Efficient cDNA-Based Rescue of La Crosse Bunyaviru
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病菌学杂志 2005年第16期
Abteilung Virologie, Institut für Medizinische Mikrobiologie und Hygiene, Universit?t Freiburg, D-79008 Freiburg, Germany
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 108 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.
INTRODUCTION
La Crosse virus (LACV) is an important mosquito-borne pathogen in North America, causing encephalitis and aseptic meningitis in children and young adults (26, 32, 43). The typical symptoms of LACV infection are fever, headache, and neurological sequelae, and the disease can be mistaken for herpes simplex encephalitis or enteroviral meningitis (26). Around 75 to 100 cases requiring hospitalization are reported annually (9, 10), and up to 57% of these patients need to be admitted to the intensive care unit (26). Although the case fatality rate of LACV encephalitis is low, 10 to 15% of hospitalized patients will have severe neurological deficits (26, 27) which can last lifelong and cause significant economic costs (38). In addition, serological survey studies suggest that the majority of infections are subclinical, and it has been estimated that there may be more than 300,000 infections annually in the midwestern United States alone (8).
LACV belongs to the California serogroup of the genus Orthobunyavirus, family Bunyaviridae (3). Bunyaviruses are a large group of viruses which are mainly transmitted by arthropods (14). Some members are human pathogens and can cause encephalitis, febrile illnesses or hemorrhagic fevers, among them LACV, Oropouche virus, Hantaan virus, Rift Valley fever virus, and Crimean-Congo hemorrhagic fever virus (15, 41). Bunyaviruses are enveloped and have a trisegmented single-stranded RNA genome of negative or ambisense polarity, replicate in the cytoplasm, and bud into the Golgi apparatus. They encode four common structural proteins: the viral polymerase (L) on the large (L) segment, two glycoproteins (Gn and Gc) on the medium (M) segment, and the viral nucleocapsid protein (N) on the smallest (S) segment.
Viruses within some genera also encode nonstructural proteins, either on the M segment (termed NSm) or on the S segment (NSs). While the function of the NSm protein remains to be elucidated, we and others have recently shown that the NSs proteins of Bunyamwera virus (BUNV) and Rift Valley fever virus are important virulence factors. Although completely different in size and sequence, both NSs proteins suppress the production of interferons alpha and beta (IFN- and -?) in infected cells (1, 4, 39) by inhibiting host cell mRNA synthesis (24, 37). This allows the virus to escape the powerful innate immune system (42). Also, it has been shown that BUNV NSs inhibits IRF-3-mediated apoptosis (21), whereas overexpression of LACV NSs can induce apoptosis (11) and counteract small interfering RNA-induced RNA silencing (35).
The general features of bunyavirus transcription and RNA replication are similar to those of other negative-stranded RNA viruses (14). The genomic RNA segments contain noncoding sequences on both the 3' and 5' ends that serve as promoters for replication of the segment and transcription of the encoded reading frames. They are encapsidated by N protein and associate with L protein both intracellularly and in the virion, and only these ribonucleoprotein particles are functional templates for mRNA synthesis and RNA replication by the viral polymerase.
The ability to rescue a virus entirely from cloned cDNA plasmids is the prerequisite for targeted mutagenesis of the viral genome ("reverse genetics") (12, 29). The first rescue of a negative-strand RNA virus with a segmented genome was achieved for the bunyavirus BUNV (5). This rescue protocol involved infection of mammalian cells with a T7 RNA polymerase-expressing vaccinia virus and transfection of six T7-driven plasmids. Three of those plasmids (support plasmids) encoded the viral proteins, and the three other plasmids (antigenomic plasmids) served to generate plus-sense copies of the viral RNA segments. The artificial viral RNAs were encapsidated and packaged by the proteins expressed from the support plasmids, giving rise to recombinant virus particles. In a subsequent step, the vaccinia helper virus was removed by passaging through insect cells which only support growth of bunyaviruses.
Recently, the original protocol for BUNV was significantly improved by use of the T7 polymerase-expressing cell line BSR-T7/5 (7) instead of the vaccinia helper virus (25). This allowed recovery of BUNV directly from the transfected cells and without a subsequent insect cell passage. Interestingly, it was also found that in BSR-T7/5 cells the antigenomic plasmids provided low levels of support proteins which are sufficient for an efficient rescue (25). Thus, in contrast to other negative-strand RNA viruses (12, 29), recombinant BUNV can be generated by transfecting just the antigenomic plasmids.
The Bunyaviridae contain several members of medical and economic importance (15, 41). Nevertheless, plasmid-based reverse genetics systems have only been reported for the benign BUNV. Possibly, rescue of the other bunyaviruses requires experimental conditions which are not met by the protocols established so far. In this study, we adopted the helper virus-free approach of Lowen et al. (25) for the successful rescue of LACV. As for BUNV, transfection of the three antigenomic plasmids was sufficient. Surprisingly, however, cotransfection of support plasmids completely abrogated the rescue, indicating that the use of support plasmids may be a reason for the paucity of bunyavirus rescue systems. To demonstrate the utility of the LACV system, we also generated a virus which is deficient in expression of the nonstructural protein NSs. Using these genetically matched viruses, we studied the influence of LACV NSs on virus growth in cell culture and on host cell gene expression.
MATERIALS AND METHODS
Cells and viruses. Vero, BHK-21, Huh7, A549, and BSR-T7/5 cells were cultivated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum. BSR-T7/5 cells (7), which constitutively express the bacteriophage T7 RNA polymerase, were additionally provided with 1 mg/ml geneticin (G418). The parental LACV strain was kindly provided by Ramaswamy Raju, Meharry Medical College, Nashville, TN.
Plasmid constructs. All plasmids were generated using standard molecular cloning techniques. PCR was carried out with AccuPrime Pfx DNA Polymerase (Invitrogen; for cloning purposes) or Taq DNA polymerase (Eppendorf; for diagnostic purposes and addition of single adenosines for TA cloning). TA cloning was done using the pcDNA3.1/V5-His TOPO TA expression kit (Invitrogen) according to the manufacturer's instructions. The preparation of LACV cDNA has been described previously (2). For primer sequences used in the subsequent cloning procedures, see Table 1.
The LACV M segment coding region was assembled from three overlapping TA-cloned fragments (encoded on pTOPO-LACVM1, pTOPO-LACVM2, and pTOPO-LACVM3). These fragments were generated by PCR using primer pairs LACVM5'_BpiI/LACVM1945RV, LACVM1286FW/LACVM3852RV, and LACVM2597FW/LACVM3'_BpiI, respectively. The fragments were consecutively cloned into plasmid pTM1 (28) using the restriction sites for BamHI and XhoI (LACVM1), BstZ17I and XhoI (LACVM3), and BstZ17I (LACVM2), resulting in plasmid pTM-LACVM, which contains the full-length M segment coding region flanked by primer-encoded BpiI sites and pTM1-encoded BamHI and XhoI sites. The open reading frame was further subcloned into the eukaryotic high-level expression plasmid pI.18 (kindly provided by Jim Robertson, National Institute for Biological Standards and Control, Hertfordshire, United Kingdom), using the restriction enzymes BamHI and XhoI (yielding plasmid pI.18-LACVM) and finally into pT7cLACVMPro (see below), using enzyme BpiI, to generate the full-length antigenome-encoding plasmid pT7ribo-LACV-cM.
