Gga-miR-30c-5p Suppresses Avian Reovirus (ARV) Replication by Inhibition of ARV-Induced Autophagy via Targeting ATG5

ABSTRACT

Avian reovirus (ARV) causes viral arthritis, chronic respiratory diseases, retarded growth, and malabsorption syndrome. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression posttranscriptionally by silencing or degrading their targets, thus playing important roles in the host response to pathogenic infection. However, the role of miRNAs in host response to ARV infection is still not clear. In this study, we show that ARV infection markedly increased gga-miR-30c-5p expression in DF-1 cells and that transfection of cells with gga-miR-30c-5p inhibited ARV replication while knockdown of endogenous gga-miR-30c-5p enhanced viral growth in cells. Importantly, we identified the autophagy related 5 (ATG5), an important proautophagic protein, as a bona fide target of gga-miR-30c-5p. Transfection of DF-1 cells with gga-miR-30c-5p markedly reduced ATG5 expression accompanied with reduced conversion of ARV-induced-microtubule-associated protein 1 light chain 3 II (LC3-II) from LC3-I, an indicator of autophagy in host cell, while knockdown of endogenous gga-miR-30c-5p enhanced ATG5 expression as well as ARV-induced conversion of LC3-II, facilitating viral growth in cells. Furthermore, knockdown of ATG5 by RNA interference (RNAi) or treatment of cells with autophagy inhibitors (3-MA and wortmannin) markedly reduced ARV-induced LC3-II and syncytium formation, suppressing viral growth in cells, while overexpression of ATG5 increased ARV-induced LC3-II and syncytium formation, promoting viral growth in cells. Thus, gga-miR-30c-5p suppressed viral replication by inhibition of ARV-induced autophagy via targeting ATG5. These findings unraveled the mechanism of how host cells combat against ARV infection by self-encoded small RNA and furthered our understanding of the role of microRNAs in host response to pathogenic infection.

IMPORTANCE Avian reovirus (ARV) is an important poultry pathogen causing viral arthritis, chronic respiratory diseases, and retarded growth, leading to considerable economic losses to the poultry industry across the globe. Elucidation of the pathogenesis of ARV infection is crucial to guiding the development of novel vaccines or drugs for the effective control of these diseases. Here, we investigated the role of miRNAs in host response to ARV infection. We found that infection of host cells by ARV remarkably upregulated gga-miR-30c-5p expression. Importantly, gga-miR-30c-5p suppressed ARV replication by inhibition of ARV-induced autophagy via targeting autophagy related 5 (ATG5) accompanied by suppression of virus-induced syncytium formation, thus serving as an important antivirus factor in host response against ARV infection. These findings will further our understanding of how host cells combat against ARV infection by self-encoded small RNAs and may be used as a potential target for intervening ARV infection.

INTRODUCTION

Avian reovirus (ARV) is a ubiquitous pathogen among poultry flocks, and its infection causes multiple types of clinical diseases such as viral arthritis, tenosynovitis, growth retardation, malabsorption syndrome, and chronic respiratory disease in chickens (1). Infection by ARVs is associated with immunosuppression and leads to an increased susceptibility to other infectious diseases and vaccination failure (2). ARV, a member of the Orthoreovirus genus in the Reoviridae family, is a nonenveloped virus with RNA genome consisting of 10 fragments (L1–3, M1–3, and S1–4) that encode at least 10 structural proteins (λA–C, μA, μB, μBC, μBN, and σA–C) and 4 nonstructural proteins (μNS, p10, p17, and σNS) (3). λA is major component of viral inner core shell (4). Viral RNA polymerase λB and its cofactor μA are presumed to be transcriptase complex (5). λC, a viral capping enzyme, is involved in the stabilization, transport, and translation of viral mRNA (6). μB and its cleavage products (μBC and μBN) play a key role in virus penetration and transcriptase activation (7). μNS can form inclusions and is required for virus factory formation (8). σA binds to dsRNA and displays anti-interferon activity (9, 10). As the major viral outer capsid protein, σB induces production of group-specific neutralizing antibodies (11, 12). σC is able to induce virus-specific neutralizing antibodies and cell apoptosis (13, 14). p10 is responsible for virus-induced cell fusion and apoptosis (15, 16). p17 induces autophagosome formation and cell cycle arrest (17–19). σNS is thought to act as an RNA chaperone and be involved in the packaging of RNA (20, 21).

MicroRNAs (miRNAs) are a class of endogenous noncoding RNAs with a length of 20 to 24 nucleotides (22). As important posttranscriptional regulatory molecules, miRNAs affect mRNA stability or inhibit translation typically by binding to the 3′ untranslated region (UTR) of target gene mRNAs, thereby silencing target gene expression (23). The important role of miRNA in host-virus interactions has gradually been revealed. In our previous study, we screened ARV-infected DF-1 cells for the potential host miRNA response to ARV infection by deep sequencing (24) and found that a number of differentially expressed miRNAs are predicted to be involved in regulating various aspects of host response, but their roles in the pathogenesis of ARV infection and underlying mechanisms have not been unraveled yet.

In this study, we found that the expression of gga-miR-30c-5p, a chicken microRNA among our previous sequencing data, significantly increased in ARV-infected DF-1 cells. Our data show that gga-miR-30c-5p acts as an antagonist against ARV infection via suppressing ARV-induced autophagy, syncytium formation, and viral replication. Importantly, we identified autophagy related 5 (ATG5), a crucial proautophagic protein, as a bona fide target of gga-miR-30c-5p, and knockdown of ATG5 or treatment of cells with autophagy inhibitors inhibited virus replication and virus-induced syncytium formation, indicating that gga-miR-30c-5p plays an important role in the host response to ARV infection.

RESULTS

Infection of DF-1 cells with ARV strain S1133 enhances gga-miR-30c-5p expression.

In our previous study, we found numerous differentially expressed miRNAs involved in the process of ARV infection using deep sequencing analysis (Gene Expression Omnibus [GEO] database accession number GSE181193) (24). Among these differentially expressed miRNAs, gga-miR-30c-5p attracted our attention due to its marked change in expression in ARV-infected cells. To further determine the effect of ARV infection on gga-miR-30c-5p expression, we infected DF-1 cells with ARV and examined the expression of gga-miR-30c-5p in cells at different time points postinfection. As a result, the expression of miR-30c-5p in cells markedly increased post ARV infection in a dose-dependent manner (Fig. 1A), as well as 12 and 24 h post ARV infection (Fig. 1B), suggesting that gga-miR-30c-5p might play a role in host response to ARV infection.

FIG 1.

Infection of DF-1 cells with ARV strain S1133 increases gga-miR-30c-5p expression. (A) Examination of gga-miR-30c-5p expression in DF-1 cells infected with ARV at different doses. DF-1 cells were mock-infected or infected with ARV at an MOI of 0.2, 1, or 5. Twenty-four hours after infection, total RNA was extracted and qRT-PCR was performed to detect gga-miR-30c-5p transcripts. The expression of U6 was used as an internal control. The relative levels of gga-miR-30c-5p expression were calculated as follows: (miR-30c-5p expression in ARV-infected cells)/(miR-30c-5p expression in mock-infected controls). (B) Examination of gga-miR-30c-5p expression in DF-1 cells at different time points post ARV infection. DF-1 cells were mock-infected or infected with ARV at an MOI of 5. At different time points (12, 24, 36, and 48 h) after ARV infection, total RNA was extracted and qRT-PCR was performed to detect gga-miR-30c-5p transcripts. The relative levels of gga-miR-30c-5p expression were calculated as described above. Data are representative of three independent experiments and are presented as means ± standard deviation (SD). ***, P < 0.001; **, P < 0.01.

Gga-miR-30c-5p inhibits ARV replication in DF-1 cells.

