Alphaviruses are positive stranded RNA viruses that replicate to extremely high titers. Sindbis and Semliki Forest viral vectors are widely used tools for high-level production of recombinant proteins. Recent studies have broadened their scope to vaccine production, gene therapy, and analysis of cell function. Here we discuss the development of non-cytopathic and inducible expression vectors which can be applied to bioprocess development strategies. Furthermore, a Sindbis-based expression cloning system has been developed that allows for the rapid identification of genes encoding proteins with a selected functional activity.

We have developed a widely applicable functional genomics strategy based on alphavirus expression vectors. The technology allows for rapid identification of genes encoding a functional activity such as binding of a defined ligand. Complementary DNA (cDNA) libraries were expressed in mammalian cells following infection with recombinant Sindbis virus (SIN replicon particles), a member of the Alphavirus genus. Virus-infected cells that specifically bound a ligand of choice were isolated using fluorescence-activated cell sorting (FACS). Replication-competent, infective SIN replicon particles harboring the corresponding cDNA were amplified in a next step. Within one round of selection, viral clones encoding proteins recognized by monoclonal antibodies or Fc-fusion molecules could be isolated and sequenced. Moreover, using the same viral libraries, a plaque-lift assay was established that allowed the identification of secreted, intracellular, and membrane proteins.

We present a temperature-regulated, alphavirus replicon-based DNA expression system. The system is regulated by a viral temperature-sensitive RNA-dependent RNA replicase, creating a temperature-dependent RNA amplification loop. Because of this positive feedback, the system exhibits both low background and high inducibility. We observed 700-fold induction in transiently transfected cells, and over 104-fold induction in stably transfected cells. The high stringency of inducibility allowed the generation of stable cell lines expressing a highly toxic protein upon temperature shift. These data suggest that the present expression system could simplify bioprocess engineering strategies, especially in situations where the cloned protein has detrimental effects on host cell metabolism.

Somatic gene therapy as a potential strategy for the treatment of myocardial diseases relies on an efficient gene transfer into cardiac muscle cells. The difficulty of delivering genes into adult cardiomyocytes exists not only in vivo but also in primary culture systems. Therefore, possibilities for ex vivo gene transfer and the in vitro study of physiological processes by reverse genetics are limited. We investigated the potential of an alphavirus-based vector system to transduce adult rat cardiomyocytes (ARC) in culture using a replication-deficient Sindbis virus (SIN) encoding beta-galactosidase (SIN-LacZ). Transduction efficiency depended on the virus concentration used, with expression of the reporter gene being detectable in up to 80% of cultured ARC as early as 24 h after infection. We observed a remarkably lower cytotoxicity of this viral vector in ARC than in other cells such as fibroblasts and neonatal cardiomyocytes. Additionally, no perceptible changes in the morphology of the nuclei or cytoskeleton were found in ARC 48 h after infection with SIN-LacZ. We conclude that SIN vectors are useful for gene delivery into adult cardiomyocytes and believe that improved versions of this viral system may be useful for cardiovascular gene therapy in the future.