The backbone plasmid for expression of the viral antigenome, pT7riboGB, was generated by PCR amplification of plasmid pT7ribo (5) using primers pT7riboGB5' and pT7riboGB3'. These primers are designed to replace the original SmaI and StuI cloning sites of pT7ribo with two Esp3I restriction sites, thereby allowing exact sticky-end insertion of viral cDNA sequences between the T7 promoter and the hepatitis delta virus ribozyme. To religate the linear PCR-amplified plasmid, the PCR fragment was digested with KpnI, whose recognition sequence was contained within the PCR primers, resulting in complementary 5' and 3' ends. The cleaved DNA was agarose gel purified, religated, and transformed into competent Escherichia coli. Successful insertion of the new Esp3I cloning sites was confirmed by DNA sequencing.
For construction of the precursor plasmids pT7ribo-LACV-cMPro (M segment) and pT7ribo-LACV-cLPro (L segment), overlapping oligonucleotides LACVMProFW1, LACVMProFW2, LACVMProRV1, and LACVMProRV2 (plasmid pT7ribo-LACV-cMPro) or LACVLProFW1, LACVLProFW2, LACVLProRV1, and LACVLProRV2 (plasmid pT7ribo-LACV-cLPro) were used in a PCR that gave rise to a fragment containing the 5' and 3' noncoding regions of the respective LACV antigenomic segment, linked by a sequence which encodes two flanking BpiI restriction sites and one central KpnI site. The PCR products were subcloned into the TA cloning vector pcDNA3.1/V5-His TOPO (Invitrogen), and several plasmid clones were sequenced. Inserts with correct 5' and 3' ends were recovered by digestion at the primer-encoded Esp3I sites and cloned into partially Esp3I-digested pT7riboGB. The partial digests were necessary because of two additional Esp3I sites in the pT7ribo backbone. They were accomplished by incubating 5 μg DNA with 3 units enzyme for 30 min and subsequent dephosphorylation with shrimp alkaline phosphatase (Roche).
A slightly different approach was used for the construction of the S segment plasmid pT7ribo-LACV-cSPro. Due to the length of the noncoding sequences of the LACV S segment, the first PCR with primers LACVSProFW2, LACVSProFW3, LACVSProRV2, and LACVSProRV3 was followed by a second one using the amplicon of the first PCR as the template together with primers LACVSProFW1 and LACVSProRV1. The primer-encoded linker sequence allows the exact insertion of viral DNA sequences between the segment-specific 5' and 3' noncoding regions upon cleavage at the BpiI sites.
For the generation of constructs which encode the full-length LACV segments in antigenomic orientation, the open reading frames of the S, M, and L segments were cloned into their respective promoter-harboring plasmids via BpiI restriction sites. The two-step cloning strategy (first step, insertion of 5' and 3' noncoding sequences; second step, insertion of the open reading frame) is outlined in Fig. 2. In the case of the S segment, the open reading frame was PCR amplified from viral cDNA using primers LACVS5'_BpiI and LACVS3'_BpiI, cleaved with enzyme BpiI, and cloned into BpiI-digested pT7ribo-LACV-cSPro. This resulted in plasmid pT7ribo-LACV-cS, which was subsequently subjected to two primer-directed mutagenesis reactions (QuikChange; Stratagene).
The first PCR with primers LACVSnoEcoFW and LACVSnoEcoRV erased the single EcoRI restriction site located on the LACV S segment by changing the A nucleotide at position 573 to G. The absence of the EcoRI site in the resulting plasmid, pT7ribo-LACV-cSnoEco, did not change the amino acid sequence and served only as a molecular marker to distinguish between parental and recombinant viruses. A second PCR with primers LACVdelNSsFW and LACVdelNS sRV introduced three point mutations which turn the two start codons of the NSs reading frame into threonine codons and the following serine codon into a stop codon. This resulted in plasmid pT7ribo-LACV-cNnoEco, which lacks the EcoRI site and encodes only a functional N protein because it has no NSs protein translation initiation codons (compare Fig. 4A). The generation of plasmid pT7ribo-LACV-cM has been described above. Analogous to the M segment, the LACV L segment open reading frame was cloned into pTM1 and subsequently inserted into plasmid pT7ribo-LACV-cLPro using enzyme BpiI. This open reading frame was modified to equal the consensus amino acid sequence of GenBank entries 22256023, 22212781, and 562069.
LACV glycoprotein fusion assay. Huh7 cells grown on coverslips were cotransfected with 1 μg pI.18-LACVM and 0.5 μg pEGFP-C1 (Clontech) using 4 μl DAC-30 transfection reagent (Eurogentec) in 200 μl serum-free medium (OptiMEM; Gibco-BRL). At 16 h posttransfection, the DMEM was replaced with fusion buffer (Earle's balanced salt solution without sodium bicarbonate, 10 mM HEPES, 0.2% bovine serum albumin, pH 5.5) for 5 min (19). Cells were washed three times with phosphate-buffered saline and incubated in DMEM with 10% fetal calf serum for another 4 h at 37°C. Then, cells were fixed with 3% paraformaldehyde and permeabilized with 0.5% Triton X-100 in phosphate-buffered saline. In a parallel experiment, permeabilization was omitted. Cells were analyzed by immunofluorescence for LACV Gc or green fluorescent protein (GFP) expression as well as for the formation of syncytia. LACV Gc protein was detected with a monoclonal mouse antibody (diluted 1:400) which was kindly provided by Francisco Gonzáles-Scarano (University of Pennsylvania School of Medicine, Philadelphia). In GFP-transfected control cells, nuclei were visualized by the DNA staining agent To-Pro3 iodide (diluted 1:1,000).
Rescue of recombinant LACV. Subconfluent layers of BSR-T7/5 cells were grown on six-well plates. Each well was transfected with 0.5 μg of plasmids pT7ribo-LACV-cM, pT7ribo-LACV-cL and pT7Ribo-LACV-cSnoEco (for rLACV) or pT7ribo-LACV-cNnoEco (for rLACVdelNSs), using 5 μl DAC-30 transfection reagent in 200 μl serum-free medium. For 5 days, supernatants were collected after 24-h intervals and the cells were provided with fresh DMEM. In order to screen for the presence of recombinant virus, Vero cells grown on six-well plates were inoculated for 1 h with 200 μl/well of day 4 supernatants at 37°C and then observed for the following days. Appearance of a cytopathic effect on these cells indicated successful rescue of LACV. To evaluate the rescue kinetics of transfected cells, titers (PFU/ml) of collected supernatants were determined by plaque assay on Vero cells.
Plaque assay. Vero cells grown on six-well plates to 90% confluence were inoculated with 10-fold serial dilutions of supernatants from transfected BSR-T7/5 cells or infected Vero cells in DMEM with 2% fetal calf serum. After 1 h incubation at 37°C, the inoculum was removed and the cells were overlaid with 3 ml DMEM with 2% fetal calf serum, 0.02% DEAE-dextran, and 0.3% Noble agar (Difco) and further incubated for 72 h at 37°C. Cells were fixed and stained with 1% crystal violet, 3.6% formaldehyde, 1% methanol, and 20% ethanol, and titers were calculated from the plaque numbers according to the dilution.
Metabolic labeling of infected and noninfected cells. Infected BHK-21 cells were incubated with [35S]methionine/cysteine (Amersham) at different time points to label newly synthesized proteins. The label process was preceded by three washing steps with phosphate-buffered saline, followed by a 1-hour starvation period with DMEM lacking methionine and cysteine. Then, cells were incubated with 200 μl DMEM lacking methionine and cysteine and supplemented with 50 μCi [35S]methionine/cysteine mix for another 2 h. The cells were lysed in 200 μl lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 25 U/ml Benzonase [Novagen]) and 10 μl of the lysates were separated on a 20% sodium dodecyl sulfate-polyacrylamide gel. The gel was dried on a Whatman paper and radiolabeled proteins were visualized on BioMax MR film (Kodak).
