Alphaviruses to the rescue?

The ability to genetically engineer animal viruses enables their application to medical research. The development of non-retroviral RNA viruses as vectors to deliver foreign genes for vaccination and gene therapy is particularly interesting, especially the domestication of the alphavirus group. Two papers in this field have recently been published1xTwo-helper RNA system for production of recombinant Semliki Forest virus particles. Smerdou, C. and Liljestrom, P. J. Virol. 1999; 73: 1092–1098PubMedSee all References, 2xNoncytopathic Sindbis virus RNA vectors for heterologous gene expression. Agapov, E.V. et al. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12689–12994Crossref | PubMed | Scopus (155)See all References.Alphaviruses offer several advantages as expression vectors: they have a broad host range, high levels of expression and a small genome that is easy to manipulate. Vectors based on Semliki Forest virus (SFV), Sindbis virus and Venezuelan equine encephalitis have been constructed and deliver and express their cargo efficiently. However, the generation of wild-type virus, owing to recombination between the artificial genome and a helper genome, remains a problem in many recombinant viral systems. This biosafety issue is a concern, especially if vectors are to be used in vaccine delivery, and methods have been developed to reduce this risk. In the SFV system, Smerdou and Liljestrom1xTwo-helper RNA system for production of recombinant Semliki Forest virus particles. Smerdou, C. and Liljestrom, P. J. Virol. 1999; 73: 1092–1098PubMedSee all References1 have prevented the production of wild-type virus by using two, independent helper RNAs; this system also circumvents the requirement of a viral protease and this portion of the genome has been mutated, conferring additional biosafety. Thus, at least two recombination events and one reversion are required to generate replication-competent virus particles. Extensive analysis has failed to demonstrate the presence of wild-type viruses, emphasizing the biosafety of the two-helper system.The expression strategy of alphaviruses imposes a severe burden on normal cellular biochemistry and infected cells die quickly, restricting the use of these vectors when long-term gene expression is required. However, Agapov et al.2xNoncytopathic Sindbis virus RNA vectors for heterologous gene expression. Agapov, E.V. et al. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12689–12994Crossref | PubMed | Scopus (155)See all References2 have developed noncytotoxic Sindbis virus vectors, which produce large amounts of foreign protein, by transfecting Sindbis virus replicons that express a puromycin N-acetyltransferase into cells grown in the presence of puromycin. Cells that survive in the presence of the drug carry replicons that are noncytotoxic and express the puromycin-inactivating enzyme. Such replicons can then be engineered to express other foreign genes for long periods in a noncytotoxic manner, which could be useful in both vaccinology and in the development of gene therapy.