**2. Alphavirus Vectors**

Alphaviruses are single stranded RNA viruses possessing a positive strand polarity [9]. The genome is encapsulated in a capsid protein structure covered by a membrane protein envelope structure. After the release of the alphavirus RNA genome in infected cells, the non-structural alphavirus proteins (nsP1-4) forms the RNA replicase complex responsible for extensive RNA replication. In expression vectors, which were first engineered for RNA replicon and replicon particle delivery, the alphavirus structural genes were replaced by the foreign gene of interest [10]. This approach required the in vitro transcription of RNA from a plasmid DNA construct, which then was directly transfected into host cells for immediate transgene expression. Alternatively, co-transfection of in vitro transcribed RNA from an alphavirus vector carrying the alphavirus structural genes allowed packaging of replication-deficient recombinant alphavirus particles. These so-called "suicide particles" are capable of one round of infection of a broad range of host cells generating high levels of transgene expression.

To be able to use alphavirus-based plasmid DNA vectors for direct immunization, a mammalian host cell compatible eukaryotic RNA polymerase II type promoter such as CMV was engineered upstream of the replicon genes [11]. DNA-based alphavirus vectors provide high biosafety levels with no risk of production of new viral progeny, but still generating high levels of transgene expression due to the presence of the alphavirus replicon. However, the host cell range is dependent on the efficacy of available transfection methods. Another issue related to plasmid DNA delivery concerns the improvement of transfer to the nucleus by the introduction of nuclear localization signals (NLS) in the vector [12].
