Passage of Classical Swine Fever Virus in Cultured Swine Kidney Cells Selects Virus Variants That Bind to Heparan Sulfate due to a Single Amino Acid Change in Envelope Protein E ^rns

Abstract

Infection of cells with Classical swine fever virus (CSFV) is mediated by the interaction of envelope glycoprotein Erns and E2 with the cell surface. In this report we studied the role of the cell surface glycoaminoglycans (GAGs), chondroitin sulfates A, B, and C (CS-A, -B, and -C), and heparan sulfate (HS) in the initial binding of CSFV strain Brescia to cells. Removal of HS from the surface of swine kidney cells (SK6) by heparinase I treatment almost completely abolished infection of these cells with virus that was extensively passaged in swine kidney cells before it was cloned (clone C1.1.1). Infection with C1.1.1 was inhibited completely by heparin (a GAG chemically related to HS but sulfated to a higher extent) and by dextran sulfate (an artificial highly sulfated polysaccharide), whereas HS and CS-A, -B, and -C were unable to inhibit infection. Bound C1.1.1 virus particles were released from the cell surface by treatment with heparin. Furthermore, C1.1.1 virus particles and CSFV E rns purified from insect cells bound to immobilized heparin, whereas purified CSFV E2 did not. These results indicate that initial binding of this virus clone is accomplished by the interaction of E rns with cell surface HS. In contrast, infection of SK6 cells with virus clones isolated from the blood of an infected pig and minimally passaged in SK6 cells was not affected by heparinase I treatment of cells and the addition of heparin to the medium. However, after one additional round of amplification in SK6 cells, infection with these virus clones was affected by heparinase I treatment and heparin. Sequence analysis of the E rns genes of these virus clones before and after amplification in SK6 cells showed that passage in SK6 cells resulted in a change of an Ser residue to an Arg residue in the C terminus of E rns (amino acid 476 in the polyprotein of CSFV). Replacement of the E rns gene of an infectious DNA copy of C1.1.1 with the E rns genes of these virus variants proved that acquisition of this Arg was sufficient to alter an HSindependent virus to a virus that uses HS as an E rns receptor.

CD46 Is a Cellular Receptor for Bovine Viral Diarrhea Virus

“…The binding of CSFV strain Brescia (9, 10) and BVDV strain PE515 (12) was demonstrated for GAGs, e.g., heparin or heparan sulfate (9). Glycoprotein E rns was identified as a primary ligand.…”

“…In either case, a single point mutation (CSFV: Ser 476 Arg) in E rns was the reason for the high affinity for GAGs. Very likely this point mutation is a result of tissue culture adaptation of the particular virus isolates (9,10,12), but GAGs also may serve with a low affinity as alternative receptors. An alternative receptor(s) may account for the residual susceptibility (Ϸ1%) of MDBK cells to BVDV NADL, since its entry was not inhibited even by high concentrations of anti-CD46 bov antibodies (data not shown).…”

The E2 Glycoprotein of Classical Swine Fever Virus Is a Virulence Determinant in Swine

“…A correlation between plaque size in vitro and pathogenesis in vivo has yet to be established. Hulst et al (11,12) serially passaged CSFV Brescia in SK6 cells to generate heparan sulfate binding-dependent small-plaque variants that contained mutations in the E rns protein; however, these varients had an unaltered virulence phenotype in pigs (11,12).…”

Heparan sulphate mediates swine vesicular disease virus attachment to the host cell

“…Thus, HS is considered as an initial receptor for a number of viruses (Compton et al, 1993;Mettenleiter et al, 1990;Okazaki et al, 1991;WuDunn & Spear, 1989;Zhu et al, 1995). While many enveloped viruses have been associated with HS binding (Asagoe et al, 1997;Bernard et al, 2000;Byrnes & Griffin, 1998;Chung et al, 1998;Hulst et al, 2000Hulst et al, , 2001Jusa et al, 1997;Krusat & Streckert, 1997;Mondor et al, 1998;Summerford & Samulski, 1998), the number of non-enveloped viruses reported to use HS for attachment to the host cell is limited. Among the Picornaviridae family, it has been shown that foot-and-mouth disease virus (FMDV) binds to HS, although it reflects an adaptation to in vitro cultures (Fry et al, 1999;Jackson et al, 1996;Sa-Carvalho et al, 1997).…”