The recent epidemic of Zika virus highlights the importance of establishing reverse genetic approaches to develop vaccines and/or therapeutic strategies. Here, we describe the protocol to rescue an infectious recombinant Zika virus from a full-length cDNA clone assembled in a bacterial artificial chromosome under the control of the human cytomegalovirus immediate-early promoter.
The association of Zika virus (ZIKV) infection with neurological complications during the recent worldwide outbreak and the lack of approved vaccines and/or antivirals have underscored the urgent need to develop ZIKV reverse genetic systems to facilitate the study of ZIKV biology and the development of therapeutic and/or prophylactic approaches. However, like with other flaviviruses, the generation of ZIKV full-length infectious cDNA clones has been hampered due to the toxicity of viral sequences during its amplification in bacteria. To overcome this problem, we have developed a nontraditional approach based on the use of bacterial artificial chromosomes (BACs). Using this approach, the full-length cDNA copy of the ZIKV strain Rio Grande do Norte Natal (ZIKV-RGN) is generated from four synthetic DNA fragments and assembled into the single-copy pBeloBAC11 plasmid under the control of the human cytomegalovirus (CMV) immediate-early promoter. The assembled BAC cDNA clone is stable during propagation in bacteria, and infectious recombinant (r)ZIKV is recovered in Vero cells after transfection of the BAC cDNA clone. The protocol described here provides a powerful technique for the generation of infectious clones of flaviviruses, including ZIKV, and other positive-strand RNA viruses, particularly those with large genomes that have stability problems during bacterial propagation.
ZIKV is a mosquito-borne member of the Flavivirus genus within the Flaviviridae family that currently constitutes a global public health emergency1. Like other flaviviruses, ZIKV is an enveloped RNA virus with an icosahedral-like structure that contains a positive sense, single-stranded RNA molecule of about 10.8 kb (Figure 1)2. The viral genome encodes a large polyprotein of approximately 3,423 amino acids that is processed by viral and cellular proteases into three structural proteins (capsid [C], premembrane/membrane [prM/M], and envelope [E]) that are involved in the formation of the viral particles and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) that participate in genome replication, virus assembly, and evasion of the host immune response (Figure 1)3.
Historically, ZIKV infection has been associated with a mild febrile illness4,5. However, the explosive recent pandemic of ZIKV infections throughout South and Central America, the South Pacific, and the Caribbean6,7,8, and its association with the occurrence of Guillain-Barré syndrome and microcephaly9,10,11,12,13, have changed the historic perception and potentiated the relevance of ZIKV as an important human pathogen. In this sense, the development of molecular tools, such as infectious cDNA clones, will facilitate the study of viral pathogenesis and the development of genetically defined vaccines and the identification of antiviral drugs for the treatment of ZIKV infection. As described for other flaviviruses, the generation of ZIKV infectious clones is difficult due to the presence of cryptic bacterial promoters in the viral genome14 that allow the leaky expression of toxic viral proteins during the propagation of the cDNA clones in bacteria using standard high-copy-number plasmids15,16,17. To overcome this toxicity problem, several nontraditional approaches have been implemented successfully in the last two years18. These include the use of low-copy-number plasmids19,20, the insertion of introns to disrupt the toxic regions21,22,23, the in vitro ligation of cDNA fragments24,25, mutational silencing of cryptic bacterial promoters present in the viral genome26,27, infectious subgenomic amplicons (ISA)28,29, the Gibson assembly method30, and the use of circular polymerase extension reaction (CPER)31.
