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. 2022 Feb;37(1):48-59.
doi: 10.1016/j.virs.2022.01.004. Epub 2022 Jan 12.

Nucleolin interacts with the rabbit hemorrhagic disease virus replicase RdRp, nonstructural proteins p16 and p23, playing a role in virus replication

Jie Zhu  1 , Qiuhong Miao  2 , Hongyuan Guo  1 , Aoxing Tang  1 , Dandan Dong  1 , Jingyu Tang  1 , Fang Wang  3 , Guangzhi Tong  4 , Guangqing Liu  5
Affiliations

Nucleolin interacts with the rabbit hemorrhagic disease virus replicase RdRp, nonstructural proteins p16 and p23, playing a role in virus replication

Jie Zhu et al. Virol Sin. 2022 Feb.

Erratum in

Abstract

Rabbit hemorrhagic disease virus (RHDV) is a member of the Caliciviridae family and cannot be propagated in vitro, which has impeded the progress of investigating its replication mechanism. Construction of an RHDV replicon system has recently provided a platform for exploring RHDV replication in host cells. Here, aided by this replicon system and using two-step affinity purification, we purified the RHDV replicase and identified its associated host factors. We identified rabbit nucleolin (NCL) as a physical link, which mediating the interaction between other RNA-dependent RNA polymerase (RdRp)-related host proteins and the viral replicase RdRp. We found that the overexpression or knockdown of NCL significantly increased or severely impaired RHDV replication in RK-13 ​cells, respectively. NCL was identified to directly interact with RHDV RdRp, p16, and p23. Furthermore, NCL knockdown severely impaired the binding of RdRp to RdRp-related host factors. Collectively, these results indicate that the host protein NCL is essential for RHDV replication and acts as a physical link between viral replicase and host proteins.

Keywords: Nucleolin; RNA-dependent RNA polymerase (RdRp); Rabbit hemorrhagic disease virus (RHDV); Virus replication.

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Conflict of interest statement

Conflict of interest The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Affinity purification of solubilized RHDV replicase RdRp-associated host factors. A Schematic of RHDV-His/HA replicon constructs. The HA and 6 ​× ​His peptide sequences are inserted into the RdRp aa sequence at 25 or 82, 442 or 483 sites, respectively. 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 hpt. Data are shown as mean with SD. Student t-tests and analysis of variance were used for statistical analyses. 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 Western blotting of recombinant RHDV replicons in RK-13 ​cells with the antibodies indicated. β-actin was used as an internal control. D After two-step affinity purification, the eluted proteins were resolved by SDS-PAGE. Specifically, enriched protein bands (arrows) in the pRHDV-His1/HA1-luc sample were identified by mass spectrometry. Mainly, host proteins and identified viral proteins in the bands indicated are shown. Host factors selected for further study are marked in red. RHDV, rabbit hemorrhagic disease virus; RdRp, RNA-dependent RNA polymerase; hpt, hours post-transfection; SD, standard deviation.
Fig. 2
Fig. 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.
Fig. 3
Fig. 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.
Fig. 4
Fig. 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.
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