The making of infectious viral RNA: No size limit in sight

Despite the disadvantage of having an RNA genome, which is more difficult than DNA to be genetically tinkered, the reverse genetics of RNA viruses actually originated at about the same time as the dawning of the genomic manipulation of DNA viruses. The first RNA virus to be genetically modified was Qβ phage (1). Initially, the RNA molecules were chemically modified during RNA replication in vitro; the procedures were cumbersome and the range of RNA mutations was limited. Nevertheless, the potential power of reverse genetics as a tool for studying RNA viruses was transparently clear in a pioneering series of site-specific mutagenesis studies from C. Weissmann's laboratory (2, 3). The advent of recombinant DNA technology in the 1970s prompted RNA virologists to convert viral RNA genomes into complementary DNA copies and replicate them as plasmid inserts in bacterial hosts for easier genetic manipulation. Amazingly, the plasmid containing the complete cDNA of the Qβ phage RNA was fully infectious when introduced into bacterial hosts and was capable of completing the full viral replication cycle (4). Presumably, transcription of the viral RNA was randomly initiated, and the RNA was processed mysteriously to the correct viral sequence. Later, this technique was applied to several other viruses, including poliovirus (5) and viroids (6). Infectious poliovirus cDNA constructs remained the staples of poliovirus genetics for many years after that. Subsequently, another technique was developed whereby RNA was made by in vitro transcription of viral cDNA templates linked to a promoter recognized by Escherichia coli or phage DNA-dependent RNA polymerases (7). When the RNA transcribed in vitro was transfected into cells, it led to viral RNA replication (8). The first virus thus studied was brome mosaic virus, a relatively small plant virus containing three RNA segments of 3.2, 2.8, and 2.1 kb. In contrast to the cDNA transfection approach, the RNA molecules generated by using the RNA transfection approach were engineered so that they had well defined ends that matched the natural viral RNA sequences. This elegant approach empowered the virologists working with viruses containing relatively small RNA genomes or multiple RNA segments with the tools of reverse genetics. In all of these approaches, the size of the viral RNA was a major limitation. The Qβ phage RNA is 4.5 kb, and the poliovirus RNA is 7.5 kb. Over time, these approaches have been refined to enable the cloning of progressively larger RNAs. With some exceptions, most viral RNAs up to 15 kb long can now realistically be cloned. The report by Almazan et al. in this issue of PNAS (9) represents a further quantum leap, i.e., the successful cloning of a 27-kb long RNA derived from a coronavirus porcine transmissible gastroenteritis virus (TGEV), a task previously thought to be unachievable. This accomplishment is an intellectual and engineering tour de force. Because coronavirus contains the longest viral RNA genome by far (and is probably one of the longest stable RNAs in nature), this approach seems to pave the way for the reverse genetics studies for all RNA viruses.