The NS4A Protein of Classical Swine Fever Virus Suppresses RNA Silencing in Mammalian Cells

ABSTRACT

RNA interference (RNAi) is a significant posttranscriptional gene silencing mechanism and can function as an antiviral immunity in eukaryotes. However, numerous viruses can evade this antiviral RNAi by encoding viral suppressors of RNA silencing (VSRs). Classical swine fever virus (CSFV), belonging to the genus Pestivirus, is the cause of classical swine fever (CSF), which has an enormous impact on animal health and the pig industry. Notably, little is known about how Pestivirus blocks RNAi in their host. In this paper, we uncovered that CSFV NS4A protein can antagonize RNAi efficiently in mammalian cells by binding to double-stranded RNA and small interfering RNA. In addition, the VSR activity of CSFV NS4A was conserved among Pestivirus. Furthermore, the replication of VSR-deficient CSFV was attenuated but could be restored by the deficiency of RNAi in mammalian cells. In conclusion, our studies uncovered that CSFV NS4A is a novel VSR that suppresses RNAi in mammalian cells and shed new light on knowledge about CSFV and other Pestivirus.

IMPORTANCE It is well known that RNAi is an important posttranscriptional gene silencing mechanism that is also involved in the antiviral response in mammalian cells. While numerous viruses have evolved to block this antiviral immunity by encoding VSRs. Our data demonstrated that the NS4A protein of CSFV exhibited a potent VSR activity through binding to dsRNA and siRNA in the context of CSFV infection in mammalian cells, which are a conservative feature among Pestivirus. In addition, the replication of VSR-deficient CSFV was attenuated but could be restored by the deficiency of RNAi, providing a theoretical basis for the development of other important attenuated Pestivirus vaccines.

INTRODUCTION

RNA interference (RNAi) is a significant posttranscriptional gene silencing mechanism that plays an important role in the antiviral immune pathway in eukaryotic organisms (1–3). In the process of virus replication, the viral replicative intermediate double-stranded RNAs (dsRNAs) can be recognized and processed into virus-derived small interfering RNAs (vsiRNAs) about 21- to 23-nucleotide (nt) by the host endoribonuclease Dicer protein. Then these vsiRNAs are packed into the Argonaute (AGO) proteins, forming the RNA-induced silencing complexes (RISCs) to mediate the cleavage of homologous viral genomic RNAs (2, 4, 5). Increasing evidence exists to demonstrate that RNAi can function as an antiviral response in mammals (6–9).

However, viruses can encode viral suppressors of RNAi (VSRs) to suppress this RNAi-mediated innate immunity through different mechanisms (10, 11). For instance, the B2 of Flock house virus (FHV) is a classic VSR capable of binding to dsRNA and protecting it from being processed by Dicer, as well as binding to siRNAs (12–14). Moreover, the B2 of Wuhan nodavirus can interact with Dicer-2 (15, 16). Furthermore, the 1A of cricket paralysis virus possesses the capacity to inhibit the endonuclease activity of AGO2 in Drosophila (17). In summary, these VSRs suppress RNAi by binding to long dsRNAs and/or siRNAs and/or by protecting them from Dicer cleavage or preventing them from loading into AGO, and some can interfere with the activities of Dicer or AGO (10).

It has been proven that several mammalian viruses are also able to encode VSRs: examples include Ebola virus VP35 (18), human immunodeficiency virus-1 (HIV-1) Tat (19), hepatitis C virus (HCV) NS2 and core (20–22), coronavirus 7a and nucleocapsid (23–25), semliki forest virus (SFV), rubella virus (RuV), and yellow fever virus (YFV) capsid (26–28). Moreover, some viral proteins encoded by mammalian viruses, including the 3A of enterovirus A71 (EV-A71), NS1 of influenza A virus (IAV), B2 of Nodamura virus (NoV), and NS2A of Dengue virus (DENV) can function as validated VSRs to suppress the RNAi pathway during the process of viral infections (6, 9, 29, 30), highlighting the significance of VSRs activity during viral life cycles.

Classical swine fever (CSF) coursed by classical swine fever virus (CSFV) is one of the notifiable viral diseases and leads to a significant impact on the health of pigs and the associated industry (31–33). Although systematic prophylactic vaccination has been applied to control or eradicate CSFV in many countries worldwide, it is effective only when vaccination coverage reaches over 90% among the swine population at any time (34).

CSFV is an enveloped, positive-stranded RNA virus belonging to the genus Pestivirus that also contains bovine viral diarrhea virus (BVDV), border disease virus (BDV), and other members (31). The CSFV genome contains one large open reading frame (ORF) flanked by two untranslated regions (5′ UTR and 3′ UTR). The ORF encodes a polyprotein that is then cleaved to form mature viral proteins containing four structural proteins (capsid, E^rns, E1, and E2) and eight nonstructural proteins (N^pro, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) by viral and host proteases (35, 36). CSFV NS4A, an 8 kDa protein localized in the cell nucleus and cytoplasm, acts as a necessary cofactor for the NS3 protease activity through its C-terminal region and can also mediate IL-8 production by stimulating the MAVS pathway (37–40). However, the roles of NS4A in the CSFV life cycle are not well studied. In this paper, we demonstrated that CSFV NS4A has a potent in vitro VSR activity that antagonizes the RNAi pathway by binding to dsRNA and siRNA in mammalian cells. Furthermore, we uncovered the fact that CSFV NS4A can function as a VSR to suppress RNAi in the course of CSFV infection in mammalian cells.

