Abstract
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.
Highlights
Rabbit hemorrhagic disease virus (RHDV) is a non-enveloped, single-stranded, positive sense RNA virus, belonging to the Caliciviridae family [1], 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]
The results of animal experiments 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
We demonstrated that rabbit nucleolin (NCL) efficiently mediates RHDV internalization by interacting with the RHDV capsid protein (VP60)
Summary
Rabbit hemorrhagic disease virus (RHDV) is a non-enveloped, single-stranded, positive sense RNA virus, belonging to the Caliciviridae family [1], 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 [4] 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 [3]. Studies on the viral entry of RHDV have relied on the self-assembly of the capsid protein into virus-like particles when expressed in Escherichia coli or insect cells. RHDV has been shown to bind to oligosaccharides H type 2 and A type 2, which are histo-blood group antigens (HBGAs) expressed on the surfaces of cells lining the duodenal surface and trachea of rabbits, thereby presenting two possible viral entry points [5]. RHDV isolates from six different genetic groups bind to different HBGAs, which act as attachment factors that facilitate infection [6,7]
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