Herein, we describe the detailed protocol for the engineering of a full-length cDNA clone of the ZIKV strain ZIKV-RGN13, using a BAC to overcome the toxicity problem, and the rescue of infectious rZIKV by direct transfection of the BAC cDNA clone into Vero cells32, a cell line approved by the Food and Drug Administration (FDA) for the development of human vaccines33. In this system, the full-length cDNA copy of the viral genome is assembled in the BAC plasmid pBeloBAC1134 (Figure 2A), a low-copy-number plasmid (one to two copies per cell) derived from the Escherichia coli F-factor35, which minimizes the toxicity of flavivirus sequences during its propagation in bacteria. The cDNA of the ZIKV genome is assembled in pBeloBAC11 under the control of the human CMV immediate-early promoter, to allow the expression of the viral (v)RNA in the nucleus of transfected cells by cellular RNA polymerase II, and flanked at the 3'-end by the hepatitis delta virus (HDV) ribozyme (RZ), followed by the sequences of the bovine growth hormone (BGH) termination and polyadenylation signals to produce synthetic RNAs bearing authentic 5'- and 3'-ends of the viral genome, respectively (Figure 2B). This cDNA-launched system results in the intracellular expression of capped viral RNA, allowing the recovery of infectious ZIKV without the need for an in vitro transcription step. The BAC approach provides a powerful methodology applicable to constructing stable and fully functional infectious cDNA clones for other flaviviruses, as well as other positive-stranded RNA viruses36,37,38,39,40,41.
1. Construction of a ZIKV Infectious cDNA Clone in a BAC
NOTE: The strategy for the assembly of ZIKV in BACs is described for the RGN strain13 (accession number KU527068) (Figure 2).
2. Preparation of High-purity pBAC-ZIKV for the Rescue of Infectious rZIKV
NOTE: The large-scale preparation of an ultrapure pBAC-ZIKV infectious clone, suitable for the transfection and rescue of infectious viruses, is performed by alkaline lysis with a commercial kit specifically developed for BAC purification (see the Table of Materials). The kit must include an ATP-dependent exonuclease digestion step that removes bacterial genomic DNA contamination, allowing the isolation of BAC cDNA with a grade of purity similar to that obtained with the cesium chloride method.
3. Rescue of Infectious rZIKV from the BAC cDNA Clone by Transfection of Vero Cells
NOTE: Infectious rZIKV is recovered by the transfection of Vero cells with the pBAC-ZIKV cDNA clone, using a commercial cationic lipid transfection reagent (see the Table of Materials; Figure 3).
4. Titration of Recovered rZIKV
5. Confirmation of Successful rZIKV Rescue
NOTE: To further confirm the identity of the rescued virus, ZIKV E protein expression is analyzed by immunofluorescence using the mouse mAb 1176-56 specific for ZIKV E protein (Figure 4D). This mAb is specific for ZIKV E protein, contrary to the situation of the pan-flavivirus E protein mAb 4G2 (step 4.6.3). Alternatively, the virus identity can be confirmed by sequencing.
6. Amplification and Generation of Viral Stocks
NOTE: Once the identity of the rescued virus is confirmed (section 5), amplify the virus on Vero cells and generate viral stocks for further studies.
The protocol described here allows for the generation of stable ZIKV full-length cDNA infectious clones using a BAC to minimize the toxicity problems associated with several flaviviral sequences. Efficient recovery of infectious rZIKV from the BAC cDNA clone can be easily achieved after the transfection of susceptible Vero cells (Figure 2). Using this protocol, we have generated a stable full-length cDNA clone of the ZIKV strain RGN32 by sequentially cloning four overlapping cDNA fragments into the BAC plasmid pBeloBAC1134 using conventional cloning methods and unique restriction sites present in the viral genome (Figure 2). The full-length cDNA clone was flanked at the 5'-end by the human CMV promoter to allow the expression of vRNA in the nucleus of transfected cells, and at the 3'-end by the HDV RZ followed by the BGH polyadenylation and termination sequences, to produce RNAs containing the exact 3'-end of the viral genome (Figure 2). The stability in bacteria of the generated BAC cDNA clone, together with its easy manipulation using standard recombinant DNA technologies, highlights the potential of the BAC cDNA approach for the rapid and reliable generation of stable full-length cDNA clones of ZIKV and other flaviviruses or positive-stranded RNA viruses with unstable viral genomes.