RT-PCR analyses. A549 cells were infected for 16 h at 5 PFU per cell and total RNA was extracted. For reverse transcription (RT), 1 μg of DNase I-treated RNA was incubated with 200 U of Superscript II reverse transcriptase (Gibco/BRL) and 100 ng random hexanucleotides in 20 μl of 1'RT buffer (Gibco/BRL) supplied with 1 mM each of the four deoxynucleotide triphosphates, 20 U of RNasin, and 10 mM dithiothreitol. The resulting cDNA was diluted 100-fold and amplified by 30 cycles of PCR, with each cycle consisting of 1 min at 95°C, 1 min at 58°C, and 1 min at 72°C, followed by 9 min at 72°C.
The upstream and downstream primers for amplifying RNA sequences were 5'-GAC AGC TAG CAT GTC GGA TTT GGT GTT TT-3' and 5'-GAC AGC GGC CGC TTA TGG AAG CCT GAT GCC AAA TTT CTG-3'(LACV S segment), 5'-GCA TGG CCT TCC GTG TCC-3' and 5'-CCA GCC CCA GCG TCA AAG GTG-3'(glyceraldehyde-3-phosphate dehydrogenase), and 5'-CGG CTA CCA CAT CCA AGG AA-3' and 5'-GCT GGA ATT ACC GCG GCT-3'(45S rRNA), respectively.
TUNEL assay. DNA strand breaks in the nuclei of cells undergoing apoptosis were visualized with the In Situ Cell Death Detection kit (Roche). Subconfluent Huh7 cells were infected at 5 PFU per cell and fixed 20 h postinfection with 3% paraformaldehyde. The cells were permeabilized with phosphate-buffered saline containing 0.1% Triton X-100 and 0.1% sodium citrate and then incubated with 50 μl terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) reaction mixture for 1 h at 37°C. Incorporation of fluorescein-labeled dUTP was analyzed by fluorescence microscopy. Complete infection of the tissue culture was demonstrated by immunofluorescence in simultaneously infected cells using an anti-LACV-nucleoprotein antibody (diluted 1:1,000) as described above.
DNA fragmentation analysis. BHK-21 cells were infected at 5 PFU per cell and 24 h later low-molecular-weight DNA was isolated following the protocol described by Hinshaw et al. (18). The DNA was eluted in 20 μl Tris-EDTA buffer containing 1 μg/μl RNase A, incubated for 1 h at 37°C, and then separated on a 2% agarose gel.
RESULTS AND DISCUSSION
Cloning of a biologically active M segment cDNA. We have previously described a minireplicon system for LACV and demonstrated the functionality of the cloned S and L segment gene products (2). The next step towards a complete rescue system was the cloning of an M segment cDNA. The M segment of LACV encodes a polyprotein which is cotranslationally processed to the proteins Gn, NSm, and Gc. To isolate a cDNA for this polyprotein, we amplified by proofreading RT-PCR three overlapping fragments spanning the entire coding sequence, using RNA from LACV-infected cells as the template. The PCR products were assembled into a full-length cDNA and inserted into a eukaryotic expression vector (see Materials and Methods).
We tested for the ability of the M cDNA-encoded glycoproteins to mediate pH-dependent cellular fusion (19). Huh7 cells were transfected with the cDNA construct, treated with fusion buffer to induce a pH shift to 5.5, and monitored for the formation of syncytia by immunofluorescence microscopy. For easy detection of transfected cells, a GFP expression plasmid was cotransfected. Figure 1A (upper panel) shows that cells containing GFP and LACV M form syncytia after the pH shift. By contrast, cells expressing just GFP do not fuse under these conditions (Fig. 1B). To confirm authentic processing of the recombinant LACV glycoproteins, we investigated their transport to the cell surface. To this end, cells were left unpermeabilized by omitting Triton X-100 treatment during the fixation procedure. As shown in Fig. 1A (lower panel), faint but specific staining by the anti-LACV Gc antibody was obtained on the surface of the fused cells. Since the intracellular translocation of the orthobunyavirus Gc protein is dependent on the Gn protein (34), we conclude that both recombinant viral glycoproteins are correctly expressed and transported to the cell surface.
Plasmids for the expression of full-length antigenomic RNAs. The protocols for rescue of BUNV involved blunt-end cloning of a cDNA cassette into the T7 expression vector pT7ribo (5, 25). Whereas the 3' ends of pT7ribo transcripts are correctly trimmed by the hepatitis delta virus ribozyme, the 5' ends contain two additional G nucleotides (5). The T7 polymerase initiates predominantly with an encoded G nucleotide at position +1 (23), but the 5'-terminal sequences of bunyavirus antigenomes start with A or U (14). Thus, the two extra G nucleotides at the 5' ends of the artificial antigenomes may reduce recognition by the LACV polymerase.
We therefore constructed a rescue vector in which cDNA fragments can be inserted via sticky ends and in which the number of nonviral G nucleotides at the 5' end is reduced to one (see Materials and Methods). Basically, we followed the strategy described by Flick and Petterson for constructing a bunyavirus minireplicon (16). The multistep cloning strategy is outlined for the antigenomic S segment plasmid in Fig. 2. A derivative of pT7ribo which contained sites for the restriction enzyme Esp3I was constructed. These restriction sites were used to introduce a short DNA cassette which contained the segment-specific promoter ends of LACV in the antigenomic orientation, flanking a linker with two BpiI sites. In a final step, BpiI digestion served to introduce the coding sequence of the respective LACV segments. The resulting plasmids allow T7-mediated synthesis of viral segments with correct 3' ends and one extra G nucleotide at the 5' end.
Following this strategy, we cloned the newly isolated LACV M cDNA as well as consensus LACV S and LACV L cDNAs as full-length antigenomic sequences into the modified pT7ribo, resulting in plasmids pT7ribo-LACV-cL, pT7ribo-LACV-cM, and pT7cLACV-cSnoEco. In addition, we constructed support plasmids for the viral proteins, using the T7-driven expression vector pTM1 which contains an internal ribosome entry site for efficient translation of the transcribed RNAs (see Materials and Methods).
Rescue of recombinant La Crosse virus. In analogy to the protocol shown to improve the rescue of BUNV (22, 25), we attempted to recover recombinant LACV in a helper virus-free system which is based on the BSR-T7/5 cell line. Initially, we transfected cells in six-well plates either with the full set of antigenomic and support plasmids or with just the three antigenomic plasmids. On day 4 posttransfection, supernatants were collected and aliquots were transferred to Vero cells to monitor development of a cytopathic effect. As shown in Table 2, the three-plasmid approach resulted in more than 50% positive dishes, indicating efficient and reproducible production of recombinant LACV (rLACV). Strikingly, the addition of support plasmids completely abrogated the rescue. These findings suggest that, as for BUNV, the antigenomic cDNA constructs can be translated and are therefore sufficient for recovery of LACV. In contrast to BUNV, however, additional expression of viral proteins from support plasmids was inhibitory, indicating that the intracellular replication of LACV is highly sensitive to elevated levels of viral proteins. In line with this, we have previously shown that overexpression of either L or N results in downregulation of LACV polymerase activity (2), whereas for BUNV it was shown that overexpression of L or N appears to be much better tolerated (13, 40).
