1887

Abstract

Thus far, systems developed for heterologous gene expression from the genomes of nidoviruses (arteriviruses and coronaviruses) have relied mainly on the translation of foreign genes from subgenomic mRNAs, whose synthesis is a key feature of the nidovirus life cycle. In general, such expression vectors often suffered from relatively low and unpredictable expression levels, as well as genome instability. In an attempt to circumvent these disadvantages, the possibility to express a foreign gene [encoding enhanced green fluorescent protein (eGFP)] from within the nidovirus replicase gene, which encodes two large polyproteins that are processed proteolytically into the non-structural proteins (nsps) required for viral RNA synthesis, has now been explored. A viable recombinant of the arterivirus , EAV-GFP2, was obtained, which contained the eGFP insert at the site specifying the junction between the two most N-proximal replicase-cleavage products, nsp1 and nsp2. EAV-GFP2 replication could be launched by transfection of cells with either -generated RNA transcripts or a DNA launch plasmid. EAV-GFP2 displayed growth characteristics similar to those of the wild-type virus and was found to maintain the insert stably for at least eight passages. It is proposed that EAV-GFP2 has potential for arterivirus vector development and as a tool in inhibitor screening. It can also be used for fundamental studies into EAV replication, which was illustrated by the fact that the eGFP signal of EAV-GFP2, which largely originated from an eGFP–nsp2 fusion protein, could be used to monitor the formation of the membrane-bound EAV replication complex in real time.

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2007-04-01
2019-11-13
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vol. , part 4, pp. 1196 – 1205

imaging analysis of Vero-E6 cells infected with EAV-GFP2

Vero-E6 cells were grown to 70 % confluence in a glass-bottomed 10 cm culture dish (MatTek Corporation) and were infected with EAV-GFP2 at an m.o.i. of 10. After 1 h, the inoculum was removed and 5 ml prewarmed Dulbecco's modified Eagle's medium containing 2 % fetal calf serum was added. The dish was placed in the 37 °C observation chamber (containing 5 % CO ) of a Leica TCS SP5 confocal microscope operating under the Leica Application Suite Advanced Fluorescence software. From 3 to 18 h p.i., a stack of five images (total thickness, 6.5 µm) was recorded every 5 min with the pinhole set at 3 Airy units. A 488 nm laser was used for eGFP excitation and emission was recorded at 500–650 nm. Subsequently, movie frames were created by merging the five images of the series captured at each time point and were processed into a movie of six frames s by using QuickTime Pro.

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