To discover the host factors that were involved in RHDV replication, we attempted to purify the viral RHDV replicase and replicase-related host factors during viral replication and identify the associated host factors. Previously, the researchers successfully identified hepatitis C virus (HCV) replicase-associated replication complex (RC) components by inserting His and HA tags into the HCV replicon replicase NS5A and NS5B (RdRp) for affinity purification (Yi et al. 2016). Here, we aim to purify RdRp-related host factors by introducing two different tags into RdRp. We generated a recombinant replicon by simultaneously introducing His and HA tag into RdRp (position: 25 or 82 aa for His, 442 or 483 for HA, respectively) of the RHDV replicon (Fig. 1A). Fluc activity and IB analyses showed that RHDV-luc-His1/HA1 and RHDV-luc-His1/HA2 replicated similarly to the untagged RHDV replicon in RK-13 cells (P > 0.05, P > 0.05, respectively) (Fig. 1B, 1C). The SWISS-MODEL online tool (https://swissmodel.expasy.org/) predicated that insertion of the His and/or HA tag into these sites does not change the general structure of RdRp, so it is speculated that the insertion into these sites might not significantly affect RdRp activity (Supplementary Fig. S1A). Fluc activity and IB analysis showed that RHDV-luc-His1, RHDV-luc-HA1, and RHDV-luc-HA2 replicated similarly to the untagged RHDV replicon (Supplementary Fig. S1B, S1C). However, the replication level of RHDV-luc-His2 was significantly reduced. We speculate that this site has an important effect on RdRp enzyme activity, and the specific mechanism needs to be further studied.
Figure Supplementary Fig. S1 . Tagging of RHDV RdRp in the viral replicon. A RHDV RdRp protein structure analysis. For clarity, the structure of the RHDV RdRp was obtained from the Protein Data Bank (PDB) under the identification number 1khv (https://www.rcsb.org/). The structure of the RdRp mutation, as predicted by the SWISS-MODEL online tool (https://www.swissmodel.expasy.org/) and based on homology molecules found in the PDB. The orange portion indicated by the red arrow is the inserted label. B Effect of the inserted tag on viral replicon activity. RK-13 cells were transfected with recombinant RHDV replicons. Luciferase activity in cell lysates was measured at 48 h post-transfection. Statistical analysis was performed by Student t-tests. **P < 0.01. Data are shown as mean with SD. Replicate 1, 2, 3 means three independent experiments, each experiment contains three technical replicate values. The number of cells used in all replicate experiments were similar. C Western blotting of recombinant RHDV replicons in RK-13 cells with the antibodies indicated. β-actin was used as an internal control. RHDV, rabbit hemorrhagic disease virus; RdRp, RNA-dependent RNA polymerase; SD, standard deviation.
RK-13 cells were transfected with RHDV-luc-His1/HA1 replicon, which replication level is closest to the untagged replicon, and the cell lysates were sequentially purified using the HA and His tags at 48 hpt. The untagged RHDV replicon acted as a negative control. After two-step affinity purification, the eluted protein complexes were resolved by SDS-PAGE and the protein bands were visualized with silver staining. In total, 11 specific or enriched bands were sliced from the RHDV-luc-His1/HA1 lane and the proteins they contained were identified using MS (Table 1). The identified host proteins were associated with cytoskeleton components, intracellular transport, chaperone, ribonucleoprotein (RNP) components, and translation machine-related proteins. Among these proteins, numerous proteins have been shown to interact with some single-stranded positive-strand RNA viral proteins to regulate viral replication, such as HnRNPK, HSPA8, DDX5, ANXA2, and PI4KA (Hsieh et al. 1998; Saxena et al. 2012; Kovalev and Nagy 2014; Zhang et al. 2014; Dorobantu et al. 2015) (Fig. 1D and Table 1).
