Contribution of DEAD-Box RNA Helicase 21 to the Nucleolar Localization of Porcine Circovirus Type 4 Capsid Protein

Abstract

Porcine circovirus type 4 (PCV4) is a newly emerging pathogen which might be associated with diverse clinical signs, including respiratory and gastrointestinal distress, dermatitis, and various systemic inflammations. The host cellular proteins binding to PCV4 capsid (Cap) protein are still not clear. Herein, we found that the PCV4 Cap mediated translocation of DEAD-box RNA helicase 21 (DDX21) to the cytoplasm from the nucleolus and further verified that the nucleolar localization signal (NoLS) of the PCV4 Cap bound directly to the DDX21. The NoLS of PCV4 Cap and 763GSRSNRFQNK772 residues at the C-terminal domain (CTD) of DDX21 were required for this PCV4 Cap/DDX21 interaction. Further studies indicated that the PCV4 Cap NoLS exploited DDX21 to facilitate its nucleolar localization. In summary, our results firstly demonstrated that DDX21 binds directly to the NoLS of the PCV4 Cap thereby contributing to the nucleolar localization of the PCV4 Cap protein.

The Network of Interactions between the Porcine Epidemic Diarrhea Virus Nucleocapsid and Host Cellular Proteins

“…The ExFect transfection reagent (T101-01/02; Vazyme Biotechnology, Nanjing, China) was used for HEK293T cell transfection as described in the manufactures’ protocols. The procedures for plasmid construction and cell transfection assays were conducted as described elsewhere [ 30 , 31 ].…”

“…Membranes were then incubated with primary antibodies overnight at 4 °C, followed by incubation with HRP-labeled secondary antibodies at room temperature for 1.0 h. The membranes were then incubated with an enhanced chemiluminescence reagent (34096; Thermo Scientific, Waltham, MA, USA), and the immunoreactive protein bands were visualized using AI800 Images (Cytiva Sweden AB, Uppsala, Sweden). SDS-PAGE and WB assays were performed as previously described [ 30 , 31 ].…”

“…The beads or agarose were washed with NP-40 buffer and resolved using standard SDS-PAGE. Co-IP assays were performed as described previously [ 30 , 31 ].…”

Advances in Crosstalk between Porcine Circoviruses and Host

“…The viral particles (nucleocapsid) then bind to nucleolar phosphoprotein nucleophosmin-1 (NPM1) through the NLS motif on viral Cap protein, move along acetylated and polymerized microtubules by the interaction between the dynein light chain (DYNLL1, LC8) and LRLQT motif near the CT of the viral Cap, and are finally transferred to the nucleus [ 38 , 39 , 45 , 52 , 57 , 58 , 59 , 60 , 61 ]. In addition, the entry of PCV4 into the nucleus is also associated with the interaction between host DEAD-box RNA helicase 21 (DDX21) and NLS of viral Cap [ 62 ]. Thereafter, the viral protein is separated from the viral DNA, and the viral genome replicates via a rolling-circle replication mechanism by a replicase complex [ 61 , 63 , 64 , 65 ].…”

“…The NLS of PCV Cap is a conserved and functionally exchangeable domain [ 60 , 67 , 71 , 72 ]. The NLS of Cap (the amino acid residues 1–37) can interact with the C-terminal domain (CTD, 763GSRSNRFQNK772 residues) of DDX21, resulting in the translocation of DDX21 to the nucleolus from the cytoplasm and the enhancement of viral replication [ 62 ]. In the nucleus, the viral NLS binds to the NPM1, leading to the translocation of NPM1 from the nucleus to the cytoplasm and facilitating the viral replication [ 59 , 60 , 72 , 73 ].…”