FMDV replicons encoding green fluorescent protein are replication competent

Abstract

The study of replication of viruses that require high bio-secure facilities can be accomplished with less stringent containment using non-infectious 'replicon' systems. The FMDV replicon system (pT7rep) reported by Mclnerney et al. (2000) was modified by the replacement of sequences encoding chloramphenicol acetyl-transferase (CAT) with those encoding a functional L proteinase (L(pro)) linked to a bi-functional fluorescent/antibiotic resistance fusion protein (green fluorescent protein/puromycin resistance, [GFP-PAC]). Cells were transfected with replicon-derived transcript RNA and GFP fluorescence quantified. Replication of transcript RNAs was readily detected by fluorescence, whilst the signal from replication-incompetent forms of the genome was >2-fold lower. Surprisingly, a form of the replicon lacking the L(pro) showed a significantly stronger fluorescence signal, but appeared with slightly delayed kinetics. Replication can, therefore, be quantified simply by live-cell imaging and image analyses, providing a rapid and facile alternative to RT-qPCR or CAT assays.

Keywords: FMDV; Fluorescence; Replication; Replicon.

Fig. 1

Replicon constructs. The structure of the FMDV genome is shown together with replicon plasmid constructs. Polyprotein domains are shown as boxed areas, together with the ‘primary’ processing products L^pro (Lab^pro and Lb^pro forms), [P1-2A], [2BC] and P3 ([3AB_1–3CD]). The original CAT replicon (pT7Rep; Ellard et al., 1999, Mclnerney et al., 2000) was modified to re-insert the L proteinase sequences and the CAT reporter replaced with a GFP-PAC fusion protein (pGFP-PAC). This plasmid was modified to create a replication incompetent form by deletion of the 3D polymerase (pGFP-PAC-Δ3D). A replication attenuated form was created by deletion of the L proteinase (pLL-GFP-PAC; similar to that described by Piccone et al., 1995).

Fig. 2

Cleavage of eIF4G and GFP in cells transfected with replicon RNA. Extracts were prepared from pGFP-PAC (panels A and C) and pLL-GFP-PAC (panel B) replicon-transfected BHK-21 cells at the time points indicated. Extracts were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes, and analysed by western blotting with anti-eIF4G (panels A and B), anti-GFP (panel C) and anti-β-tubulin antibodies (panels A–C). The sequence flanking the L^pro/1A, eIF4G and predicted GFP L^pro cleavage sites are shown (Panel D).

Fig. 3

GFP expression in FMDV replicon-transfected BHK-21 cells. Transcript RNAs from the pGFP-PAC, pLL-GFP-PAC and pGFP-PAC-Δ3D replicons were transfected into cell monolayers, and fluorescent images captured at 2 h intervals over a 24 h period using the IncuCyte ZOOM imaging system. A representative of the nine images (captured for each well at each time point) is shown.

Fig. 4

Time course of FMDV replicon-derived GFP fluorescence. Data from mock-transfected BHK-21 cells are shown (×), together with cells transfected with transcript RNA derived from the pGFP-PAC replicon (◆), ‘leaderless’ replicon pLL-GFP-PAC (■) and polymerase deletion pGFP-PAC-Δ3D (▴) constructs. At the time points indicated images were captured and the GFP fluorescence quantified for each replicon construct: data shown as the green object count/mm² (Panel A) or the integrated GFP fluorescence intensity, ×10⁴ (Panel B). Data points/error bars shown are derived from three independent transfections, with four replicates for each transfection.