VEGAS: A Platform for Continuous Directed Evolution in Mammalian Cells (RNAseq)

Sindbis virus is a mutagenic ssRNA arbor virus. The structural genome component of this virus can be exchanged for any transgene of < 6kb in size. Here we sequence transgenic elements inserted to the virus. Our EGFP transgene was analyzed as a function of time during viral replication to quantify the mutagenic rate of the virus. Our nanobody transgene was analyzed to assess cDNA library complexity and identity. In this study we packaged transgenic virus carrying either EGFP or a library of nanobody cDNA sequences obtained from a 5HT2A receptor immunized llama. The EGFP packaged virus was applied to BHK21 (hamster) cells harboring the plasmid pCMV-SSG, a mammalian expression plasmid to drive constitutive production of the sindbis virus structural genome (SSG). This allows the virus to freely proliferate in the transfected culture. We collected virus from cell culture at 3, 6, 24, and 36 hours after infection and amplified the EGFP transgene. The amplicons were sequenced and sequence integrity of individual nucleotide positions was determined. The nanobody-containing Sindbis genome was directly amplified without culturing and sequenced. The resulting sequences were aligned to the FASTA files provided in this submission to determine if the FASTA deposited nanobody sequences were present in the originating viral titer.