Category and band no. Protein score Mass (kDa) Gene name Protein description No. of unique peptides No. of peptides SC (%) Category and band no. Protein score Mass (kDa) Gene name Protein description No. of unique peptides No. of peptides SC (%) RHDV protein Cytoskeleton 6 273.3 57.8 RdRp RHDV RdRp 3 4 21.1 11 105.5 14.3 CFL1 Cofilin 1 2 6 16.8 10 186.4 25.1 p23 RHDV p23 2 3 15.5 3 316.5 102.9 ACTN1 Actinin alpha 1 2 7 32.4 11 165.8 16.2 p16 RHDV p16 2 4 10.4 4 286.3 87.4 ACTN4 Actinin alpha 4 2 6 28.5 Transport 8 357.8 41.7 ACTB Actin, cytoplasmic 1 2 5 19.0 8 57.1 39.2 ANXA2 Annexin A2 4 7 10.4 7 419.4 53.6 VIM Vimentin 13 13 25.3 1 201.3 233.6 PI4KA Phosphatidylinositol 4-kinase alpha 6 6 30.5 Chaperon 5 303.5 68.9 ALB Serum albumin 8 10 15.2 7 65.2 45.1 CSNK2A1 Casein kinase Ⅱ subunit alpha 5 5 7.5 11 287.6 15.6 HBA hemoglobin subunit alpha 5 5 40.4 4 236.2 71.0 HSPA8 Heat shock cognate 71 kDa protein 7 7 14.2 6 173.2 59.7 ATP5A1 F-type H+-transporting ATPase subunit beta 3 3 7.8 9 153.9 30.7 PYCR2 Pyrroline-5-carboxylate reductase 4 5 16.8 11 365.6 16.1 HBB Hemoglobin subunit beta 7 9 58.7 3 70.1 116.8 DSG1 Desmoglein 1 2 5 9.0 11 365.6 16.1 HBB Hemoglobin subunit beta 7 9 58.7 11 50.3 14.7 LYZ Lysozyme C 2 3 11.5 9 98.3 32.9 SLC25A5 Solute carrier family 25 3 3 11.2 4 53.5 73.5 HSPA9 Eat shock protein family A (Hsp70) member 9 2 2 13.8 11 99.8 12.6 FABP5 Fatty acid-binding protein 5 3 4 13.5 10 152.2 26.2 NUDT21 Nudix hydrolase 21 3 3 24.5 RNP complex 8 142.8 40.3 PDCD2L Programmed cell death protein 2-like 4 4 28.7 10 78.5 25.7 HNRNPA1 Heterogeneous nuclear ribonucleoprotein A1 3 7 13. 6 11 108.3 16.0 FAM207A Family with sequence similarity 207 member A 5 8 19.6 10 67.9 30.3 HNRNPAB Heterogeneous nuclear ribonucleoprotein A/B 2 6 8.9 Translation machines 7 127.2 50.9 HNRNPK Heterogeneous nuclear ribonucleoprotein K 4 9 11.6 7 94.2 50.0 EEF1A Elongation factor 1-alpha 7 7 9.3 5 87.0 67.5 DDX5 DEAD-box helicase 5 2 4 8.3 11 135.4 17.7 RPS18 Ribosomal protein S18 4 4 18.7 5 90.6 69.4 NCL Nucleolin 8 10 28.8 11 113.3 16.3 AAES 40S ribosomal protein S14 3 3 28.3 2 117.8 130.7 SORBS2 Sorbin and SH3 domain containing 2 5 5 14.7 10 174.3 20.2 RPL11 Ribosomal protein L11 4 4 20.1 10 135.6 26.2 NUDT21 cleavage and polyadenylation specificity factor subunit 5 3 3 10.4 10 283.1 21.2 RPL12 Ribosomal protein L12 5 5 33.5 5 99.1 70.9 PABPC1 Polyadenylate-binding protein 1 4 4 16.3 11 164.9 12.8 RPL30 60S ribosomal protein L30 4 4 50.5 9 128.4 36.0 YBX1 Nuclease sensitive element-binding protein 1 5 6 22.1 11 68.3 15.5 RPS17 40S ribosomal protein S17 3 3 29.6 6 112.1 57.4 PTBP1 Polypyrimidine tract-binding protein 1 4 7 6.7 10 323.2 22.9 RPS5 Ribosomal protein S5 7 7 18.5 6 65.2 52.1 SRSF1 Splicing factor, arginine/serine-rich 1 6 7 8.8 9 258.2 31.1 RPS3 40S ribosomal protein S3 9 9 25.8 5 75.9 63.5 CPSF6 Cleavage and polyadenylation specificity factor subunit 6/7 3 10 10.9 7 74.4 45.3 EIF4A1 Eukaryotic initiation factor 4A-I 4 6 9.7 6 75.2 62.5 DDX41 Probable ATP-dependent RNA helicase 3 12 14.5 11 122.6 13.7 RPS25 Ribosomal protein S25 5 5 21.3 11 75.3 14.0 RPS15A Ribosomal protein S15a 2 5 18.3 a Protein lists for each of the proteins identified in Fig. 2B. SC (%) refers to the percent sequence coverage for the protein.
