Respiratory syncytial virus grown in Vero cells contains a truncated attachment protein that alters its infectivity and dependence on glycosaminoglycans - PubMed (original) (raw)

Respiratory syncytial virus grown in Vero cells contains a truncated attachment protein that alters its infectivity and dependence on glycosaminoglycans

Steven Kwilas et al. J Virol. 2009 Oct.

Abstract

Human respiratory syncytial virus (RSV) contains a heavily glycosylated 90-kDa attachment glycoprotein (G). Infection of HEp-2 and Vero cells in culture depends largely on virion G protein binding to cell surface glycosaminoglycans (GAGs). This GAG-dependent phenotype has been described for RSV grown in HEp-2 cells, but we have found that it is greatly reduced by a single passage in Vero cells. Virions produced from Vero cells primarily display a 55-kDa G glycoprotein. This smaller G protein represents a post-Golgi compartment form that is lacking its C terminus, indicating that the C terminus is required for GAG dependency. Vero cell-grown virus infected primary well-differentiated human airway epithelial (HAE) cell cultures 600-fold less efficiently than did HEp-2 cell-grown virus, indicating that the C terminus of the G protein is also required for virus attachment to this model of the in vivo target cells. This reduced infectivity for HAE cell cultures is not likely to be due to the loss of GAG attachment since heparan sulfate, the primary GAG used by RSV for attachment to HEp-2 cells, is not detectable at the apical surface of HAE cell cultures where RSV enters. Growing RSV stocks in Vero cells could dramatically reduce the initial infection of the respiratory tract in animal models or in volunteers receiving attenuated virus vaccines, thereby reducing the efficiency of infection or the efficacy of the vaccine.

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Figures

FIG. 1.

Importance of cell surface GAGs for infection and size of the G protein in virions produced in HEp-2 and Vero cells. (A) GAG dependency of recombinant RSVs from strain A2 expressing different combinations of viral glycoproteins that were grown in HEp-2 or Vero cells. Virus titers were determined on CHO K1 and CHO A745 cells and compared as follows: CHO K1/CHO A745 = GAG dependency index. (B) Western blot assay of sucrose-purified virions (15,000 cpm) probed with MAbs L9 (G protein) and D14 (N protein). All lanes are from the same gel, from which irrelevant lanes were removed. (C) The percentage of total virion G protein in the 90-kDa form was divided by the amount of virion N protein to normalize the amount of G protein in the virions produced by HEp-2 and Vero cells.

FIG. 2.

Pulse-chase of G protein in RSV-infected cells. (A) HEp-2 and (B) Vero cells were pulsed with Tran35S-label for 15 min. Cells were lysed and then IP with MAb L9 at the times indicated (in minutes). After IP, the samples were treated with 500 U of Endo H for 2 h at 37°C (+) or left untreated (−). After SDS-PAGE separation, the gel was fixed and dried. The gel was exposed to film at −80°C for 1 week before film development. Mon, monensin.

FIG. 3.

Glycan maturity and presence of the C terminus in virion G protein. (A) Schematic of the RSV G protein, an N-terminally anchored type II glycoprotein. The soluble form of the G protein (sG) starts at Met-48 and is released from the cell surface by cleavage. A central conserved domain with four conserved cysteines (CCCC) is followed by a predicted HBD. The G protein is modified by N-linked glycans (N) and many O-linked glycans (potential sites are indicated by stalks with filled circles). The binding areas for MAbs L9, 130-2G, and 232-1F are designated by arrows. The six-His tag at the COOH terminus is also shown. TM, transmembrane domain. (B) Western blot assay of viruses treated with Endo H and probed with MAb L9. Samples containing each virus (15,000 cpm) were treated with 500 U of Endo H for 2 h at 37°C. (C) HEp-2 and Vero cell-grown viruses were probed with MAb L9. The same blot was stripped, blocked, and reprobed with MAb 130-2G. (D) Western blot assay of rgRSV-G-6His purified virions (15,000 cpm) from HEp-2 and Vero cells probed with MAb L9 or 5His.

FIG. 4.

Comparison of G proteins from total cell lysates, the cell surface, and purified virions. Lanes 1, 4, 5, and 8 were stained with MAb L9. Lanes 2, 3, 6, and 7 were processed separately to detect the biotinylated cell surface proteins that had been IP with MAb L9 and developed with streptavidin-HRP and a chemiluminescent substrate.

FIG. 5.

Forms of the G protein in virions produced by other cell lines. (A) Western blot assay of purified rgRSV-G-6His virions probed with MAbs L9 and D14. (B) Ratio of the total G protein versus N protein and the percent 90-kDa G protein. Ratios were determined from triplicate blots. (C) GAG dependency of rgRSV-G-6His grown in different cells. Virus titers on CHO K1/CHO A745 = GAG dependency index.

FIG. 6.

(A) GAG dependency indexes of RSVs produced in HEp-2, Vero, and HAE cells. Virus titers were determined on CHO K1 and CHO A745 cells and compared as follows: CHO K1/CHO A745 = GAG dependency index. (B) Western blot assay of crude virus released from HAE cell cultures, probed with MAb L9. HEp-2 virus initial inoculum bound to HAE cell culture at 0 days p.i. (lane 1), HAE cell virus culture no. 1 (lane 2), HAE cell virus culture no. 2 (lane 3). (C) Western blot assay of rgRSV-G-6His virions from HEp-2 cells and HAE cell cultures probed with MAb L9 or 5His.

FIG. 7.

Infection of primary HAE cell cultures inoculated with rgRSV-SGF grown in HEp-2 and Vero cells and rgRSV-F grown in HEp-2 cells (6.2 × 106 PFU in 100 μl [∼MOI, 0.05]). The percent GFP was calculated from five random pictures obtained from each HAE cell culture. Each time point represents three HAE cell cultures.

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Respiratory syncytial virus grown in Vero cells contains a truncated attachment protein that alters its infectivity and dependence on glycosaminoglycans - PubMed (original) (raw)