Once the BAC cDNA clone was assembled (Figure 2), infectious virus could be easily recovered after the direct transfection of susceptible Vero cells with the BAC cDNA clone using cationic liposomes(Figure 3). This cDNA-launched system allowed the intracellular expression of the capped vRNA, allowing the recovery of infectious viruses without the need for an in vitro transcription step. Using this approach, we were able to rescue rZIKV-RGN with titers higher than 106 PFU/mL at 5 days posttransfection (Figure 4A). In addition, the rescued virus induced a clear CPE (Figure 4B), generated homogeneous plaques of about 2 mm in size (Figure 4C), and its identity was confirmed by sequencing (data not shown) and immunofluorescence analysis using the mAb specific for ZIKV E protein, 1176-56 (Figure 4D). In vitro data indicate that the recovered rZIKV-RGN replicated efficiently in Vero cells and to levels compared to a natural ZIKV isolate32 (data not shown). Overall, these results demonstrate that infectious rZIKV can be rescued from full-length cDNA clones assembled in a BAC.
Figure 1: ZIKV genome organization and virion structure. (A) Genome organization: ZIKV contains a positive single-stranded RNA that is translated as a single polyprotein. The translated polyprotein was cleaved by viral (arrows) and host (diamonds) proteases to produce the structural proteins capsid (C, blue), matrix (M, brown), and envelop (E, green), and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The 5' and 3' untranslated regions (UTRs) at the end of the viral genome are indicated with black lines. (B) Virion structure: ZIKV virions were decorated with the E and M proteins, anchored in a lipid bilayer with an icosahedral-like structure. Under the viral envelop was the viral nucleocapsid composed of the C protein associated with the viral genomic RNA. This figure has been adapted from Ávila-Pérez et al.18. Please click here to view a larger version of this figure.
Figure 2: Assembly of the ZIKV full-length infectious cDNA clone in a BAC. (A) Schematic representation of the pBeloBAC11 BAC:The regulatory genes parA, parB, parC, and repE,the F-factor replication origin (OriS), the chloramphenicol resistant gene (Cmr), and the lacZ gene are indicated. The relevant restriction sites used to assemble the infectious ZIKV cDNA clone are underlined. (B) Assembly of ZIKV full-length infectious cDNA clone into the pBeloBAC11 BAC:Four overlapping DNA fragments (Z1-Z4), covering the entire ZIKV genome (Figure 1) and flanked by the indicated restriction sites, were generated by chemical synthesis and sequentially cloned into pBeloBAC11 to generate the infectious ZIKV cDNA clone pBAC-ZIKV. The full-length ZIKV infectious cDNA was assembled under the control of the human cytomegalovirus immediate-early promoter (CMV) and flanked at the 3'-end by the HDV ribozyme (RZ) and the bovine growth hormone (BGH) termination and polyadenylation sequences. The acronyms for the viral genes and regulatory elements are as described in Figure 1. Please click here to view a larger version of this figure.
Figure 3: Workflow to generate rZIKV from the BAC cDNA clone. Vero cells at 90% of confluence were transfected in monolayer with the ZIKV full-length infectious cDNA clone pBAC-ZIKV (Figure 2) using cationic liposomes. At 4-6 days posttransfection, when CPE was evident, tissue culture supernatants containing rZIKV were collected and evaluated for the presence of virus (Figure 4) and used for viral amplification in Vero cells. Please click here to view a larger version of this figure.
