identify the domain of the NP that interacts with SQSTM1, we constructed five truncated NPs (Fig. 4A) according to the previously reported structure of RVFV NP (28). All five truncated mutants were successfully expressed at comparable levels (Fig. 4B). Pulldown experiments showed that deletion of the C-terminal region from position 128 to 207 or position 208 to 245 of the NP (NPΔ128–207 and NPΔ208–245, respectively) strongly impaired the interaction of NP with SQSTM1 (Fig. 4B). To further evaluate the region of the NP responsible for triggering autophagy, we transiently transfected HeLa cells with constructs expressing truncated NPs and eGFP-LC3B and then performed immunofluorescence assays. The truncated constructs NPΔ128–207 and NPΔ208–245 did not induce eGFP-LC3B punctum formation, whereas other truncations induced eGFP-LC3B punctum formation, similar to the full-length NP and rapamycin treatment control (Fig. 4C). Furthermore, Western blotting (WB) analysis showed that the expression of NPΔ128–207 and NPΔ208–245 did not result in the accumulation of LC3B-II, whereas the expression of other truncations and the full-length NP led to the accumulation of LC3B-II (Fig. 4D). Collectively, these results revealed that the C-terminal 128–245 region of the RVFV NP interacts with SQSTM1 to promote autophagy flux.

FIG 4.

The 128–245 region of the RVFV NP interacts with SQSTM1 to promote autophagy flux. (A) Schematic diagram of the full-length and five truncated NPs, with the secondary structure elements (α-helices) shown at the top. (B) Pulldown assay analysis of the interaction between truncated NP and SQSTM1 in HEK293T cells. Flag-tagged SQSTM1 or eGFP and Strep-tagged truncated NP were coexpressed in HEK293T cells as indicated. After 24 h, the cell lysates were subjected to pulldown assays with streptavidin MagBeads. NPs were detected by the anti-Strep antibody, and eGFP or SQSTM1 was detected by the anti-Flag antibody. (C) IFA analysis of exogenously expressed truncated NPs for autophagy activation in HeLa cells. eGFP-LC3B and empty vector or Strep-tagged truncated NPs were coexpressed in HeLa cells. After 24 h the cells were processed and assessed via IFA. NPs were labeled with anti-Strep (red) antibody. Bars, 10 μm. The graph shows quantitation of autophagosomes by taking the average number of eGFP-LC3B dots in 50 cells. (D) WB analysis of truncated NPs for autophagy activation in HEK293T cells. Strep-tagged truncated NPs were expressed in HEK293T cells. After 24 h, the cells were processed and assessed via WB. The gray values of LC3B-II bands were measured (graph). Data were analyzed using Student’s t test and are presented as means ± SD. ***, P < 0.001; ns, nonsignificant difference.

RVFV-triggered autophagy facilitated viral replication in macrophages.

To investigate whether RVFV-triggered autophagy affected viral infection, we analyzed RVFV infection in cells depleted of several key autophagy-related genes, including ATG5, ATG7, and SQSTM1. THP-1^PMA cells were depleted of ATG5, ATG7, or SQSTM1 through CRISPR-Cas9-mediated knockout. WB showed that the intracellular level of viral NP was strongly decreased in the RVFV-infected ATG5, ATG7, or SQSTM1 knockout cells compared with that in the control cells (Fig. 5A to C). Furthermore, plaque assays showed that depletion of these autophagy-related genes strongly reduced the production of infectious virions in RVFV-infected THP-1^PMA cells (Fig. 5D to F). Reverse transcription-quantitative real-time PCR (RT-qPCR) analysis showed that the level of intracellular viral mRNA was strongly reduced in ATG5-, ATG7-, and SQSTM1-depleted THP-1^PMA cells (Fig. 5G to I), indicating inhibition of viral replication at an early stage in these cells.

FIG 5.

