Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Yun, Sang-Im; Song, Byung-Hak; Kim, Jin-Kyoung; Lee, Young-Min

For RNA virologists, the advent of recombinant DNA technology in the late 1970s made it possible to convert viral RNA genomes into cDNA clones, which could then be propagated as plasmids in bacteria for the genetic manipulation of RNA viruses. 1 The first RNA virus to be molecularly cloned was bacteriophage Qβ, a positive-strand RNA virus that infects Escherichia coli. A plasmid containing a complete cDNA copy of the Qβ genomic RNA gave rise to infectious Qβ phages when introduced into E. coli. 2 Shortly thereafter, this technique was applied to poliovirus, a positive-strand RNA virus of humans and animals. A plasmid bearing a full-length cDNA of the poliovirus genomic RNA was infectious when transfected into mammalian cells and capable of producing infectious virions. 3 In this "DNA-launched" approach, the cloned cDNAs should be transcribed intracellularly to initiate viral RNA replication; however, it is unclear how the transcription is initiated and how the transcripts are processed to the correct viral sequence. This concern has led to the development of an alternative "RNA-launched" approach, whereby a complete cDNA copy of the viral RNA genome is cloned under a promoter recognized by an E. coli or phage RNA polymerase for the production of synthetic RNAs in vitro with defined 5' and 3' termini, which undergo the complete viral replication cycle when introduced into host cells. 4, 5 The first success with this approach was reported for brome mosaic virus, 6 ,7 a positive-strand RNA virus of plants. Since then, the RNA-launched approach has been developed for a wide range of positive-strand RNA viruses, including caliciviruses, alphaviruses, flaviviruses, arteriviruses, and coronaviruses. 1, 4, 5, 8 In both the DNA-and RNA-launched reverse genetics systems, the construction of a full-length cDNA clone is the key to generating infectious DNA or RNA of positive-strand RNA viruses, but it becomes a considerable technical challenge as the size of the viral genome increases. [9] [10] [11] [12] [13] [14] [15] [16] [17] In particular, a large RNA genome of ~10-32 kb presents three major obstacles to the cloning of a full-length functional cDNA. 18 The first difficulty is the synthesis of a faithful cDNA copy, since the fidelity of RT-PCR is inversely proportional to the length of the viral RNA. The second hurdle is the presence of potentially toxic sequences, since long RNA molecules are more likely to contain unexpected sequences capable of making the cDNA fragment in plasmids unstable in E. coli. The third and most critical issue is the availability of a suitable vector, since it is difficult to find a cloning vector that can house a viral cDNA insert of >10 kb. Over the past three decades, these barriers have been overcome by several advances in enzymology, methodology, and vectorology. 1, 4, 5, 8 Of these, the most promising and innovative development is the cloning of large positive-strand RNA viruses as infectious bacterial artificial chromosomes (BACs). The BAC vector is a low-copy cloning plasmid (1-2 copies/cell) based on the E. coli fertility factor, with an average DNA insert size of ~120-350 kb. 19 2. Assemble a set of the four modified, overlapping cDNAs into a single full-length SA 14 -14-2 BAC (pBAC/SA 14 -14-2) by joining at three natural restriction sites (BsrG I, BamH I, and Ava I) in a sequential manner using the five-step cloning procedures detailed in Protocols 3.1-3.5 ( Figure 1E) ) in a 6-well plate. 7. After 4-6 hr