RESULTS

CSFV NS4A protein is a potential VSR.

We examined whether CSFV has any protein that exhibits the VSR activity by conducting a reversal-of-silencing experiment in 293T cells. In brief, cells were cotransfected with the plasmid encoding enhanced green fluorescent protein (EGFP), EGFP-specific short hairpin RNA (shEGFP), and the plasmid encoding CSFV proteins (Fig. 1A). At 48 h posttransfection (hpt), the level of EGFP mRNA was detected by Northern blotting. As expected, EGFP transcript and protein can be eliminated by EGFP-specific shRNA (Fig. 1A, lane 2). SFV capsid, which is a confirmed VSR, was used as a positive control; as expected, this rescued EGFP mRNA and protein (Fig. 1A, lane 3) (28). The expression of viral proteins was detected by Western blotting with anti-Flag antibody (Fig. 1B). Our data showed that CSFV NS4A protein could effectively rescue the expression of RNAi-silenced EGFP (Fig. 1A, lane 5), indicating that CSFV NS4A protein exhibits the VSR activity.

FIG 1.

CSFV NS4A protein is a potential VSR. 293T cells were cotransfected with a plasmid encoding EGFP (0.1 μg), shEGFP (0.3 μg) and either empty plasmid or a plasmid encoding CSFV protein (1 μg for each). At 48 hpt, the expression of EGFP reporter was analyzed. (A) total RNAs were extracted and the level of EGFP mRNA was examined via Northern blotting. SFV capsid (SFV_capsid) was used as a positive control. 18s rRNA was used as a loading control. (B) The expression of CSFV protein was detected by Western blotting with anti-Flag antibody. (C and D) The reversal-of-silencing assay was performed in PK-15 cells, and Northern blotting and Western blotting were performed to determine the expression of EGFP at 48 hpt. (E and F) 293T were transfected with SFV capsid, or CSFV NS4A as indicated, and then infected with SFV_WT or VSR-deficient SFV (SFV_K124A/K128A) at an MOI of 1. At 6, 12, and 24 hpi, the total RNAs were extracted for Northern blotting with DIG-labeled RNA probe targeting the nt 10637 to 11163 of SFV genome RNA (E), or qRT-PCR (F), and the level of SFV_K124A/K128A RNA in 293T cells at 6 hpi was defined as 1. All data represent the means and standard deviations of three independent experiments. **, P < 0.01; ***, P < 0.001 (as measured by two-way ANOVA; GraphPad Prism). (G) The expression of SFV capsid and CSFV NS4A was detected by Western blotting. (H) A plasmid encoding CSFV, BVDV, or BDV NS4A (1 μg for each) were transfected into PK-15 cells in the reversal-of-silencing assay. Northern blotting and Western blotting were performed to determine the expression of EGFP at 48 hpt. (I) PK-15 cells were cotransfected with a plasmid encoding EGFP (0.1 μg) and CSFV, BVDV, or BDV NS4A (1 μg for each). At 48 hpt, the expression of EGFP was determined by Northern blotting and Western blotting. (J) The expression of CSFV, BVDV, BDV NS4A was detected by Western blotting with anti-Flag antibody.

To provide further evidence regarding the VSR activity of CSFV NS4A, PK-15 cells were cotransfected with the plasmid encoding EGFP, CSFV NS4A, and shEGFP. As shown in Fig. 1C, ectopic expression of CSFV NS4A could also effectively inhibit the shRNA-induced RNAi in PK-15 cells. Moreover, we found that VSR activity of CSFV NS4A was dosage dependent (Fig. 1D). We also examined whether CSFV NS4A could rescue the replication of SFV_K124A/K128A, which is a VSR-deficient virus that has a defective self-replication (28). In brief, 293T cells were transfected with plasmid encoding SFV capsid or CSFV NS4A and then infected with SFV_WT or SFV_K124A/K128A; viral RNA accumulation was then determined at 6, 12, and 24 h postinfection (hpi), respectively. Our data indicated that ectopic expression of CSFV NS4A could rescue the replication of SFV_K124A/K128A, as with SFV capsid (Fig. 1E, lane 3 and 4; Fig. 1F). The expression of CSFV NS4A and SFV capsid was detected by Western blotting with anti-Flag antibody (Fig. 1G).

NS4A protein is a highly conserved protein within Pestivirus, suggesting a conservative function. Therefore, we tested the VSR activity of NS4A protein from BDV and BVDV via the reversal-of-silencing assays in PK-15 cells. Our findings showed that NS4A of BDV and BVDV also had the ability to suppress RNAi (Fig. 1H, lane 5 and 6), indicating that the in vitro VSR activity of NS4A proteins is a general characteristic of Pestivirus. Moreover, ectopic expression of Pestivirus NS4A in PK-15 cells did not affect the transcription and translation level of EGFP (Fig. 1I). The expression of BDV, BVDV, and CSFV NS4A was detected by Western blotting with anti-Flag antibody (Fig. 1J).

