Abstract
We have previously shown that Classical Swine Fever Virus (CSFV) p7 is an essential nonstructural protein with a viroporin activity, a critical function in the progression of virus infection. We also identified p7 domains and amino acid residues critical for pore formation. Here, we describe how p7 specifically interacts with host protein CAMLG, an integral ER transmembrane protein involved in intracellular calcium release regulation and signal response generation. Detection of interaction as well as the identification of p7 areas mediating interaction with CAMLG was performed by yeast two-hybrid. p7-CAMLG interaction was further confirmed by confocal microscopy in eukaryotic cells, co-expressing both proteins. Mutant forms of p7 having substituted native residues identified as mediating interaction with CAMLG showed a decreased co-localization compared with the native forms of p7. Furthermore, it is shown that native p7, but not the mutated forms of p7 that fail to interact with CAMLG, efficiently mediates calcium permeability in the ER. Interestingly, viruses harboring some of those mutated forms of p7 have been previously shown to have a significantly decreased virulence in swine.
Highlights
Classical swine fever virus (CSFV) is the causative agent of a highly contagious economically significant viral disease of domestic and wild pigs
The resulting plasmid pBD-p7 was transformed into yeast strain AH109, along with a negative control -pGADT7, the empty library vector containing the GAL4 Activation Domain (AD) to confirm that there was no self-activation of pBD-p7
Classical Swine Fever Virus (CSFV) nonstructural protein p7 has been previously shown to be a virally encoded pore forming protein, or viroporin, which is essential for virus replication as well as for virulence in swine [4]. p7 has been further characterized in terms of the protein domains and critical residues involved in pore forming activities [5,6,7]
Summary
Classical swine fever virus (CSFV) is the causative agent of a highly contagious economically significant viral disease of domestic and wild pigs. CSFV is a member of the genus Pestivirus in the family Flaviviridae along with two other viruses of significant veterinary importance—bovine viral diarrhea virus (BVDV) and border disease virus (BDV) [1]. 12 kb length, comprises of only one open reading frame encoding a unique polyprotein that, after posttranslational processing, originates 12 individual proteins: NH2-Npro-C-Erns-E1-E2p7-NS2-NS3-NS4A-NS4BNS5A-NS5B-COOH [2,3]. The nonstructural protein p7 is an essential small hydrophobic transmembrane protein of approximately 6–7 kDa. Structurally, p7 possesses a short area of charged residues that is flanked by stretches of hydrophobic amino acids, which are predicted to constitute a cytosolic loop and two transmembrane helices, respectively [4]. Our laboratory has shown that p7 is a class II viroporin
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