Mutational Analysis of the Uukuniemi Virus ( Bunyaviridae Family) Promoter Reveals Two Elements of Functional Importance

Abstract

We have performed an extensive mutational analysis of the proposed promoter region of the phlebovirus Uukuniemi (UUK), a member of the Bunyaviridae family. This was achieved by using a recently developed RNA polymerase I (Pol I)-driven reverse genetics system (R. Flick and R. F. Pettersson, J. Virol. 75: 1643-1655, 2001). Chimeric cDNAs containing the coding region for the reporter chloramphenicol acetyltransferase (CAT) in an antisense orientation were flanked by the 5′- and 3′-terminal nontranslated regions of the UUK virus-sense RNA (vRNA) derived from the medium-sized (M) RNA segment. The chimeric cDNAs (Pol I expression cassettes) were cloned between the murine Pol I promoter and terminator, and the plasmids were transfected into BHK-21 cells. CAT activity was determined after cotransfection with viral expression plasmids encoding the RNA-dependent RNA polymerase (L) and the nucleoprotein (N) or, alternatively, after superinfection with UUK virus helper virus. Using oligonucleotide-directed mutagenesis, single point mutations (substitutions, deletions, and insertions) were introduced into the viral promoter region. Differences in CAT activities were interpreted to reflect the efficiency of mRNA transcription from the mutated promoter and the influence on RNA replication. Analysis of 109 mutants allowed us to define two important regulatory regions within the proximal promoter region (site A, positions 3 to 5 and 2 to 4 ; site B, positions 8 and 8 , where underlined nucleotides refer to positions in the vRNA 3′ end). Complementary double nucleotide exchanges in the proximal promoter region, which maintained the possibility for base pairing between the 5′ and 3′ ends, demonstrated that nucleotides in the two described regions are essential for viral polymerase recognition in a base-specific manner. Thus, mere preservation of panhandle base pairing between the 5′ and 3′ ends is not sufficient for promoter activity. In conclusion, we have been able to demonstrate that both ends of the M RNA segment build up the promoter region and are involved in the specific recognition by the viral polymerase.