Table 1. Categories of host factors found to be associated with RHDV replicasea.
NCL is a phosphoprotein that is ubiquitously and abundantly expressed in many eukaryotic cells and is highly conserved during evolution, as it is involved in a remarkably large number of cellular activities (Jia et al. 2017). It has been confirmed that NCL also plays important roles in the replication and intracellular trafficking of multiple viruses (Tuteja and Tuteja 1998; Bicknell et al. 2005; Hirano et al. 2005; Becherel et al. 2006; Mongelard and Bouvet 2007; Abdelmohsen and Gorospe 2012; Durut and Saez-Vasquez 2015). To determine if NCL was required for RHDV replication, RK-13 cells were co-transfected with an RHDV replicon, NCL siRNA or Flag-tagged NCL plasmids and internal control plasmid encoding an Rluc gene. The reporter luciferase activity was evaluated using a dual-luciferase reporter assay system with cell lysates that were harvested at 24 and 48 hpt. Fluc activity was normalized with respect to a co-transfected plasmid encoding an Rluc. Similar results were obtained in three independent experiments. The results showed that there is a positive correlation between the expression level of Fluc and NCL. The Fluc activity decreased with increasing NCL siRNA transfection dose and increased with increased dose of Flag-NCL transfection (Fig. 2A, 2B). In previous study, we found that the RHDV replicon activity reached a maximum value at 24 hpt and then declined obviously at 48 hpt (Wang et al. 2013). In this study, it was found that after overexpression of NCL, the replication level of RHDV replicon at 48 hpt is close to the 24 hpt level. Therefore, the expression of NCL promotes RHDV replication in a dose-dependent manner. Subsequently, we examined the effect of NCL on mRHDV, which could proliferate in RK-13 cells (Zhu et al. 2017). We also used NCL siRNA or Flag-tagged NCL to change the expression level of NCL, and then infected the cell with mRHDV (MOI = 1). At 48 h post-infection (hpi), the replication level of mRHDV was detected by Western blotting (WB) and qPCR. The results were similar to RHDV replicons. As shown in Fig. 2C and 2D, the replication level of mRHDV increased with increased dose of Flag-NCL and decreased with increasing NCL siRNA. In addition, we successfully constructed an RK-NCL cell line, which overexpressed the NCL gene, using a lentiviral packaging system (Fig. 2E). To evaluate the replication of RHDV in RK-NCL cells, the cells were co-transfected with an RHDV replicon and internal control plasmid encoding an Rluc gene. The reporter luciferase activity was evaluated at 24 hpi. The results showed that the expression level of Fluc in RK-NCL cells was significantly higher than that in control cells (RK-GFP cells and RK-13 cells) (Fig. 2F). Collectively, these data suggest that NCL is involved in RHDV replication.
