FMDV replicons encoding green fluorescent protein are replication competent

TY - JOUR

T1 - FMDV replicons encoding green fluorescent protein are replication competent

AU - Tulloch, Fiona

AU - Pathania, Uday

AU - Luke, Garry A.

AU - Nicholson, John

AU - Stonehouse, Nicola J.

AU - Rowlands, David J.

AU - Jackson, Terry

AU - Tuthill, Toby

AU - Haas, Juergen

AU - Lamond, Angus I.

AU - Ryan, Martin D.

PY - 2014/12/1

Y1 - 2014/12/1

N2 - 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 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 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.

AB - 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 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 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.

UR - https://www.scopus.com/pages/publications/84907521743

U2 - 10.1016/j.jviromet.2014.08.020

DO - 10.1016/j.jviromet.2014.08.020

M3 - Article

C2 - 25194890

AN - SCOPUS:84907521743

SN - 0166-0934

VL - 209

SP - 35

EP - 40

JO - Journal of Virological Methods

JF - Journal of Virological Methods

ER -