To characterize the three-plasmid rescue of LACV in more detail, we measured the development of virus titers in the supernatants of transfected cells for 5 days. The rescue kinetics of two representative experiments are shown in Fig. 3A. Typically, infectious virus appeared 48 h posttransfection, and titers increased to up to 108 PFU/ml after 3 more days. To verify that the virus was derived from the transfected cDNAs, we had introduced a silent mutation into the S segment, destroying an EcoRI site at position 572. RNA was extracted from infected cells, and a DNA fragment containing the mutated site was amplified by RT-PCR. Figure 3B shows that rLACV indeed contained the introduced mutation, whereas the parental virus (LACV) had the EcoRI site, as expected. Thus, taken together, the experimental procedures employing just the three antigenomic constructs and the BSR-T7/5 cell line stably expressing the T7 polymerase resulted in an efficient rescue of recombinant LACV.
Rescue of La Crosse virus with a deleted NSs gene. To test whether our rescue system can be used for introduction of functionally important mutations into the LACV genome, we generated a mutant virus with an inactivated NSs gene. Because the N and NSs reading frames overlap (14), complete deletion of the NSs gene is not possible without affecting the N gene. We therefore applied the strategy which was used for abrogation of BUNV NSs gene expression (6) and changed the tandem ATG start codons of NSs to ACG and the third codon to a stop codon (see Fig. 4A). The resulting antigenomic LACV S construct, pT7ribo-LACV-cNnoEco, expresses an unaltered N protein but no NSs.
For virus rescue, BSR-T7/5 cells were transfected with the plasmids pT7ribo-LACV-cL, pT7ribo-LACV-cM, and the mutant construct pT7ribo-LACV-cNnoEco as outlined above. Time course titrations (Fig. 4B) indicated that rescue of the mutant virus, which we termed rLACVdelNSs, occurred with an efficiency similar to that for rescue of the wild-type virus (compare Fig. 3A). To establish that rLACVdelNSs is derived from the cDNA plasmids, we confirmed the absence of the EcoRI site on the S segment (Fig. 4C). Furthermore, since mutagenesis of the NSs start site created a unique recognition site for the restriction enzyme TaiI, we were able to distinguish wild-type and delNSs mutant S segment by TaiI restriction analysis (Fig. 4D). The results of those analyses confirmed the rescue of a recombinant LACV containing the desired rLACVdelNSs mutation in the S segment.
Phenotype of the recombinant viruses in cell culture. We performed an initial phenotypic characterization of the rescued viruses. With respect to plaque formation on Vero cells, rLACV was indistinguishable from the parental wild-type LACV, as expected (Fig. 5A). rLACVdelNSs, by contrast, exhibited a turbid-plaque phenotype, whereas the overall plaque size was not strikingly different from that of the NSs-expressing viruses. This is in contrast to the NSs-deficient mutant of BUNV, which was found to form significantly smaller plaques than wild-type BUNV (6). However, subsequent analyses revealed that plaque size differences also occur for different wild-type BUNV strains and are the result of promoter variations rather than the absence of NSs (20).
Infection with bunyaviruses is known to induce a strong shutoff of host cell protein synthesis (31, 33). To investigate this for the recombinant viruses, BHK cells were infected at 5 PFU per cell and labeled with [35S]methionine/cysteine at early (3 h) and late (23 h) time points after infection, and cell extracts were analyzed by gel electrophoresis and autoradiography. Both rLACV and the parental LACV induce a significant shutoff of host cell protein synthesis at 23 h postinfection (Fig. 5B, lanes 1 to 4). The delNSs virus, by contrast, displayed a weaker shutoff, although a considerable reduction in host cell protein synthesis still occurs, as is evident by the comparison with mock-infected cells (Fig. 5B, lanes 5 to 8). Thus, as with BUNV (6), the shutoff imposed by LACV appears to consist of two different components, the one caused by the NSs protein and a second, not yet characterized mechanism.
We have recently shown that the NSs protein of BUNV inhibits phosphorylation of the C-terminal domain of RNA polymerase II, thus aborting transcription of host cell mRNAs (37). In a similar but not identical way, the NSs protein of Rift Valley fever virus blocks host cell mRNA synthesis (1, 4) by preventing the assembly of TFIIH, an essential RNA polymerase II transcription factor (24). To investigate this for LACV NSs, we assessed the accumulation of cellular RNAs in infected cells, using semiquantitative RT-PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA and 45S rRNA were used as markers for RNA polymerase II and RNA polymerase I transcripts, respectively. As shown in Fig. 5C, infection with rLACV strongly reduced the amount of GAPDH mRNA but not of 45S rRNA, whereas the delNSs mutant had only a slight impact on the GAPDH mRNA. This mirrors the effects of rLACV and rLACVdelNSs on host cell protein synthesis (see Fig. 5B) and indicates that a major part of the LACV NSs-induced shutoff is due to inhibition of RNA polymerase II. Thus, similar to the NSs proteins of BUNV and Rift Valley fever virus, the NSs of LACV appears to be able to downregulate host cell RNA polymerase II.
To evaluate whether the lack of NSs-induced shutoff has consequences for virus replication, we compared the growth of rLACV and rLACVdelNSs in cell culture. For BUNV it was previously shown that the rLACVdelNSs mutant grows in Vero cells to titers about 1 log lower than the wild-type virus (36). To investigate this for LACV, Vero cells were infected with 0.01 PFU per cell, and virus titers in the supernatants were determined at 24 h and 48 h postinfection. Surprisingly, despite the differences in plaque phenotype and host cell shutoff (see Fig. 5), both viruses replicated with similar efficiencies (Fig. 6). This indicates that, at least in Vero cells, NSs does not confer a significant growth advantage for LACV. In line with this, we found that rLACV is not able to outcompete rLACVdelNSs in coinfection studies (data not shown).
It is known that BUNV NSs has the ability to block apoptosis induction by the transcription factor IRF-3 (21). LACV, by contrast, is strongly apoptogenic (30), possibly due to NSs (11). LACV kills cells faster, replicates faster, and reaches higher titers than BUNV (our unpublished observations). Therefore, the possibility exists that, unlike BUNV, the fast-growing LACV does not delay programmed cell death but rather enhances it by NSs. To clarify this, we monitored the induction of apoptosis by the recombinant viruses. Cells were infected and observed for the appearance of DNA breaks, a hallmark of apoptosis, using TUNEL assays and DNA fragmentation analysis. Figure 7A shows that many more TUNEL-positive cells were detectable with rLACV than with rLACVdelNSs. Consequently, a strong fragmentation of the cellular DNA occurred in rLACV-infected cells, whereas rLACVdelNSs-infected cells did not develop DNA fragmentation (Fig. 7B). Thus, we can conclude that, unlike the NSs of BUNV (21), the NSs of LACV indeed promotes apoptosis, as previously observed in overexpression studies (11).
The need for reverse genetics systems to study the biology of highly virulent bunyaviruses is generally recognized (17, 35). Here, we have adopted the three-plasmid system developed for BUNV (25) to rescue the human-pathogenic LACV. Cotransfection of support plasmids, which is a common practice in reverse genetics systems (12, 29), was found to be absolutely detrimental for LACV rescue. Such an untypical requirement for virus rescue may explain the absence of reverse genetics systems for other members of the Bunyaviridae family. Our rescue system now enables us to manipulate the genome of LACV in order to characterize its virulence factors and develop potential vaccine candidates.
ACKNOWLEDGMENTS
We are indebted to Karl-Klaus Conzelmann, Richard Elliott, Francisco Gonzáles-Scarano, Ramaswamy Raju, and Jim Robertson for kindly providing reagents which were essential for this work. We thank Otto Haller for support and helpful comments, Georg Kochs, Alain Kohl, Christine Krempl, Martin Spiegel, and Peter Staeheli for critically reading the manuscript, and Richard Elliott for sharing information on Bunyamwera virus rescue prior to publication.