RVFV-triggered autophagy facilitated viral replication in macrophages. (A to C) WB analysis of the indicated proteins in Scramble (a non-targeting CRISPR-cas9 control sgRNA), ATG5 (A), ATG7 (B), and SQSTM1 (C) knockout THP-1^PMA cells mock infected or infected with RVFV (MOI = 5) for 24 h. The gray values of NP bands were measured (right). (D to F) Extracellular viral titers from Scramble, ATG5 (D), ATG7 (E), and SQSTM1 (F) knockout THP-1^PMA cells infected with RVFV (MOI = 5) at 24 hpi were determined by plaque assays. (G to I) Cell lysates from Scramble, ATG5 (G), ATG7 (H), or SQSTM1 (I) knockout THP-1^PMA cells infected with RVFV (MOI = 5) were collected at 24 hpi. Total RNA was extracted, and the mRNA levels of NP were measured by RT-qPCR. 18S rRNA was used as a control for normalization. (J and K) The effect of autophagy inhibitor BAFA1 on RVFV replication was determined by WB. THP-1^PMA cells (J) and mBMDMs (K) were mock infected or infected with RVFV (MOI = 5) for 2 h and then incubated with BAFA1 at the indicated concentrations for an additional 24 h. Cell lysates were harvested and analyzed by WB. The gray values of NP bands were measured (bottom). (L and M) RT-qPCR analysis of NP mRNA in THP-1^PMA cells (L) from panel J and mBMDMs (N) from panel K. (N and O) Extracellular viral titers from supernatants were determined by plaque assays in THP-1^PMA cells (N) from panel J and mBMDMs (O) from panel K. ***, P < 0.001. The data are representative of three independent experiments. Statistical analysis was performed with Student’s t test.

To verify the effects of autophagy on RVFV infection, we performed infection in the presence of the autophagy inhibitor BAFA1. Two types of macrophages, THP-1^PMA cells and mBMDMs, were infected with RVFV for 2 h and then incubated with BAFA1 at the indicated concentrations for an additional 24 h. Notably, intracellular viral NP levels were decreased by BAFA1 treatment in a concentration-dependent manner in both THP-1^PMA cells (Fig. 5J) and mBMDMs (Fig. 5K). RVFV-triggered degradation of SQSTM1 and LC3B-II was also inhibited by BAFA1 treatment, consistent with the inhibition of autophagy (Fig. 5J and K). RT-qPCR and plaque assays showed that the intracellular mRNA levels of the viral NP gene (Fig. 5L and M) and production of extracellular infectious virus (Fig. 5N and O) were significantly reduced by BAFA1 treatment in THP-1^PMA cells and mBMDMs. These results suggested that RVFV-triggered autophagy facilitated viral replication in macrophages.

Since blocking autophagy inhibits the early stage of RVFV infection, we explored whether blocking autophagy affected viral entry. For this purpose, ATG5 and ATG7 knockout THP-1^PMA cells were chilled at 4°C for 15 min and then incubated with RVFV at 4°C for 1 h to synchronize the virus-binding process. Next, ammonium chloride was added to prevent low-pH-dependent membrane fusion, blocking viral entry at the fusion step at 37°C for 3 h. The unbound virions were removed by washing with phosphate-buffered saline three times, and the viral RNA was harvested and analyzed via RT-qPCR (29) (Fig. S3A). The results showed that virus binding or internalization was not affected in these autophagy-defective cells (Fig. S3B), indicating that autophagy facilitated viral infection downstream of the virus entry event.

RVFV-triggered autophagy was dispensable for virus replication in Huh7 cells and HUVECs.

We next analyzed whether RVFV-triggered autophagy affected viral infection in other target cell types. A major target organ of RVFV replication in vivo is the liver, and RVFV infection can lead to serious liver damage in patients (2, 30). Blood vessels are another target tissue for RVFV, the infection of which is potentially associated with hemorrhage (31). Therefore, we analyzed the effects of blocking autophagy on RVFV infection in Huh7 cells, a human hepatocellular cell line, and human umbilical vein endothelial cells (HUVECs), a human umbilical vein endothelial cell line. WB confirmed the efficient depletion of ATG5 in both cell types (Fig. 6A and B). Conversion of endogenous LC3B-I to LC3B-II was strongly decreased in ATG5 knockout Huh7 cells and HUVECs, confirming the inhibition of the autophagy pathway. In contrast to RVFV-infected THP-1^PMA cells, RVFV-infected ATG5 knockout cells showed similar levels of intracellular viral NP with the control cells, as demonstrated by WB (Fig. 6A and B). Next, the levels of intracellular NP mRNA and extracellular infectious-virus production were measured using RT-qPCR and plaque assays, respectively. ATG5 depletion in Huh7 cells and HUVECs did not affect RVFV intracellular NP mRNA abundance (Fig. 6C and D) or infectious virus production compared with those in the control (Fig. 6E and F). These results showed that unlike the findings in THP-1^PMA cells, impairing autophagy did not affect RVFV replication in these cell types.