As ARV infection increased gga-miR-30c-5p expression, we proposed that gga-miR-30c-5p might play a role in host response to ARV infection. To test this hypothesis, we first synthesized gga-miR-30c-5p mimics and inhibitors and measured their effects on endogenous miR-30c-5p expression. As shown in Fig. 2A, miR-30c-5p mimics could effectively increase the expression of miR-30c-5p relative to that of miRNA controls, whereas miR-30c-5p inhibitor markedly knocked down the expression of endogenous miR-30c-5p relative to that of miRNA inhibitor controls. Then, we ectopically expressed miR-30c-5p in DF-1 cells by transfection, infected these cells with ARV at an multiplicity of infection (MOI) of 5, and examined the effect of gga-miR-30c-5p on ARV replication. As a result, overexpression of miR-30c-5p significantly inhibited the mRNA expression of multiple viral genes, including σA, σB, σC, and p10 (P < 0.01) (Fig. 2B), and reduced expression of σB, a structural protein of ARV, at a protein level compared to that of controls (P < 0.01) (Fig. 2C and D), suggesting that miR-30c-5p inhibits ARV replication. Furthermore, we measured ARV growth in miR-30c-5p-transfected cells at different time points (12, 24, and 48 h) after ARV infection using 50% tissue culture infective dose (TCID₅₀) assays. As shown in Fig. 2E, transfection of DF-1 cells with miR-30c-5p markedly inhibited ARV replication in cells compared to that of controls. On the contrary, knockdown of endogenous miR-30c-5p in DF-1 cells significantly promoted mRNA expressions of σA, σB, σC, and p10 in ARV-infected cells (Fig. 3A) and increased protein expression of σB post ARV infection (Fig. 3B and C). Consistently, knockdown of endogenous miR-30c-5p markedly enhanced ARV growth (Fig. 3D). These data clearly show that gga-miR-30c-5p inhibits ARV replication in DF-1 cells, suggesting that gga-miR-30c-5p may serve as an important antivirus component of host response against ARV infection.

FIG 2.

Transfection of DF-1 cells with Gga-miR-30c-5p inhibits ARV replication. (A) Measurement of gga-miR-30c-5p expression in cells transfected with gga-miR-30c-5p mimics or inhibitor. DF-1 cells were transfected with miR-30c-5p mimics (100 nM), inhibitors (In; 100 nM), or miRNA controls. Twenty-four hours after transfection, total RNA was extracted, and qRT-PCR was performed to detect gga-miR-30c-5p transcripts. The expression of U6 was used as an internal control. The relative levels of gga-miR-30c-5p expression were calculated as follows: (gga-miR-30c-5p expression in miRNA- or miRNA inhibitor-transfected cell sample)/(gga-miR-30c-5p expression in normal cells). (B) Transfection of DF-1 cells with gga-miR-30c-5p reduced mRNA expressions of ARV σA, σB, σC, and p10 after ARV infection. DF-1 cells were transfected with gga-miR-30c-5p mimics or controls at 100 nM for 24 h, followed by infection with ARV at an MOI of 5. Twenty-four hours after infection, total RNA was extracted and viral gene transcripts were detected by qRT-PCR. The expression of GAPDH was used as an internal control. The relative levels of gene expression were calculated as follows: (mRNA expression of σA, σB, σC, or p10 in miR-30c-5p- or miRNA control-transfected cells infected with ARV)/(mRNA expression of respective viral genes in normal cells infected with ARV). (C and D) Overexpression of miR-30c-5p inhibited σB expression in ARV-infected cells. DF-1 cells were transfected with gga-miR-30c-5p mimics or miRNA controls at 100 nM for 24 h, followed by infection with ARV at an MOI of 5. (C) Twenty-four hours after infection, cell lysates were prepared and examined by Western blotting using anti-σB antibodies. (D) The band densities of ARV σB shown in panel C were quantitated by densitometry. Endogenous GAPDH expression was examined as an internal control. The relative levels of σB were calculated as follows: (protein expression of σB in gga-miR-30c-5p mimics or miRNA control-transfected cells)/(protein expression of σB in normal cells). (E) Analysis of the effect of miR-30c-5p on ARV replication by TCID₅₀ assay. DF-1 cells were transfected with miRNA controls, miR-30c-5p mimics, or medium only. Twenty-four hours after transfection, cells were infected with ARV at an MOI of 5. At different time points (12, 24, and 48 h) after ARV infection, the viral loads in the cell cultures were determined by TCID₅₀ assays in 96-well plates. Data are representative of three independent experiments and are presented as means ± SD. ***, P < 0.001; **, P < 0.01; *, P < 0.05.

FIG 3.

Knockdown of endogenous gga-miR-30c-5p enhances ARV replication in DF-1 cells. (A) Knockdown of endogenous gga-miR-30c-5p in DF-1 cells enhanced mRNA expression of ARV σA, σB, σC, and p10 post ARV infection. DF-1 cells were transfected with miR-30c-5p inhibitors (Inh) or miRNA inhibitor control (miR-Inh-ctrl) at 100 nM for 24 h, followed by infection with ARV at an MOI of 5. Twenty-four hours after infection, total RNA was extracted and qRT-PCR was performed to detect viral genes transcripts. The expression of GAPDH was used as an internal control. The relative levels of gene expression were calculated as follows: (mRNA expression of σA, σB, σC, or p10 in miR-30c-5p Inh- or miR-Inh-ctrl-transfected cells with ARV infection)/(mRNA expression of σA, σB, σC, or p10 in normal control cells with ARV infection). (B and C) Inhibition of endogenous gga-miR-30c-5p in DF-1 cells enhanced σB expression in ARV-infected cells. DF-1 cells were transfected with gga-miR-30c-5p Inh or miR-Inh-ctrl at 100 nM for 24 h or without any transfection as normal control, followed by infection with ARV at an MOI of 5. (B) Twenty-four hours after infection, cell lysates were prepared and examined by Western blotting using anti-σB antibodies. (C) The band densities of ARV σB in panel B were quantitated by densitometry. Endogenous GAPDH expression was examined as an internal control. The relative levels of σB were calculated as follows: (protein expression of σB in cells transfected with gga-miR-30c-5p Inh or miR-Inh-ctrl)/(protein expression of σB in normal cells). (D) Knockdown of endogenous gga-miR-30c-5p in DF-1 cells enhanced ARV replication. DF-1 cells were transfected with miR-30c-5p Inh, miR-Inh-ctrl, or medium only. Twenty-four hours after transfection, cells were infected with ARV at an MOI of 5. At different time points (12, 24, and 48 h) after ARV infection, the viral loads in the cell cultures were determined by TCID₅₀ assays in 96-well plates. Data are representative of three independent experiments and are presented as means ± SD. **, P < 0.01; *, P < 0.05.

ATG5 gene is a direct target of gga-miR-30c-5p.

Since gga-miR-30c-5p inhibits ARV replication, it is intriguing to explore the underlying molecular mechanism. The potential targets of gga-miR-30c-5p in host cells were predicted by using TargetScan, PicTar, and RNA22 (version 2) databases, and the region of the autophagy related 5 (ATG5) 3′-UTR at 534 bp was found to contain the target site for gga-miR-30c-5p (Fig. 4A). As ATG5, a well-characterized proautophagic protein, plays an important role in the formation of autophagosomes (25) and it was found that ARV induced autophagy in host cells, affecting viral replication (26, 27), we assumed that gga-miR-30c-5p might inhibit ARV-induced autophagy by targeting ATG5, thereby inhibiting viral replication. To test this hypothesis, the predicted target site in ATG5 as well as mutant control construct with mutations in the seed region (Fig. 4A) were cloned into luciferase reporter gene vector pGL3-control, and the effect of miR-30c-5p on reporter gene (pGL3-ATG5-WT and pGL3-ATG5-Mut) activity was examined by dual-luciferase reporter gene assay. As shown in Fig. 4B, luciferase activity was significantly reduced in DF-1 cells transfected with miR-30c-5p together with pGL3-ATG5-WT (P < 0.01); however, this reduction could be completely abolished in cells transfected with pGL3-ATG5-Mut. On the contrary, knockdown of endogenous miR-30c-5p by application of specific inhibitors significantly increased the luciferase activity of the wild-type (WT) reporter gene but not that of its cognate mutant (P < 0.05) (Fig. 4C). These data demonstrate that miR-30c-5p inhibits ATG5 expression by specifically binding to the sequence in the 3′ UTR of ATG5 gene.