Work in the authors' laboratory is supported by grants WE 2616/2-1 and WE 2616/2-2 from the Deutsche Forschungsgemeinschaft.
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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 108 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.
INTRODUCTION
La Crosse virus (LACV) is an important mosquito-borne pathogen in North America, causing encephalitis and aseptic meningitis in children and young adults (26, 32, 43). The typical symptoms of LACV infection are fever, headache, and neurological sequelae, and the disease can be mistaken for herpes simplex encephalitis or enteroviral meningitis (26). Around 75 to 100 cases requiring hospitalization are reported annually (9, 10), and up to 57% of these patients need to be admitted to the intensive care unit (26). Although the case fatality rate of LACV encephalitis is low, 10 to 15% of hospitalized patients will have severe neurological deficits (26, 27) which can last lifelong and cause significant economic costs (38). In addition, serological survey studies suggest that the majority of infections are subclinical, and it has been estimated that there may be more than 300,000 infections annually in the midwestern United States alone (8).
LACV belongs to the California serogroup of the genus Orthobunyavirus, family Bunyaviridae (3). Bunyaviruses are a large group of viruses which are mainly transmitted by arthropods (14). Some members are human pathogens and can cause encephalitis, febrile illnesses or hemorrhagic fevers, among them LACV, Oropouche virus, Hantaan virus, Rift Valley fever virus, and Crimean-Congo hemorrhagic fever virus (15, 41). Bunyaviruses are enveloped and have a trisegmented single-stranded RNA genome of negative or ambisense polarity, replicate in the cytoplasm, and bud into the Golgi apparatus. They encode four common structural proteins: the viral polymerase (L) on the large (L) segment, two glycoproteins (Gn and Gc) on the medium (M) segment, and the viral nucleocapsid protein (N) on the smallest (S) segment.
Viruses within some genera also encode nonstructural proteins, either on the M segment (termed NSm) or on the S segment (NSs). While the function of the NSm protein remains to be elucidated, we and others have recently shown that the NSs proteins of Bunyamwera virus (BUNV) and Rift Valley fever virus are important virulence factors. Although completely different in size and sequence, both NSs proteins suppress the production of interferons alpha and beta (IFN- and -?) in infected cells (1, 4, 39) by inhibiting host cell mRNA synthesis (24, 37). This allows the virus to escape the powerful innate immune system (42). Also, it has been shown that BUNV NSs inhibits IRF-3-mediated apoptosis (21), whereas overexpression of LACV NSs can induce apoptosis (11) and counteract small interfering RNA-induced RNA silencing (35).
The general features of bunyavirus transcription and RNA replication are similar to those of other negative-stranded RNA viruses (14). The genomic RNA segments contain noncoding sequences on both the 3' and 5' ends that serve as promoters for replication of the segment and transcription of the encoded reading frames. They are encapsidated by N protein and associate with L protein both intracellularly and in the virion, and only these ribonucleoprotein particles are functional templates for mRNA synthesis and RNA replication by the viral polymerase.
The ability to rescue a virus entirely from cloned cDNA plasmids is the prerequisite for targeted mutagenesis of the viral genome ("reverse genetics") (12, 29). The first rescue of a negative-strand RNA virus with a segmented genome was achieved for the bunyavirus BUNV (5). This rescue protocol involved infection of mammalian cells with a T7 RNA polymerase-expressing vaccinia virus and transfection of six T7-driven plasmids. Three of those plasmids (support plasmids) encoded the viral proteins, and the three other plasmids (antigenomic plasmids) served to generate plus-sense copies of the viral RNA segments. The artificial viral RNAs were encapsidated and packaged by the proteins expressed from the support plasmids, giving rise to recombinant virus particles. In a subsequent step, the vaccinia helper virus was removed by passaging through insect cells which only support growth of bunyaviruses.
Recently, the original protocol for BUNV was significantly improved by use of the T7 polymerase-expressing cell line BSR-T7/5 (7) instead of the vaccinia helper virus (25). This allowed recovery of BUNV directly from the transfected cells and without a subsequent insect cell passage. Interestingly, it was also found that in BSR-T7/5 cells the antigenomic plasmids provided low levels of support proteins which are sufficient for an efficient rescue (25). Thus, in contrast to other negative-strand RNA viruses (12, 29), recombinant BUNV can be generated by transfecting just the antigenomic plasmids.
The Bunyaviridae contain several members of medical and economic importance (15, 41). Nevertheless, plasmid-based reverse genetics systems have only been reported for the benign BUNV. Possibly, rescue of the other bunyaviruses requires experimental conditions which are not met by the protocols established so far. In this study, we adopted the helper virus-free approach of Lowen et al. (25) for the successful rescue of LACV. As for BUNV, transfection of the three antigenomic plasmids was sufficient. Surprisingly, however, cotransfection of support plasmids completely abrogated the rescue, indicating that the use of support plasmids may be a reason for the paucity of bunyavirus rescue systems. To demonstrate the utility of the LACV system, we also generated a virus which is deficient in expression of the nonstructural protein NSs. Using these genetically matched viruses, we studied the influence of LACV NSs on virus growth in cell culture and on host cell gene expression.
MATERIALS AND METHODS
Cells and viruses. Vero, BHK-21, Huh7, A549, and BSR-T7/5 cells were cultivated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum. BSR-T7/5 cells (7), which constitutively express the bacteriophage T7 RNA polymerase, were additionally provided with 1 mg/ml geneticin (G418). The parental LACV strain was kindly provided by Ramaswamy Raju, Meharry Medical College, Nashville, TN.
Plasmid constructs. All plasmids were generated using standard molecular cloning techniques. PCR was carried out with AccuPrime Pfx DNA Polymerase (Invitrogen; for cloning purposes) or Taq DNA polymerase (Eppendorf; for diagnostic purposes and addition of single adenosines for TA cloning). TA cloning was done using the pcDNA3.1/V5-His TOPO TA expression kit (Invitrogen) according to the manufacturer's instructions. The preparation of LACV cDNA has been described previously (2). For primer sequences used in the subsequent cloning procedures, see Table 1.
The LACV M segment coding region was assembled from three overlapping TA-cloned fragments (encoded on pTOPO-LACVM1, pTOPO-LACVM2, and pTOPO-LACVM3). These fragments were generated by PCR using primer pairs LACVM5'_BpiI/LACVM1945RV, LACVM1286FW/LACVM3852RV, and LACVM2597FW/LACVM3'_BpiI, respectively. The fragments were consecutively cloned into plasmid pTM1 (28) using the restriction sites for BamHI and XhoI (LACVM1), BstZ17I and XhoI (LACVM3), and BstZ17I (LACVM2), resulting in plasmid pTM-LACVM, which contains the full-length M segment coding region flanked by primer-encoded BpiI sites and pTM1-encoded BamHI and XhoI sites. The open reading frame was further subcloned into the eukaryotic high-level expression plasmid pI.18 (kindly provided by Jim Robertson, National Institute for Biological Standards and Control, Hertfordshire, United Kingdom), using the restriction enzymes BamHI and XhoI (yielding plasmid pI.18-LACVM) and finally into pT7cLACVMPro (see below), using enzyme BpiI, to generate the full-length antigenome-encoding plasmid pT7ribo-LACV-cM.
The backbone plasmid for expression of the viral antigenome, pT7riboGB, was generated by PCR amplification of plasmid pT7ribo (5) using primers pT7riboGB5' and pT7riboGB3'. These primers are designed to replace the original SmaI and StuI cloning sites of pT7ribo with two Esp3I restriction sites, thereby allowing exact sticky-end insertion of viral cDNA sequences between the T7 promoter and the hepatitis delta virus ribozyme. To religate the linear PCR-amplified plasmid, the PCR fragment was digested with KpnI, whose recognition sequence was contained within the PCR primers, resulting in complementary 5' and 3' ends. The cleaved DNA was agarose gel purified, religated, and transformed into competent Escherichia coli. Successful insertion of the new Esp3I cloning sites was confirmed by DNA sequencing.
