title: Systems to establish bunyavirus genome replication in the absence of transcription.
abstract: The L polymerase of bunyaviruses replicates and transcribes the viral genome. While replication products are faithful copies of the uncapped genomic RNA, transcription products contain capped 5' extensions which had been cleaved from host cell mRNAs. For La Crosse virus (LACV; genus Orthobunyavirus), the nuclease responsible for host cell mRNA cleavage is located at the N terminus of the L protein, with an active site of five conserved amino acids (H34, D52, D79, D92, and K94) surrounding two Mn(2+) ions (J. Reguera, F. Weber, and S. Cusack, PLoS Pathog. 6:e1001101, 2010). Here, we present reverse genetics systems and L mutants enabling us to study bunyaviral genome replication in the absence of transcription. Transcription was evaluated with an enhanced minigenome system consisting of the viral polymerase L, nucleocapsid protein N, a negative-sense minigenome, and--to alleviate antiviral host responses--a dominant-negative mutant (PKRΔE7) of the antiviral kinase protein kinase R (PKR). The transcriptional activity was strongly reduced by mutation of any of the five key amino acids, and the H34K, D79A, D92A, and K94A LACV L mutants were almost entirely silent in transcription. The replication activity of the L mutants was measured by packaging of progeny minigenomes into virus-like particles (VLPs). All mutant L proteins except K94A retained full replication activity. To test the broader applicability of our results, we introduced the homolog of mutation D79A (D111A) into the L sequence of Rift Valley fever virus (RVFV; genus Phlebovirus). As for LACV D79A, the RVFV D111A was incapable of transcription but fully active in replication. Thus, we generated mutants of LACV and RVFV L polymerases that are specifically deficient in transcription. Genome replication by bunyavirus polymerases can now be studied in the absence of transcription using convenient reverse genetics systems.
authors.affiliation: Institute for Virology, Philipps-University Marburg, Marburg, Germany.
authors.forename: Carolin
authors.initials: C
authors.lastname: Klemm
authors.forename: Juan
authors.initials: J
authors.lastname: Reguera
authors.forename: Stephen
authors.initials: S
authors.lastname: Cusack
authors.forename: Florian
authors.initials: F
authors.lastname: Zielecki
authors.forename: Georg
authors.initials: G
authors.lastname: Kochs
authors.forename: Friedemann
authors.initials: F
authors.lastname: Weber
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000502:physiology*; D016679:Genome, Viral / Q000235:genetics*; D018062:La Crosse virus / Q000235:genetics*; D008156:Luciferases; D020125:Mutation, Missense / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D059386:Reverse Genetics / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012260:Ribonucleases / Q000235:genetics / Q000502:physiology*; D012296:Rift Valley fever virus / Q000235:genetics; D014158:Transcription, Genetic / Q000502:physiology; D014779:Virus Replication / Q000235:genetics / Q000502:physiology*
chemical_list: D017931:DNA Primers; D008156:Luciferases; D012321:DNA-Directed RNA Polymerases; D012260:Ribonucleases