FIG 4.

ATG5 gene is a direct target of gga-miR-30c-5p. (A) Diagram of predicted target site for gga-miR-30c-5p in ATG5 gene. The seed sequence of miR-30c-5p is underlined and was mutated as indicated by the arrow. (B and C) Overexpression of miR-30c-5p in DF-1 cells reduced expression of ATG5 but not its mutant. DF-1 cells were cotransfected with indicated miRNAs, controls, and luciferase reporter vectors. Forty-eight hours posttransfection, cells were lysed, and ATG5 expression was measured by a luciferase reporter gene assay. The relative levels of luciferase activity (Rel Luc Act) were calculated as follows: (luciferase activity of cells transfected with the reporter gene plasmids together with miR-30c-5p mimics or inhibitors)/(luciferase activity of cells cotransfected with the reporter gene plasmids and miRNA controls). (D) Overexpression of gga-miR-30c-5p in DF-1 cells reduced mRNA expression of ATG5. DF-1 cells were transfected with miR-30c-5p mimics or miRNA controls. Twenty-four hours after transfection, qRT-PCR analysis was performed to examine the mRNA expression of ATG5. GAPDH was used as an internal control. The relative levels of gene expression were calculated as follows: (mRNA expression of ATG5 in cells transfected with miR-30c-5p mimics or miRNA controls)/(mRNA expression of ATG5 in normal cell controls). (E and F) Overexpression of gga-miR-30c-5p in DF-1 cells reduced the expression of ATG5 at protein level. DF-1 cells were transfected with miR-30c-5p mimics or miRNA controls at 100 nM. Forty-eight hours after transfection, (E) cell lysates were prepared and ATG5 protein expression was examined by Western blotting using anti-ATG5 antibodies, and (F) the band densities of ATG5 in panel E were quantitated by densitometry. Endogenous GAPDH expression was examined as an internal control. The relative levels of ATG5 protein were calculated as follows: (band density of ATG5/band density of GAPDH in the same sample). (G) Knockdown of endogenous gga-miR-30c-5p enhanced mRNA expression of ATG5. DF-1 cells were transfected with miR-30c-5p Inh or miRNA inhibitor controls (miR-Inh ctrl) at 100 nM. Twenty-four hours after transfection, qRT-PCR analysis was performed to examine the mRNA expression of ATG5, and the relative levels of gene expression were calculated as described in panel D. (H and I) Knockdown of endogenous gga-miR-30c-5p enhanced the expression of ATG5 protein. DF-1 cells were transfected with miR-30c-5p Inh or miR-Inh-ctrl as described in panel G. Forty-eight hours after transfection, the expression of ATG5 protein was examined as described in panel E, (I) the band densities of ATG5 shown in panel H were quantitated by densitometry, and the relative levels of ATG5 expression were calculated as described in panel F. (J and K) ARV infection reduced ATG5 expression in host cells. DF-1 cells were mock-infected or infected with ARV at an MOI of 0.2, 1, or 5. Twenty-four hours after ARV infection, the expression of ATG5 protein was examined as described in panel E. The band densities of ATG5 shown in panel J were quantitated by densitometry (K) and the relative levels of ATG5 expression were calculated as described above in panel F. Data are representative of three independent experiments and are presented as means ± SD. ***, P < 0.001; **, P < 0.01; *, P < 0.05.

To consolidate these findings, the effect of gga-miR-30c-5p on ATG5 expression was examined at both mRNA and protein levels. As shown in Fig. 4D to F, at both mRNA and protein levels, the expression of ATG5 was markedly reduced in DF-1 cells transfected with miR-30c-5p, while expression of ATG5 was enhanced in DF-1 cells transfected with miR-30c-5p inhibitors compared to that of controls (P < 0.01) (Fig. 4G to I). Furthermore, considering that ARV infection increased gga-miR-30c-5p expression and that gga-miR-30c-5p inhibited ARV replication and ATG5 expression, we assumed that ARV infection might reduce ATG5 expression in host cells. To test this hypothesis, we infected DF-1 cells with different doses of ARV and examined ATG5 expression at 24 h postinfection. As a result, the expression of ATG5 in host cells markedly decreased post ARV infection in a dose-dependent manner (P < 0.01) (Fig. 4J and K). These data suggest that gga-miR-30c-5p suppresses viral replication by targeting ATG5 gene, inhibiting autophagy in ARV-infected cells.

Gga-miR-30c-5p inhibits microtubule-associated protein 1 LC3-II formation in DF-1 cells.

As we found that gga-miR-30c-5p suppressed viral replication by targeting ATG5 gene, it was reported that ARV infection triggered autophagy affecting virus production (26, 28), and miR-30d acted as an autophagy inhibitor promoting apoptosis in tumor cells (29), we hypothesized that gga-miR-30c-5p suppressed ARV replication by inhibiting autophagy in host cells. Thus, we set out to determine the role of gga-miR-30c-5p in autophagy in DF-1 cells by examining the conversion of microtubule-associated protein 1 light chain 3 II (LC3-II) from LC3-I, because conversion of soluble LC3-I to lipidated LC3-II is considered to be a hallmark of autophagy induction (30). We transfected DF-1 cells with gga-miR-30c-5p mimics or inhibitors and examined LC3-II at different time points posttransfection using Western blotting. As shown in Fig. 5A and B, transfection of cells with gga-miR-30c-5p markedly reduced LC3-II compared to that of controls at 48 h posttransfection (P < 0.05), while inhibition of endogenous miR-30c-5p by its inhibitors significantly increased LC3-II (P < 0.01) (Fig. 5C and D). As the accumulated LC3-IIs (punctate dot formation) located in autophagosomes are commonly used as markers for the indication of autophagosomes (31), we examined the effect of gga-miR-30c-5p on the accumulation of LC3-II. Consistent with above results, we found that gga-miR-30c-5p overexpression decreased the number of LC3 puncta, while miR-30c-5p inhibition increased the number of LC3 puncta in DF-1 cells (Fig. 5E and F). These results indicate that gga-miR-30c-5p inhibits autophagy in DF-1 cells in a cell-autonomous manner.

FIG 5.