For construction of the precursor plasmids pT7ribo-LACV-cMPro (M segment) and pT7ribo-LACV-cLPro (L segment), overlapping oligonucleotides LACVMProFW1, LACVMProFW2, LACVMProRV1, and LACVMProRV2 (plasmid pT7ribo-LACV-cMPro) or LACVLProFW1, LACVLProFW2, LACVLProRV1, and LACVLProRV2 (plasmid pT7ribo-LACV-cLPro) were used in a PCR that gave rise to a fragment containing the 5' and 3' noncoding regions of the respective LACV antigenomic segment, linked by a sequence which encodes two flanking BpiI restriction sites and one central KpnI site. The PCR products were subcloned into the TA cloning vector pcDNA3.1/V5-His TOPO (Invitrogen), and several plasmid clones were sequenced. Inserts with correct 5' and 3' ends were recovered by digestion at the primer-encoded Esp3I sites and cloned into partially Esp3I-digested pT7riboGB. The partial digests were necessary because of two additional Esp3I sites in the pT7ribo backbone. They were accomplished by incubating 5 μg DNA with 3 units enzyme for 30 min and subsequent dephosphorylation with shrimp alkaline phosphatase (Roche).
A slightly different approach was used for the construction of the S segment plasmid pT7ribo-LACV-cSPro. Due to the length of the noncoding sequences of the LACV S segment, the first PCR with primers LACVSProFW2, LACVSProFW3, LACVSProRV2, and LACVSProRV3 was followed by a second one using the amplicon of the first PCR as the template together with primers LACVSProFW1 and LACVSProRV1. The primer-encoded linker sequence allows the exact insertion of viral DNA sequences between the segment-specific 5' and 3' noncoding regions upon cleavage at the BpiI sites.
For the generation of constructs which encode the full-length LACV segments in antigenomic orientation, the open reading frames of the S, M, and L segments were cloned into their respective promoter-harboring plasmids via BpiI restriction sites. The two-step cloning strategy (first step, insertion of 5' and 3' noncoding sequences; second step, insertion of the open reading frame) is outlined in Fig. 2. In the case of the S segment, the open reading frame was PCR amplified from viral cDNA using primers LACVS5'_BpiI and LACVS3'_BpiI, cleaved with enzyme BpiI, and cloned into BpiI-digested pT7ribo-LACV-cSPro. This resulted in plasmid pT7ribo-LACV-cS, which was subsequently subjected to two primer-directed mutagenesis reactions (QuikChange; Stratagene).
The first PCR with primers LACVSnoEcoFW and LACVSnoEcoRV erased the single EcoRI restriction site located on the LACV S segment by changing the A nucleotide at position 573 to G. The absence of the EcoRI site in the resulting plasmid, pT7ribo-LACV-cSnoEco, did not change the amino acid sequence and served only as a molecular marker to distinguish between parental and recombinant viruses. A second PCR with primers LACVdelNSsFW and LACVdelNS sRV introduced three point mutations which turn the two start codons of the NSs reading frame into threonine codons and the following serine codon into a stop codon. This resulted in plasmid pT7ribo-LACV-cNnoEco, which lacks the EcoRI site and encodes only a functional N protein because it has no NSs protein translation initiation codons (compare Fig. 4A). The generation of plasmid pT7ribo-LACV-cM has been described above. Analogous to the M segment, the LACV L segment open reading frame was cloned into pTM1 and subsequently inserted into plasmid pT7ribo-LACV-cLPro using enzyme BpiI. This open reading frame was modified to equal the consensus amino acid sequence of GenBank entries 22256023, 22212781, and 562069.
LACV glycoprotein fusion assay. Huh7 cells grown on coverslips were cotransfected with 1 μg pI.18-LACVM and 0.5 μg pEGFP-C1 (Clontech) using 4 μl DAC-30 transfection reagent (Eurogentec) in 200 μl serum-free medium (OptiMEM; Gibco-BRL). At 16 h posttransfection, the DMEM was replaced with fusion buffer (Earle's balanced salt solution without sodium bicarbonate, 10 mM HEPES, 0.2% bovine serum albumin, pH 5.5) for 5 min (19). Cells were washed three times with phosphate-buffered saline and incubated in DMEM with 10% fetal calf serum for another 4 h at 37°C. Then, cells were fixed with 3% paraformaldehyde and permeabilized with 0.5% Triton X-100 in phosphate-buffered saline. In a parallel experiment, permeabilization was omitted. Cells were analyzed by immunofluorescence for LACV Gc or green fluorescent protein (GFP) expression as well as for the formation of syncytia. LACV Gc protein was detected with a monoclonal mouse antibody (diluted 1:400) which was kindly provided by Francisco Gonzáles-Scarano (University of Pennsylvania School of Medicine, Philadelphia). In GFP-transfected control cells, nuclei were visualized by the DNA staining agent To-Pro3 iodide (diluted 1:1,000).
Rescue of recombinant LACV. Subconfluent layers of BSR-T7/5 cells were grown on six-well plates. Each well was transfected with 0.5 μg of plasmids pT7ribo-LACV-cM, pT7ribo-LACV-cL and pT7Ribo-LACV-cSnoEco (for rLACV) or pT7ribo-LACV-cNnoEco (for rLACVdelNSs), using 5 μl DAC-30 transfection reagent in 200 μl serum-free medium. For 5 days, supernatants were collected after 24-h intervals and the cells were provided with fresh DMEM. In order to screen for the presence of recombinant virus, Vero cells grown on six-well plates were inoculated for 1 h with 200 μl/well of day 4 supernatants at 37°C and then observed for the following days. Appearance of a cytopathic effect on these cells indicated successful rescue of LACV. To evaluate the rescue kinetics of transfected cells, titers (PFU/ml) of collected supernatants were determined by plaque assay on Vero cells.
Plaque assay. Vero cells grown on six-well plates to 90% confluence were inoculated with 10-fold serial dilutions of supernatants from transfected BSR-T7/5 cells or infected Vero cells in DMEM with 2% fetal calf serum. After 1 h incubation at 37°C, the inoculum was removed and the cells were overlaid with 3 ml DMEM with 2% fetal calf serum, 0.02% DEAE-dextran, and 0.3% Noble agar (Difco) and further incubated for 72 h at 37°C. Cells were fixed and stained with 1% crystal violet, 3.6% formaldehyde, 1% methanol, and 20% ethanol, and titers were calculated from the plaque numbers according to the dilution.
Metabolic labeling of infected and noninfected cells. Infected BHK-21 cells were incubated with [35S]methionine/cysteine (Amersham) at different time points to label newly synthesized proteins. The label process was preceded by three washing steps with phosphate-buffered saline, followed by a 1-hour starvation period with DMEM lacking methionine and cysteine. Then, cells were incubated with 200 μl DMEM lacking methionine and cysteine and supplemented with 50 μCi [35S]methionine/cysteine mix for another 2 h. The cells were lysed in 200 μl lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 25 U/ml Benzonase [Novagen]) and 10 μl of the lysates were separated on a 20% sodium dodecyl sulfate-polyacrylamide gel. The gel was dried on a Whatman paper and radiolabeled proteins were visualized on BioMax MR film (Kodak).
