Iduronic Acid-Containing Glycosaminoglycans on Target Cells Are Required for Efficient Respiratory Syncytial Virus Infection

Abstract

Respiratory syncytial virus (RSV) is an important human respiratory pathogen, particularly in infants. Glycosaminoglycans (GAGs) have been implicated in the initiation of RSV infection of cultured cells, but it is not clear what type of GAGs and GAG components are involved, whether the important GAGs are on the virus or the cell, or what the magnitude is of their contribution to infection. We constructed and rescued a recombinant green fluorescent protein (GFP)-expressing RSV (rgRSV) and used this virus to develop a sensitive system to assess and quantify infection by flow cytometry. Evaluation of a panel of mutant Chinese hamster ovary cell lines that are genetically deficient in various aspects of GAG synthesis showed that infection was reduced up to 80% depending on the type of GAG deficiency. Enzymatic removal of heparan sulfate and/or chondroitin sulfate from the surface of HEp-2 cells also reduced infection, and the removal of both reduced infection even further. Blocking experiments in which RSV was preincubated with various soluble GAGs revealed the relative blocking order of: heparin > heparan sulfate > chondroitin sulfate B. Iduronic acid is a component common to these GAGs. GAGs that do not contain iduronic acid, namely, chondroitin sulfate A and C and hyaluronic acid, did not inhibit infection. A role for iduronic acid-containing GAGs in RSV infection was confirmed by the ability of basic fibroblast growth factor to block infection, because basic fibroblast growth factor binds to GAGs containing iduronic acid. Pretreatment of cells with protamine sulfate, which binds and blocks GAGs, also reduced infection. In these examples, infection was reduced by pretreatment of the virus with soluble GAGs, pretreatment of the cells with GAG-binding molecules, pretreatment of the cells with GAG-destroying enzymes or in cells genetically deficient in GAGs. These results establish that the GAGs involved in RSV infection are present on the cell rather than on the virus particle. Thus, the presence of cell surface GAGs containing iduronic acid, like heparan sulfate and chondroitin sulfate B, is required for efficient RSV infection in cell culture.

Phosphatidylinositol inhibits respiratory syncytial virus infection

“…The high-affi nity interaction of PI with RSV blocks the attachment of the virus to epithelial cell plasma membrane and, consequently, infection. Several molecules have been proposed as receptors for RSV including plasma membrane nucleolin and glycosaminoglycans among others (32)(33)(34)(35). The inhibition of viral infection in vitro is signifi cant with concentrations of PI of 200 g/ml reducing infection by a factor of >10 3 .…”

Mucosal delivery of a double-stapled RSV peptide prevents nasopulmonary infection

“…rgRSV was obtained from M. Peeples (Nationwide Children's Hospital, Columbus, Ohio, USA), and the virus was propagated and plaques imaged as described previously (41). RSV-A2, an A-subtype RSV, was 4% paraformaldehyde in PBS.…”

Protein-Protein Interactions in RSV Assembly: Potential Targets for Attenuating RSV Strains

“…RSV infection of host cells is initiated when the large glycoprotein G attaches to glycosaminoglycans in cultured cells [3][4][5]; the receptor used in human infection in the field is still unknown. After attachment, the viral F (fusion) protein triggers fusion of cell and virus membranes, releasing the nucleocapsid into the cytoplasm where genomic RNA is transcribed and translated to give rise to individual viral proteins and replicated to form progeny genomes.…”

“…Although the cellular receptor for field isolates of RSV still unknown, in cell culture, G glycoprotein mediates attachment via binding to cell surface glycosaminoglycans [5]. G protein is translated as a 32 kDa protein which acquires N-linked and O-linked glycosylation as it passes through the cellular glycoprotein secretory pathway, growing to 85-90 kDa in size.…”