Considered collectively, our findings demonstrated that CSFV NS4A protein can act as a potential VSR to inhibit RNAi in mammalian cells.

NS4A protein inhibited Dicer-mediated siRNA generation by sequestrating dsRNA.

RNAi is a vital mechanism that regulates the eukaryotic posttranscriptional gene silencing through small RNA-induced sequence-specific RNA degradation. dsRNA can be processed by Dicer into mature siRNAs, which were then packed into AGO proteins. To determine whether CSFV NS4A can interfere with this process, small RNAs harvested from PK-15 cells cotransfected with the plasmids encoding EGFP, shEGFP and CSFV NS4A were subjected to small RNAs Northern blotting. The probe was DIG-labeled and targeted EGFP siRNA processed from shRNA. As shown in Fig. 2A, the accumulation of mature siRNA was reduced in cells that expressing NS4A compared to that in cells expressing EGFP and shEGFP (Fig. 2A, lane 1); this demonstrates that CSFV NS4A can inhibit Dicer-mediated siRNA generation, which is in accord with the previous data that CSFV NS4A rescued EGFP levels in PK-15 cells (Fig. 1C).

FIG 2.

CSFV NS4A suppressed Dicer-mediated siRNA production by sequestrating dsRNA. (A) The reversal-of-silencing assay was performed in PK-15 cells. At 48 hpt, the total RNAs were extracted for small RNAs Northern blotting. U6 was used as a loading control. (B) SDS-PAGE of purified recombinant CSFV NS4A (MBP-NS4A). BSA was used as a quantity control. (C and D) Increasing amounts (0 to 4 μM or 0 to 0.5 μM) of MBP fusion NS4A (MBP-NS4A) were incubated with 0.5 μM 200-nt DIG-labeled dsRNA at 26°C for 40 min. The reaction mixtures were subjected to 1.5% native-TBE agarose gel, transferred to membranes, and then detected with anti-DIG antibody conjugated to alkaline phosphatase. MBP was used as a negative control. (E) Increasing amounts (0 to 2 μM) of MBP-NS4A were incubated with 1 μM 200-nt DIG-labeled dsRNA at 26°C for 30 min. 1 U of RNase III was added into the reaction system at 37°C for 30 or 60 min (lane 4). Complexes were subjected to 7 M urea–15% PAGE analysis.

Furthermore, we aimed to explore whether CSFV NS4A suppresses siRNA production by directly binding to long dsRNA. To do so, we obtained the recombinant maltose-binding protein (MBP)-fusion NS4A protein (MBP-NS4A) (Fig. 2B, lane 2 and 3) and performed electrophoretic mobility shift assay (EMSA) by incubating the MBP-NS4A with the in vitro transcribed DIG-labeled 200-nt dsRNA. As shown in Fig. 2C and D, the combinative amount of labeled dsRNAs was increased by MBP-NS4A in a dose-dependent manner.

Subsequently, we aimed to investigate whether MBP-NS4A can protect dsRNA from being processed by Dicer through an in vitro RNase III experiment (41). It has been previously described that RNase III has been widely used to substitute Dicer to explore the function of VSR (15). In brief, MBP-NS4A was incubated with DIG-labeled 200-nt dsRNA for 30 min and then RNase III was added into this reaction at 37°C for 30 or 60 min. As shown in Fig. 2E, dsRNAs efficiently escaped from RNase III cleavage in a dose-dependent manner by MBP-NS4A (lane 5 to 8). When dsRNA was digested by RNase III for 60 min, MBP-NS4A could also protect dsRNA from Dicer digestion (Fig. 2E, lane 4). While dsRNA incubated with MBP alone was cleaved into siRNA (Fig. 2E, lane 3).

Taken together, our data suggested that MBP-NS4A can block RNAi by sequestrating dsRNA from Dicer digestion.

CSFV NS4A suppressed siRNA-induced RNAi.

The mature siRNAs are loaded into RISC to mediate the cleavage of cognate mRNAs during the process of RNAi. Given that CSFV NS4A could inhibit RNAi by binding to dsRNA, we next sought to examine whether NS4A could suppress siRNA-induced RNAi by binding to siRNA. We then transfected PK-15 cells with plasmids expressing EGFP, in vitro synthesized EGFP-specific siRNA (siEGFP) and CSFV NS4A. As shown in Fig. 3A, ectopic expression of CSFV NS4A could also effectively inhibit the siRNA-induced RNAi and restore the level of EGFP mRNA and protein in PK-15 cells. We subsequently investigated whether NS4A protein had siRNA-binding activity. In brief, we conducted EMSA by incubating MBP-NS4A together with DIG-labeled synthetic 22-nt siRNA. As shown in Fig. 3B and C, NS4A could interact with siRNA in a dose-dependent manner. In summary, our data suggested that CSFV NS4A can suppress RNAi by sequestrating siRNA.

FIG 3.

CSFV NS4A inhibited siRNA-induced RNAi. (A) The reversal-of-silencing assay was performed in PK-15 cells, and Northern blotting and Western blotting were performed to determine the expression of EGFP at 48 hpt. (B and C) Increasing amounts (0 to 5 μM or 0 to 2 μM) of MBP fusion NS4A (MBP-NS4A) were incubated with 1 μM 22-nt DIG-labeled siRNA at 26°C for 40 min. The siRNA-protein complexes were subjected to1.5% native-TBE agarose gel, transferred to membranes, and then incubated with anti-DIG antibody conjugated to alkaline phosphatase. MBP was used as a negative control.

