The Sindbis genome encodes a positive-stranded 11.7 kb RNA that is both capped and polyadenylated. Unlike most eukaryotic RNA molecules, the Sindbis genome is bicistronic, containing two open reading frames, separated by stop codons. The 59 ORF encodes a ��nonstructural�� polyprotein that is translated and cleaved into the four subunits of the Sindbis replicase, an RNA-dependent RNA polymerase. ORF2 encodes a ��structural�� polyprotein containing the virus glycoproteins, as well as an RNAbinding capsid protein. Sequences at the end of the genome are important for packaging the genome into the virus particle, while sequences at the end are important for the initiation of replication by the RdRP. For viral replication to occur, the RdRP first produces a full-length, complementary copy of the genome in orientation. In order for the second ORF to be translated, an additional, shorter message in orientation must be generated by the viral replicase, through initiation at an internal ����RNA promoter���� on the antigenome. Different gene products and reporter genes have been inserted into ORF2 of Sindbis replicons to study RdRP-dependent viral replication quantitatively, or to express foreign proteins at high levels. The Sindbis replication cycle takes place in the cytoplasm of the host cell, where it is subject to MK-5172 cellular defense pathways, both in vertebrates, as well as in insects. Because of its powerful molecular genetic tools, Drosophila represents an attractive model for studying cellular defenses against viruses. Many RNA viruses replicate in Drosophila, including Vesicular Stomatitis Virus, Cricket Paralysis Virus, Drosophila C Virus, ��Yellow Rift Fever Virus��, and Sindbis. Important roles for several cellular pathways in suppressing or promoting viral replication have been identified in Drosophila, like the imd pathway, the Jak/Stat signaling pathway, autophagy, and RNA interference. While these studies used purified virus particles injected into the hemolymph, transgenic replicons in combination with real-time qPCR was used to quantify the role of the imd pathway, antimicrobial peptides, as well as the Akt/Pi3K pathway on Metformin replicon expression. Hence, the transgenic replicon technique serves as a promising alternative for the genetic dissection of factors affecting viral replication in vivo, by increasing both reproducibility and tissue-specificity.