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Journal of Virology, November 2000, p. 10811-10815, Vol. 74, No. 22
Department of Biology, Georgia State
University, Atlanta, Georgia 30303
Received 15 May 2000/Accepted 22 August 2000
Rubella virus (RUB) is a small plus-strand RNA virus
classified in the Rubivirus genus of the family
Togaviridae. Live, attenuated RUB vaccines have been
successfully used in vaccination programs for over 25 years, making RUB
an attractive vaccine vector. In this study, such a vector was
constructed using a recently developed RUB infectious cDNA clone
(Robo). Using a standard strategy employed to produce expression
and vaccine vectors with other togaviruses, the subgenomic
promoter was duplicated to produce a recombinant construct (termed
dsRobo) that expressed reporter genes such as chloramphenicol
acetyltransferase and green fluorescent protein (GFP) under control of
the second subgenomic promoter. However, expression of the reporter
genes, as exemplified by GFP expression by dsRobo/GFP virus,
was unstable during passaging, apparently due to homologous
recombination between the subgenomic promoters leading to deletion of
the GFP gene. To improve the stability of the vector, the internal
ribosome entry site (IRES) of a picornavirus, encephalomyocarditis virus, was used instead of the second
subgenomic promoter to eliminate homology. Construction was initiated
by first replacing the subgenomic promoter in the parent Robo
infectious clone with the IRES. Surprisingly, viable virus resulted;
this virus did not synthesize a subgenomic RNA. The subgenomic promoter was then reintroduced in an orientation such that a single subgenomic RNA was produced, GFP was the initial gene on this RNA, while the RUB
structural protein open reading frame was downstream and under control
of the IRES element. GFP expression by this vector was significantly
improved in comparison to dsRobo/GFP. This strategy should be
applicable to increase the stability of other togavirus vectors.
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Development of a Rubella Virus Vaccine Expression
Vector: Use of a Picornavirus Internal Ribosome Entry Site
Increases Stability of Expression

*
Corresponding author. Department of Biology, Georgia
State University, 24 Peachtree Center Ave., Atlanta, GA 30303. Phone and fax: (404) 651-3105. E-mail: tfrey{at}gsu.edu.
Present address: Oravax, Inc., Cambridge, MA 02139.
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