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Journal of Virology, February 1999, p. 1092-1098, Vol. 73, No. 2
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Two-Helper RNA System for Production of Recombinant Semliki Forest Virus Particles

C. Smerdou1 and P. Liljeström1,2,*

Microbiology and Tumor Biology Center, Karolinska Institute, S-17177 Stockholm,1 and Department of Vaccine Research, Swedish Institute for Infectious Disease Control, S-17182 Solna,2 Sweden

Received 18 September 1998/Accepted 30 October 1998

Alphavirus expression systems based on suicidal virus particles carrying recombinant replicons have proven to be a very efficient way to deliver genes for heterologous protein expression. However, present strategies for production of such particles have biosafety limitations due to the generation, by RNA recombination, of replication-proficient viruses (RPVs). Here we describe a new packaging system for Semliki Forest virus (SFV) based on a the use of a two-helper system in which the capsid and spike proteins of the C-p62-6K-E1 polyprotein are expressed from two independent RNA molecules. The capsid gene contains a translational enhancer and therefore that sequence was also engineered in front of the spike sequence p62-6K-E1. A sequence coding for the foot-and-mouth disease virus 2A autoprotease was inserted in frame between the capsid translational enhancer and the spike genes. This allows production of the spike proteins at high levels with cotranslational removal of the enhancer sequence and normal biosynthesis of the spike complex. The autoprotease activity of the capsid protein was abolished by mutation, further increasing the biosafety of the system. Cotransfection of cells with both helper RNAs and an SFV vector replicon carrying the LacZ gene led to production of recombinant particles with titers of up to 8 × 108 particles per 106 cells. Extensive analysis failed to demonstrate the presence of any RPVs, emphasizing the high biosafety of the system based on two-helper RNAs.

* Corresponding author. Mailing address: Microbiology and Tumor Biology Center, Karolinska Institute, Box 280, S-17177 Stockholm, Sweden. Phone: 46-8-457 2550. Fax: 46-8-310848. E-mail: peter.liljestrom{at}mtc.ki.se.

Journal of Virology, February 1999, p. 1092-1098, Vol. 73, No. 2
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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Copyright © 1999 by the American Society for Microbiology. All rights reserved.