Gga-miR-30c-5p inhibits autophagy in DF-1 cells. (A and B) Transfection of DF-1 cells with gga-miR-30c-5p mimics inhibited LC3-II in cells. DF-1 cells were transfected with miR-30c-5p mimics or miRNA controls at 100 nM. (A) Twenty-four or 48 h posttransfection, LC3-II protein expression was examined by Western blotting using anti-LC3B antibodies, and (B) the band densities of LC3-II in panel A were quantitated by densitometry. Endogenous GAPDH expression was examined as an internal control. The relative levels of LC3-II were calculated as follows: (band density of LC3-II)/(band density of GAPDH). (C and D) Knockdown of endogenous gga-miR-30c-5p by specific miRNA inhibitor promoted endogenous LC3-II in DF-l cells. DF-1 cells were transfected with gga-miR-30c-5p inhibitor (Inh) or miRNA inhibitor controls (miR-Inh-ctrl) at 100 nM. (D) Twenty-four or 48 h after transfection, the expression of LC3-II protein was examined as above described in panel A, the band densities of LC3-II shown in panel C were quantitated by densitometry, and the relative levels of LC3-II expression were calculated as above described in panel B. (E and F) Gga-miR-30c-5p inhibited the formation of punctate GFP-LC3 in DF-1 cells. DF-1 cells were seeded on 24-well plates and cultured overnight, followed by transfection with GFP-LC3. Twelve hours after transfection, cells were transfected with miR-30c-5p mimics, inhibitors, or miRNA controls. Thirty-six hours after transfection, cells were fixed with 4% paraformaldehyde. After washes, the cell nuclei were counterstained with DAPI (blue). (E) The cell samples were observed with a confocal laser scanning microscope. GFP-LC3 punctate are indicated by arrows. The average number of GFP-LC3 puncta per cell was calculated and shown in panel F. The scale bar in the picture represents 10 μm. Data are representative of three independent experiments and are presented as means ± SD. **, P < 0.01; *, P < 0.05.

To determine the role of gga-miR-30c-5p in ARV-induced autophagy in host cells, we transfected DF-1 cells with gga-miR-30c-5p mimics, inhibitors, or miRNA controls and examined conversion of LC3-II at different time points (12 and 24 h) post ARV infection. As a result, overexpression of gga-miR-30c-5p markedly inhibited ARV-induced LC3-II conversion in DF-1 cells (P < 0.01) (Fig. 6A and B), while inhibition of endogenous miR-30c-5p significantly enhanced ARV-induced LC3-II conversion in DF-1 cells (P < 0.05) (Fig. 6C and D). Furthermore, transfection of DF-1 cells with gga-miR-30c-5p reduced the number of LC3 puncta, while knockdown of endogenous miR-30c-5p by its inhibitors increased the number of LC3 puncta in DF-1 cells infected with ARV (Fig. 6E and F). These data clearly show that gga-miR-30c-5p inhibits ARV-induced autophagy in host cells.

FIG 6.

Gga-miR-30c-5p inhibits ARV-induced autophagy in DF-1 cells. (A and B) Gga-miR-30c-5p inhibited ARV-induced LC3-II in DF-l cells. DF-1 cells were transfected with miR-30c-5p mimics or miRNA controls at 100 nM. Twenty-four hours after transfection, cells were mock-infected or infected with ARV at an MOI of 5. (A) Twelve or 24 h post ARV infection, LC3-II protein expression was examined by Western blotting using anti-LC3B antibodies, and (B) the band densities of LC3-II in panel A were quantitated by densitometry. Endogenous GAPDH expression was examined as an internal control. The relative levels of LC3-II were calculated as follows: (band density of LC3-II)/(band density of GAPDH). (C and D) Knockdown of endogenous gga-miR-30c-5p by specific miRNA inhibitor promoted ARV-induced LC3-II in DF-l cells. DF-1 cells were transfected with gga-miR-30c-5p inhibitor (Inh) or miRNA inhibitor controls (miR-Inh-ctrl) at 100 nM. Twenty-four hours after transfection, cells were mock-infected or infected with ARV at an MOI of 5. Twelve or 24 h post ARV infection, the expression of LC3-II protein was examined as described in panel A, (D) the band densities of LC3-II shown in panel C were quantitated by densitometry, and the relative levels of LC3-II expression were calculated as described in panel B. (E and F) Gga-miR-30c-5p inhibited ARV-induced formation of GFP-LC3 punctate in DF-1 cells. DF-1 cells were seeded on 24-well plates and cultured overnight, followed by transfection with GFP-LC3. Twelve hours after transfection, cells were transfected miR-30c-5p mimics, inhibitors, or miRNA controls. Twenty-four hours after transfection, cells were mock-infected or infected with ARV at an MOI of 5. Twelve hours after infection, cells were fixed with 4% paraformaldehyde and probed with mouse anti-σB antibody followed by TRITC-conjugated goat anti-mouse antibodies (red). Nuclei were counterstained with DAPI (blue). (E) The cell samples were observed under a confocal laser scanning microscope. GFP-LC3 punctate are indicated by arrows. The average number of GFP-LC3 puncta per cell was calculated and shown in panel F. The scale bar in the picture represents 10 μm. Data are representative of three independent experiments and are presented as means ± SD. **, P < 0.01; *, P < 0.05.

Inhibition of autophagy suppresses viral replication.

As gga-miR-30c-5p suppresses viral replication and ARV-induced autophagy by targeting ATG5 in ARV-infected cells, it is highly possible that ATG5 is involved in host response to ARV infection and that knockdown of ATG5 in ARV-infected cells may affect ARV replication. Therefore, we made ATG5 RNAi construct and found that it could effectively lower the cellular level of ATG5 without causing discernible changes in cell morphology (Fig. 7A to C). As expected, knockdown of ATG5 by RNAi in ARV-infected cells significantly reduced the expression of LC3-II and σB, a structural protein of ARV, compared to that of RNAi controls (P < 0.01) (Fig. 7D and E), suggesting that inhibition of autophagy suppresses viral replication. Furthermore, knockdown of ATG5 markedly reduced viral titers in ARV-infected cells compared to that of RNAi controls (P < 0.05) (Fig. 7F). In contrast, the expression of LC3-II and σB in pRK5-flag-ATG5-transfected cells with ARV infection significantly increased compared to that in controls (Fig. 7G and H), and so did the viral loads in ATG5-overexpressed cell culture (Fig. 7I). These data suggest that ATG5-mediated autophagy plays an important role in viral replication.

FIG 7.

Knockdown of ATG5 inhibits ARV replication in DF-1 cells. (A) Effects of ATG5 RNAi on the cell morphology. DF-1 cells were transfected with siRNA constructs or controls. Double transfections were performed at a 24-h interval. Twenty-four hours after the second transfection, the pictures were taken under a light microscope at 100× magnification, respectively. Scale bar, 100 μm. (B and C) Effects of ATG5 RNAi on the expression of endogenous ATG5 in DF-1 cells. DF-1 cells were transfected with siRNA constructs or controls. Double transfections were performed at a 24-h interval. (B) Twenty-four hours after the second transfection, ATG5 protein expression was examined by Western blotting using anti-ATG5 antibodies, and (C) the band densities of ATG5 in panel B were quantitated by densitometry. Endogenous GAPDH expression was examined as an internal control. The relative levels of ATG5 were calculated as follows: (band density of ATG5)/(band density of GAPDH). (D and E) Knockdown of ATG5 by RNAi reduced ARV σB expression in host cells. DF-1 cells were treated with ATG5 RNAi or control RNAi, followed by mock infection or infection with ARV at an MOI of 5. (E) Twenty-four hours after infection, cell lysates were examined by Western blotting using anti-σB, anti-ATG5, or anti-LC3B antibodies. (D) The band densities of ARV σB shown in panel E were quantitated by densitometry. Endogenous GAPDH expression was examined as an internal control. The relative levels of σB were calculated as follows: (protein expression of σB in cells transfected with ATG5 siRNA or siRNA controls)/(protein expression of normal cells). (F) Knockdown of ATG5 by RNAi suppressed ARV replication in host cells. DF-1 cells were treated with ATG5 RNAi or RNAi control, followed by infection with ARV at an MOI of 5. Twenty-four hours postinfection, viral titers in the cell cultures were determined by TCID₅₀. (G and H) Overexpression of ATG5 promoted ARV σB expression in host cells. DF-1 cells were transfected with pRK5-flag-ATG5 or pRK5-flag as a control. Twenty-four hours after transfection, cells were mock-infected or infected with ARV at an MOI of 5. Twenty-four hours post ARV infection, the expression of σB protein was examined as described in panel E, (H) the band densities of σB shown in panel G were quantitated by densitometry, and the relative levels of σB expression were calculated as above described in panel D. (I) Overexpression of ATG5 promoted ARV replication in host cells. DF-1 cells were transfected with pRK5-flag-ATG5 or pRK5-flag as a control, followed by infection with ARV at an MOI of 5. Twenty-four hours postinfection, viral titers in the cell cultures were determined by TCID₅₀. Data are representative of three independent experiments and presented as means ± SD. ***, P < 0.001; **, P < 0.01; *, P < 0.05.

