Respiratory syncytial virus G glycoprotein expressed using the Semliki Forest virus replicon is biologically active

Respiratory syncytial virus G glycoprotein expressed using the Semliki Forest virus replicon is... The respiratory syncytial virus (RSV) G glycoprotein mediates attachment of RSV to cells via an unknown receptor. To study G glycoprotein function we have cloned two variants of the RSV G gene into a Semliki Forest virus (SFV) expression vector, a full length (rG) and soluble (srG) G glycoprotein variant. By immunofluorescence microscopy, rG was found to be predominantly membrane associated, while srG was mostly cytoplasmic. The rG (80–85 kDa) and srG (75–80 kDa) constructs produced heavily glycosylated proteins, however they were slightly smaller than the G glycoprotein expressed in RSV infected HEp-2 cells (85–90 kDa). The biological activity of purified srG was tested by its ability to bind to RSV permissive cells. Purified srG bound to HEp-2 cells and the amount bound increased linearly with the quantity added. Binding was not saturable with the small quantities of protein available. Binding of srG to HEp-2 cells was inhibited (67–68%) by MAb 30 and neutralising anti-G MAb 29. Nonpermissive SF9 insect cells bound 20–50 times less srG than HEp-2 cells. SFV expressed recombinant RSV G glycoprotein should be useful for studying interactions between the RSV G glycoprotein and cells. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Archives of Virology Springer Journals

Respiratory syncytial virus G glycoprotein expressed using the Semliki Forest virus replicon is biologically active

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Publisher
Springer Journals
Copyright
Copyright © Wien by 1999 Springer-Verlag/
Subject
Legacy
ISSN
0304-8608
eISSN
1432-8798
D.O.I.
10.1007/s007050050488
Publisher site
See Article on Publisher Site

Abstract

The respiratory syncytial virus (RSV) G glycoprotein mediates attachment of RSV to cells via an unknown receptor. To study G glycoprotein function we have cloned two variants of the RSV G gene into a Semliki Forest virus (SFV) expression vector, a full length (rG) and soluble (srG) G glycoprotein variant. By immunofluorescence microscopy, rG was found to be predominantly membrane associated, while srG was mostly cytoplasmic. The rG (80–85 kDa) and srG (75–80 kDa) constructs produced heavily glycosylated proteins, however they were slightly smaller than the G glycoprotein expressed in RSV infected HEp-2 cells (85–90 kDa). The biological activity of purified srG was tested by its ability to bind to RSV permissive cells. Purified srG bound to HEp-2 cells and the amount bound increased linearly with the quantity added. Binding was not saturable with the small quantities of protein available. Binding of srG to HEp-2 cells was inhibited (67–68%) by MAb 30 and neutralising anti-G MAb 29. Nonpermissive SF9 insect cells bound 20–50 times less srG than HEp-2 cells. SFV expressed recombinant RSV G glycoprotein should be useful for studying interactions between the RSV G glycoprotein and cells.

Journal

Archives of VirologySpringer Journals

Published: Jan 1, 1999

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