Reverse genetics of negative-strand RNA viruses: closing the circle.

The study of viruses and their interactions with host cells and organisms has benefited greatly from the ability to engineer specific mutations into viral genomes, a technique known as reverse genetics. Genome manipulations of DNA viruses, either by transfecting cells with plasmids encoding the viral genome (1) or by heterologous recombination of plasmids bearing viral sequences with the virus genome (2–4), were the first to be performed. Positive-strand RNA virus genome manipulation followed quickly, partly because the viral genome is also mRNA sense. Simply transfecting plasmids, or RNA transcribed from plasmids, containing the poliovirus genome into susceptible cells resulted in the recovery of infectious poliovirus (5, 6). The negative-strand RNA viruses include a number of human and animal pathogens such as influenza A, B, and C viruses, hantaviruses, Lassa virus, rabies virus, Ebola virus, Marburg virus, measles virus, canine distemper virus, rinderpest virus, respiratory syncytial virus, mumps virus, human parainfluenza virus types 1–4, and Nipah virus (which recently emerged in Malaysia, causing respiratory distress and encephalitis in pigs and humans). However, the genomes of the negative-strand RNA viruses have been less amenable to artificial manipulation for several reasons: (i) precise 59 and 39 ends are required for replication and packaging of the genomic RNA; (ii) the viral RNA polymerase is essential for transcribing both mRNA and complementary, positive-sense antigenome template RNA; and (iii) both genomic and antigenomic RNAs exist as viral ribonucleoprotein (RNP) complexes (reviewed in ref. 7). The segmented genomes of influenza viruses, bunyaviruses, and arenaviruses allowed some genetic manipulation through the isolation of reassortant viruses, but manipulation of the complete genome of segmented negative-strand RNA viruses has progressed slowly, hampered by the very fact that the genome is segmented. In this issue of the Proceedings, Neumann and coworkers (8) have come full circle on recovering recombinant, segmented negative-strand RNA viruses with the production of influenza virus entirely from plasmid DNA and driven only by the host cell transcription and translation machinery. Coming nearly 10 years after the first published reports of influenza virus genome manipulation (9) and after another Proceedings article describing the generation of recombinant bunyaviruses wholly from cDNA by using a recombinant vaccinia virus-driven system (10), virologists finally have acquired the tools necessary to perform sophisticated and comprehensive investigations of the role of all influenza virus proteins and RNA elements in replication and pathogenesis. The influenza virus RNPs, upon their release into the cytoplasm of an infected cell, enter the cell nucleus, and the influenza virus polymerase complex, consisting of the PA, PB1, and PB2 proteins, begins to transcribe the genomic RNA into mRNA and a positive-sense antigenome RNA that serves as the template for the production of genome RNA. Although influenza virus was the first negative-strand RNA virus to have individual virus genes replaced by artificially manipulated segments, the difficulty in dealing with a segmented RNA genome, as well as the use of labor-intensive and selectiondependent techniques to drive reverse genetics has hindered the application of this technology. Nonetheless, many important discoveries pertaining to individual influenza virus proteins as well as demonstrating the use of influenza virus to serve as a viral expression vector have been obtained by application of the existing reverse genetics technology (reviewed in refs. 7 and 11). Neumann and coworkers (8) have established a system that conscripts the host cell into making the equivalent of newly released RNPs by cotransfecting eight plasmids encoding each of the influenza virus genomic RNA segments under control of the RNA polymerase type I (pol I) promoter and transcription terminator along with four plasmids encoding the polymerase complex proteins and nucleoprotein (NP) cDNAs under control of an RNA polymerase type II (pol II) promoter. Although the concept of cotransfecting multiple plasmids to reconstitute a biochemical activity was pioneered for studying herpes virus DNA replication (12), the daunting nature of this 12–17 plasmid transfection (a likely record for most plasmids transfected into one cell) still results in approximately 1 in 1,000 cells producing infectious virus. The lack of a helper influenza virus allows the virus from the initial transfection to be characterized immediately, thus limiting the chance of viruses containing reversions or second-site mutations from becoming significant contaminants. One can only speculate as to how quickly our knowledge of influenza virus will progress, now that every nucleotide of the viral genome can be mutated and engineered back into the genome, in nearly endless combinations with other mutations. As with most important scientific advances, the work of Neumann and coworkers builds on a large body of experiments that have identified the basic requirements for replicating and packaging influenza virus RNA segments. The technique used first to introduce a new, artificial RNA segment into influenza virus (13) and refined subsequently to create influenza viruses containing neuraminidase (NA) proteins derived from plasmid cDNAs (9) relied on reconstitution of viral RNPs from in vitro-transcribed RNA and purified nucleocapsid proteins (Fig. 1). The protein-RNA complex was transfected into cells, followed by infection with a helper influenza virus. The application of a selection pressure against the helper virus facilitates the detection of progeny virus containing the plasmid DNA-derived RNA segment. Although a tour de force of molecular biology at the time, the technique requires the purification of large amounts of viral nucleocapsid proteins and is most efficient when a strong selection pressure can be applied against the helper virus. The use of pol I transcripts to produce artificial influenza virus RNA segments was pioneered by Hobom and colleagues (14–16). Unlike the mRNA transcripts produced by pol II, the primary RNA transcripts synthesized by pol I are ribosomal RNAs that possess neither a 59 cap structure nor a 39 poly(A) tail. Zobel and coworkers (16) successfully produced artificial influenza virus RNA segments with precise 59 and 39 ends, and