RT-PCR analyses. A549 cells were infected for 16 h at 5 PFU per cell and total RNA was extracted. For reverse transcription (RT), 1 μg of DNase I-treated RNA was incubated with 200 U of Superscript II reverse transcriptase (Gibco/BRL) and 100 ng random hexanucleotides in 20 μl of 1'RT buffer (Gibco/BRL) supplied with 1 mM each of the four deoxynucleotide triphosphates, 20 U of RNasin, and 10 mM dithiothreitol. The resulting cDNA was diluted 100-fold and amplified by 30 cycles of PCR, with each cycle consisting of 1 min at 95°C, 1 min at 58°C, and 1 min at 72°C, followed by 9 min at 72°C.
The upstream and downstream primers for amplifying RNA sequences were 5'-GAC AGC TAG CAT GTC GGA TTT GGT GTT TT-3' and 5'-GAC AGC GGC CGC TTA TGG AAG CCT GAT GCC AAA TTT CTG-3'(LACV S segment), 5'-GCA TGG CCT TCC GTG TCC-3' and 5'-CCA GCC CCA GCG TCA AAG GTG-3'(glyceraldehyde-3-phosphate dehydrogenase), and 5'-CGG CTA CCA CAT CCA AGG AA-3' and 5'-GCT GGA ATT ACC GCG GCT-3'(45S rRNA), respectively.
TUNEL assay. DNA strand breaks in the nuclei of cells undergoing apoptosis were visualized with the In Situ Cell Death Detection kit (Roche). Subconfluent Huh7 cells were infected at 5 PFU per cell and fixed 20 h postinfection with 3% paraformaldehyde. The cells were permeabilized with phosphate-buffered saline containing 0.1% Triton X-100 and 0.1% sodium citrate and then incubated with 50 μl terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) reaction mixture for 1 h at 37°C. Incorporation of fluorescein-labeled dUTP was analyzed by fluorescence microscopy. Complete infection of the tissue culture was demonstrated by immunofluorescence in simultaneously infected cells using an anti-LACV-nucleoprotein antibody (diluted 1:1,000) as described above.
DNA fragmentation analysis. BHK-21 cells were infected at 5 PFU per cell and 24 h later low-molecular-weight DNA was isolated following the protocol described by Hinshaw et al. (18). The DNA was eluted in 20 μl Tris-EDTA buffer containing 1 μg/μl RNase A, incubated for 1 h at 37°C, and then separated on a 2% agarose gel.
RESULTS AND DISCUSSION
Cloning of a biologically active M segment cDNA. We have previously described a minireplicon system for LACV and demonstrated the functionality of the cloned S and L segment gene products (2). The next step towards a complete rescue system was the cloning of an M segment cDNA. The M segment of LACV encodes a polyprotein which is cotranslationally processed to the proteins Gn, NSm, and Gc. To isolate a cDNA for this polyprotein, we amplified by proofreading RT-PCR three overlapping fragments spanning the entire coding sequence, using RNA from LACV-infected cells as the template. The PCR products were assembled into a full-length cDNA and inserted into a eukaryotic expression vector (see Materials and Methods).
We tested for the ability of the M cDNA-encoded glycoproteins to mediate pH-dependent cellular fusion (19). Huh7 cells were transfected with the cDNA construct, treated with fusion buffer to induce a pH shift to 5.5, and monitored for the formation of syncytia by immunofluorescence microscopy. For easy detection of transfected cells, a GFP expression plasmid was cotransfected. Figure 1A (upper panel) shows that cells containing GFP and LACV M form syncytia after the pH shift. By contrast, cells expressing just GFP do not fuse under these conditions (Fig. 1B). To confirm authentic processing of the recombinant LACV glycoproteins, we investigated their transport to the cell surface. To this end, cells were left unpermeabilized by omitting Triton X-100 treatment during the fixation procedure. As shown in Fig. 1A (lower panel), faint but specific staining by the anti-LACV Gc antibody was obtained on the surface of the fused cells. Since the intracellular translocation of the orthobunyavirus Gc protein is dependent on the Gn protein (34), we conclude that both recombinant viral glycoproteins are correctly expressed and transported to the cell surface.
Plasmids for the expression of full-length antigenomic RNAs. The protocols for rescue of BUNV involved blunt-end cloning of a cDNA cassette into the T7 expression vector pT7ribo (5, 25). Whereas the 3' ends of pT7ribo transcripts are correctly trimmed by the hepatitis delta virus ribozyme, the 5' ends contain two additional G nucleotides (5). The T7 polymerase initiates predominantly with an encoded G nucleotide at position +1 (23), but the 5'-terminal sequences of bunyavirus antigenomes start with A or U (14). Thus, the two extra G nucleotides at the 5' ends of the artificial antigenomes may reduce recognition by the LACV polymerase.
We therefore constructed a rescue vector in which cDNA fragments can be inserted via sticky ends and in which the number of nonviral G nucleotides at the 5' end is reduced to one (see Materials and Methods). Basically, we followed the strategy described by Flick and Petterson for constructing a bunyavirus minireplicon (16). The multistep cloning strategy is outlined for the antigenomic S segment plasmid in Fig. 2. A derivative of pT7ribo which contained sites for the restriction enzyme Esp3I was constructed. These restriction sites were used to introduce a short DNA cassette which contained the segment-specific promoter ends of LACV in the antigenomic orientation, flanking a linker with two BpiI sites. In a final step, BpiI digestion served to introduce the coding sequence of the respective LACV segments. The resulting plasmids allow T7-mediated synthesis of viral segments with correct 3' ends and one extra G nucleotide at the 5' end.
Following this strategy, we cloned the newly isolated LACV M cDNA as well as consensus LACV S and LACV L cDNAs as full-length antigenomic sequences into the modified pT7ribo, resulting in plasmids pT7ribo-LACV-cL, pT7ribo-LACV-cM, and pT7cLACV-cSnoEco. In addition, we constructed support plasmids for the viral proteins, using the T7-driven expression vector pTM1 which contains an internal ribosome entry site for efficient translation of the transcribed RNAs (see Materials and Methods).
Rescue of recombinant La Crosse virus. In analogy to the protocol shown to improve the rescue of BUNV (22, 25), we attempted to recover recombinant LACV in a helper virus-free system which is based on the BSR-T7/5 cell line. Initially, we transfected cells in six-well plates either with the full set of antigenomic and support plasmids or with just the three antigenomic plasmids. On day 4 posttransfection, supernatants were collected and aliquots were transferred to Vero cells to monitor development of a cytopathic effect. As shown in Table 2, the three-plasmid approach resulted in more than 50% positive dishes, indicating efficient and reproducible production of recombinant LACV (rLACV). Strikingly, the addition of support plasmids completely abrogated the rescue. These findings suggest that, as for BUNV, the antigenomic cDNA constructs can be translated and are therefore sufficient for recovery of LACV. In contrast to BUNV, however, additional expression of viral proteins from support plasmids was inhibitory, indicating that the intracellular replication of LACV is highly sensitive to elevated levels of viral proteins. In line with this, we have previously shown that overexpression of either L or N results in downregulation of LACV polymerase activity (2), whereas for BUNV it was shown that overexpression of L or N appears to be much better tolerated (13, 40).
To characterize the three-plasmid rescue of LACV in more detail, we measured the development of virus titers in the supernatants of transfected cells for 5 days. The rescue kinetics of two representative experiments are shown in Fig. 3A. Typically, infectious virus appeared 48 h posttransfection, and titers increased to up to 108 PFU/ml after 3 more days. To verify that the virus was derived from the transfected cDNAs, we had introduced a silent mutation into the S segment, destroying an EcoRI site at position 572. RNA was extracted from infected cells, and a DNA fragment containing the mutated site was amplified by RT-PCR. Figure 3B shows that rLACV indeed contained the introduced mutation, whereas the parental virus (LACV) had the EcoRI site, as expected. Thus, taken together, the experimental procedures employing just the three antigenomic constructs and the BSR-T7/5 cell line stably expressing the T7 polymerase resulted in an efficient rescue of recombinant LACV.
