Flexibility of bunyavirus genomes: creation of an orthobunyavirus with an ambisense S segment.

unlabelledThe Bunyamwera (BUNV) orthobunyavirus NSs protein has proven a challenge to study in the context of viral infection. NSs is encoded in a reading frame that overlaps that of the viral nucleocapsid (N) protein thus limiting options for mutagenesis. In addition, NSs is poorly immunogenic, and antibodies only work in certain techniques while the protein itself is subject to proteasomal degradation. In order to generate a virus that expresses NSs independently of N, an ambisense S RNA segment was designed by mutating the 5'- and 3'-terminal nucleotide sequences. These mutations were previously shown to alter promoter activity so that both replication and transcription were promoted from both the genome and the antigenome RNAs (J. N. Barr et al., J. Virol. 79: 12602-12607, 2005). As proof of principle, a recombinant BUNV was created that expressed green fluorescent protein (GFP) in the ambisense orientation. GFP expression was detected throughout at least 10 passages. Recombinant BUNV encoding epitope-tagged versions of NSs in the ambisense orientation expressed NSs via a subgenomic mRNA, and two viruses grew to titers only modestly lower than parental rBUNdelNSs2 virus. The ambisense viruses were temperature sensitive, and NSs was shown to localize to both the nucleus and the cytoplasm during infection. These viruses will be useful in further studies on structure-function relationships of the orthobunyavirus NSs protein. IMPORTANCE: Bunyamwera virus (BUNV) is the type species and model system for both the family Bunyaviridae and the genus Orthobunyavirus, a group that includes many significant human and animal pathogens. Studying the basic molecular biology of these viruses is of great importance to underpin research into vaccines and antivirals. We demonstrate here the plasticity of the BUNV genome by generating recombinant viruses where the normal negative-sense S segment has been converted into an ambisense segment, allowing independent expression of either a foreign gene (green fluorescent protein) or the viral nonstructural NSs protein. These new reagents will allow detailed investigation of NSs, the orthobunyavirus interferon antagonist.