Nucleolin is essential for rabbit hemorrhagic disease virus replication by providing a physical link in replication complex formation

Rabbit hemorrhagic disease virus (RHDV) is an important member of the Caliciviridae family and cannot be propagated in vitro, which has greatly impeded 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 required for the formation of RHDV replication complexes (RCs), by mediating the interaction between other host proteins and the viral RNA replicase, RNA-dependent RNA polymerase (RdRp). We found that RHDV RdRp uses an amino acid (aa) region spanning residues 448–478 to directly interact with NCL’s RNA-recognition motif 2. We also found that the viral p16 protein uses a highly conserved region (35Cys–Ile–Arg–Ala38 or CIRA motif) to specifically bind the N-terminal region of NCL (aa 1–110) and that RHDV p23 uses a specific domain (aa 90–145) to bind NCL’s RNA-recognition motif 1. Disrupting these protein–protein interactions severely weakened viral replication. Furthermore, NCL overexpression or knockdown significantly increased or severely impaired, respectively, RHDV replication. Collectively, these results indicate that the host protein NCL is essential for RHDV replication and plays a key role in the formation of RHDV RCs. The mechanisms by which NCL promotes viral replicase assembly reported here shed light on viral RC biogenesis and may inform antiviral therapies. Author summary Rabbit hemorrhagic disease virus (RHDV) is the causative agent of highly contagious and lethal hemorrhagic disease in the European rabbit, but the host factors involved in RHDV replication remain poorly understood. In the present study, we isolated RHDV replication complex (RC) for the first time and identified its main components. We found that nucleolin (NCL) plays a key role in the formation of the RHDV RC. NCL not only interacts with viral replicase (RdRp), it also specifically binds to other important host factors. In addition, we proved that NCL is necessary for RHDV replication because the level of RHDV replication is significantly affected by knocking down the NCL gene in cells. Together, our data suggest that RHDV completes its replication by hijacking NCL to recruit other viral proteins and host factors, thereby assembling the RC of RHDV.


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Rabbit hemorrhagic disease virus (RHDV) is the causative agent of rabbit hemorrhagic 59 disease (RHD), which primarily infects the wild and domestic European rabbit (Orcytolagus 60 cuniculus) [1] and is characterized by liver degeneration, diffuse hemorrhaging and high 61 mortality [2,3]. However, the molecular mechanisms responsible for RHDV replication 62 remain poorly understood, mainly due to the lack of a robust cell culture system for 63 propagation of the virus. 64 RHDV is a nonenveloped positive-sense single-stranded RNA virus, which belongs to the 120 122 To discover the host factors that are involved in RHDV replication, we attempted to 123 purify the viral RCs formed during viral replication and identify the associated host factors. 124 Previously, the researchers successfully identified hepatitis C virus (HCV) replicase-125 associated RC components by inserting His and HA tags into the HCV replicon replicase 126 NS5A and NS5B (RdRp) for affinity purification [16]. Here, we aimed to affinity tag RdRp 127 with two different tags to facilitate tandem affinity purification. We generated a recombinant 128 replicon by introducing a His or HA tag into RdRp (aa sites: 25, 82, 442, or 483, respectively) 129 of the RHDV replicon (Fig. 1A). Moreover, as predicated using the SWISS-MODEL online 130 tool (https://swissmodel.expasy.org/), we found that insertion of the His and/or HA tag into 131 these sites would have no effect on the structure of RdRp (Fig. S1). Fluc activity analysis 132 showed that RHDV-luc-His 25 , RHDV-luc-HA 442 , and RHDV-luc-HA 483 replicated similarly 133 to the untagged RHDV replicon, whereas the replication ability of RHDV-luc-His 82 was 134 significantly inhibited in RK-13 cells (Fig. 1B). The same results were obtained in 135 immunoblotting (IB) detection (Fig. 1C). The luciferase activity from the replicon lacking the 136 RdRp gene has previously been shown to be approximately 4-logs lower than that of the wild 137 type replicon [14]. In RHDV replicon, the viral sequence was generated as a consequence of 138 polymerase II transcription from the cytomegalovirus (CMV) promoter, and the authentic 3' 139 end of the viral genome was under controlled by a hepatitis delta virus ribozyme [17,18].  Subsequently, we introduced HA and His peptides into RdRp simultaneously to obtain a 145 double-affinity-tagged replicon (Fig. 1A). Fluc activity and IB analyses showed that RHDV-146 luc-His 25 /HA 442 and RHDV-luc-His 25 /HA 483 replicated similarly to the untagged RHDV 147 replicon in RK-13 cells ( Fig. 1D and 1E). Therefore, we used RHDV-luc-His 25 /HA 442 in 148 affinity purification assays.   Flag-NCL and decreased with increasing NCL siRNA.

