Reverse Genetics System for Uukuniemi Virus ( Bunyaviridae ): RNA Polymerase I-Catalyzed Expression of Chimeric Viral RNAs

Abstract

We describe here the development of a reverse genetics system for the phlebovirus Uukuniemi virus, a member of the Bunyaviridae family, by using RNA polymerase I (pol I)-mediated transcription. Complementary DNAs containing the coding sequence for either chloramphenicol acetyltransferase (CAT) or green fluorescent protein (GFP) (both in antisense orientation) were flanked by the 5-and 3-terminal untranslated regions of the Uukuniemi virus sense or complementary RNA derived from the medium-sized (M) RNA segment. This chimeric cDNA (pol I expression cassette) was cloned between the murine pol I promoter and terminator and the plasmid transfected into BHK-21 cells. When such cells were either superinfected with Uukuniemi virus or cotransfected with expression plasmids encoding the L (RNA polymerase), N (nucleoprotein), and NSs (nonstructural protein) viral proteins, strong CAT activity or GFP expression was observed. CAT activity was consistently stronger in cells expressing L plus N than following superinfection. No activity was seen without superinfection, nor was activity detected when either the L or N expression plasmid was omitted. Omitting NSs expression had no effect on CAT activity or GFP expression, indicating that this protein is not needed for viral RNA replication or transcription. CAT activity could be serially passaged to fresh cultures by transferring medium from CAT-expressing cells, indicating that recombinant virus containing the reporter construct had been produced. In summary, we demonstrate that the RNA pol I system, originally developed for influenza virus, which replicates in the nucleus, has strong potential for the development of an efficient reverse genetics system also for Bunyaviridae members, which replicate in the cytoplasm.

Functional L polymerase of La Crosse virus allows in vivo reconstitution of recombinant nucleocapsids

“…This is similar to the situation with the related BUNV (Dunn et al, 1995) and suggests that bunyavirus L and N have regulatory functions. In contrast, an approximately linear dose-dependency of minireplicon activity has been described for Uukuniemi phlebovirus (Flick & Pettersson, 2001), suggesting a less tight regulation of polymerase activity.…”

“…Genome fragments of most human pathogenic bunyaviruses have been cloned and sequenced previously (Elliott, 1996). Reverse genetic systems, however, have so far been established only for the human pathogenic RVFV (Lopez et al, 1995) and the less pathogenic BUNV (Dunn et al, 1995), Uukuniemi virus (Flick & Pettersson, 2001) and Toscana virus (Accardi et al, 2001). In fact, BUNV was the first negative-stranded RNA virus with a segmented genome to be reconstituted entirely from cDNA plasmids (Bridgen & Elliott, 1996).…”

Recombinant lymphocytic choriomeningitis virus expressing vesicular stomatitis virus glycoprotein

“…Pol-I-based vectors were first applied for influenza virus reverse genetics (24), including rescue of infectious influenza viruses (28). The application of a Pol-I-driven plasmid for the generation of a recombinant cytoplasmic negative-strand (NS) RNA virus, however, is novel and illustrates the wide potential of this approach so far exploited only for nuclear viruses (28) and MG of cytoplasmic viruses (11,29). It may represent an efficient alternative approach to rescue cytoplasmic NS RNA viruses including LCMV entirely from cDNA, although with potential limitations for viral genomes containing splicing signals.…”

A reverse genetics system for Borna disease virus

“…The 59-and 39-termini in our BDV MG correspond to those determined for BDV strain He80 Pleschka et al, 2001). We selected murine pol-Ip because it has been shown to work well in BHK-21 cells (Flick & Pettersson, 2001;Pinschewer et al, 2003), a cell line that is easy to transfect and supports BDV multiplication. Upon encapsidation of the primary pol I transcript, BDV polymerase reconstituted intracellularly is expected to use it as a template for synthesis of full-length anti-MG (aMG) RNA (replicate) and subgenomic CAT mRNA (transcript) (Fig.…”

“…For this purpose, the sequence of a BDV minigenome (MG) was cloned in the polymerase I (pol I) expression vector pRF42 (Flick & Pettersson, 2001). In the resulting pol I-MG construct ( Fig.…”