To consolidate these findings, we further determined the role of autophagy in ARV replication. We treated DF-1 cells with 3-MA or wortmannin, two autophagy inhibitors commonly used to inhibit autophagy (32), infected these cells with ARV at an MOI of 5, and examined the viral replication 24 h after ARV infection. As shown in Fig. 8A to D, treatment of cells with 3-MA or wortmannin markedly inhibited LC3-II and σB expressions in cells with ARV infection compared to that of controls (P < 0.01), indicating that inhibition of autophagy in ARV-infected cells is unfavorable to viral replication. Consistently, inhibition of autophagy in ARV-infected cells by wortmannin or 3-MA significantly reduced viral replication compared to that of controls (P < 0.05) (Fig. 8E and F). These results further demonstrate that inhibition of autophagy suppresses ARV replication.

FIG 8.

Inhibition of autophagy by inhibitors impairs ARV replication in DF-1 cells. (A to D) Inhibition of autophagy by 3-MA or wortmannin reduced ARV σB expression in cells. DF-1 cells were treated with (A) 5 mM 3-MA, (C) 20 nM wortmannin (Wort), or corresponding solvent (PBS for 3-MA or dimethyl sulfoxide [DMSO] for wortmannin) as controls for 4 h, followed by mock infection or infection with ARV at an MOI of 5. Two hours postinfection, cells were retreated with 3-MA (5 mM), Wort (20 nM), or corresponding solvent as controls. Twenty-four hours after infection, cell lysates were prepared and examined by Western blotting using anti-σB or anti-LC3B antibodies. The band densities of ARV σB shown in panels A and C were quantitated by densitometry (B and D, respectively). Endogenous GAPDH expression was examined as an internal control. The relative levels of σB were calculated as follows: (band density of σB in cells treated with 3-MA, Wort, or corresponding solvent)/(band density of σB in normal cells). (E and F) Inhibition of autophagy by 3-MA or wortmannin suppressed ARV replication in DF-1 cells. (E) DF-1 cells were treated with 3-MA, (F) Wort, or corresponding solvents, followed by infection with ARV as described above, and 24 h after infection, viral titers in the cell cultures were determined by TCID₅₀. Data are representative of three independent experiments and presented as means ± SD. **, P < 0.01; *, P < 0.05.

Inhibition of endogenous gga-miR-30c-5p restored ARV replication that was suppressed by ATG5 RNAi.

The fact that gga-miR-30c-5p inhibits ARV replication and ARV-induced autophagy and directly targets ATG5 and that inhibition of autophagy by knocking down ATG5 suppresses ARV replication suggest that miR-30c-5p might suppress ARV replication by targeting ATG5 and inhibiting ARV-induced autophagy, and that inhibition of miR-30c-5p by inhibitors would, therefore, restore viral replication suppressed by knockdown of ATG5 in ARV-infected cells. To test this hypothesis, we transfected ATG5 RNAi alone or together with miR-30c-5p inhibitor, followed by infection with ARV, and examined the effect of inhibition of miR-30c-5p on viral replication suppressed by knockdown of ATG5. As a result, inhibition of endogenous miR-30c-5p restored ARV σB and LC3-II expression that was suppressed by ATG5 RNAi (Fig. 9A and B). Consistently, inhibition of endogenous miR-30c-5p restored viral titers that was reduced by ATG5 RNAi (Fig. 9C). These data demonstrate that gga-miR-30c-5p suppresses viral replication by targeting ATG5 gene, inhibiting autophagy in ARV-infected cells.

FIG 9.

Inhibition of endogenous gga-miR-30c-5p restored ARV replication that was suppressed by ATG5 RNAi. (A and B) Inhibition of endogenous gga-miR-30c-5p restored ARV σB expression that was inhibited by ATG5 RNAi. DF-1 cells were transfected with ATG5 RNAi or control RNAi. Twenty-four hours after transfection, DF-1 cells were transfected with miR-30c-5p inhibitors (Inh) or miRNA inhibitor control (miR-Inh-ctrl) for 24 h, followed by mock infection or infection with ARV at an MOI of 5. (A) Twenty-four hours after infection, cell lysates were prepared and examined by Western blotting using anti-σB, anti-ATG5, or anti-LC3B antibodies. (B) The band densities of ARV σB shown in panel A were quantitated by densitometry. Endogenous GAPDH expression was examined as an internal control. The relative levels of σB were calculated as follows: (protein expression of σB in cells transfected with gga-miR-30c-5p Inh or miR-Inh-ctrl)/(protein expression of normal cells). (C) Inhibition of endogenous gga-miR-30c-5p-restored viral titers that was reduced by ATG5 RNAi. DF-1 cells were treated with ATG5 RNAi, miR-30c-5p inhibitors, or controls, followed by infection with ARV as described above. Twenty-four hours after infection, viral titers in the cell cultures were determined by TCID₅₀. Data are representative of three independent experiments and presented as means ± SD. **, P < 0.01.

gga-miR-30c-5p suppresses syncytium formation in ARV-infected cells by inhibiting ARV-induced autophagy.

As ARV infection caused syncytium formation in host cells that facilitates viral spread (15, 16, 33, 34), autophagy promoted virus-induced syncytium formation (35), and our data indicate that gga-miR-30c-5p inhibited autophagy and viral replication by targeting ATG5, it would be intriguing to determine the role of gga-miR-30c-5p in syncytium formation in ARV-infected cells. Hence, we altered gga-miR-30c-5p expression in DF-1 cells by transfection with gga-miR-30c-5p mimics or inhibitors, infected these cells with ARV, and examined ARV-induced syncytium formation using Giemsa stain. As shown in Fig. 10A and D, overexpression of gga-miR-30c-5p significantly reduced syncytium formation in ARV-infected cells, while knockdown of endogenous miR-30c-5p markedly enhanced ARV-induced syncytium formation compared to that of controls (P < 0.01), indicating that gga-miR-30c-5p inhibits syncytium formation in ARV-infected cells. The fact that gga-miR-30c-5p inhibits ARV replication by targeting ATG5 and inhibits syncytium formation in ARV-infected cells prompted us to examine the effect of autophagy on syncytium formation in ARV-infected cells. Thus, we treated DF-1 cells with autophagy inhibitors (3-MA and wortmannin), infected these cells with ARV, and examined syncytium formation in cells 24 h postinfection. As a result, inhibition of autophagy by 3-MA or wortmannin significantly reduced syncytium formation in ARV-infected cells compared to that in controls (P < 0.05) (Fig. 10B and E). Likewise, inhibition of autophagy by knockdown of ATG5 significantly inhibited ARV-induced syncytium formation, while induction of autophagy by overexpression of ATG5 markedly enhanced ARV-induced syncytium formation in DF-1 cells compared to that of controls (P < 0.05) (Fig. 10C and F). These data demonstrate that gga-miR-30c-5p suppressed syncytium formation in ARV-infected cells by inhibiting ARV-induced autophagy.

FIG 10.