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In addition, we successfully constructed an RK-NCL cell line, which overexpressed the 203 NCL gene, using a lentiviral packaging system (Fig. 3E). To evaluate the replication 204 dynamics of mRHDV in RK-NCL cells, the cells were infected with mRHDV (MOI=1), and 205 subsequently the expression level of VP60 was evaluated with qRT-PCR and WB at 48 hpi.

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The results showed that the expression level of VP60 in RK-NCL cells was significantly 207 higher than that in control cells (RK-GFP cells and RK-13 cells) ( Fig. 3E and 3F).

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Collectively, these data suggest that NCL is involved in RHDV replication. The results of an immunoprecipitation (IP) assay performed with cell lysates using NCL mAb 219 showed that regardless of whether the cell lysates were treated with RNase, NCL interacted 220 with RdRp, p16, and p23 in infected cells, but did not in uninfected cells (Fig. 4B).

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Moreover, an immunofluorescence assay (IFA) was performed using mAbs against NCL and 228 RdRp in RK-13 cells infected with mRHDV at 24 hours post infection (hpi). As shown in Fig.   229 4D, NCL was co-localized with RHDV RdRp in the RK-13 cell cytoplasm. In addition, the 230 distribution of NCL in the cytoplasm increased after RHDV infection.

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The multifunctionality of NCL mainly results from its multidomain structure, which is RBD and GST-NCL-RRM2 interact with RdRp whereas the other proteins did not (Fig. 4F).

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These findings indicate that NCL interacts with RHDV RdRp via the RRM2 motif. GST-RdRp  bound to NCL whereas the other proteins did not (Fig. 4I). These results

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suggest that NCL directly and specifically interacts with the C-terminal aa residues 448-478 262 of RHDV RdRp.

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In addition, as predicated using the SWISS-MODEL online tool, we found that there is a

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"key" and "lock" structure formed by the amino acid sequence 448-478 of RdRp and RRM2 265 of NCL, providing space for the interaction between NCL and RdRp (Fig. 4J). Moreover, 266 analysis of the RdRp sequence of GI.1a-GI.1d genotypes of RHDV showed that the C-267 terminal aa residues 448-478 were highly conserved (Fig. S3A). Together, these observations 268 confirm that RHDV RdRp interacts with the RRM2 motif of NCL via the C-terminal aa 269 residues 448-478, which is a conserved sequence in RHDV.  In this study, we purified viral replicase and identified the replicase-associated host 456 factors using an RHDV replicon system in which two different affinity tags were 457 simultaneously inserted in-frame into RdRp. We determined that NCL plays a key role in the 458 formation of RHDV RCs. On the one hand, NCL binds to RHDV replicase (RdRp) (Fig. 4). hypothesize that these host factors may also be involved in the replication of RHDV. 471 We also identified that NCL interacts with nonstructural proteins (p16 and p23) of RHDV 472 (Figs. 5, 6). By blocking the interaction of NCL with p16 and p23, we found that these 473 interactions have important roles in RHDV replication (Fig. 7) we revealed that NCL interacts with p16 of RHDV via the NTD domain (Fig. 5) and that this 482 interaction plays a role in RHDV replication (Fig. 7) In conclusion, we identified the components of the RHDV RC, for the first time. We 515 found that NCL acts as a link to recruit host factors, viral replicase (RdRp), and nonstructural 516 proteins (p16 and p23), thereby forming a complex and ordered RHDV RC (Fig. 9).

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Elucidation of the molecular mechanism by which NCL regulates viral replicase assembly 518 may lead to new insights into viral RC biogenesis and novel antiviral strategies. The pRHDV-luc plasmid, in which the VP60 and partial VP10 genes are replaced with the 530 Fluc gene, was generated in our previous study [14]. To generate pRHDV-luc-HA1, 531 pRHDV-luc-HA2, pRHDV-luc-His1, and pRHDV-luc-His2, the nucleotide sequence were simultaneously inserted in-frame into RdRp, were generated by fusion PCR (Fig. 1A). including residue numbers, expression vectors, and tags, are summarized in Table S1. In 577 addition, the primers used in this research are listed in Table S2.  The cDNA samples were subjected to real-time PCR with SYBR Premix Ex Taq Tli RNase H 648 Plus (Takara) using an ABI 7500 Fast Real-Time PCR system (Applied Biosystems, USA).

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The primers are listed in Table S2. The relative RNA levels were determined according to the 650 2 -∆∆CT method. The amount of mRNA in each sample was normalized to that of GAPDH.     We thank LetPub for its linguistic assistance during the preparation of this manuscript.   are labeled in yellow. Note: the network was generated using the data presented in Table 1.

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The interaction network was generated using the STRING online tool and then presented