A Respiratory Syncytial Virus Replicon That Is Noncytotoxic and Capable of Long-Term Foreign Gene Expression

Abstract

Respiratory syncytial virus (RSV) infection of most cultured cell lines causes cell-cell fusion and death. Cell fusion is caused by the fusion (F) glycoprotein and is clearly cytopathic, but other aspects of RSV infection may also contribute to cytopathology. To investigate this possibility, we generated an RSV replicon that lacks all three of its glycoprotein genes and so cannot cause cell-cell fusion or virus spread. This replicon includes a green fluorescent protein gene and an antibiotic resistance gene to enable detection and selection of repliconcontaining cells. Adaptive mutations in the RSV replicon were not required for replicon maintenance. Cells containing the replicon could be cloned and passaged many times in the absence of antibiotic selection, with 99% or more of the cells retaining the replicon after each cell division. Transient expression of the F and G (attachment) glycoproteins supported the production of virions that could transfer the replicon into most cell lines tested. Since the RSV replicon is not toxic to these cultured cells and does not affect their rate of cell division, none of the 8 internal viral proteins, the viral RNA transcripts, or the host response to these molecules or their activities is cytopathic. However, the level of replicon genome and gene expression is controlled in some manner well below that of complete virus and, as such, might avoid cytotoxicity. RSV replicons could be useful for cytoplasmic gene expression in vitro and in vivo and for screening for compounds active against the viral polymerase.

The Interferon Type I/III Response to Respiratory Syncytial Virus Infection in Airway Epithelial Cells Can Be Attenuated or Amplified by Antiviral Treatment

“…The MT4 cell line was obtained from the NIH AIDS Research and Reference Reagent Program (Germantown, MD) and cultured in RPMI 1640 medium (Irvine Scientific) supplemented with 10% FBS, 100 units/ml penicillin, 100 units/ml streptomycin, and 2 mM L-glutamine. RSV replicon BHK cells (19) were grown in MEM (1ϫ) containing GlutaMAX with Earle's salts (Life Technologies), 10% HI-FBS, 10 units/ml penicillin, 10 g/ml streptomycin, and 50 g/ml blasticidin S. Air-liquid-interface differentiated primary normal human airway epithelial (HAE) cells were obtained from MatTek Corporation (AIR-100 kit). The HAE cells were derived from trachea and mainstem bronchi of lung tissue donated with informed consent for research purposes at the time of death.…”

“…BHK cells containing a stably transformed subgenomic RSV replicon expressing a green fluorescent protein (GFP) reporter cassette were licensed from Apath, LLC (19). Replicon cells were seeded at a density of 10,000 cells/well in black, flat, clear-bottom 96-well plates (Corning) and cultured overnight in growth media containing 50 g/ml blasticidin S. The growth medium was aspirated and replaced with 200 l of 3-fold serially diluted compounds prepared in 2% HI-FBS media containing 50 g/ml blasticidin S. After 3 days at 37°C, the cells were fixed using 4% paraformaldehyde (PFA) supplemented with Hoechst dye (Invitrogen).…”

Novel Vaccine Regimen Elicits Strong Airway Immune Responses and Control of Respiratory Syncytial Virus in Nonhuman Primates

“…However, high levels of the hRSV-F protein, as well as its secreted form, could be expressed from a codon-optimized hRSV-F sequence. For the immunization in the presented study, the secreted form of hRSV-F was used as a vaccine antigen since expression of a fusion active full-length hRSV-F was shown to be cytotoxic (18). Consistent with other studies (19,20), we had previously observed that immunization with DNA or adenoviral vectors (AdV) vaccines expressing the soluble hRSV-F protein, resulted in a superior immune response and protective efficacy in mice, which opened the possibility to further explore these genetic vaccines against hRSV in NHPs (21,22).…”

High-Throughput Hit Screening Cascade to Identify Respiratory Syncytial Virus (RSV) Inhibitors

“…21 The replicon cell line used in this screen uses a modified viral genome encoding these proteins essential for viral replication. Viral proteins associated with viral entry and RSV-induced cytotoxicity such as the viral fusion (F) protein, attachment glycoprotein (G), and small hydrophobic (SH) glycoprotein are removed and replaced with a luciferase reporter gene.…”