Effects of gga-miR-30c-5, autophagy inhibitors, and ATG5 on ARV-induced syncytium formation. (A and D) Gga-miR-30c-5p inhibited ARV-induced syncytium formation in DF-1 cells. DF-1 cells were transfected with miR-30c-5p mimics (100 nM), inhibitors (Inh; 100 nM), or miRNA controls. Twenty-four hours after transfection, cells were mock-infected or infected with ARV at an MOI of 5. (A) Twenty-four hours after infection, cells were fixed and stained with Giemsa stain to visualize syncytia, and the average number of syncytial nuclei per field is shown in panel D. (B and E) Inhibition of autophagy by pharmaceutical inhibitors suppressed ARV-induced syncytium formation in host cells. DF-1 cells were treated with 5 mM 3-MA, 20 nM wortmannin (Wort), or corresponding solvent as a control for 4 h, followed by mock infection or infection with ARV at an MOI of 5. Two hours postinfection, the cells were retreated with 3-MA (5 mM), Wort (20 nM), or corresponding solvent as a control in fresh DMEM. (B) Twenty-four hours after infection, cells were fixed and stained with Giemsa stain to visualize syncytia, and the average number of syncytial nuclei per field is shown in panel E. (C and F) ATG5 promoted ARV-induced syncytium formation in host cells. DF-1 cells were treated with ATG5 RNAi, control RNAi, pRK5-flag-ATG5, or empty vectors, followed by mock infection or infection with ARV at an MOI of 5. (C) Twenty-four hours after infection, cells were fixed and stained with Giemsa stain to visualize syncytia, and the average number of syncytial nuclei per field is shown in panel F. Syncytia are indicated by arrows, and the scale bar in the picture represents 50 μm. Data are representative of three independent experiments and presented as means ± SD. **, P < 0.01; *, P < 0.05.

Taken together, our data show that gga-miR-30c-5p, whose expression increased in host cells in response to ARV infection, inhibited ARV-induced autophagy via targeting ATG5 accompanied by suppression of virus-induced syncytium formation and viral replication, serving as an important antidefense factor in host response against ARV infection.

DISCUSSION

ARV is an important pathogen causing various clinical chicken diseases such as arthritis, tenosynovitis, growth retardation, chronic respiratory diseases, and so on, endangering the development of the poultry industry (36). ARV infection involves a series of host responses, such as apoptosis (13, 15, 16), autophagy (18, 26), and cell cycle arrest (37). However, the exact molecular mechanism of host response to ARV infection is still not clear. In the present study, first, our data show that the expression of gga-miR-30c-5p markedly increased in cells with ARV infection. Second, overexpression of gga-miR-30c-5p significantly suppressed ARV replication and autophagy in DF-1 cells, while knockdown of gga-miR-30c-5p enhanced viral replication and autophagy in ARV-infected cells. Third, gga-miR-30c-5p inhibited ARV-induced autophagy by directly targeting cellular proautophagic protein ATG5, suppressing viral replication. Fourth, inhibition of autophagy by knockdown of ATG5 or using pharmaceutical inhibitors markedly suppressed virus replication. Finally, ARV-induced syncytium formation could be inhibited by gga-miR-30c-5p or inhibition of autophagy. These results clearly show that gga-miR-30c-5p inhibits ARV-induced autophagy by targeting ATG5, suppressing viral replication. Thus, gga-miR-30c-5p may serve as an important component of host antidefense against ARV infection, highlighting a critical role of miRNAs in host defense against pathogenic infection.

Autophagy is an evolutionarily conserved degradative process that is required to maintain homeostasis (38). There are three main autophagy routes: microautophagy, macroautophagy, and chaperone-mediated autophagy (39). In this report, the autophagy refers specifically to macroautophagy, which includes 4 key steps (38). The first step is the induction of autophagy, which is regulated by the key molecules mTOR, ULK1/2, ATG13, BECN1, and Vps34 (39–41). The second step is the formation of autophagosomes, involving key molecules ATG12, ATG5, and ATG16 (42, 43). The third step is the formation of autolysosome, fusion-related proteins LAMP2, RAB and STX17 are involved in this process (44, 45), and the fourth step is the degradation of the contents in the autolysosome, which is dependent mainly on acid hydrolase (46). As a part of host stress responses, autophagy plays an important role in pathogenic infections. It was reported that ARV infection induces autophagy in vivo and in vitro, and inhibition of autophagy significantly reduced the virus titer (26, 27). It was found that ARV P17 activated phosphatase and tension homology deleted on chromosome 10 (PTEN), AMP-activated protein kinase (AMPK), and double-stranded RNA (dsRNA)-dependent protein kinase RNA (PKR)/eIF2 signaling pathways, accompanied by downregulation of Akt/mTORC1 pathway, thereby triggering autophagy (19). Furthermore, not only could P17 bind to CDK2 to inhibit CDK2/cyclin A2 complex, but it also cooperated with σA to degrade ribosomal proteins, disrupted mTORC2-robosome association, further inhibited phosphorylation of Akt, and promoted autophagy (47). However, the exact mechanisms underlying host response to ARV-induced autophagy remain elusive. In this study, we found that gga-miR-30c-5p inhibited ARV-induced autophagy by targeting ATG5 accompanied by suppressing viral replication and syncytium formation, revealing the role of miRNA in regulating ARV-induced autophagy.

In our previous studies, we found numerous differentially expressed miRNAs involved in the process of ARV infection by deep sequencing analysis and found that gga-miR-29a-3p suppressed viral replication by targeting caspase-3 (24). In the present study, we further investigated the role of miRNAs in host response to ARV infection and found that gga-miR-30c-5p suppressed viral replication by inhibition of autophagy via targeting ATG5. These findings indicate that the roles of miRNAs in host response are directly related to functions of their target molecules. In the previous studies of other animal viruses by our laboratory and others, it was found that miRNAs might play dual roles in host response to infectious bursal disease virus (IBDV) infection, some miRNAs suppressing viral replication (48, 49), with others facilitating virus replication (50, 51). For example, gga-miR-130b inhibited IBDV replication by targeting IBDV segment A and cellular suppressors of cytokine signaling 5 (SOCS5) (48), gga-miR-27b-3p inhibited IBDV replication by targeting SOCS3&6 (49), while gga-miR-16-5p promoted IBDV replication by targeting BCL-2 (50), and gga-miR-9* promoted IBDV replication by targeting interferon regulatory factor 2 (IRF-2) (51). Similarly, miR-376b-3p promoted porcine reproductive and respiratory syndrome virus (PRRSV) replication by targeting replication restriction factor TRIM22 (52). However, there are no miRNAs reported so far that facilitate ARV replication.

The interaction between autophagy and viral infection is complicated. On the one hand, autophagy can directly degrade viral protein and nucleic acid or presented viral components to activate innate immunity to exert antiviral effects (53). For instance, picornaviruses, including poliovirus, can be detected by galectin 8 and be restricted to replicate by initiating the autophagic degradation of the viral RNA genome (54). However, in an ongoing evolutionary arms race, viruses have acquired the potent ability to escape autophagy degradation and hijack autophagy for facilitating their replication (53). It was found that after ARV infection, the viral components σC and p17 colocalized with autophagy marker LC3, but p17 and σC proteins did not coimmunoprecipitate with LC3 (19), suggesting that ARV may use the bilayer membrane structure of autophagosomes as a replication platform. Converting the autophagosome to their home for replication seems to be a common strategy for multiple RNA viruses (55). Related research on other nonenveloped RNA viruses, polioviruses, has proved that the double-membrane compartments formed during autophagy provide a scaffold for viral RNA replication (56). Double-layer membrane structure is believed to not only locally concentrate the intermediates needed for replication but also protect viral RNA from detection by innate immune sensors and degradation (53). However, in this study, we found that overexpression of gga-miR-30c-5p reduced the number of ARV-induced LC3 puncta, suggesting that gga-miR-30c-5p inhibits virus replication by reducing the formation of autophagosomes. It seems that as viruses evolve, the host also needs to develop corresponding antiviral strategies to strengthen its defense against virus infection. It is highly possible that host employs miRNAs to inhibit viral replication by reducing the formation of autophagosomes as an anti-virus replication platform.

