HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Supplementary movie Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by van den Born, E. Articles by Snijder, E. J. Search for Related Content PubMed PubMed Citation Articles by van den Born, E. Articles by Snijder, E. J. Agricola Articles by van den Born, E. Articles by Snijder, E. J.

An infectious recombinant equine arteritis virus expressing green fluorescent protein from its replicase gene

¹ Molecular Virology Laboratory, Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands
² Section Electron Microscopy, Department of Molecular Cell Biology, Leiden University Medical Center (LUMC), PO Box 9600, 2300 RC Leiden, The Netherlands

Correspondence
Eric J. Snijder
e.j.snijder{at}lumc.nl

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 Equine arteritis virus, 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 in vitro-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.

The GenBank/EMBL/DDBJ accession number for the sequence of pDE-GFP2 is EF115507.

A movie showing in vivo imaging analysis of EAV-GFP2 replicase expression in Vero-E6 cells is available as supplementary material in JGV Online.

This article has been cited by other articles:

				D. Horst, D. van Leeuwen, N. P. Croft, M. A. Garstka, A. D. Hislop, E. Kremmer, A. B. Rickinson, E. J. H. J. Wiertz, and M. E. Ressing Specific Targeting of the EBV Lytic Phase Protein BNLF2a to the Transporter Associated with Antigen Processing Results in Impairment of HLA Class I-Restricted Antigen Presentation J. Immunol., February 15, 2009; 182(4): 2313 - 2324. [Abstract] [Full Text] [PDF]

				E. F. Donaldson, B. Yount, A. C. Sims, S. Burkett, R. J. Pickles, and R. S. Baric Systematic Assembly of a Full-Length Infectious Clone of Human Coronavirus NL63 J. Virol., December 1, 2008; 82(23): 11948 - 11957. [Abstract] [Full Text] [PDF]

				X. Zhang and D. L. Nuss A host dicer is required for defective viral RNA production and recombinant virus vector RNA instability for a positive sense RNA virus PNAS, October 28, 2008; 105(43): 16749 - 16754. [Abstract] [Full Text] [PDF]

				M. C. Verweij, D. Koppers-Lalic, S. Loch, F. Klauschies, H. de la Salle, E. Quinten, P. J. Lehner, A. Mulder, M. R. Knittler, R. Tampe, et al. The Varicellovirus UL49.5 Protein Blocks the Transporter Associated with Antigen Processing (TAP) by Inhibiting Essential Conformational Transitions in the 6+6 Transmembrane TAP Core Complex J. Immunol., October 1, 2008; 181(7): 4894 - 4907. [Abstract] [Full Text] [PDF]

				M. A. Tijms, D. D. Nedialkova, J. C. Zevenhoven-Dobbe, A. E. Gorbalenya, and E. J. Snijder Arterivirus Subgenomic mRNA Synthesis and Virion Biogenesis Depend on the Multifunctional nsp1 Autoprotease J. Virol., October 1, 2007; 81(19): 10496 - 10505. [Abstract] [Full Text] [PDF]