Classical Swine Fever Virus p7 Protein Is a Viroporin Involved in Virulence in Swine

Abstract

The nonstructural protein p7 of classical swine fever virus (CSFV) is a small hydrophobic polypeptide with an apparent molecular mass of 6 to 7 kDa. The protein contains two hydrophobic stretches of amino acids interrupted by a short charged segment that are predicted to form transmembrane helices and a cytosolic loop, respectively. Using reverse genetics, partial in-frame deletions of p7 were deleterious for virus growth, demonstrating that CSFV p7 function is critical for virus production in cell cultures. A panel of recombinant mutant CSFVs was created using alanine scanning mutagenesis of the p7 gene harboring sequential three- to six-amino-acid residue substitutions spanning the entire protein. These recombinant viruses allowed the identification of the regions within p7 that are critical for virus production in vitro. In vivo , some of these viruses were partially or completely attenuated in swine relative to the highly virulent parental CSFV Brescia strain, indicating a significant role of p7 in CSFV virulence. Structure-function analyses in model membranes emulating the endoplasmic reticulum lipid composition confirmed that CSFV p7 is a pore-forming protein, and that pore-forming activity resides in the C-terminal transmembrane helix. Therefore, p7 is a viroporin which is clearly involved in the process of CSFV virulence in swine.

Viroporins: structure, function and potential as antiviral targets

“…Thus, whilst clearly an indirect measure of channel activity, such indirect assays conducted upon multiple channels [e.g. IAV M2, HCV p7, classical swine fever virus (CSFV) p7, HPV E5, respiratory syncytial virus (RSV) SH and picornavirus 2B] have provided important insights into their activity as well as their inhibition by small molecules, with results generally consistent with those observed in culture (Agirre et al, 2002;Aldabe et al, 1996;Atkins et al, 2014;Carter et al, 2010;Gladue et al, 2012;Guinea & Carrasco, 1994;Lama & Carrasco, 1992;Perez et al, 1997;Sanz et al, 1994;StGelais et al, 2007;Wetherill et al, 2012;Wozniak et al, 2010).…”

“…As described above, BIT225 was derived from a bacterial screen versus genotype 1a p7 and has been shown to exert an antiviral effect against the pestivirus BVDV (Luscombe et al, 2010). However, the mode of action for this inhibitor is unknown and activity against HCV in culture has not been published; this may be of concern given recently reported differences in pestivirus p7 function (Gladue et al, 2012;Guo et al, 2013). Nevertheless, BIT225 appears to have a reasonable safety profile and preliminary findings in small patient studies appear encouraging, with larger studies planned (see http://www.…”

“…In this regard, the ability of p7 to mediate proton conductance within infected Huh7 cells remains the only activity for which a biologically relevant function has been assigned within the HCV life cycle (Wozniak et al, 2010), although roles for other observed conductances cannot be ruled out. Interestingly, p7 from the related pestivirus CSFV was recently shown to behave as an amlodipine-sensitive Ca 2+ channel (Gladue et al, 2012;Guo et al, 2013), illustrating that not all 'p7' sequences necessarily behave similarly and that genetic divergence, such as that observed between some HCV genotypes, may significantly affect channel functions.…”

Selection of Classical Swine Fever Virus with Enhanced Pathogenicity Reveals Synergistic Virulence Determinants in E2 and NS4B

“…This is reflected in numerous reports in which mutations of different functional domains in the structural proteins C, E rns , E1, and E2 and in the nonstructural proteins p7 and NS4B were shown to reduce the pathogenicity in pigs, indicating that these genes may be involved in determining CSFV virulence (7,8,9,10,11,12,13,32,36,37,38,39,40,46,54).…”

“…Comparison of the genomes of the GPE Ϫ vaccine virus and the parent ALD virus revealed 225 nucleotide substitutions and 46 amino acid differences (22), which did not provide any conclusive information on the molecular basis of the attenuation. Numerous reports suggest that the viral proteins N pro , C, E rns , E1, E2, p7, and NS4B may be involved in determining the virulence of CSFV in pigs (7,8,9,10,11,12,13,32,36,37,38,39,40,43,46,54). So far, however, all conclusions on CSFV virulence determinants rely on mutations that result in downregulation of pathogenicity.…”