The formation of syncytia induced by ARV is necessary for the spread and replication of the virus, and viral protein P10 plays a key role in ARV-induced cell fusion (15, 33, 34). In the present study, we found that gga-miR-30c-5p inhibits syncytium formation and that inhibition of autophagy by ATG5 RNAi or inhibitors reduced ARV-induced syncytium formation, suggesting that gga-miR-30c-5p inhibits syncytium formation by inhibiting autophagy via targeting ATG5, leading to decreased syncytium formation. As far as we know, this is the first study to show that ARV-induced autophagy is related to syncytium formation. There are similar results in some other viruses that can induce syncytium formation. It was found that autophagy is beneficial to membrane fusion and syncytium formation induced by F and HN proteins of Newcastle disease virus (NDV) (57). In measles virus infection, knockdown of key autophagy molecule ATG7 resulted in a decrease in the number of virus-induced syncytia independently of virus replication (35). Thus, it is very likely that virus-induced autophagy may facilitate virus replication by promoting the efficiency of viral cell-to-cell spread. However, in this study, there might be another possibility that gga-miR-30c-5p reduces syncytium formation by suppressing ARV replication, thereby reducing viral fast protein P10 expression, which may affect syncytial formation. As knockdown of ATG5 by RNAi markedly reduced ARV-induced syncytium formation, it is highly possible that gga-miR-30c-5p plays a major role in suppression of syncytium formation via targeting ATG5.

In summary, our data show that gga-miR-30c-5p expression markedly increased in cells post ARV infection and that gga-miR-30c-5p suppressed viral replication in host cells. Importantly, our results demonstrate that gga-miR-30c-5p inhibited ARV-induced autophagy by directly targeting ATG5 and that inhibition of autophagy by knockdown of ATG5 or by inhibitors suppressed viral replication as well as ARV-induced syncytium formation. Thus, gga-miR-30c-5p suppresses ARV replication by inhibiting autophagy via directly targeting ATG5, serving as an important antivirus factor in host response against ARV infection. These findings will further the understandings of the molecular mechanism underlying host-virus interaction at an RNA level.

MATERIALS AND METHODS

Cells and virus.

DF-1 cells were obtained from the ATCC and cultured in Dulbecco’s modified Eagle medium (DMEM; Macgene Biotechnology, Beijing, China) supplemented with 10% fetal bovine serum (FBS) in a 5% CO₂ incubator at 37°C. ARV S1133 strain was kindly provided by Jingliang Su (China Agricultural University, Beijing, China).

Reagents.

Monoclonal antibody against ARV-σB (EU0212) was obtained from CAEU Biological Company (Beijing, China). Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; AC002) mouse antibodies and anti-ATG5 (A19677) rabbit antibodies were obtained from ABclonal Technology (Wuhan, China). Anti-LC3B (14600-1-AP) rabbit antibodies were obtained from Proteintech Group (USA). Tetramethyl rhodamine isothiocyanate (TRITC)-conjugated goat anti-mouse IgG and horseradish peroxidase (HRP)-conjugated goat anti-mouse and anti-rabbit IgG antibodies were purchased from Ding Guo Sheng Wu (Beijing, China). Lipofectamine 3000 reagent was obtained from Invitrogen (USA). A dual-specific luciferase assay kit was purchased from Promega (USA). 4′,6-Diamino-2-phenylindole (DAPI) and Giemsa stain solution were purchased from Solarbio (Beijing, China). Autophagy inhibitors 3-MA and wortmannin were obtained from Selleck (USA). pEGFP-C1 and pRK5-flag vectors were obtained from Clontech.

Plasmid construction.

Gallus LC3B (GenBank accession No. NM_001031461) and ATG5 (GenBank accession No. NM_001006409.2) were cloned from DF-1 cells with following primers: for LC3B, sense primer 5′-GCAAGCTTATGCCCTCGGAGAAGAGCTTC-3′ and antisense primer 5′-GCGTCGACCTAGACGGAAGATTGCACTCCG-3′; for ATG5, sense primer 5′-GGACGACGATGACAAGGGATCCATGACAGATGACAAAGAT-3′ and antisense primer 5′-GGCCAAGCTTCTGCAGGTCGACTCAATCAGTAGGTCGGGG-3′. The primers were synthesized by Sangon Company (Shanghai, China). pEGFP-C1-LC3B and pRK5-flag-ATG5 expression plasmids were constructed by standard molecular biology techniques.

Sequences of miRNA mimics or inhibitors.

Mimics/inhibitors for miRNA were synthesized by GenePharma (Shanghai, China). The sense sequences are as follows: for gga-miR-30c-5p mimics, 5′-UGUAAACAUCCUACACUCUCAGCU-3′; for gga-miR-30c-5p inhibitors, 5′-AGCUGAGAGUGUAGGAUGUUUACA-3′; for mimic negative controls, 5′-UUCUCCGAACGUGUCACGUTT-3′; for inhibitor negative controls, 5′-CAGUACUUUUGUGUAGUACAA-3′.

miRNA target prediction.

miRNA targets in host cells were predicted by RNA22, version 2 (https://cm.jefferson.edu/rna22), TargetScan (http://www.targetscan.org/vert_70/), miRDB (http://www.mirdb.org/), and PicTar (https://pictar.mdc-berlin.de) (58).

RNA isolation and qRT-PCR analysis.

Total RNA and miRNAs were prepared from DF-1 cells using an EASYspin plus kit and RNA microRNA minikit (Aidlab, China), respectively, per the manufacturer’s instructions. Quantitative reverse transcription-PCR (qRT-PCR) was performed using a PrimeScript RT reagent kit (TaKaRa) on a Light Cycler 480 II (Roche, USA). Primers used for qRT-PCR were as follows: for ARV σA, sense primer 5′-TTTCGGGAATCGTGGTCTAGCG-3′ and antisense primer 5′-ACGAACCTGGATAGGGTGCTTC-3′; for ARV σB, sense primer 5′-ACTTTTGGACTTACCCGCTTGA-3′ and antisense primer 5′-TTGCATTTGACCAAAGACGGAA-3′; for ARV σC, sense primer 5′-TACGGTTGACGGAAACTCCAC-3′ and antisense primer 5′-ACACTAAGCGGAGGCGAAAA-3′; for ARV p10, sense primer 5′-TCCCGGTTCGTGTAACGGTG-3′ and antisense primer 5′-GCCGCTAGATAAGGCCAA-3′; for ATG5, sense primer 5′-GGCACCGACCGATTTAGT-3′ and antisense primer 5′-GCTGATGGGTTTGCTTTT-3′; for chicken GAPDH (chGAPDH), sense primer 5′-CAACTACATGGTTTACATGTTCC-3′ and antisense primer 5′-GGACTGTGGTCATGAGTCCT-3′. All primers were designed and synthesized by Sangon Company. Thermal cycling parameters were as follows: 94°C for 2 min, 40 cycles of 94°C for 20 s, 55°C for 20 s, and 72°C for 20 s, and 1 cycle of 95°C for 30 s, 60°C for 30 s, and 95°C for 30 s. Gga-miR-30c-5p expression was examined by qRT-PCR using an RT-PCR quantitation kit (GenePharma, China). Thermal cycling parameters for miRNAs were as follows: 95°C for 3 min, 40 cycles of 95°C for 12 s and 62°C for 40 s, and 1 cycle of 95°C for 30 s, 60°C for 30 s, and 95°C for 30 s. The final step was to obtain a melting curve for the PCR products to determine the specificity of the amplification. All samples were used in triplicate on the same plate, and the GAPDH gene or U6 snRNA was utilized as the reference gene. The expression levels of genes were calculated relative to that of the GAPDH gene or U6 snRNA and are presented as fold increases or decreases relative to the control sample level.

