Insertion of EGFP into the replicase gene of Semliki Forest virus results in a novel, genetically stable marker virus.
Alphavirus-based vector and replicon systems have been extensively used experimentally and are likely to be used in human and animal medicine. Whilst marker genes can be inserted easily under the control of a duplicated subgenomic promoter, these constructs are often genetically unstable. Here, a novel alphavirus construct is described in which an enhanced green fluorescent protein (EGFP) marker gene is inserted into the virus replicase open reading frame between nsP3 and nsP4, flanked by nsP2 protease-recognition sites. This construct has correct processing of the replicase polyprotein, produces viable virus and expresses detectable EGFP fluorescence upon infection of cultured cells and cells of the mouse brain. In comparison to parental virus, the marker virus has an approximately 1 h delay in virus RNA and infectious virus production. Passage of the marker virus in vitro and in vivo demonstrates good genetic stability. Insertion of different markers into this novel construct has potential for various applications.
Tamberg
Nele
N
Estonian Biocentre, Tartu, Estonia.
Lulla
Valeria
V
Estonian Biocentre, Tartu, Estonia.
Fragkoudis
Rennos
R
Centre for Infectious Diseases, College of Medicine and Veterinary Medicine, University of Edinburgh, Edinburgh, UK.
Lulla
Aleksei
A
Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia.
Fazakerley
John K
JK
Centre for Infectious Diseases, College of Medicine and Veterinary Medicine, University of Edinburgh, Edinburgh, UK.
Merits
Andres
A
Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia.|Estonian Biocentre, Tartu, Estonia.
D000818:Animals; D001921:Brain / Q000821:virology; D002460:Cell Line; D006224:Cricetinae; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics* / Q000502:physiology; D012672:Semliki forest virus / Q000235:genetics* / Q000254:growth & development; D013194:Staining and Labeling; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000502:physiology; D014779:Virus Replication
D017361:Viral Nonstructural Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D012324:RNA-Dependent RNA Polymerase