Figure 4: Recovery and in vitro characterization of rZIKV. (A) Rescue of infectious rZIKV from the BAC cDNA clone: Vero cells at 90% of confluence (6-well plate format, triplicates) were mock-transfected or transfected with 4 µg/well of pBAC-ZIKV (Figure 3), and at the indicated days posttransfection, virus titers in tissue culture supernatants were determined by plaque assay (PFU/mL). The error bars indicate standard deviations from three different transfection experiments.The dotted black line indicates the limit of detection (50 PFU/mL). (B) Viral CPE:Vero cells at 90% confluence (6-well plate format, triplicates) were mock-infected (top) or infected (MOI of 0.5 PFU/cell) with rZIKV, and at 48 h postinfection, the presence of CPE was evaluated by light microscopy. Scale bars = 100 µm. (C) Viral plaque assay and immunostaining: Vero cells at 90% confluence (12-well plate format) were infected with rZIKV, and at 72 h postinfection, viral plaques were visualized by crystal violet staining (left) or by immunostaining (right) using the pan-flavivirus E protein mAb 4G2. Scale bars = 5 mm. (D) Immunofluorescence:Vero cells at 90% confluence (24-well plate format, triplicates) were infected (MOI of 0.5 PFU/cell) with rZIKV and, at 48 h postinfection, analyzed by immunofluorescence using the mAb 1176-56, specific for ZIKV E protein. Cell nuclei were stained with DAPI. A representative merged image of ZIKV-infected Vero cells is shown. The white square in the top right represents an enlarged image of ZIKV-infected Vero cells. Scale bars = 100 µm. Please click here to view a larger version of this figure.
Infectious cDNA clones constitute essential molecular tools for basic research of RNA viruses and the development of vaccines and/or the identification of antiviral strategies. However, for many positive-stranded RNA viruses, including flaviviruses, the generation of infectious cDNA clones are difficult due to the instability of the cloned cDNAs when propagated in bacteria using standard high-copy-number plasmids. In the case of ZIKV and other flaviviruses, this instability is mainly due to the leaky expression of toxic viral proteins from cryptic bacterial promoters present in the viral genome14,15,16,17. Here, we describe an alternative and powerful protocol to generate a stable ZIKV full-length infectious cDNA clone as a single plasmid, based on the use of the BAC plasmid pBeloBAC1134 (Figure 2A) to overcome the toxicity problem, the use of the CMV promoter to allow the expression of the vRNA in the nucleus of transfected cells, and the HDV RZ to generate vRNAs with accurate 3'-ends (Figure 2B). Using this method, we have successfully generated a fully stable infectious clone of the ZIKV strain RGN that allows the efficient and reliable recovery of infectious rZIKV after the direct transfection of susceptible Vero cells (Figure 3 and Figure 4).
A huge effort has been made in the last few years to overcome the instability problems associated with ZIKV infectious cDNA clones, and several approaches have been successfully implemented18, including the in vitro ligation of cDNA fragments24,25, low-copy plasmids19,20, the inactivation of cryptic bacterial promoters by the introduction of silent mutations26,27, intron insertion21,22,23, the Gibson assembly method30, the ISA method28,29, and the use of CPER31. Although these approaches overcome the toxicity problem and are useful to generate ZIKV infectious cDNA clones, some of them are laborious and present several disadvantages, including the need for in vitro ligation and transcription steps that reduce virus recovery efficiency or the introduction of a high number of silent mutations to inactivate cryptic bacterial promoter that could affect viral fitness, among others. The approach described in this protocol presents the following advantages. i) The BAC plasmid pBeloBAC1134 has a strictly controlled replication, keeping one or two copies of plasmid per cell, which minimizes toxicity and allows stable maintenance in bacteria of instable cDNAs. ii) The propagation and modification of BAC plasmids are almost similar to those of conventional plasmids, considering the slight modifications described in this protocol to manipulate large-size BAC-DNA fragments and low-copy plasmids. Notably, the BAC cDNA clone can also be modified into E. coli by homologous recombination using the Red recombination system42,43,44. iii) The use of CMV promoter allows the intracellular expression of capped ZIKV vRNA and the recovery of infectious viruses without requiring an in vitro transcription step. iv) Infectious rZIKV is generated after the direct transfection of susceptible cells (e.g., Vero) with the BAC cDNA clone. Since DNA transfection in mammalian cells is more efficient than RNA transfection, the virus recovery efficiency with the BAC approach is higher than that observed using RNA transcripts, reducing the number of passages in culture cells to generate a viral stock and, consequently, limiting the introduction of unwanted mutations by cell culture adaptation.
