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

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 39 and 59 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.

La Crosse Bunyavirus Nonstructural Protein NSs Serves To Suppress the Type I Interferon System of Mammalian Hosts

“…The eukaryotic expression plasmid pI.18-HA-LACV-NSs was constructed by ligating a PCR fragment encompassing the LACV NSs sequence with a 5Јterminal hemagglutinin (HA) tag into the cloning vector pI.18 (kindly provided by Jim Robertson, National Institute for Biological Standards and Control, Hertfordshire, United Kingdom). The T7 promoter-driven expression constructs pTM-LACV-L and pTM-LACV-N were described previously (5). The firefly luciferase (FF-Luc) reporter plasmid for monitoring IFN-␤ promoter activation (p-125Luc) was kindly provided by Takashi Fujita, Institute for Virus Research, Kyoto University, Japan (58).…”

Functional Analysis of the Noncoding Regions of the Uukuniemi Virus ( Bunyaviridae ) RNA Segments

“…The terminal nucleotides are genus specific and highly conserved, and because of their partial inverted complementarities they can form double-stranded regions leading to circular RNAs (17), providing the functional promoter region for interaction with the viral polymerase (12). The function of the remaining nucleotides of the NCRs is still not well understood, despite the fact that several reverse genetics and minigenome rescue systems for different bunyaviruses have recently been developed (3,4,7,10,11,13,18,24). However, an encapsidation site has been characterized within the 5Ј NCR of the S vRNA segment, using the Orthobunyavirus Bunyamwera as a model (21).…”

Rift Valley Fever Virus Nonstructural Protein NSs Promotes Viral RNA Replication and Transcription in a Minigenome System

“…Another method for viral protein expression is to infect cells with recombinant vaccinia viruses, each of which encodes a virus protein. Studies using minigenomes and other systems re-vealed that the coexpression of N and L proteins was required for the RNA synthesis of viruses that belong to the Bunyaviridae family (1,3,13,14,16,21).…”

“…The expressed minigenome RNA transcripts undergo RNA replication and transcription in the presence of coexpressed viral proteins or coinfected helper virus; the levels of reporter expression are a measure of the efficiency of minigenome RNA replication and transcription. Minigenome RNA transcripts are expressed by either host RNA polymerase I or T7 RNA polymerase, the latter of which is often provided by vaccinia virus (1,3,13) or Sindbis virus (31). The expression of viral proteins is accomplished by transfecting either an expression plasmid that carries a eukaryotic promoter sequence followed by a viral structural gene or a T7 expression plasmid carrying a T7 promoter sequence in place of the eukaryotic promoter of the former; in the latter case, vaccinia virus expressing T7 RNA polymerase is frequently used to provide T7 RNA polymerase and capping of expressed RNA transcripts.…”