FIG 6.

RVFV-triggered autophagy was dispensable for virus replication in Huh7 cells or HUVECs. (A and B) WB analysis of the indicated proteins in Scramble or ATG5 knockout Huh7 cells (A) and HUVECs (B) mock infected or infected with RVFV (MOI = 2) at 24 hpi. The gray values of NP bands were measured (right). (C and D) RT-qPCR analysis of NP mRNA in ATG5 knockout Huh7 cells (C) or HUVECs (D) infected with RVFV (MOI =2) at 24 hpi. (E and F) Extracellular viral titers from supernatants in ATG5 knockout Huh7 (E) and HUVECs (F) were determined by plaque assays. The data are representative of three independent experiments. Statistical analysis was performed with Student’s t test. ns, nonsignificant difference.

RVFV-triggered autophagy dampened the antiviral innate immune response to promote viral replication.

The finding that the autophagy pathway was important for RVFV infection in macrophages but not in other cell types suggested that RVFV-triggered autophagy may be involved in regulating antiviral innate immune responses. Therefore, we assessed the expression levels of genes involved in immune signaling in RVFV-infected ATG5 or ATG7 knockout THP-1^PMA cells. ATG5 or ATG7 knockout and control THP-1^PMA cells were infected with RVFV, and cell lysates were harvested at 6 and 9 hpi, followed by RNA extraction. RT-qPCR analysis showed that the mRNA levels of IFN-α, IFN-β, ISG15, ISG54, ISG56, retinoic acid-inducible gene I (RIG-I), RNA-dependent protein kinase (PKR), radical S-adenosyl methionine domain-containing 2 (RSAD2), and cytidine/UMP kinase 2 (CMPK2) were significantly upregulated in RVFV-infected ATG5 or ATG7 knockout THP-1^PMA cells compared with those in the control (Fig. 7A and B). Similar to the results above, the intracellular viral NP mRNA level was significantly decreased in ATG5 or ATG7 knockout THP-1^PMA cells compared with that in control THP-1^PMA cells (Fig. 7C and D). These results also indicated that the upregulated ISGs in ATG5 or ATG7 knockout THP-1^PMA cells was not due to higher RVFV replication. WB further showed that the protein levels of ISGs, including ISG15, ISG54, and RSAD2, were markedly increased in RVFV-infected ATG5 and ATG7 knockout THP-1^PMA cells compared with those in control cells at 12 hpi (Fig. 7E and F). Consistent with the above results, plaque assays showed that the production of extracellular infectious virions was markedly reduced in ATG5 and ATG7 knockout THP-1^PMA cells (Fig. 7G and H).

FIG 7.

RVFV-triggered autophagy dampened the antiviral innate immune responses to promote viral replication. (A to D) RT-qPCR analysis of IFN-α, IFN-β, ISG15, ISG54, ISG56, RIG-I, PKR, RSAD2 and CMPK2 in Scramble, ATG5 (A), or ATG7 (B) knockout THP-1^PMA cells infected with RVFV (MOI = 2) at 6 hpi and 9 hpi. RT-qPCR analysis of NP mRNA in Scramble, ATG5 (C), or ATG7 (D) knockout THP-1^PMA cells infected with RVFV (MOI = 2) at 6 hpi and 9 hpi. (E and F) WB analysis of ISG54, RSAD2 and ISG15 from ATG5 (E) and ATG7 (F) knockout THP-1^PMA cells mock infected or infected with RVFV (MOI = 2) at 12 hpi. The gray values were measured (right). (G and H) Extracellular viral titers from ATG5 (G) and ATG7 (H) knockout THP-1^PMA cells infected with RVFV (MOI = 2) at 12 hpi were determined by plaque assays. **, P < 0.01; ***, P < 0.001; ns, nonsignificant difference. The data are representative of three independent experiments.