Finally, the potential of the BAC approach is supported by the successful use of this method (with slight modifications) to engineer infectious cDNA clones of other flaviviruses, including dengue virus36, and several coronaviruses of high impact in human and animal health, such as transmissible gastroenteritis coronavirus37 (TGEV), feline infectious peritonitis virus38 (FIPV), human coronavirus OC4339 (HCoV-OC43), severe acute respiratory syndrome coronavirus40 (SARS-CoV), and Middle East respiratory syndrome coronavirus41 (MERS-CoV), among others.
In the protocol described here, there are two critical steps that should be considered. One important consideration is identifying appropriate unique restriction sites in the viral genome that are absent in the BAC plasmid. If no adequate restriction sites are available, new restriction sites can be generated during the cloning design by the introduction of silent nucleotide mutations. Another important issue is that the BAC plasmids are present in only one or two copies per cell, and therefore, low yields of BAC plasmids with a high contamination of bacterial genomic DNA are obtained using standard protocols designed for high- and medium-copy-number plasmids. This potential problem is easily overcome using large culture volumes and purifying the BAC plasmid with a commercial kit specifically developed for BAC purification.
In summary, we have developed a powerful ZIKV reverse genetic approach based on the use of a BAC that could be adapted to generate stable and fully functional infectious cDNA clones of other positive-stranded RNA viruses to facilitate the study of the biology of these viruses and the development of vaccines and/or to facilitate the identification of antiviral drugs.
The authors have nothing to disclose.
The authors would like to thank Carla Gómez for her technical assistance in the BAC cDNA clone generation and Snezhana Dimitrova for helping with the video preparation. This work was supported in part by grants from the Spanish Ministry of Economy and Competitiveness (MINECO, grant number BFU2016-79127-R) to F.A.T. and the National Institutes of Health (NIH, grant number 1R21AI120500) to L.M.S. and F.A.T.
1. Molecular Biology Reagents | |||
Afe I | New England BioLabs | R0652S | 10,000 units/mL |
AmpliTaq DNA Polymerase | ThermoFisher Scientific (Applied Biosystems) | N8080161 | 5,000 Units/mL |
ApaL I | New England BioLabs | R0507S | 10,000 units/mL |
Asc I | New England BioLabs | R0558S | 10,000 units/mL |
BamH I | New England BioLabs | R0136S | 10,000 units/mL |
BstB I | New England BioLabs | R0519S | 20,000 units/mL |
Chloramphenicol | Sigma-Aldrich | C0378 | |
ElectroMAX DH10B Cells | ThermoFisher Scientific (Invitrogen) | 18290015 | Electocompetent DH10B cells |
Electroporation Cuvettes, 0.2 cm | Bio-Rad | 165-2086 | |
Ethanol | Merck | 100983 | Flamable |
Isopropanol | Merck | 109634 | Flamable |
Large-Construct Kit (10) | QIAGEN | 12462 | For high-purity BAC preparation |
LB Broth | ThermoFisher Scientific (Invitrogen) | 12780029 | Can be homemade as well |
LB with Agar | ThermoFisher Scientific (Invitrogen) | 22700041 | Can be homemade as well |
Methanol | Merck | 106009 | Flamable |