Figure 2. NCL is involved in RHDV replication. A The effect of NCL eukaryotic plasmids on viral replicon activity. Relative luciferase activity was evaluated in RK-13 cells carrying pRHDV-luc, and trans-supplemented NCL eukaryotic plasmids pFlag-NCL (0.2 μg, 0.4 μg) at 24 h post-transfection (hpt) and 48 hpt. The p3×Flag-CMV-14 vector acted as negative control (–). The luciferase activity in RK-13 cells was evaluated by measuring Fluc activity. Rluc activity was measured to normalize the transfection efficiency. B The effect of NCL siRNA on viral replicon activity. The RK-13 cells, co-transfected with pRHDV-luc and NCL siRNA (20 pmol, 40 pmol, 60 pmol, or 80 pmol), were lysed at 24 hpt and 48 hpt, and Fluc activity was measured based on RLUs and normalized according to the results obtained for a co-transfected pLTK plasmid encoding Rluc. The nonspecific siRNA acted as negative control. C–D The effect of NCL on mRHDV replication. The RK-13 cells, transfected with pFlag-NCL (1 μg, 2 μg) or NCL siRNA (40 pmol, 80 pmol), were infected with mRHDV (MOI = 1) at 24 hpt, and the level of mRHDV replication were evaluated by Western blotting and qRT-PCR at 48 hpi. The p3×Flag-CMV-14 vector (EV) and nonspecific siRNA (–) acted as negative control. RK-13 cell acted as mock control. E The expression level of NCL in RK-NCL cells at 10 passages was determined by Western blotting analysis with anti-Flag mAb. F The RHDV replicon replication levels in RK-NCL cells were evaluated by measure Luc at 24 hpi. RK-GFP cells acted as negative controls; RK-13 cells acted as blank controls. Statistical analysis was performed by Student t-tests. *P < 0.05, **P < 0.01 and ***P < 0.001. Data are shown as mean with SD. Replicate 1, 2, 3 means three independent experiments, each experiment contains three technical replicate values. The number of cells used in all replicate experiments was similar. RHDV, rabbit hemorrhagic disease virus; NCL, nucleolin; hpt, hours post-transfection; SD, standard deviation.
To determine if NCL regulates RHDV replication through interaction with viral nonstructural proteins, we used M2H assays to screen the interaction between NCL and viral nonstructural proteins. As shown in Fig. 3A, NCL interacted with RdRp, p16, and p23. Moreover, an IFA was performed using NCL mAbs, RdRp mAbs, p16 polyclonal antibody and p23 polyclonal antibody in RK-13 cells infected with mRHDV at 24 hpi. As shown in Fig. 3B, NCL was co-localized with RHDV RdRp, p16 and p23 in the RK-13 cell cytoplasm. In addition, the distribution of NCL in the cytoplasm increased after RHDV infection. To determine whether endogenous NCL binds to these viral nonstructural proteins, during RHDV genome replication, we assessed the interaction between NCL and these viral proteins in RK-13 cells, in the presence and absence of mRHDV infection for 24 h at 37 ℃. The results of an IP assay performed with cell lysates using NCL mAb showed that regardless of whether the cell lysates were treated with RNase or not, NCL interacted with RdRp, p16, and p23 in infected cells, but did not in uninfected cells (Fig. 3C). To prove NCL interacts with RdRp, p16, and p23, a series of Co-IP assays were used with a myc mAb in RK-13 cells, which were co-transfected with pRdRp-myc, p16-myc, p23-myc and pNCL-Flag eukaryotic expression plasmids. We showed that overexpressed NCL-Flag was present in the anti-myc immunocomplex (Fig. 3D). These results showed that NCL could interact with RHDV RdRp, p16, and p23.
Figure 3. NCL interacts with RHDV replicase RdRp, nonstructural proteins p16 and p23. A M2H interaction of NCL with RHDV nonstructural proteins. Statistical analysis was performed by Student t-tests. *P < 0.05. Data are shown as mean with SD. Replicate 1, 2, 3 means three independent experiments, each experiment contains three technical replicate values. The number of cells used in all replicate experiments was similar. B Confocal microscopy analysis of NCL (green), RdRp (red), p16 (red) and p23 (red) in RK-13 cells infected with mRHDV at 24 h post-infection with mAbs against NCL and RdRp. The small white boxes represent amplified random co-localization spots within the merged image, and the co-localization spots are indicated with white arrowheads. C NCL binds to RdRp, p16 and p23 during RHDV replication. An IP assay was performed on cell lysates using NCL mAb in RK-13 cells that were infected or uninfected with mRHDV, then immunoblotted with Abs against NCL, RdRp, p16 or p23. β-actin was used as an internal control. Cells uninfected with mRHDV served as negative controls. D Validation of the interaction of RHDV RdRp, p16 and p23 with NCL in a Co-IP assay. RK-13 cells were co-transfected with the indicated plasmids (+) or empty vectors (–). At 48 h post-transfection, cells were lysed, and IP of myc-fused proteins was performed using anti-myc mAb. Lysates (input) and IPs were analyzed with IB using antibody against myc or Flag. β-actin was used as an internal control. RHDV, rabbit hemorrhagic disease virus; RdRp, RNA-dependent RNA polymerase; SD, standard deviation; NCL, nucleolin; IP, immunoprecipitation; IB, immunoblotting; mAb, monoclonal antibody.
