Bunyamwera virus (BUNV) is the prototype virus for both the genus Orthobunyavirus and the family Bunyaviridae. BUNV has a tripartite, negative-sense RNA genome. The coding region of each segment is flanked by untranslated regions (UTRs) that are partially complementary. The UTRs play an important role in the virus life cycle by promoting transcription, replication, and encapsidation of the viral genome. Using reverse genetics, we generated recombinant viruses that contained deletions within the 3= and/or 5= UTRs of the L or M segments to determine the minimal UTRs competent for virus viability. We then generated viruses carrying deleted UTRs in all three segments. These viruses were grossly attenuated in tissue culture, being significantly impaired in their ability to produce plaques in BHK cells, and had a reduced capacity to cause host cell protein shutoff. After serial passage in tissue culture, some viruses partially recovered fitness, generating higher titers and producing larger plaques. We determined the complete nucleotide sequence for each virus. The deleted UTR sequences were maintained, and no amino acid changes were observed in the nonstructural proteins (NSs and NSm), the nucleocapsid protein (N), or the Gn glycoprotein. One virus had a single amino acid substitution in Gc. Three viruses contained amino acid changes in the viral polymerase that mostly occurred in the C-terminal domain of the L protein. Although the role of this domain remains unknown, we suggest that those changes might be involved in the evolution of the polymerase to recognize the deleted UTRs more efficiently.
T he genusOrthobunyavirus and the family Bunyaviridae take their name from Bunyamwera virus (BUNV), the prototype bunyavirus, which was isolated from mosquitoes of Aedes species in the Semliki Forest in Uganda (25). The BUNV genome is composed of three single-stranded RNA segments of negative polarity, named large (L), medium (M), and small (S), which encode four structural proteins. The L segment codes for the RNA-dependent RNA polymerase or L protein; the M segment codes for the two envelope glycoproteins, Gn and Gc; and the S segment codes for the nucleocapsid protein (N). Nonstructural proteins are encoded on the M and S segment and are named NSm and NSs, respectively (reviewed in reference 10). The NSm protein was shown to be involved in virion assembly (24), while NSs plays a role in counteracting the host cell innate immune response through a general block in transcription and translation (4,16,26,27). Each genome segment is encapsidated by numerous copies of the N protein and is associated with a few copies of the L protein to form ribonucleoprotein (RNP) complexes that are the functional templates for both transcription and replication.The coding regions are flanked by untranslated regions (UTRs), whose size and sequence vary greatly between segments, but as a general rule, the 3= UTR is shorter than the 5= UTR. BUNV 3= and 5= UTRs on the L and M segments are about 50 nucleotides (nt) and 100 nt, respectively, whil...