R38/K53 of CSFV NS4A were critical for the VSR activity.

After confirming the VSR activity of CSFV NS4A, we aimed to ascertain the critical residues necessary for its RNAi inhibition activity. NS4A is a small molecular protein consisting of 64 amino acids, the structure of which has not yet been completely analyzed. Based on multiple sequences alignments of NS4A encoded by BDV, BVDV, and CSFV (Fig. 4A), we subjected the conserved positively charged residues (lysine [K] and arginine [R]) to single-point mutations to alanine (A), and then explored the VSR activity of these mutant NS4A proteins via the reversal-of-silencing assay in PK-15 cells. Even though the single-point mutations still held the VSR activity (Fig. 4A, lane 5 to 10), the double-point mutations R38A/K53A (NS4A_R38A/K53A) significantly lost the activity to suppress RNAi (Fig. 4C, lane 7; Fig. 4E). The expression of CSFV NS4A mutations was detected by Western blotting with anti-Flag antibody (Fig. 4B and D). It is well known that CSFV NS4A is a necessary cofactor for CSFV NS3 protease activity; moreover, our findings showed that NS4A_R38A/K53A could still interact with NS3 as with wild-type NS4A_WT (Fig. 4F and G), indicating that the double-point mutations of NS4A did not affect the NS3 protease activity which plays a significant role in CSFV morphogenesis. In addition, NS4A_R38A/K53A also disrupted the VSR activity of suppressing siRNA-induced RNAi in PK-15 cells (Fig. 4H). Furthermore, we found that the R38A/K53A mutation (MBP-NS4A_R38A/K53A) (Fig. 5A, lane 2) abolished the ability to bind to either dsRNA or siRNA (Fig. 5B and C).

FIG 4.

R38/K53 of CSFV NS4A were critical for the VSR activity. (A to E) The reversal-of-silencing assay was performed in PK-15 cells, and Northern blotting, Western blotting and qRT-PCR were performed to determine the expression of EGFP at 48 hpt (A, C, and E); The expression of CSFV NS4A single-point or double-point mutations was detected by Western blotting with anti-Flag antibody (B and D). All data represent the means and standard deviations of three independent experiments. *, P < 0.05 (as measured by two-way ANOVA; GraphPad Prism). (F and G) PK-15 cells were cotransfected with indicated plasmids individually. At 48 hpt, total cell lysates were immunoprecipitated (IP) with an anti-His or anti-Flag antibody, and bound proteins were then detected by immunoblot (IB) analysis using an anti-Flag or anti-His antibody. (H) The reversal-of-silencing assay was performed in PK-15 cells, and Northern blotting and Western blotting were performed to determine the expression of EGFP at 48 hpt.

FIG 5.

R38/K53 of the CSFV NS4A were critical for the dsRNA and siRNA binding activity. (A) SDS-PAGE of purified MBP-fusion CSFV NS4A_R38A/K53A. (B and C) MBP-NS4A_WT or MBP-NS4A_R38A/K53A was incubated with 0.5 μM 200-nt DIG-labeled dsRNA (B) or 1 μM 22-nt siRNA (C) at 26°C for 40 min. The bound complexes were subjected to 1.5% native-TBE agarose gel, transferred to membranes, and then incubated with anti-DIG antibody conjugated with alkaline phosphatase. MBP was used as a negative control.

Taken together, our findings demonstrated that the CSFV NS4A R38/K53 residues are crucial for its dsRNA/siRNA-binding and VSR activities.

Construction and recovery of VSR-deficient CSFV.

To determine whether CSFV NS4A protein authentically inhibited antiviral RNAi in the context of virus infection, the R38A/K53A mutations of NS4A were introduced into the infectious clone of CSFV (Fig. 6A). We successfully rescued the wild-type (CSFV_WT) and R38A/K53A mutant (CSFV_R38A/K53A) viruses and immunofluorescence assays were conducted to confirm the virus titration of these two viruses (Fig. 6B). In addition, the one-step growth curve of these two viruses was confirmed in PK-15 cells, indicating that CSFV_R38A/K53A displayed a weaker growth pattern than CSFV_WT in PK-15 cells (Fig. 6C). Taken together, our data showed that the defective VSR led to the reduced replication of CSFV.

FIG 6.

Construction and recovery of VSR-deficient CSFV. (A) CSFV genome and the mutation sites of R38A/K53A. (B) Rescue of CSFV_WT and CSFV_R38A/K53A. PK-15 cells infected with 10-fold serial dilution of CSFV_WT or CSFV_R38A/K53A were analyzed by immunofluorescence (IF) staining with an NS3-specific antibody at 72 hpi. The microscopy images were captured at 20× magnification. (C) PK-15 cells were infected with CSFV_WT or CSFV_R38A/K53A at an MOI of 5, respectively. At 12, 24, and 48 hpi, the viral titers in supernatant were measured. All data represent the means and standard deviations of three independent experiments. *, P < 0.05; **, P < 0.01 (as measured by two-way analysis of variance [ANOVA]; GraphPad Prism).