ATG5 knockout and control Huh7 cells were infected with RVFV, and similar analyses with THP-1^PMA cells were performed. In contrast to the results in THP-1^PMA cells, ATG5 depletion in Huh7 cells did not affect the expression levels of genes involved in immune signaling, including IFN-α, IFN-β, ISG15, ISG54, and ISG56 (Fig. S4A). Consistent with the fact that Huh7 cells are immunocompromised, RVFV infection did not induce the upregulation of selected ISGs, including ISG54 and RSAD2 (Fig. S4B). The expression level of intracellular viral NP and the production of extracellular infectious virions were also comparable between RVFV-infected ATG5 knockout and control Huh7 cells (Fig. S4B and C). Similar results were also observed in the ATG5 knockout and control HUVECs (Fig. S5A and B). These results together suggested that RVFV-triggered autophagy dampens antiviral innate immune responses to promote viral replication.

DISCUSSION

RVFV is a mosquito-borne virus transmitted mainly through mosquito bites (7, 32, 33). Macrophages are likely among the first cell types encountered by RVFV through this viral transmission route. Establishing productive infection in these cells, which can induce robust antiviral innate immune responses, is therefore important for successful replication of RVFV in the host. RVFV encodes a strong inhibitor of antiviral innate immune responses, the nonstructural protein NSs, which impedes IFN production by preventing activation of the IFN-β promoter (34), targeting PKR for degradation (35, 36) and interfering with the assembly of the RNA polymerase II preinitiation complex transcription factor II H (37). Here, we found that the RVFV NP could trigger autophagy to dampen antiviral immune responses in infected macrophages. This additional layer of autophagy-dependent inhibition of innate immune responses, in combination with the inhibitory mechanisms of NSs, may further promote efficient RVFV replication in these immunocompetent cell types.

Viral infections can trigger autophagy through different mechanisms. A recent study reported that the NP of SFTSV binds to Beclin1 to trigger Beclin1-dependent autophagy (13). Beclin1 is an essential factor in autophagy initiation and is inhibited by its association with the negative regulator BCL2 (38). The interaction between SFTSV-NP and Beclin1 leads to the dissociation of Beclin1 and BCL2 and initiation of autophagy. Here, we found that RVFV NP did not bind to Beclin1; instead, it interacted with the autophagy receptor SQSTM1 and enhanced the interaction between SQSTM1 and LC3B to promote LC3B punctum formation and autophagy flux. SQSTM1 is an autophagy adaptor that binds polyubiquitinated cargo and LC3B to mediate cargo degradation (39, 40). Because RVFV NP-triggered autophagy led to a reduction in the innate immune response, future studies could investigate whether the NP-SQSTM1-LC3B complex leads to the degradation of certain molecules that mediate immune signaling. Notably, impairing autophagy in other cell types, including Huh7 cells and HUVECs, showed no effect on RVFV replication. Unlike macrophages, Huh7 cells and HUVECs have compromised immune responses (41–44). This further indicates that immune suppression is the major mechanism underlying the autophagy-dependent promotion of RVFV replication, at least in macrophages.

Several viruses have been reported to trigger autophagy to dampen the antiviral IFN-I immune responses. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) M protein triggers mitophagy to impair the functionality of mitochondrial networks, thereby suppressing the antiviral IFN-I signaling to enhance viral replication (45). Influenza A virus (IAV) PB1-F2 protein also induces mitophagy to suppress the IFN-I signaling by promoting autophagy-mediated degradation of the MAVS protein (46). It would be interesting to investigate whether RVFV NP-promoted autophagy also facilitates mitophagy to dampen the antiviral immune response during RVFV infection.

An earlier study reported that RVFV-triggered autophagy showed inhibitory effects on RVFV infection (47). Flies with depletion of the core autophagy gene ATG18, ATG5, or ATG7 showed increased RVFV replication and enhanced lethality (47). The same study also reported that depletion of ATG5 or ATG7 in U2OS human osteosarcoma cells increased virus production (47). In this study, we employed cell models of differentiated macrophages (THP-1^PMA cells), Huh7 hepatocytes, and HUVECs. Our results showed that RVFV successfully infected macrophages (Fig. S6A) and hepatocytes (Fig. S6B) in a mouse model, consistent with other studies (48). Vein endothelia are also a target tissue type of RVFV infection in vivo (49). The differences in model cell types between our study and the previous study may explain the different results obtained in these studies and indicate the need to investigate this pathway in different, and potentially more physiologically relevant, cell models. Further investigations in RVFV-infected animal models may be necessary for in-depth interpretation of the physiological relevance of virus-triggered autophagy in regulating viral infection and pathogenesis.

