Reverse genetics of negative-strand RNA viruses: Closing the circle

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Abstract
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 5′ and 3′ 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 …