The replication defect of VSR-deficient CSFV could be restored by the deficiency of RNAi in PK-15 cells.

To further investigate the VSR activity of CSFV NS4A in the course of viral infection, PK-15 cells were infected with CSFV_WT or CSFV_R38A/K53A at an MOI of 5. The viral genomic RNA accumulations were determined at 12, 24, and 48 hpi, respectively. As shown in Fig. 7A, CSFV_R38A/K53A had a lower viral RNA accumulation than that of CSFV_WT in PK-15 cells. The genetic ablation of the RNAi pathway by Dicer knockdown in PK-15 cells rescued the replication of CSFV_R38A/K53A (Fig. 7A). The expression of Dicer was analyzed by Western blotting with anti-Dicer antibody and qRT-PCR (Fig. 7B and C). It is interesting to note that enoxacin could reduce the RNA accumulation of CSFV_R38A/K53A in PK-15 cells compared to that in the controlled PK-15 cells (Fig. 7D). As we know, enoxacin is a doubtless RNAi pathway enhancer and plays a part in steps after siRNA generation by Dicer in the RNAi (2, 8, 42, 43).

FIG 7.

The deficiency of RNAi restored the defective replication of VSR-deficient CSFV. (A) PK-15 or PK-15 Dicer-knockdown cells were transfected with either empty plasmid or a plasmid encoding SFV capsid, CSFV NS4A, or NS4A_R38A/K53A as indicated. At 24 hpt, the cells were infected with CSFV_WT or CSFV_R38A/K53A at an MOI of 5. At 12, 24, and 48 hpi, the levels of CSFV genomic RNAs in cells were detected by qRT-PCR, and the level of CSFV_R38A/K53A RNA in PK-15 cells at 12 hpi was defined as 1. (B and C) PK-15 cells were transfected with Dicer-specific shRNA (shDicer) or shRNA cotrol (0.6 μg for each). At 24 hpt, the expression of Dicer was confirmed by qRT-PCR (B) and Western blotting (C). (D) Enoxacin (100 μM) was added into PK-15 cells for 1 h and then cells were infected with CSFV_R38A/K53A at an MOI of 5. At 12, 24, and 48 hpi, the accumulations of CSFV genomic RNAs in cells were detected by qRT-PCR, and the level of CSFV_R38A/K53A RNA in PK-15 cells treated with DMSO at 12 hpi was defined as 1. (E) The expression of SFV capsid, CSFV NS4A, or NS4A_R38A/K53A in PK-15 cells was detected by Western blotting. All data represent the means and standard deviations of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (as measured by two-way ANOVA; GraphPad Prism).

Moreover, the ectopic expression of SFV capsid or CSFV NS4A could rescue the viral RNA accumulation of CSFV_R38A/K53A in PK-15 cells, although this was not the case for the VSR-inactive mutant of NS4A (NS4A_R38A/K53A) (Fig. 7A). The expression of SFV capsid, NS4A, and NS4A_R38A/K53A was confirmed by Western blotting (Fig. 7E). These data demonstrated that the ectopic expression of foreign VSR rescuing the replication of CSFV_R38A/K53A is indeed dependent on RNAi.

To eliminate the potential effect of the IFN-I pathway, PK-15 or PK-15 Dicer-knockdown cells were handled with ruxolitinib, which can inhibit the JAK1 and JAK3 pathway to block IFN-I, and then infected with CSFV_R38A/K53A or CSFV_WT. As shown in Fig. 8A, the interferon response was indeed suppressed by ruxolitinib. The replication of CSFV_R38A/K53A could still be enhanced by the ectopic expression of a VSR or a deficiency of Dicer in ruxolitinib-treated cells, demonstrating that the rescuing effect is not dependent on IFN-I (Fig. 8B). The expression of SFV capsid, NS4A, and NS4A_R38A/K53A was confirmed by Western blotting (Fig. 8C). In addition, when IFN-I pathway was blocked by ruxolitinib, enoxacin could also reduce the RNA accumulation of CSFV_R38A/K53A in PK-15 cells compared to that in the controlled PK-15 cells (Fig. 8D), indicating that enoxacine enhancing RNAi pathway was irrespective of IFN-I.

FIG 8.

