Efficient cDNA-Based Rescue of La Crosse Bunyaviruses Expressing or Lacking the Nonstructural Protein NSs

Abstract

La Crosse virus (LACV) belongs to the Bunyaviridae family and causes severe encephalitis in children. It has a negative-sense RNA genome which consists of the three segments L, M, and S. We successfully rescued LACV by transfection of just three plasmids, using a system which was previously established for Bunyamwera virus (Lowen et al., Virology 330:493-500, 2004). These cDNA plasmids represent the three viral RNA segments in the antigenomic orientation, transcribed intracellularly by the T7 RNA polymerase and with the 3 ends trimmed by the hepatitis delta virus ribozyme. As has been shown for Bunyamwera virus, the antigenomic plasmids could serve both as donors for the antigenomic RNA and as support plasmids to provide small amounts of viral proteins for RNA encapsidation and particle formation. In contrast to other rescue systems, however, transfection of additional support plasmids completely abrogated the rescue, indicating that LACV is highly sensitive to overexpression of viral proteins. The BSR-T7/5 cell line, which constitutively expresses T7 RNA polymerase, allowed efficient rescue of LACV, generating approximately 10 8 infectious viruses per milliliter. The utility of this system was demonstrated by the generation of a wild-type virus containing a genetic marker (rLACV) and of a mutant with a deleted NSs gene on the S segment (rLACVdelNSs). The NSsexpressing rLACV formed clear plaques, displayed an efficient host cell shutoff, and was strongly proapoptotic. The rLACVdelNSs mutant, by contrast, exhibited a turbid-plaque phenotype and a less-pronounced shutoff and induced little apoptosis. Nevertheless, both viruses grew in Vero cells to similar titers. Our reverse genetics system now enables us to manipulate the genome of LACV in order to characterize its virulence factors and to develop potential vaccine candidates.

Interferon Antagonist NSs of La Crosse Virus Triggers a DNA Damage Response-like Degradation of Transcribing RNA Polymerase II

“…Aedes albopictus C6/36 were grown at 30°C without CO 2 in Leibowitz L-15 medium (Invitrogen) supplemented with 10% FCS, antibiotics, and 4% tryptose phosphate broth (Difco). The recombinant virus strains rLACV and rLACVdelNSs were grown and maintained as described previously (9). ␣-Amanitin and ZVAD-fluoromethyl ketone were purchased from AppliChem and Bachem, respectively, and cycloheximide, actinomycin D, doxorubicin, and MG132 were purchased from Sigma.…”

“…The resulting plasmid, which carries a 3ϫFLAG sequence tag upstream of the multiple cloning site, was designated pI.18/3ϫFLAG. To construct the expression vector pI.18/3ϫFLAG_LACV NSs, the NSs sequence was PCR-amplified from the LACV S segment rescue plasmid (9) by using a forward primer containing a 5Ј BamHI site (5Ј-GAG AGG ATC CTC GCA TCA ACA GGT GCA AAT GGA-3Ј) and a reverse primer containing an XhoI site (5Ј-GAC ACT CGA GCT AAA TAC CCA GAT AAT CTG TGG AT-3Ј). The PCR fragment was cloned into pI.18/3ϫFLAG via BamHI/XhoI sites present in both insert and vector.…”

“…A recombinant virus mutant unable to express NSs, rLACVdelNSs (9), was shown to be a strong inducer of IFN transcription both in cell culture and in vivo (10 -12). The corresponding NSs-expressing wt virus (rLACV), by contrast, efficiently suppressed transcriptional up-regulation of IFNs.…”

A Recombinant Chimeric La Crosse Virus Expressing the Surface Glycoproteins of Jamestown Canyon Virus Is Immunogenic and Protective against Challenge with either Parental Virus in Mice or Monkeys

“…A silent change, G4081A, was left in the L segment to allow for differentiation of wild-type and recombinant viruses (see Table S1 in the supplemental material). The subcloned fragments were reconstructed into full S, M, and L segments on separate plasmids with each genome segment flanked by a T7 polymerase promoter sequence and the hepatitis delta virus ribozyme as previously described (6,10). To generate an rLACV virus expressing the glycoproteins and NS M of JCV virus, the M ORF of LACV was replaced with that of JCV/61/CO-cl using standard molecular cloning techniques.…”

Deletion Mutants of Schmallenberg Virus Are Avirulent and Protect from Virus Challenge

“…Like the NSs protein of the prototype orthobunyavirus Bunyamwera virus (BUNV), La Crosse virus (LACV), or Akabane virus (AKAV), NSs of SBV was also found to be nonessential for virus viability in mammalian and insect cells (16)(17)(18)(19)(20). However, in infected mammalian cells, the NSs protein counteracts the shutoff of host cell protein synthesis and the induction of interferon (IFN) similar to BUNV (21), but it is also involved in other functions like regulation of translation, apoptosis, and viral polymerase activity (19,(22)(23)(24). In both in vitro and in vivo mouse experiments, NSs deletion mutants displayed an attenuated phenotype in IFN-competent cells and animals, but not in systems lacking the IFN receptor (16,25).…”