Taken together, these findings demonstrated that activation of the autophagy pathway upon RVFV infection in macrophages plays a proviral role, supporting efficient viral replication. We conclude that the RVFV NP interacted with the autophagy receptor SQSTM1 to promote autophagy flux and inhibit the antiviral innate immune response (Fig. 8). Our study provides novel insights into the mechanisms of RVFV NP-triggered autophagy and may help to identify targets of the autophagy flux for the development of antiviral strategies against RVFV infection.

FIG 8.

Proposed model of RVFV-triggered autophagy in regulating virus replication. RVFV infection results in accumulation of endogenous LC3B puncta and triggers autophagosome formation, followed by the fusion of autophagosome and lysosome. The 128–245 region of the RVFV NP binds to the autophagy receptor SQSTM1, promoting the interaction between SQSTM1 and LC3B and inducing complete autophagy. In immunocompetent cells, such as THP-1^PMA cells, RVFV-triggered autophagy dampens the antiviral innate immune responses to promote viral replication.

MATERIALS AND METHODS

Cells.

HEK293T cells, HeLa cells, Huh7 cells, HUVECs, MEFs, BHK-21 cells, and Vero cells were cultured in Dulbecco’s modified Eagle medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) and 100 U/mL penicillin-streptomycin. Human monocytic THP-1 cells were grown in RPMI 1640–l-glutamine (Gibco) with 10% FBS and treated with 40 ng/mL PMA (Sigma) for 2 days to induce differentiation to THP-1^PMA macrophages maintained in RPMI 1640 medium. Primary mBMDMs were derived from 10-week-old male BALB/c mice (Charles River Laboratories, China) and cultured for 3 to 4 days with 10 ng/mL recombinant murine macrophage colony-stimulating factor (M-CSF; PeproTech, Austria). THP-1 stable cell lines were generated using a standard selection protocol with 2 μg/mL of puromycin (50).

Viruses.

RVFV was rescued by a T7 RNA polymerase-driven reverse genetics system as previously described (51). RVFV was propagated in BHK-21 cells, and the viral titration was measured by plaque assays using Vero cells with crystal violet staining. For infection, cell monolayers were washed once with phosphate-buffered saline (PBS), and then RVFV was added at the indicated multiplicity of infection (MOI). For mock-infected controls, cells were washed with PBS and incubated with complete medium without virus in parallel during virus infection. All RVFV experiments were performed at the biosafety level 3 (BSL-3) facility of the Wuhan Institute of Virology, Chinese Academy of Sciences (Wuhan, China), according to institutional guidelines.

Antibodies and reagents.

The primary antibodies used in this study were as follows: monoclonal rabbit anti-LC3B (3868S), anti-ATG5 (12994S), anti-ATG7 (8558S), and monoclonal mouse anti-LC3B (E5Q2K) from Cell Signaling Technology; polyclonal rabbit anti-SQSTM1/p62 (18420-1-AP), anti-Beclin1 (11306-1-AP), anti-ISG15 (15981-1-AP), anti-ISG54 (12604-1-AP), anti-RSAD2 (28089-1-AP), anti-α-tubulin (11224-1-AP), and anti-DYKDDDDK tag (Flag; 20543-1-AP) from Proteintech; monoclonal mouse anti-ATG5 (66744-1-Ig), anti-ATG7 (67341-1-Ig), and anti-GFP tag (66002-1-Ig) from Proteintech; monoclonal mouse anti-LAMP2 (sc-18822) from Santa Cruz Biotechnology; monoclonal mouse anti-streptavidin tag (A01732) and polyclonal rabbit anti-streptavidin tag (A00626) from GenScript; and polyclonal rabbit anti-RVFV NP from our laboratory.

Horseradish peroxidase (HRP)-conjugated AffiniPure goat anti-mouse IgG (H+L) (SA00001-1) and goat anti-rabbit IgG (H+L) (SA00001-2) secondary antibodies were from Proteintech. Fluorescence-labeled secondary antibodies, including Alexa Fluor 488 goat anti-mouse IgG (H+L) (A-11001), Alexa Fluor 488 goat anti-rabbit IgG (H+L) (A-11008), Alexa Fluor 568 goat anti-mouse IgG (H+L) (A-11004), Alexa Fluor 568 goat anti-rabbit IgG (H+L) (A-11036), Alexa Fluor 647 goat anti-mouse IgG (H+L) (A-21235), and Alexa Fluor 647 goat anti-rabbit IgG (H+L) (A-21245), were from Thermo Fisher.

