Recombination of nonreplicative RNA precursors of Sindbis virus in infected cells overexpressing murine-inducible nitric oxide synthase

The Sindbis virus-based SINrep5 expression system is one of the most efficient vectors for gene transfer leading to fast and high expression of the gene of interest. This system was used to transfect vascular endothelial and smooth muscle cells using murine inducible nitric oxide synthase (miNOS) as a reporter gene. Infection of both cell types leads to high expression levels of miNOS. In addition, the harvested supernatant of these infected cells was used for further rounds of infections, demonstrating that recombination of the parental RNA with the helper RNA takes place and results in the production of infectious particles. As shown by RT-PCR, after recombination the miNOS gene is located in between the nonstructural and structural viral genes. This study demonstrates that despite claims in other publications, the Sindbis virus-based SINrep5 expression system leads to recombination and is thus not a safe system for in vitro and in vivo applications.