The positive-strand RNA viruses share a conserved replication mechanism in which viral proteins induce host membrane modification to assemble membrane-associated viral replication complex (RCs) (den Boon and Ahlquist 2010). Viruses hijack host factors to facilitate this energy-unfavorable process (Nagy and Pogany 2011). Therefore, the components of the viral RC are numerous and complex. NCL is capable of binding to nonstructural proteins (RdRp, p16, and p23) of RHDV. To test the hypothesis that NCL acted as a platform for the RdRp to be attracted to the p16 and p23 proteins, a series of HA tag affinity purification analyses were performed. RHDV-luc-His1/HA1 replicon was co-transfected with NCL siRNA, or non-specific siRNA, in RK-13 cells, respectively, or was transfected in RK-NCL cells. Using IB to detect the purified RdRp-associated protein, we found that the RdRp-associated protein content was significantly reduced in NCL siRNA-treated cells and significantly increased in RK-NCL cells (Fig. 4A). To investigate the specific role of NCL in the formation of RHDV RCs, M2H assays were used to screen the interactions between viral nonstructural proteins and multiple host factors in RCs. As shown in Fig. 4B, there are complex interactions between viral nonstructural proteins and host factors in RCs. For example, p16 interacts with itself, helicase, p29, protease, and NCL; p23 binds to protease and NCL; p29 binds to helicase and VPg; helicase interacts with itself; VPg binds to protease; protease interacts with itself and RPS5; RdRp interacts with RPS5; and NCL binds to HnRNPK, CSNK2A1, RPS5, and RPL11. We subsequently used a series of Co-IP assays with a myc mAb in RK-13 cells, which were co-transfected with bait (myc fusion protein) and prey (Flag fusion protein) eukaryotic expression plasmids. IB analysis using a mAb against Flag showed the specific band corresponding to prey proteins in the myc Co-IP assay (Fig. 4C). These results reveal that RHDV replicase RdRp cannot directly bind to other nonstructural proteins of the virus. It is noteworthy that NCL directly interacts with RHDV RdRp and nonstructural proteins (p16 and p23). Together, these data suggest that RHDV completes its replication process by hijacking NCL to recruit other viral proteins and host factors.
Figure 4. Identification of interactions between RHDV nonstructural proteins and host factors of RCs. A NCL siRNA inhibited the formation of the RHDV RC. After HA tag affinity purification, the eluted proteins were resolved by SDS-PAGE. The protein bands were visualized with silver staining. PBS acted as a negative control; β-actin acted as an internal control and was detected by IB with mAb against β-actin. B Identification of these interactions by M2H assays. Bait and prey plasmids were co-transfected with pG5luc plasmids into subconfluent HEK-293T cells at a molar ratio of 1:1:1 for the pACT: pBIND: pG5luc vector. At 48 h post-transfection (hpt), the HEK-293T cells were lysed, and Rluc and Fluc activities were evaluated using the Promega Dual-Luciferase Reporter Assay System. All experimental groups were compared with the negative control group (ACT-Bind). Statistical analysis was performed by Student t-tests. *P < 0.05 and **P < 0.01. Data are shown as mean with SD. Replicate 1, 2, 3 means three independent experiments, each experiment contains three technical replicate values. The number of cells used in all replicate experiments was similar. C These interactions were verified using Co-IP assays. RK-13 cells were co-transfected with bait and prey plasmids. Cell lysates were prepared 48 hpt and the proteins were subjected to IP followed by IB analysis. myc fusion proteins acted as bait proteins and Flag fusion proteins acted as prey proteins. RHDV, rabbit hemorrhagic disease virus; RC, replication complex; IP, immunoprecipitation; IB, immunoblotting; mAb, monoclonal antibody; SD, standard deviation.