Mlu I | New England BioLabs | R0198S | 10,000 units/mL |
Oligonucleotides | IDT | N/A | |
Plasmid pBeloBAC11 | New England BioLabs | ER2420S (E4154S) | |
Plasmid Midi Kit (25) | QIAGEN | 12143 | For midle-scale preparation of BAC plasmids |
Pml I | New England BioLabs | R0532S | 20,000 units/mL |
Polypropylene tubes (10 mL) | DeltaLab | 175724 | Other commercial sources are acceptable |
QIAEX II Gel Extraction Kit (150) | QIAGEN | 20021 | Gel-clean-up kit optimized for DNA fragments larger than 10 kb |
Shrimp AlKaline Phosphatase (rSAP) | New England BioLabs | M0371S | 1,000 units/mL |
SOC Medium | ThermoFisher Scientific (Invitrogen) | 15544034 | Can be homemade as well |
Synthesis of cDNA fragments | Bio Basic | N/A | |
T4 DNA Ligase | Sigma-Aldrich (Roche) | 10481220001 | 1,000 units/mL |
2. Cell Culture Reagents | |||
6-Well Plates | ThermoFisher Scientific (Nunc) | 140675 | |
12-Well Plates | ThermoFisher Scientific (Nunc) | 150628 | |
24-Well Plates | ThermoFisher Scientific (Nunc) | 142485 | |
Agar Noble | VWR | 214230 | |
Alexa Fluor 488 Conjugate ant-mouse secondary antibody | Varies | N/A | |
Biotinylated Anti-Mouse Secondary Antibody | Varies | N/A | |
Cell Culture Dishes (100×21 mm) | ThermoFisher Scientific (Nunc) | 172931 | |
Conical Tubes (15 mL) | VWR | 525-0150 | |
Conical Tubes (50 mL) | VWR | 525-0155 | |
Crystal Violet | Sigma-Aldrich | C6158 | |
DAPI | Sigma-Aldrich | D9542 | Toxic and carcinogenic |
DEAE-Dextran | Sigma-Aldrich | D9885 | |
DMEM | ThermoFisher Scientific (Gibco) | 11995065 | |
Fetal Bovine Serum (FBS) | ThermoFisher Scientific (HyClone)) | SV30160.03 | |
Formaldehyde | Sigma-Aldrich | F8775 | Toxic and carcinogenic |
L-Glutamine | ThermoFisher Scientific (Gibco) | 25030081 | |
Lipofectamine 2000 | ThermoFisher Scientific (Invitrogen) | 11668019 | Transfection reagent |
Nonessential amino acids | ThermoFisher Scientific (Gibco) | 11140035 | |
Opti-MEM I Reduced Serum Medium | ThermoFisher Scientific (Gibco) | 31985070 | Transfection medium |
Pan-flavivirus E protein mAb 4G2 | BEI Resources | NR-50327 | |
Paraformaldehyde | Electron Microscopy Sciences | 15710-S | Toxic and carcinogenic |
PBS | ThermoFisher Scientific (Gibco) | 14190144 | |
Penicillin/Streptomycin | ThermoFisher Scientific (Gibco) | 15140122 | |
ProLong Gold Antifade Reagent | ThermoFisher Scientific (Invitrogen) | P10144 | |
Triton-X-100 | Sigma-Aldrich | T8787 | |
Vectastain ABC Kit | Vector Laboratories Inc | PK-4010 | Avidin/biotin-based peroxidase kit |
Vero Cells | ATCC | CCL-81 | |
ZIKV E Protein mAb 1176-56 | BioFront Technologies | BF-1176-56 | |
3. Equipment | |||
Agarose Gel Electrophoresis System | Bio-Rad | 1704468 | Other commercial sources are acceptable |
Class II Biosafety CO2 Cabinet | Varies | N/A | Other commercial sources are acceptable |
Desktop Refrigrated Centrifuge | Varies | N/A | |
Fluorescence Microscope | Varies | N/A | |
High-Speed Refrigrated Centrifuge | Varies | N/A | |
MicroPulser Electroporator | Bio-Rad | 1652100 | Other machines are acceptable |
SimpliAmp Thermal Cycler | ThermoFisher Scientific (Applied Biosystems) | A24811 | Other machines are acceptable |
Vortexer | Varies | N/A |