Derivation of a novel SARS-coronavirus replicon cell line and its application for anti-SARS drug screening

Abstract

The severe acute respiratory syndrome (SARS) outbreak in 2002, which had a high morbidity rate and caused worldwide alarm, remains untreated today even though SARS was eventually isolated and controlled. Development and high-throughput screening of efficacious drugs is therefore critical. However, currently there remains a lack of such a safe system. Here, the generation and characterization of the first selectable, SARS-coronavirus (SARS-CoV)-based replicon cell line which can be used for screening is described. Partial SARS-CoV cDNAs and antibiotic resistance/reporter gene DNA were generated and assembled in vitro to produce the replicon transcription template, which was then transcribed in vitro to generate the replicon RNA. The latter was introduced into a mammalian cell line and the transfected cells were selected for by antibiotic application. For the antibiotic-resistant cell lines thus generated, the expression of reporter gene was ensured by continued monitoring using fluorescent microscopy and flow cytometry. The suitability of this replicon cell line in drug screening was demonstrated by testing the inhibitory effect of several existing drugs and the results demonstrate that the SARS-CoV replicon cell lines provide a safe tool for the identification of SARS-CoV replicase inhibitors. The replicon cell lines thus developed can be applied to high-throughput screening for anti-SARS drugs without the need to grow infectious SARS-CoV.

Fig. 1

SARS–CoV replicon and the strategy for its construction. The structural relationship of the SARS–CoV genome and SARS–CoV replicon cDNA is shown. Blue box represents SARS–CoV transcription regulatory sequences (TRSs) site. The 5′-caps and 3′-polyadenine tails of the SARS–CoV genome and replicon RNAs are omitted. Gb, green fluorescent protein–blasticidin deaminase fusion gene; L, leader sequence; S, spike gene; N, nucleocapsid gene; T7, T7 promoter.

Fig. 2

Analysis of replicon RNA-containing cells. (A) Combined green fluorescence and phase-contrast microscopic images of SCR-1 cells are shown. (B) Presence of SARS–CoV replicon and sub-replicon RNAs in replicon-carrying cells as detected by Northern blot analysis. Total RNA preparations from BHK-21 (BHK) and SCR-1 cells were analyzed as indicated. Arrows indicate full-length replicon RNA and replicon RNA-derived transcripts encoding GFP and N. (C) A flow cytometry analysis of SCR-1 cells is shown. Samples of SCR-1 cells were analyzed at passage number P10, P20, P30 and P40 under blasticidin selection and the parent BHK-21 (BHK) cells were co-analyzed. Indicated values represent the percentages of green fluorescent cells. The results are presented as histograms with green fluorescence intensity in exponential scale (horizontal axis) against cell number in linear scale (vertical axis).

Fig. 3

Inhibition of SARS–CoV replication. SCR-1 cells containing SARS–CoV-based replicon RNA that mediates GFP expression were used to assess the inhibitory effect of the compounds by (A) flow cytometry analysis. One representative experiment out of four is shown. Bars indicate GFP-expressing cells in flow cytometry analyses. The GFP expression of untreated cells was set at 100%. (B) Fluorescence microscopy analysis. Quadruple wells of untreated and treated SCR-1 cells are shown. The parent BHK-21 (BHK) cells were co-analyzed. (C) Quantitative real-time RT–PCR. Copy number of SARS replicon RNA in SCR-1 cells before and after inhibitor treatments are shown. The inhibitors used are indicated below. Data are the means of three independent experiments. Bar: 95% confidence intervals.

Fig. 4

Generation of sub-replicon RNAs through discontinuous transcription of SARS–CoV replicon RNA in the replicon-carrying cells. The black box represents the 72-nt leader RNA sequence, derived from the 5′ end of the replicon, located at the 5′ end of each sub-replicon RNA. The size of each RNA shown is exclusive of the poly-A tail.