GREATLY INCREASED EFFICIENCY OF RABIES VIRUS PRODUCTION FROM DNA

Multiplexed monosynaptic tracing entails the use of barcoded rabies virus libraries to separately identify the inputs to individual postsynaptic cells. Typical barcoded rabies virus libraries are plagued by markedly inhomogeneous barcode distributions, with a small subset of overrepresented barcode clones dominating a given library. This is because of the low efficiency of the first step of producing nonsegmented negative-strand (NNS) RNA viruses such as rabies virus: the so-called "rescue" of infectious virus particles from plasmid DNA. This inefficiency results in the earliest successfully-rescued clones spreading throughout the producer cell plates, producing many more copies than do later-rescued clones.
Rescuing NNS viruses requires transfecting cells with plasmids expressing viral genes as well as an mRNA transcript containing the desired vector genome (or, more commonly, "antigenome", the genome's reverse complement), flanked by self-cleaving ribozymes to create authentic ends of the viral (anti)genome, allowing it to be packaged by the viral nucleoprotein. Recently, a novel family of highly-efficient ribozymes – the so-called "twister" ribozymes – was found to be widespread in eukaryotes and bacteria. Here we show that a "twister" ribozyme on the 3' end of the rabies viral antigenome increases rescue efficiency 37-fold over that of the most commonly-used 3' ribozyme (the standard hepatitis delta virus ribozyme) and 4.5-fold over that of the best previously-used 3' ribozyme (the "super cutter" hepatitis delta virus ribozyme). This increased efficiency should considerably increase the uniformity of barcode distribution in rabies virus preparations for multiplexed monosynaptic tracing.