2. Prior To Adding Antiviral Compounds

Replicon RNAs are autonomously replicating RNAs encoding: (i) all replicative proteins required for the expression of a functional replication complex, and (ii) cis-acting elements required for the recognition of the replicon RNA by the replicase complex. Usually replicon RNAs are devoid of sequences leading to production of progeny particles. Coronavirus replicon RNAs differ from those of other positive-stranded RNA viruses in that they have to encode the nucleocapsid protein, which has been shown to be important for efficient coronavirus RNA replication (8,9.

It has been shown for HCoV-229E and SARS-CoV replicons that stable cell lines can be generated if the replicon RNA mediates the expression of a selection marker (4,10. Two selection markers, conferring neomycin/G418 (4 or blasticidin (10 resistance, have been used successfully for establishing stable coronavirus replicon cell lines. The HCoV-229E replicon RNA encodes the neomycin resistance gene inserted downstream of the nonstructural protein (Nsp) 1 and a sequence encoding a “2A-like” autoprocessing peptide. The 2A-like autoprocessing peptide mediates a co-translational liberation of a slightly modified Nsp1 carboxyterminus and subsequent translation of the neomycin resistance gene. In order to ensure translation of the remaining Nsps of the replicase gene (Nsps 2-16), an internal ribosomal entry site (IRES) derived from the encephalomyocarditis virus (EMCV) has been placed upstream of the Nsp2-coding sequence.

The SARS-CoV replicon RNA contains a gene encoding a fusion protein comprising the green fluorescent protein (GFP) and the blasticidin deaminase (GFP-BlaR) that has been cloned downstream of the replicase gene as a separate transcription unit under the control of the transcription regulatory sequence (TRS) of the SARS-CoV spike gene. In both cases, transfection of in vitro synthesized replicon RNA into eukaryotic cells and subsequent selection using G418 or blasticidin resulted in the establishment of stable cell lines containing actively replicating coronavirus replicon RNAs. To facilitate the detection of replicon-containing cell lines, green fluorescence resulting from replicon-mediated GFP expression has been used. For the SARS-CoV replicon RNA this has been achieved by the use of the GFP-BlaR fusion protein (10. To achieve GFP expression by the HCoV-229E replicon RNA, the GFP gene has been inserted as a separate transcription unit downstream from the replicase gene, driven by the TRS of the HCoV-229E spike gene (4.

Coronavirus replicon cell lines can be used as a noninfectious system to analyze coronavirus replication and transcription or to identify and evaluate replicase inhibitors. The following protocols describe the generation of corona- virus replicon cell lines and their use in the evaluation of coronavirus replicase inhibitors.

3.4.1 Generation of Coronavirus Replicon Cell Lines

1. Based on a full-length coronavirus cDNA cloned in vaccinia virus, a replicon RNA-encoding cDNA can be generated using vaccinia virus-mediated homologous recombination as described in Section 3.2.

2. Generate replicon RNA by in vitro transcription as described in Section 3.3.1.

3. Introduce the replicon RNA into a host cell line of choice (see Note 16) by electroporation as described in Section 3.3.2 (steps 1-5).

4. Plate the transfected cells in normal growth medium. Change the medium after 3-6 h when cells have attached to the bottom of the culture dish and continue to culture the cells for 1-2 days in growth medium without selection pressure. Split if necessary.

5. Start the selection of stable lines at antibiotic concentrations only slightly above the level at which nontransfected cells die (see Note 17).

6. Increase the antibiotic concentration gradually during the following 2-3 weeks until resistant colonies appear.

7. Pick colonies for subculture in separate wells and test them for maintenance of replicon RNA. Expression of a reporter protein, such as GFP, by the replicon RNA facilitates the screening of replicon RNA-containing resistant colonies.

8. When stable clones have been obtained, further culturing can be done under low selection pressure (see note 18). Replicon cells can be stored in liquid nitrogen.

3.4.2 Identification and Evaluation of Coronavirus Replicase Inhibitors Using Replicon Cell Lines

1. Seed the replicon cells in selection medium so that they are 50-70% confluent on the next day. You can use 96- , 24- , or 6-well dishes.

2. Prior to adding antiviral compounds, wash the cells and culture them in standard medium without selection drugs.

3. Add graded doses of antiviral compound(s) to the cells and culture them for 1-3 days (see Note 19). For comparison include nontreated cells and culture them under identical conditions.

4. In order to assay for cytotoxicity of candidate inhibitors and to determine the selectivity index, include a cytotoxicity/cell viability test. This can be done with replicon cells or the respective parental cell line.

5. Determine GFP expression on days 1, 2, and 3 posttreatment by fluorescence microscopy and flow cytometry (see Note 20).