Rapamycin (S1039), bafilomycin A1 (BAFA1; S1413), and hydroxychloroquine sulfate (CQ; S4430) were purchased from Selleck. EBSS (24010043) was purchased from Thermo Fisher. DAPI (4′,6-diamidino-2-phenylindole; C1002) was purchased from Beyotime.

Plasmids construction and DNA transfection.

In order to construct a series of plasmids capable of expressing the single viral protein fused with streptavidin (Strep) or Twin-Strep (TS), the corresponding cDNA sequences of RVFV were obtained by RT-PCR. Using seamless cloning technology (Vazyme; C112-02), cDNA sequences of RdRp, Gn, and Gc were cloned into the pCAGGS-TS vector and the cDNA sequences of NSm, NP and NSs were cloned into the pRK-Strep vector. Coding sequences for ATG5, ATG7, ATG12, Beclin1, SQSTM1, and LC3B were amplified by PCR from cDNA of human THP-1 cells and inserted into the pRK-Flag vector. pRK-eGFP-LC3B was generated by inserting PCR-amplified eGFP and LC3B sequences between the HindIII and XbaI restriction sites of pRK. pRK-Flag-eGFP, pRK-eGFP-Strep, and the tandem reporter plasmid pmCherry-eGFP-LC3B were stored in our laboratory. The NP truncation plasmids (Δ2–32, Δ33–92, Δ93–127, Δ128–207, and Δ208–245) were generated by seamless cloning technology (Vazyme; C112-02). DNA transfections were performed with Lipofectamine 2000 (Invitrogen) for 24 h in the HEK293T cells and Lipofectamine 3000 (Invitrogen) in the HeLa cells according to the manufacturer’s protocol.

Virus titration.

Titration of the virus was measured by plaque assays. Briefly, confluent monolayers of Vero cells in 6-well plates were infected with 100 μL serial dilutions of either cleared cell lysate or supernatant at 37°C for 1 h. After removal of the inoculum, cells were overlaid with DMEM containing 2% FBS and 1.1% carboxymethyl cellulose (Sigma). After 7 days of incubation, the cells were stained with 1% crystal violet after being fixed with 4% formaldehyde. The plaques were counted and quantified according to standard protocol (52), and the virus titers were expressed in PFU per milliliter.

WB.

Cells were washed with PBS (pH 7.4) and then harvested in SDS lysis buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 5 mM EDTA, 1% SDS, and 10% glycerol). Cell lysates were boiled at 95°C for 10 min, subjected to 12% to 15% SDS-PAGE, and then electrotransferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). After blocking with 5% nonfat milk in Tris-buffered saline–Tween (TBS-T) for 1 h at room temperature, the membrane was further incubated with the primary antibodies overnight at 4°C, followed by HRP-conjugated secondary antibodies for 1 h at room temperature. The proteins on the membrane were detected and visualized using an enhanced chemiluminescence (ECL) kit (Millipore) on ChemiScope 6000 Exp imaging system (Clinx Science Instruments, China).

RNA extraction and RT-qPCR.

Total RNA was extracted from cells by using RNAiso Plus reagent (TaKaRa; 9109) according to the manufacturer’s protocol and were subsequently reverse-transcribed to cDNA using a HiScript II first-strand cDNA synthesis kit (with genomic DNA [gDNA] wiper) (Vazyme, China; R212-01). qPCR was performed with a ChamQ universal SYBR qPCR master mix (Vazyme, China; Q711-02) on a real-time PCR system (Roche, Germany). Three biological replications were carried out for each sample. The RT-qPCR primer sequences are listed in Table S1.

Streptavidin/Flag MagBeads pulldown assays.

After transfection with the indicated plasmids and washing with PBS, HEK293T cells were lysed using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology) at 4°C for 20 min. Lysates were centrifuged at 13,000 rpm for 20 min at 4°C to remove cell debris. Then, the supernatant was incubated with prewashed MagStrep type3 XT beads (IBA Life Sciences; 2-4090-010) or BeyoMag anti-Flag magnetic beads (Beyotime Biotechnology; P2115) overnight at 4°C. After 2 washes with lysis buffer, the bound proteins were eluted and subjected to WB using anti-Strep or anti-Flag antibody.

