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. 2003 May;84(Pt 5):1207-1214.
doi: 10.1099/vir.0.18876-0.

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

Gjon Blakqori  1 , Georg Kochs  1 , Otto Haller  1 , Friedemann Weber  1
Affiliations

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

Gjon Blakqori et al. J Gen Virol. 2003 May.

Abstract

La Crosse virus (LACV), a member of the family Bunyaviridae, is the primary cause of paediatric encephalitis in the United States. In this study, a functional RNA polymerase (L) gene of LACV was cloned and a reverse genetics system established. A reporter minireplicon mimicking the viral genome was constructed by flanking the Renilla luciferase gene with the 3' and 5' noncoding regions of the genomic M segment. These noncoding regions serve as promoters for the viral polymerase. Both L and nucleocapsid (N) genes were expressed by means of T7 RNA polymerase, which was provided by the recombinant T7-expressing modified vaccinia virus Ankara. Renilla reporter activity in transfected cells reflected reconstitution of recombinant nucleocapsids by functional L and N gene products. Time-course experiments revealed a rapid increase in minireplicon activity from 10 to 18 h after the onset of L and N expression. Minireplicon activity was found to be dependent on the correct ratio of L to N plasmids, with too much of either construct resulting in downregulation. Furthermore, a specific inhibitory effect of LACV NSs protein on minireplicon activity was found. In passaging experiments using parental helper virions, it was demonstrated that the recombinant nucleocapsids are a useful model for transcription, replication and packaging of LACV.

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