Polylactosaminoglycan modification of the respiratory syncytial virus small hydrophobic (SH) protein: A conserved feature among human and bovine respiratory syncytial viruses

Abstract

We investigated the nature of the oligosaccharide modification of the glycosylated forms of the small hydrophobic integral membrane protein, SH (previously designated 1A), of respiratory syncytial (RS) virus. Analysis of SH protein expressed in cells infected with RS virus or with a recombinant vaccinia virus revealed two glycosylated SH protein species, SH 9 and SH p, which contained N-linked carbohydrate residues. SH p migrated diffusely on polyacrylamide gels, which suggested modification by polylactosaminoglycan oligosaccharides. Polylactosaminoglycan modification of SH p was established from three lines of investigation: (1) the synthesis of SH p in a cell line (IdID) conditionally defective in the ability to add specific carbohydrate residues to N- or O-linked oligosaccharide chains required the addition of galactose, which is a component of the N-acetyllactosamine repeating unit; (2) SH p was sensitive to digestion with endo-,β-galactosidase, which cleaves the β1–4 linkage between galactose and N-acetylglucosamine of the repeated N-acetyllactosamine subunit; and (3) SH p was selected by Datura stramonium lectin (Dsl), which has specificity for polylactosaminoglycans. The presence of SH p as a component of purified human subgroups A and B and bovine RS virus particles was demonstrated by Dsl affinity selection. In addition to SH p, nonglycosylated SH 0 was selected by Dsl affinity, indicating that SH p and SH 0 may associate to form complexes within infected cells and virus particles. To identify conserved amino acid residues among the human and bovine SH glycoproteins that may function as signals for polylactosaminoglycan modification, the nucleotide sequences of the SH protein genes of a human subgroup B virus ( 8 60 ) and a bovine virus (391-2) were determined and compared to those of a human subgroup A virus (A2), a subgroup B virus (18537), and a bovine virus (A51908). A comparison of the deduced amino acid sequences of the human and bovine RS virus SH proteins indicated that a central hydrophobic region and the presence of potential N-linked glycosylation sites on either side of the central hydrophobic region were conserved features that may be required for the polylactosaminoglycan modification of SH.