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 (PKR⌬E7) 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.

A Virus-Like Particle System Identifies the Endonuclease Domain of Crimean-Congo Hemorrhagic Fever Virus

“…The endonuclease domain of LACV (genus Orthobunyavirus) is situated at the N terminus of the L protein. Sequence alignments indicated a similar N-terminal endonuclease domain for three other Bunyaviridae genera, Phlebovirus, Hantavirus, and Tospovirus (32), and for phleboviruses, this was confirmed by functional data (21,22). For the genus Nairovirus (to which CCHFV belongs), however, the N terminus of the L protein harbors the so-called OTU domain, which has immune escape function (33).…”

“…1B and D). Experiments employing a neutralizing antibody showed that the GP-mediated boost (which was also observed for LACV and RVFV systems, though in a less extreme manner [18,22]) is due to reinfection of transfected cells by the generated VLPs (data not shown). Differently from the previously published minireplicon system (14), we obtained very little background activity by the minigenome plasmid if transfected without L polymerase (data not shown).…”

“…Compared to systems for other bunyaviruses like LACV and RVFV (18,22), the reporter activity of the CCHFV tc-VLPs is rather low (data not shown). One measure to obtain a robust VLP system was to employ the minireplicon with the stronger L promoter (Fig.…”

“…Protocols and primers for real-time reverse transcription-PCR (RT-PCR) were previously described (22).…”

“…The constructs encoding wild-type (wt) and RdRp-inactive CCHFV L polymerase (pCAGGS_V5_L_wt and pCAGGS_V5_L_⌬DD), CCHFV N nucleoprotein (pCAGGS_N), CCHFV-specific Gaussia luciferase minigenome (T7-vS-Gluc and T7-vL-Gluc), T7 polymerase (pCAGGS_T7), and negative-control protein (pcDNA3.1_3ϫFlag_ ⌬Mx) were described previously (14,22). The plasmids pGL3-luc and pRL-SV40, constitutively expressing firefly luciferase (FF-Luc) or Renilla luciferase (REN-Luc), respectively, were purchased from Promega.…”