The rescuing effect of VSR-deficient CSFV was not dependent on IFN-I. (A) PK-15 cells were treated with ruxolitinib (0 to 10 μM) as indicated for 1 h and then infected with CSFVWT at an MOI of 5. At 48 hpi, the levels of IFN-β RNAs in cells were confirmed by qRT-PCR, and the level of IFN-β RNAs in untreated PK-15 cells was defined as 1. (B) PK-15 or PK-15 Dicer-knockdown cells were transfected with either empty plasmid or a plasmid encoding SFV capsid, CSFV NS4A, or NS4A_R38A/K53A as indicated. At 24 hpt, the cells were treated with ruxolitinib (10 μM) for 1 h and then infected with CSFV_WT or CSFV_R38A/K53A at an MOI of 5. At 12, 24, and 48 hpi, the levels of CSFV genomic RNAs in cells were determined by qRT-PCR, the level of CSFV_R38A/K53A RNA in PK-15 cells treated with ruxolitinib at 12 hpi was defined as 1. (C) The expression of SFV capsid, CSFV NS4A, or NS4A_R38A/K53A in PK-15 cells treated with ruxolitinib was determined by Western blotting. (D) PK-15 cells were treated with ruxolitinib (10 μM) for 1 h and then treated with enoxacin (100 μM) or DMSO for another 1 h and infected with CSFVR38A/K53A at an MOI of 5. At 12, 24, and 48 hpi, the accumulations of CSFV genomic RNAs in cells were detected by qRT-PCR, and the level of CSFVR38A/K53A RNA in PK-15 cells treated with DMSO at 12 hpi was defined as 1. (E) PK-15 Dicer-knockdown cells were transfected with either empty plasmid or a plasmid encoding SFV capsid, or CSFV NS4A as indicated. At 24 hpt, the cells were infected with CSFV_R38A/K53A at an MOI of 5. At 12, 24, and 48 hpi, the levels of CSFV genomic RNAs in cells were confirmed by qRT-PCR, and the level of CSFV_R38A/K53A RNA in PK-15 cells transfected with either empty plasmid at 12 hpi was defined as 1. (F) The expression of SFV capsid and CSFV NS4A in PK-15 Dicer-knockdown cells was detected by Western blotting. (G) PK-15 cells were infected with CSFV_WT or CSFV_R38A/K53A at an MOI of 10 for 48 h, and then the total RNAs were extracted for small RNAs Northern blotting with a DIG-labeled RNA probe targeting nt 30 to 80 of antigenomic CSFV RNA. U6 was used a loading control. All data represent the means and standard deviations of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (as measured by two-way ANOVA; GraphPad Prism).

Moreover, the ectopic expression of CSFV NS4A or SFV capsid in PK-15 Dicer-knockdown was not able to restore the replication of CSFV_R38A/K53A (Fig. 8E), indicating that the ectopic expression of foreign VSR restoring the replication of CSFV_R38A/K53A does indeed rely on RNAi. The expression of SFV capsid and NS4A was confirmed by Western blotting (Fig. 8F). In addition, we detected CSFV vsiRNAs in PK-15 cells via Northern blotting. The probe was DIG-labeled and targeted the 5′-end 30 to 80 nt of the negative-stranded antigenomic RNA. As shown in Fig. 8G, the amount of CSFV vsiRNAs was obviously increased in PK-15 cells infected with CSFV_R38A/K53A, but not in those infected with CSFV_WT.

To sum up, our findings demonstrated that the replication of VSR-deficient CSFV can be rescued when the RNAi pathway is defective in PK-15 cells.

DISCUSSION

RNAi can function as an antiviral natural immunity in invertebrates and mammals. However, when challenged by the RNAi pathway, viruses can encode types of proteins called VSR to evade this antiviral immunity (2, 4). Whether or not Pestivirus possesses a VSR to suppress antiviral RNAi during viral infection in host cells remains poorly understood. In addition, it is essential to determine whether the deficiency of VSR can lead to the abortion of viral replication and whether the deficiency of the RNAi pathway can restore the replication defect of the VSR-deficient mutant virus.

In this paper, we found that the nonstructural protein NS4A of CSFV possesses VSR activity by sequestrating dsRNA and siRNA in mammalian cells, providing new insights into its multifunctional roles during viral infection. The VSR activity of CSFV NS4A restored the abortion of replication of VSR-deficient SFV_K124A/K128A (Fig. 1E and F) in mammalian cells. What’s more, the VSR deficiency of CSFV NS4A led to substantial restriction of viral replication in PK-15 cells. While the replication of VSR-deficient CSFV can be rescued by the deficiency of RNAi by either ectopically expressing foreign VSRs or blocking the RNAi pathway, and this rescuing effect is independent of IFN-I. In addition, the amount of CSFV vsiRNAs was enhanced in PK-15 cells when the NS4A-mediated RNAi suppression was genetically disabled in CSFV (Fig. 8G). Our findings demonstrated that CSFV NS4A can function as VSR to promote viral replication by sequestrating viral dsRNA from Dicer cleavage and by binding to vsiRNA to interfere with the assembly of RISC (Fig. 9).

FIG 9.

Model for CSFV NS4A protein suppressing RNAi in mammalian cells. After CSFV entry the host cells, its single-stranded genomic (+) RNAs act as the template to yield the negative genomic (−) RNAs and viral proteins. Then this (−) RNAs can also act as the template to yield more (+) RNAs. The replicative intermediate (vRI-dsRNAs) formed during viral replication can be recognized and cleaved into virus-derived siRNAs (vsiRNAs) by Dicer. Then these vsiRNAs are loaded into AGO and mediate the clearance of virus. Whereas CSFV NS4A protein can evade the RNAi by binding to dsRNA and siRNA, contributing to the survival of CSFV.

