Efficient cDNA-based rescue of La Crosse bunyaviruses expressing or lacking the nonstructural protein NSs - PubMed (original) (raw)
Efficient cDNA-based rescue of La Crosse bunyaviruses expressing or lacking the nonstructural protein NSs
Gjon Blakqori et al. J Virol. 2005 Aug.
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 NSs-expressing 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.
Figures
FIG. 1.
LACV M-induced cell fusion. (A) Huh7 cells were transfected with the eukaryotic expression plasmid pI.18-LACVM and plasmid pEGFP-C1, expressing the green fluorescent protein (GFP). At 16 h posttransfection, cells were treated with fusion buffer for 5 min and incubated for another 4 h in normal growth medium. Then, cells were fixed with paraformaldehyde and either permeabilized with 0.5% Triton X-100 (upper panel) or left unpermeabilized (lower panel). For analysis by indirect immunofluorescence, a monoclonal mouse antibody directed against LACV Gc was used (red), and GFP was detected in parallel (green). (B) Absence of fusion in the GFP control. Cells were transfected with pEGFP-C1, treated with fusion buffer, and permeabilized as described above. To visualize cell nuclei, chromosomal DNA was counterstained with To-Pro3 iodide (blue).
FIG. 2.
Generation of the full-length antigenomic LACV S plasmid. The backbone plasmid pT7riboGB can be cleaved between the T7 promoter and the hepatitis delta virus ribozyme with the enzyme Esp3I, generating noncompatible sticky ends. A DNA cassette which contains the LACV S segment-specific 5′ and 3′ promoter ends is inserted in the antisense orientation, flanking two BpiI sites separated by a KpnI spacer. Cleavage of the resulting shuttle plasmid, called pT7ribo-LACV-cSPro, with BpiI yields noncompatible sticky ends, which are used to insert the S segment open reading frame by using the attached BpiI sites. The asterisk indicates the first T7-transcribed G nucleotide, which is not encoded by LACV. The T7 promoter (T7 pro), T7 terminator (T7 term), hepatitis delta virus ribozyme (δ), viral 5′ and 3′ noncoding sequences, and S segment coding region are indicated by boxes and symbols. Only nucleotides and restriction sites which are relevant for the respective cloning steps are shown.
FIG. 3.
Rescue of rLACV. (A) Rescue kinetics. BSR-T7/5 cells grown in six-well dishes were transfected with 0.5 μg each of the antigenomic constructs pT7ribo-LACV-cL, pT7ribo-LACV-cM, and pT7ribo-LACV-cSnoEco. Supernatants were replaced every 24 h to determine virus titers. Data from two representative experiments are shown. (B) Genetic marker of rLACV. RNA from infected cells was reverse transcribed and a 789-bp fragment of the LACV S segment was amplified by PCR using primers LACVSProFW1 and LACVS3′_BpiI. EcoRI digestion of RT-PCR products from the parental nonrecombinant virus (LACV) yielded two fragments 572 bp and 217 bp in size, whereas the recombinant virus rLACV had a silent mutation which destroys the EcoRI recognition sequence.
FIG. 4.
Rescue of NSs-deleted rLACV. (A) Schematic representation of the NSs translational start site in the LACV S segment. The noncoding 5′- and 3′-end sequences are indicated by hatched boxes. The NSs reading frame is expressed from a +1-shifted reading frame within the N gene. The mutations introduced to ablate NSs translation without affecting the N gene are shown. (B) Rescue kinetics. BSR-T7/5 cells in six-well dishes were transfected with pT7ribo-LACV-cL, pT7ribo-LACV-cM, and pT7ribo-LACV-cNnoEco, and virus titers in the supernatants were determined as described for Fig. 3A. Data from two representative experiments are shown. (C) Genetic marker for rescued virus. EcoRI digestion of the RT-PCR product from rLACVdelNSs demonstrates the silent mutation used to distinguish recombinant viruses from the parental LACV, as described for Fig. 3B. (D) Genetic marker for NSs-mutated rLACV. The mutations introduced to destroy the NSs reading frame generate a new TaiI restriction site which is therefore only present in rLACVdelNSs. RNA from infected cells was reverse transcribed and a 338-bp fragment of the S segment was amplified by PCR using primers LACVSProFW1 and LACVS338RV. TaiI digestion of the RT-PCR product from rLACVdelNSs yields two fragments of 103 bp and 232 bp.
FIG. 5.
Initial phenotypic characterization. (A) Comparison of plaques produced by parental LACV, rLACV, and the rLACVdelNSs mutant. The inserts on the upper left are magnifications of single plaques. (B) Protein synthesis in infected cells. BHK cells were infected at 5 PFU per cell and labeled for 2 h with [35S]methionine/cysteine at 3 h or 23 h after infection, and cell extracts were analyzed by gel electrophoresis and autoradiography. The positions of the viral proteins Gc and N are indicated. (C) RNA accumulation analysis. A549 cells were infected at 5 PFU per cell and total RNA was extracted 16 h later. Using semiquantitative RT-PCR, the steady-state levels of LACV S segment RNA (upper panel), GAPDH (middle panel), and 45S rRNA (lower panel) were determined.
FIG. 6.
Viral growth in cell culture. Vero cells were infected with 0.01 PFU per cell of rLACV or rLACVdelNSs, and virus titers in the supernatants were determined by plaque assay at 24 h and 48 h postinfection.
FIG. 7.
Apoptosis of infected cells. (A) TUNEL assays. Huh7 cells were infected at 5 PFU per cell and fixed 20 h later. DNA breaks were visualized in single cells by labeling them with fluorescein-dUTP using terminal transferase (upper panel). Cells in parallel cultures were immunostained for LACV N protein to confirm infection. (B) DNA fragmentation analysis. BHK-21 cells were infected at 5 PFU per cell and harvested 24 h later. The low-molecular-weight DNA which appears as a result of genomic DNA breaks was isolated and separated on an agarose gel.
References
- Blakqori, G., G. Kochs, O. Haller, and F. Weber. 2003. Functional L polymerase of La Crosse virus allows in vivo reconstitution of recombinant nucleocapsids. J. Gen. Virol. 84:1207-1214. - PubMed
- Borucki, M. K., B. J. Kempf, B. J. Blitvich, C. D. Blair, and B. J. Beaty. 2002. La Crosse virus: replication in vertebrate and invertebrate hosts. Microbes Infect. 4:341-350. - PubMed
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