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. 2001 Mar;75(6):2653-9.
doi: 10.1128/JVI.75.6.2653-2659.2001.

Ross River virus glycoprotein-pseudotyped retroviruses and stable cell lines for their production

C M Sharkey  1 , C L North, R J Kuhn, D A Sanders
Affiliations

Ross River virus glycoprotein-pseudotyped retroviruses and stable cell lines for their production

C M Sharkey et al. J Virol. 2001 Mar.

Abstract

Pseudotyped retroviruses have important applications as vectors for gene transfer and gene therapy and as tools for the study of viral glycoprotein function. Recombinant Moloney murine leukemia virus (Mo-MuLV)-based retrovirus particles efficiently incorporate the glycoproteins of the alphavirus Ross River virus (RRV) and utilize them for entry into cells. Stable cell lines that produce the RRV glycoprotein-pseudotyped retroviruses for prolonged periods of time have been constructed. The pseudotyped viruses have a broadened host range, can be concentrated to high titer, and mediate stable transduction of genes into cells. The RRV glycoprotein-pseudotyped retroviruses and the cells that produce them have been employed to demonstrate that RRV glycoprotein-mediated viral entry occurs through endocytosis and that membrane fusion requires acidic pH. Alphavirus glycoprotein-pseudotyped retroviruses have significant advantages as reagents for the study of the biochemistry and prevention of alphavirus entry and as preferred vectors for stable gene transfer and gene therapy protocols.

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Figures

FIG. 1
FIG. 1
Immunoblot analysis of RRV E2E1-pseudotyped virus-producing cells and virus. Purified RRV (lane 1) and supernatant medium and lysates of gpnlslacZ cells (lanes 2 and 3, respectively) and of SafeRR-nlslacZA cells (lanes 4 and 5, respectively) were separated by SDS-PAGE and immunoblotted with polyclonal rabbit antibodies to (A) RRV E2 (PAbE2) and (B) RRV E1 as described in Materials and Methods. A cross-reactive protein of higher mobility than E1 is found in the lysates of both the gpnlslacZ and SafeRR-nlslacZA cells. Sizes are shown in kilodaltons.
FIG. 2
FIG. 2
Antibody-mediated neutralization of RRV E2E1-pseudotyped retroviral infection. RRV E2E1- (black), Mo-MuLV Env- (white), or VSV G- (gray) pseudotyped retroviruses (produced as described in Materials and Methods) were treated with the indicated dilution of anti-RRV E2 monoclonal antibody (MAb10C9) or polyclonal rabbit antiserum (PAbE2) as well as 10% guinea pig complement in PBS for 1 h at room temperature. The antibody-treated virus was diluted in DMEM-CS/PS and used to infect NIH 3T3 cells in the presence of hexadimethrine bromide (8 μg/ml). The data are presented as the percent inhibition of transduction by antibody-treated virus relative to the level of transduction by virus treated only with the 10% guinea pig complement in PBS. The data are representative of three independent experiments
FIG. 3
FIG. 3
Inhibition of transduction by RRV E2E1-pseudotyped retrovirus by lysosomotropic weak bases. NIH 3T3 cells were treated for 1 h with the indicated concentrations of (A) chloroquine or (B) ammonium chloride in PBS. Medium carrying pseudotyped nlslacZ-conveying retroviruses (RRV glycoproteins, Mo-MuLV virus envelope protein, or VSV G protein) containing the indicated concentration of base as well as hexadimethrine bromide (8 μg/ml) was incubated with the cells in a CO2 incubator at 37°C. The cells were stained with X-gal at 48 h postinfection, and blue cells were counted. Viral transduction of cells treated with the indicated concentrations of reagent is represented as a percentage of the level of transduction of untreated cells. The data are representative of three independent experiments.
FIG. 4
FIG. 4
Cell fusion in low-pH buffer. SafeRR-nlslacZA (A and C) and gpnlslacZ (B and D) cells were grown to near confluence, washed once with PBS, and overlaid with cell fusion buffer (10 mM MES 10 mM HEPES [pH 5.5]) (A and B) or neutral buffer (10 mM MES, 10 mM HEPES [pH 7.0]) (C and D) for 1 min. They were then grown for 4 h with DMEM-FBS/PS in a CO2 incubator at 37°C to allow syncytium formation.
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