CSFV is the etiological agent of classical swine fever, which results in severe diseases among domestic pigs and wild boars (31–33). It contributes to the development of antiviral therapy by understanding the interaction between CSFV and the host. CSFV belongs to the family Flaviviridae, which contains yellow fever virus (YFV), Hepatitis C virus (HCV), DENV, and others. Previous studies have identified that YFV capsid (27), HCV NS2, core and E2 (20–22), and DENV NS3 and NS4B (44, 45) displayed RNAi inhibitory activity by interfering with the proteins or RNA components in RNAi. While the VSR activity of these proteins was not verified in the context of viral infection. Despite the fact that Flavivirus uses NS2A as a bona fide VSR to evade RNAi in mammals and mosquitoes (30), the sequence of NS2A or NS4A among the genera Flavivirus and Pestivirus in the Flaviviridae family was not conservative. In addition, it has been confirmed that more than one virus has two or more VSRs, such as p20, p23, and coat protein of citrus tristeza virus (46), P1, and HcPro of potyviruses (47) and NS2, core, and E2 of HCV. In addition, we demonstrated that BVDV and BDV NS4A exhibited VSR activity, indicating NS4A protein has a conservative VSR activity among Pestivirus.

CSFV NS4A is a small molecule but multifunctional protein. In particular, the β-sheet from NS4A (residues 21 to 40) may be crucial for the stabilization of CSFV NS3 protease (37). Moreover, the C-terminal region of NS4A can also interact with a hydrophobic patch of NS3 protein (39). Indeed, the mutational analyses proved that NS4A_R38A/K53A abolished the dsRNA-/siRNA-binding activities but had no effect on the interaction with NS3 protease (Fig. 4F and G), indicating that these residues were not crucial for the interaction between NS4A and viral NS3 protease, although they may have participated in the process of interacting with RNA.

In conclusion, our findings demonstrated that CSFV NS4A acts as a VSR to promote viral replication by sequestrating dsRNA from Dicer cleavage and interfering with RISC assembly by binding to siRNA. Furthermore, this is the first time it has been shown that Pestivirus can encode a VSR to block RNAi during viral infection, adding new insight regarding the interaction between Pestivirus and the antiviral RNAi pathway.

MATERIALS AND METHODS

Plasmids and RNAs.

For the expression of the EGFP, its ORF was cloned into expressing vector pcDNA3.1-His. For the expression of capsid of the SFV and nine proteins of the CSFV, their ORFs were cloned into expressing vector pRK-Flag. To get the MBP fusion NS4A protein, its ORF was cloned into the pMal-c2X vector. The EGFP/Dicer specific shRNA was cloned into the pSuperRetro vector. The EGFP-siRNA (siEGFP) was chemically synthesized by ShengGong, Wuhan, China. The full-length CSFV cDNA clone was constructed into pCMV-Myc vector.

Cell culture and transfection.

HEK293T and PK-15 (porcine kidney 15 cell line) cells were cultivated in Dulbecco modified Eagle medium (DMEM) containing 100 μg/mL streptomycin, 100 U/mL penicillin, and 10% fetal bovine serum (FBS; GIBCO) at 37°C in incubator with 5% CO₂. The 293T or PK-15 cells were cultivated in 6-well dishes and grown overnight to reach to 70% confluence, followed by transfection with Lipofectamine 2000 transfection reagent (Invitrogen), according to the manufacturer’s protocol. Briefly, before transfection, the medium was substituted with 2% DMEM without any antibiotic. Then plasmids or siRNAs were mixed with Lipofectamine 2000 for 15 min at room temperature and added into 293T or PK-15 cells.

For the reversal-of-silencing experiment, 293T and PK-15 cells were cotransfected with a plasmid encoding EGFP (0.1 μg), shEGFP/siRNAs (0.3 μg) and either empty plasmid or a plasmid encoding CSFV protein (1 μg for each). For the Dicer-knockdown experiment, PK-15 cells were cotransfected with Dicer-specific shRNA (shDicer) or shRNA cotrol (0.6 μg for each). At last, the cells were harvested for analysis after cultivation for 48 h.

Construction and recovery of CSFV mutant virus.

R38A/K53A mutation in NS4A was introduced into the infectious CSFV cDNA clone (the CSFV Shimen strain) by PCR-mediated site-directed mutagenesis. One μg of in vitro T7 RNA polymerase-transcribed viral RNAs was transfected into PK-15 cells by using Lipofectamine 2000. The rescued viruses were harvested after transfection at 37°C for 72 h.

Virus infection.

When the 293T or PK-15 cells were grown to 80%, the medium was changed with 2% FBS DMEM, and the cells were infected with SFV or CSFV. The cells or supernatant then were harvested at 6, 12, and 24 hpi for SFV or 12, 24, and 48 hpi for CSFV to extract total RNAs for qRT-PCR and Northern blotting. For the rescue assays, we transfected PK-15 cells with the plasmid encoding the indicated proteins or Dicer-specific shRNA (shDicer). At 24 hpt, the cells were treated with ruxolitinib (10 μM, Selleck) or enoxacin (100 μM, Selleck) for 1 h and then infected with viruses.

For virus titration, PK-15 cells in 96-well plates were infected with 10-fold serial dilution of virus at 37°C for 2 h to ensure the adsorption of all the viruses. Then the supernatant was discarded, after which cells were washed with 1×PBS and cultivated at 37°C for 72 h. The cells were subjected to an immunofluorescence assay. Viral titers were calculated using the method of Reed and Muench (48).

Immunofluorescence (IF) assay.

