Date Published: October 19, 2018
Publisher: Public Library of Science
Author(s): Jie Zhu, Qiuhong Miao, Jingyu Tang, Xiaoxue Wang, Dandan Dong, Teng Liu, Ruibin Qi, Zhibiao Yang, Guangqing Liu, Jens H. Kuhn.
Rabbit hemorrhagic disease virus (RHDV) is an important member of the Caliciviridae family and a highly lethal pathogen in rabbits. Although the cell receptor of RHDV has been identified, the mechanism underlying RHDV internalization remains unknown. In this study, the entry and post-internalization of RHDV into host cells were investigated using several biochemical inhibitors and RNA interference. Our data demonstrate that rabbit nucleolin (NCL) plays a key role in RHDV internalization. Further study revealed that NCL specifically interacts with the RHDV capsid protein (VP60) through its N-terminal residues (aa 285–318), and the exact position of the VP60 protein for the interaction with NCL is located in a highly conserved region (472Asp-Val-Asn474; DVN motif). Following competitive blocking of the interaction between NCL and VP60 with an artificial DVN peptide (RRTGDVNAAAGSTNGTQ), the internalization efficiency of the virus was markedly reduced. Moreover, NCL also interacts with the C-terminal residues of clathrin light chain A, which is an important component in clathrin-dependent endocytosis. In addition, the results of animal experiments also demonstrated that artificial DVN peptides protected most rabbits from RHDV infection. These findings demonstrate that NCL is involved in RHDV internalization through clathrin-dependent endocytosis.
Rabbit hemorrhagic disease virus (RHDV) is a non-enveloped, single-stranded, positive sense RNA virus, belonging to the Caliciviridae family , and it is the causative agent of a highly contagious and lethal disease in rabbits which is strongly associated with liver degeneration and diffuse hemorrhage [2,3]. RHDV was first isolated in China in 1984  and it has been subsequently detected in rabbit populations throughout Asia, Europe, Australia, and the Americas, resulting in the death of millions of wild and domestic adult rabbits .
NCL is well known as a multifunctional protein that is mainly localized in the nucleolus, but is also found in the nucleoplasm and cytoplasm, as well as on the cell membrane . The multi-functionality of NCL mainly results from its multi-domain structure. Sequence comparison of different species revealed a high degree of evolutionary conservation of this large protein, and biophysical and biochemical studies have shown that NCL is composed of three main structural domains: the N-terminal domain, the central domain, and the C-terminal domain . The N-terminal domain of NCL contains acidic regions, rich in glutamic acid and aspartic acid, which are the sites of phosphorylation, and participate in the transcription of rRNA and interact with components of the pre-rRNA processing complex . The central domain of NCL contains four RNA-binding domains (RBDs), also known as RNA-recognition motifs, which are involved in a variety of biological processes, including RNA packaging, pre-mRNA splicing, poly-A tail synthesis and maturation, translational control, and mRNA stability . The C-terminal region of NCL contains a glycine- and arginine-rich domain, through which NCL interacts with target mRNAs and proteins, including ribosomal proteins .