Human respiratory syncytial virus glycoproteins are not required for apical targeting and release from polarized epithelial cells

Abstract

Human respiratory syncytial virus (HRSV) is released from the apical membrane of polarized epithelial cells. However, little is known about the processes of assembly and release of HRSV and which viral gene products are involved in the directional maturation of the virus. Based on previous studies showing that the fusion (F) glycoprotein contained an intrinsic apical sorting signal and that N- and O-linked glycans can act as apical targeting signals, we investigated whether the glycoproteins of HRSV were involved in its directional targeting and release. We generated recombinant viruses with each of the three glycoprotein genes deleted individually or in groups. Each deleted gene was replaced with a reporter gene to maintain wild-type levels of gene expression. The effects of deleting the glycoprotein genes on apical maturation and on targeting of individual proteins in polarized epithelial cells were examined by using biological, biochemical, and microscopic assays. The results of these studies showed that the HRSV glycoproteins are not required for apical maturation or release of the virus. Further, deletion of one or more of the glycoprotein genes did not affect the intracellular targeting of the remaining viral glycoproteins or the nucleocapsid protein to the apical membrane.

FIG. 1.

Schematic of the gene content of engineered viruses. All engineered viruses were generated from a cDNA of the A2 strain of HRSV. Viruses RSΔSH, RSΔG, and RSΔF have the missing ORFs of the SH, G, and F genes replaced with that of GFP. Viruses RSΔSH,G, RSΔSH,F, and RSΔG,F have the two glycoprotein ORFs deleted and replaced with ORFs encoding marker proteins GUS and GFP. Finally, virus RSΔSH,G,F has the ORFs encoding each of the three HSRV glycoproteins replaced with those of reporter genes GFP, CAT, and GUS, respectively.

FIG. 2.

Directional release of virus from polarized epithelial cells. (A) Release of wild-type VSV and HRSV from polarized epithelial cells quantified by plaque assay. Polarized MDCK or nonpolarized HEp-2 cells grown on filter inserts were infected with either wild-type VSV or HRSV viruses. The apical and basolateral media were collected at 3 days postinfection for HRSV and at 6 h postinfection for VSV and analyzed for infectivity by plaque assay on Vero cells as described in Materials and Methods. Error bars represent standard deviations. (B) Release of wild-type HRSV quantified by anti-N ELISA. Polarized MDCK cells or nonpolarized HEp-2 cells were infected with virus, and at 1 to 4 days postinfection the apical and basolateral supernatants were analyzed for released HRSV by using an anti-N ELISA as described in Materials and Methods. The bars represent the percentage of absorbance measured at 490 nm in the apical and basolateral chambers of total absorbance.

FIG. 3.

Directional release of HRSV with single glycoprotein gene deletions. Polarized MDCK cells and nonpolarized HEp-2 cells grown on filter inserts were infected with wild-type or engineered viruses at a multiplicity of 0.25. At 3 days postinfection the apical and basolateral supernatants were analyzed for released HRSV by using an anti-N ELISA. Error bars represent standard deviations from at least three experiments.

FIG. 4.

Intracellular localization of HRSV proteins expressed by viruses with single glycoprotein deletions. HEp-2 and MDCK cells grown on filter inserts were infected with wild-type or engineered viruses at a multiplicity of 0.1. At 2 days postinfection the cells were fixed, permeabilized, and stained for HRSV N protein (A), F glycoprotein (B), and G glycoprotein (C), as shown in red. Engineered viruses lacking a particular glycoprotein gene were still stained for that protein as a control. The nuclei were stained with Hoechst as shown in blue. All images were taken on a Zeiss confocal at ×100 magnification in the x-z plane.

FIG. 5.

Quantitative cell surface localization of HRSV proteins expressed by viruses with single glycoprotein deletions. Duplicate cultures of MDCK cells grown on filter inserts were infected with wild-type or engineered viruses at a multiplicity of 0.25. At 3 days postinfection the cells were fixed and blocked and then incubated with primary and secondary antibodies in either the apical or basolateral chamber. The amount of HRSV F glycoprotein (A) and G glycoprotein (B) localizing to the apical and basolateral membranes of infected cells was quantified by using cell ELISA as described in Materials and Methods. (C) The amount of endogenous TfnR localizing to the cell surface of uninfected cells was also quantified as a control for basolateral staining efficiency. “Mock” refers to samples incubated only with secondary antibody to control for any nonspecific secondary antibody binding. Error bars represent standard deviations from at least three experiments.

FIG. 6.

Directional release of HRSV with multiple glycoprotein deletions. Polarized MDCK cells or nonpolarized HEp-2 cells grown on filter inserts were infected with wild-type HRSV or with viruses with the SH and G genes; the SH and F, G, and F genes; or the SH, G, and F genes deleted at an MOI of 1.0. At 3 days postinfection the apical and basolateral supernatants were analyzed for released HRSV by using an anti-N ELISA as described in Materials and Methods. The percentage of virus released apically and basolaterally is shown. Error bars represent standard deviation from at least three experiments.

FIG. 7.

Intracellular localization of HRSV proteins expressed by viruses having multiple glycoprotein deletions. MDCK cells grown on filter inserts were infected with wild-type or engineered viruses at a multiplicity of 0.1. At 2 days postinfection cells were fixed, permeabilized, and stained for HRSV N protein (A), F glycoprotein (B), and G glycoprotein (C), as shown in red. Engineered viruses missing a glycoprotein were still stained for that protein as controls. The nuclei were stained with Hoechst as shown in blue. All images were taken on a Zeiss confocal at ×100 magnification in the x-z plane.

FIG. 8.

Quantitative cell surface localization of HRSV proteins expressed by viruses having multiple glycoprotein deletions. Duplicate cultures of MDCK cells grown on filter inserts were infected with engineered viruses containing F or G glycoproteins at a multiplicity of 0.25. At 3 days postinfection the cells were fixed, blocked, and stained with primary and secondary antibodies either in the apical or basolateral chamber. The amounts of HRSV F glycoprotein (A) and G glycoprotein (B) localizing to the apical and basolateral membranes of infected cells were quantified by using cell ELISA as described in Materials and Methods. Error bars represent standard deviations from at least three experiments.