Membrane orientation and oligomerization of the small hydrophobic protein of human respiratory syncytial virus.
Previous work has demonstrated that the small hydrophobic (SH) protein of human respiratory syncytial virus (RSV) A2 strain is a 64 amino acid integral membrane protein that accumulates intracellularly as an unglycosylated major species (SH0), a minor species truncated at the amino terminus and two N-glycosylated species one of which contains a further addition of polylactosamine. In this study, the membrane orientation of SH0 was mapped by trypsinization of intact RSV-infected cells followed by washout, lysis and immunoprecipitation of protected fragments with antisera specific for the protein termini. This showed that the C terminus is extracellular and the SH protein was not detectably palmitylated. Analysis of the SH protein by sedimentation on sucrose gradients showed that it rapidly assembles into a homo-oligomer that co-sediments with the F protein tetramer. Interestingly, all forms of the SH protein were found in the oligomeric fraction. Chemical cross-linking generated species which appeared to represent dimers, trimers, tetramers and pentamers as well as a minor species of 180K which might correspond to the oligomeric form detected by sucrose gradient sedimentation.
Collins
P L
PL
Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Mottet
G
G
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D015707:HN Protein; D006801:Humans; D046911:Macromolecular Substances; D008715:Methionine / Q000378:metabolism; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D010449:Peptide Mapping; D011499:Protein Processing, Post-Translational; D012136:Respiratory Syncytial Viruses / Q000378:metabolism*; D014357:Trypsin; D014759:Viral Envelope Proteins; D014764:Viral Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*
D015707:HN Protein; D046911:Macromolecular Substances; D010446:Peptide Fragments; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C116204:attachment protein G; D008715:Methionine; D014357:Trypsin