Rescue of La Crosse virus with a deleted NSs gene. To test whether our rescue system can be used for introduction of functionally important mutations into the LACV genome, we generated a mutant virus with an inactivated NSs gene. Because the N and NSs reading frames overlap (14), complete deletion of the NSs gene is not possible without affecting the N gene. We therefore applied the strategy which was used for abrogation of BUNV NSs gene expression (6) and changed the tandem ATG start codons of NSs to ACG and the third codon to a stop codon (see Fig. 4A). The resulting antigenomic LACV S construct, pT7ribo-LACV-cNnoEco, expresses an unaltered N protein but no NSs.
For virus rescue, BSR-T7/5 cells were transfected with the plasmids pT7ribo-LACV-cL, pT7ribo-LACV-cM, and the mutant construct pT7ribo-LACV-cNnoEco as outlined above. Time course titrations (Fig. 4B) indicated that rescue of the mutant virus, which we termed rLACVdelNSs, occurred with an efficiency similar to that for rescue of the wild-type virus (compare Fig. 3A). To establish that rLACVdelNSs is derived from the cDNA plasmids, we confirmed the absence of the EcoRI site on the S segment (Fig. 4C). Furthermore, since mutagenesis of the NSs start site created a unique recognition site for the restriction enzyme TaiI, we were able to distinguish wild-type and delNSs mutant S segment by TaiI restriction analysis (Fig. 4D). The results of those analyses confirmed the rescue of a recombinant LACV containing the desired rLACVdelNSs mutation in the S segment.
Phenotype of the recombinant viruses in cell culture. We performed an initial phenotypic characterization of the rescued viruses. With respect to plaque formation on Vero cells, rLACV was indistinguishable from the parental wild-type LACV, as expected (Fig. 5A). rLACVdelNSs, by contrast, exhibited a turbid-plaque phenotype, whereas the overall plaque size was not strikingly different from that of the NSs-expressing viruses. This is in contrast to the NSs-deficient mutant of BUNV, which was found to form significantly smaller plaques than wild-type BUNV (6). However, subsequent analyses revealed that plaque size differences also occur for different wild-type BUNV strains and are the result of promoter variations rather than the absence of NSs (20).
Infection with bunyaviruses is known to induce a strong shutoff of host cell protein synthesis (31, 33). To investigate this for the recombinant viruses, BHK cells were infected at 5 PFU per cell and labeled with [35S]methionine/cysteine at early (3 h) and late (23 h) time points after infection, and cell extracts were analyzed by gel electrophoresis and autoradiography. Both rLACV and the parental LACV induce a significant shutoff of host cell protein synthesis at 23 h postinfection (Fig. 5B, lanes 1 to 4). The delNSs virus, by contrast, displayed a weaker shutoff, although a considerable reduction in host cell protein synthesis still occurs, as is evident by the comparison with mock-infected cells (Fig. 5B, lanes 5 to 8). Thus, as with BUNV (6), the shutoff imposed by LACV appears to consist of two different components, the one caused by the NSs protein and a second, not yet characterized mechanism.
We have recently shown that the NSs protein of BUNV inhibits phosphorylation of the C-terminal domain of RNA polymerase II, thus aborting transcription of host cell mRNAs (37). In a similar but not identical way, the NSs protein of Rift Valley fever virus blocks host cell mRNA synthesis (1, 4) by preventing the assembly of TFIIH, an essential RNA polymerase II transcription factor (24). To investigate this for LACV NSs, we assessed the accumulation of cellular RNAs in infected cells, using semiquantitative RT-PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA and 45S rRNA were used as markers for RNA polymerase II and RNA polymerase I transcripts, respectively. As shown in Fig. 5C, infection with rLACV strongly reduced the amount of GAPDH mRNA but not of 45S rRNA, whereas the delNSs mutant had only a slight impact on the GAPDH mRNA. This mirrors the effects of rLACV and rLACVdelNSs on host cell protein synthesis (see Fig. 5B) and indicates that a major part of the LACV NSs-induced shutoff is due to inhibition of RNA polymerase II. Thus, similar to the NSs proteins of BUNV and Rift Valley fever virus, the NSs of LACV appears to be able to downregulate host cell RNA polymerase II.
To evaluate whether the lack of NSs-induced shutoff has consequences for virus replication, we compared the growth of rLACV and rLACVdelNSs in cell culture. For BUNV it was previously shown that the rLACVdelNSs mutant grows in Vero cells to titers about 1 log lower than the wild-type virus (36). To investigate this for LACV, Vero cells were infected with 0.01 PFU per cell, and virus titers in the supernatants were determined at 24 h and 48 h postinfection. Surprisingly, despite the differences in plaque phenotype and host cell shutoff (see Fig. 5), both viruses replicated with similar efficiencies (Fig. 6). This indicates that, at least in Vero cells, NSs does not confer a significant growth advantage for LACV. In line with this, we found that rLACV is not able to outcompete rLACVdelNSs in coinfection studies (data not shown).
It is known that BUNV NSs has the ability to block apoptosis induction by the transcription factor IRF-3 (21). LACV, by contrast, is strongly apoptogenic (30), possibly due to NSs (11). LACV kills cells faster, replicates faster, and reaches higher titers than BUNV (our unpublished observations). Therefore, the possibility exists that, unlike BUNV, the fast-growing LACV does not delay programmed cell death but rather enhances it by NSs. To clarify this, we monitored the induction of apoptosis by the recombinant viruses. Cells were infected and observed for the appearance of DNA breaks, a hallmark of apoptosis, using TUNEL assays and DNA fragmentation analysis. Figure 7A shows that many more TUNEL-positive cells were detectable with rLACV than with rLACVdelNSs. Consequently, a strong fragmentation of the cellular DNA occurred in rLACV-infected cells, whereas rLACVdelNSs-infected cells did not develop DNA fragmentation (Fig. 7B). Thus, we can conclude that, unlike the NSs of BUNV (21), the NSs of LACV indeed promotes apoptosis, as previously observed in overexpression studies (11).
The need for reverse genetics systems to study the biology of highly virulent bunyaviruses is generally recognized (17, 35). Here, we have adopted the three-plasmid system developed for BUNV (25) to rescue the human-pathogenic LACV. Cotransfection of support plasmids, which is a common practice in reverse genetics systems (12, 29), was found to be absolutely detrimental for LACV rescue. Such an untypical requirement for virus rescue may explain the absence of reverse genetics systems for other members of the Bunyaviridae family. Our rescue system now enables us to manipulate the genome of LACV in order to characterize its virulence factors and develop potential vaccine candidates.
ACKNOWLEDGMENTS
We are indebted to Karl-Klaus Conzelmann, Richard Elliott, Francisco Gonzáles-Scarano, Ramaswamy Raju, and Jim Robertson for kindly providing reagents which were essential for this work. We thank Otto Haller for support and helpful comments, Georg Kochs, Alain Kohl, Christine Krempl, Martin Spiegel, and Peter Staeheli for critically reading the manuscript, and Richard Elliott for sharing information on Bunyamwera virus rescue prior to publication.
Work in the authors' laboratory is supported by grants WE 2616/2-1 and WE 2616/2-2 from the Deutsche Forschungsgemeinschaft.
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