Generation of stable cell lines by lentiviral transduction.

The single guide RNA (sgRNA) (in lentiCRISPR-v2) construct with two other packaging plasmids, pCMV-dR8.91 (encoding Gag, Pol, Tat, and Rev) and pMD2.G, was cotransfected into HEK293T cells at around 80% confluence using Lipofectamine 2000. Lentivirus-containing cell culture media were harvested 60 h after transfection and filtered through a 0.45-μm filter to remove cell debris. The harvested lentivirus was added to THP-1 cells, HUVECs or Huh7 cells. At 48 h after transduction, the medium containing the viruses was replaced with fresh medium containing 2 μg/mL puromycin for selection. Single clones from a stable cell pool were then isolated and expanded. The target sequences for sgRNA used in this study are available in Table S2.

Immunofluorescence assays (IFA) and confocal microscopy.

Cells infected with RVFV were washed with PBS, fixed with 4% paraformaldehyde (PFA), permeabilized with 0.2% (vol/vol) Triton X-100, and blocked with 3% bovine serum albumin (BSA). Next, the cells were incubated with the indicated primary antibodies overnight at 4°C. The cells were sequentially washed, followed by a fluorescently labeled secondary antibody at room temperature for 1 h. Nuclei were stained with DAPI. Images were observed with a confocal microscope (Andor Dragonfly 202) with a 63× oil objective.

Culture and proliferation of mBMDMs.

Primary BMDMs were isolated from male BALB/c mice (10 weeks of age) purchased from Charles River Laboratories (Beijing, China). Briefly, the dissected femurs and tibiae were collected and bone marrow cavities were gently flushed with PBS containing 1% penicillin-streptomycin. Then, red blood cells (RBCs) from macrophages were removed using RBC lysing buffer (Sigma, Roche) followed by washing with PBS. Cells were cultured for 3 to 4 days in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin-streptomycin, and 10 ng/mL recombinant murine M-CSF. After 4 days, the differentiated mBMDMs were used for experiments.

TEM analysis.

THP-1^PMA cells seeded in 10-cm dishes were infected with RVFV (MOI of 10) for 6 hpi and 24 hpi, respectively, or treated with rapamycin (5 μM) for 6 h. The cells were fixed in 2.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M PBS (pH 7.4) for 2 h at room temperature. Then, dehydration was performed in a graded series of ethanol (30%, 50%, 70%, 90%, and 100%) for 45 min before cells were embedded in epoxy resin and polymerized for at least 48 h. The embedded samples were trimmed and sectioned for transmission electron microscopy (TEM; FEI Tecnai G2 microscope at 200 kV).

RVFV inoculation into mice.

Male 8-week-old C57BL/6J mice were obtained from Charles River Laboratories (Beijing, China) and were maintained under specific-pathogen-free conditions at the BSL-3 facility of the Central Animal Laboratory in accordance with the ethical guidelines of the Wuhan Institute of Virology. The animals were anesthetized using sevoflurane and infected with RVFV via an intraperitoneal inoculation of 10 PFU/mouse. After euthanasia at 2 days postinfection, spleen and liver samples were collected for immunohistochemical analysis.

IHC.

The collected tissue samples obtained from infected and mock-infected mice were fixed with 4% paraformaldehyde, embedded in paraffin, and cut into sections (5 μm). Sections were used for immunofluorescence staining using a PANO 7-plex immunohistochemistry (IHC) kit (Panovue, China) according to the manufacturer’s instructions. Images were observed with a confocal microscope (Andor Dragonfly 202) with a 63× oil objective.

Autophagic flux intervention.

To induce autophagy, the cells were washed three times with PBS and then cultured in complete medium with rapamycin (5 μM) or starvation medium (EBSS, GIBCO) for 6 h. Cells were treated with CQ (10 μM) for 6 h to block autophagosome-lysosome fusion. To determine the effect of RVFV infection on autophagic flux or drug treatment on RVFV replication, BAFA1 was used for cell experiments.

Statistical analysis.

Student’s t test was used for two-group comparisons. For comparisons of three or more groups, one- or two-way analysis of variance (ANOVA) was performed in GraphPad Prism version 8.4.3. For all experiments, a P value of <0.05 was considered statistically significant.