Western blotting.

To determine the effect of gga-miR-30c-5p on ARV replication, DF-1 cells were seeded on 12-well plates and cultured for 24 h before transfection with miRNA controls, gga-miR-30c-5p mimics, or miR-30c-5p inhibitors using Lipofectamine 3000. Twenty-four hours after transfection, cells were mock-infected or infected with ARV at an MOI of 5 for 24 h. Cell lysates were prepared using a lysis buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 5 mM EDTA, 1% NP-40, 10% glycerol, 1× complete cocktail protease inhibitor), boiled with SDS loading buffer for 10 min, and fractionated by electrophoresis on 10% SDS-PAGE gels. The resolved proteins were then transferred onto polyvinylidene difluoride (PVDF) membranes. After blocked with 5% skimmed milk, the membranes were incubated with anti-σB or anti-GAPDH antibodies, followed by incubation with HRP-conjugated secondary antibodies. To determine the effect of gga-miR-30c-5p on LC3 lipidation, DF-1 cells were transfected with indicated miRNAs. Twenty-four and 48 h after transfection, the cell lysates were harvested for Western blotting using anti-LC3B or anti-GAPDH antibodies, followed by detection with HRP-conjugated secondary antibodies. To determine the effect of miR-30c-5p on its target in host cells, DF-1 cells were transfected with indicated miRNAs. Forty-eight hours after transfection, the cell lysates were harvested for Western blotting using anti-ATG5 or anti-GAPDH antibodies, followed by detection with HRP-conjugated secondary antibodies. To determine the effect of ATG5 on ARV replication, DF-1 cells were transfected with indicated siRNA constructs, RNAi controls, pRK5-flag-ATG5, or empty vectors. Twenty-four hours after transfection, cells were mock-infected or infected with ARV at an MOI of 5 for 24 h. Cell lysates were harvested for Western blotting using anti-σB, anti-LC3B, or anti-GAPDH antibodies, followed by detection with HRP-conjugated secondary antibodies. To determine the effect of 3-MA or wortmannin on ARV replication, DF-1 cells were treated with 5 mM 3-MA, 20 nM wortmannin, or corresponding solvent as a control for 4 h, followed by infection with ARV at an MOI of 5 for 24 h. Cell lysates were harvested for Western blotting using anti-σB, anti-LC3B, or anti-GAPDH antibodies, followed by detection with HRP-conjugated secondary antibodies. Blots were developed using an enhanced chemiluminescence (ECL) kit (Millipore, USA) per the manufacturer’s instructions.

Measurement of ARV growth in DF-1 cells.

Normal cells or cells receiving miRNA controls, miR-30c-5p mimics, miR-30c-5p inhibitors, ATG5 RNAi, RNAi controls, pRK5-flag-ATG5, or empty vectors were infected with ARV at an MOI of 5, and cell cultures were collected at different time points (12, 24, and 48 h) postinfection. The samples were subjected to three rounds of freeze-thawed treatment and centrifuged at 6,000 × g for 10 min. The viral contents in supernatants were titrated using 50% tissue culture infective doses (TCID₅₀) in DF-1 cells. Cells were continuously cultured for 7 days at 37°C in a 5% CO₂ incubator. Tissue culture wells with cytopathic effect (CPE) were determined as positive. The titer was calculated based on a previously described method (59). To determine the role of 3-MA or wortmannin in ARV replication, DF-1 cells treated with 5 mM 3-MA, 20 nM wortmannin, or corresponding solvent as controls were infected with ARV at an MOI of 5. Twenty-four hours after infection, the viral contents in cell culture were titrated as described above.

Luciferase reporter gene assays.

DF-1 cells were seeded on 24-well plates and cultured overnight, followed by transfection with luciferase reporter gene plasmids (pGL3-target-ATG5-wt or pGL3-target-ATG5-mutant) and miRNA controls, gga-miR-30c-5p mimics, or gga-miR-30c-5p inhibitors. To normalize for transfection efficiency, another plasmid pRL-TK expressing Renilla luciferase reporter gene was added to each transfection as a control. Forty-eight hours posttransfection, luciferase reporter gene assays were performed with a dual-luciferase reporter gene assay system. Firefly luciferase activities were normalized on the basis of Renilla luciferase activities.

Confocal laser scanning microscopy assays.

DF-1 cells were seeded on 24-well plates with coverslips and cultured overnight, followed by transfection with pEGFP-LC3B (1 μg per well). Twelve hours after transfection, cells were transfected miR-30c-5p mimics, inhibitors, or miRNA controls. Thirty-six hours after transfection, cells were fixed with 4% paraformaldehyde. After washes, the cell nuclei were counterstained with DAPI (blue). For the examination of ARV-induced GFP-LC3 punctate, DF-1 cells were transfected with pEGFP-LC3B and indicated miRNA, and 24 h after transfection, cells were infected with ARV at an MOI of 5. Twelve hours after infection, DF-1 cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 for 15 min, blocked with 1% bovine serum albumin, and then probed with mouse anti-σB monoclonal antibody followed by TRITC-conjugated goat anti-mouse IgG antibodies. After washes, the cell nuclei were counterstained with DAPI. The cells were covered with ProLong Gold antifade reagent. The samples were observed under a confocal laser scanning microscope (Olympus Corporation, Tokyo, Japan). The number of GFP-LC3 puncta in each cell was counted, and the average number of GFP-LC3 puncta from 20 cells was calculated.

Knockdown of ATG5 by RNAi.

To knockdown ATG5 in DF-1 cells, siRNAs were designed with reference to previous publications (60) and synthesized by GenePharma Company (Shanghai, China). The sense sequence of siRNAs for targeting ATG5 in DF-1 cells was as follows: 5′-GGAUGUGAUUGAAGCUCAUTT-3′. DF-1 cells were seeded onto 12-well plates and cultured for 24 h before transfection with siRNA or control using lipo3000. Double transfections were performed at 24-h intervals. Twenty-four hours after the second transfection, cells were harvested for further analysis.

Examination of syncytium formation.

DF-1 cells were transfected with miRNA controls, miR-30c-5p mimics, miR-30c-5p inhibitors, ATG5 RNAi, RNAi controls, pRK5-flag-ATG5, or empty vectors. After transfection, cells were mock-infected or infected with ARV at an MOI of 5. Twenty-four hours after ARV infection, cells were fixed with 4% paraformaldehyde and stained with Giemsa stain to visualize syncytia. Syncytium formation was examined by microscopy. The syncytial nuclei in each microscopic field were counted, and the average number of syncytial nuclei from six fields was calculated. To determine the role of 3-MA or wortmannin in ARV-induced syncytium formation, DF-1 cells treated with 5 mM 3-MA, 20 nM wortmannin, or corresponding solvent as controls were mock-infected or infected with ARV at an MOI of 5. Twenty-four hours after infection, syncytium formation was examined as described above.

Statistical analysis.

The statistical analysis was performed using GraphPad Prism version 8.0. The significance of differences between miRNA mimics and controls or inhibitors in gene expression, luciferase activities, syncytium formation, and viral growth, between ATG5 RNAi and RNAi controls or pRK5-flag-ATG5 and empty vectors in gene expression, viral growth, and syncytium formation, and between autophagy inhibitor-treated cells and controls in viral growth, gene expression, and syncytium formation were determined by the Mann-Whitney test or analysis of variance (ANOVA), accordingly.