PK-15 cells were fixed with 50% vol/vol methanol/acetone at −20°C for 30 min and blocked with 3% bovine serum albumin (BSA) at 37°C for 30 min. Next, the cells were incubated with rabbit polyclonal anti-NS3 antibody (1:300) at 37°C for 2 h and secondary antibody (1:1000, Alexa 488-conjugated goat anti-rabbit IgG, Jackson, West Grove, PA, USA) at 37°C for 1 h.

Western blotting.

Cells were harvested in lysis buffer (0.25% deoxycholate, a protease inhibitor cocktail [Rhochel], 1% NP-40, 150 mM NaCl, and 50 mM Tris-HCl [pH 7.4]). Then the lysates were subjected to SDS-PAGE and Western blotting according to our standard procedures (28). The antibodies used in this assay were as follows: anti-β-actin (Protein Tech Group, 1:3000), anti-Flag (MBL, 1:2000), anti-Dicer (Abcam, 1:2000), anti-EGFP (Protein Tech Group, 1:2000), anti-His (Protein Tech Group, 1:2000).

Immunoprecipitation.

PK-15 cells were cotransfected with indicated plasmids. After 48 h, cells were washed twice with PBS and lysed in lysis buffer (0.25% deoxycholate, a protease inhibitor cocktail [Rhochel], 1% NP-40, 150 mM NaCl, and 50 mM Tris-HCl [pH 7.4]). The lysates were incubated with specific antibodies and protein A/G agarose (Roche) overnight at 4°C. The resins were collected by centrifugation and washed five times with lysis buffer. Bound proteins were then subjected to SDS-PAGE and detected with appropriate antibodies.

Northern blotting and qRT-PCR.

The total RNAs were harvested from cells using TRIzol reagent (Thermo) according to the manufacturer’s instructions and subjected to denatured 1.5% agarose gels with 2.2 M formaldehyde according to our standard procedures (28). In brief, the separated RNAs were transferred onto the Hybond-A nylon membrane (GE Healthcare) and fixed by 120°C for 15 min. Then the membranes were hybridized with digoxigenin (DIG)-labeled probes in Hybridization Ovens at 65°C overnight and incubated with anti-DIG-alkaline phosphatase (AP). The probe for the detection of EGFP or SFV genome and subgenomic RNA was complementary to nt 520 to 700 of the EGFP ORF region or complementary to nt 10637 to 11163 of SFV genome RNA, respectively. These probes were labeled with DIG-UTP (Roche) by transcription in vitro.

For detection of the small RNAs, the total RNAs were extracted using TRIzol reagent (Thermo) and subjected to 7 M urea-15% PAGE. The RNAs were next transferred to Hybond-A nylon membrane (GE Healthcare). At last, the membranes were chemically cross-linked in 1-ethly-3-(3-dimethylaminopropyl) carbodiimide (EDC) at 60°C. The probes targeting EGFP siRNA, CSFV vsiRNAs, and, U6 were synthesized by in vitro transcription, and their sequences are listed in S1 TABLE. The levels of EGFP mRNA, β-actin mRNA, and SFV/CSFV genomic mRNA were confirmed by qRT-PCR using SYBR mix (TaKaRa) according to the manufacturer’s instructions. All data represent the means and standard deviations of three independent experiments. **, P < 0.01; ***, P < 0.001 (as measured by two-way ANOVA; GraphPad Prism). All the primes are listed in S1 TABLE.

Expression and purification of NS4A protein.

The expression plasmid pMal-c2x containing the coding sequences of CSFV NS4A protein was transformed into Escherichia coli BL21 (Invitrogen). Escherichia coli BL21 were induced with 0.8 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at 26°C for 8 h after being grown to the log phase. Then the cells were harvested and resuspended with lysis buffer (5% absolute ethyl alcohol, 1 M Tris-HCl [pH 7.5], 10 mM β-mercaptoethanol, 0.2 M NaCl, 10% glycerinum, and 0.5 M EDTA [pH 8.0]), followed by ultrasonication and centrifugation. Finally, the supernatant was mixed with Amylose Resin (Biolabs) for purification according to the manufacturer’s instructions.

Electrophoretic mobility shift assay (EMSA) and RNase III cleavage assays.

MBP-fusion NS4A or mutant proteins were reacted with DIG-labeled RNAs (0.5 μM 200-nt dsRNA, 1 μM 22-nt ds-siRNA) in a 10 μL reaction buffer containing 40 mM MgCl₂, 50 mM NaCl, 25 mM HEPES (pH 7.5), 3 mM dithiothreitol (DTT), 1 U of RNase inhibitor at 26°C for 40 min. Then the mixtures were subjected to 1.5% native-TBE agarose gel and transferred to Hybond-A nylon membranes (GE Healthcare). Finally, membranes were washed with maleic acid buffer for 10 min, after which they were incubated with anti-DIG antibody conjugated with alkaline phosphatase (Roche) for 60 min.

For the RNase III cleavage experiments, 1 μM 200 nt dsRNA was incubated with MBP-fusion NS4A at 26°C for 30 min. Next, 0.5 U RNase III (Thermo) was added into the reaction commixture for 30 min or 60 min at 37°C. Finally, the mature siRNAs processed by RNase III were extracted from the reaction complex by using TRIzol reagent (Thermo) and subjected to 7 M urea-16% PAGE for Northern blotting.