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. Author manuscript; available in PMC: 2024 Aug 1.
Published in final edited form as: Virology. 2023 Jun 13;585:109–116. doi: 10.1016/j.virol.2023.05.013

An in vitro workflow to create and modify infectious clones using replication cycle reaction

Jeffrey M Marano 1,2,4,#, Chelsea Cereghino 2,4,#, Carla V Finkielstein 3,5,6, James Weger-Lucarelli 2,4,*
PMCID: PMC10528026  NIHMSID: NIHMS1910319  PMID: 37331111

Abstract

Reverse genetics systems are critical tools in combating emerging viruses which enable a better understanding of the genetic mechanisms by which viruses cause disease. Traditional cloning approaches using bacteria are fraught with difficulties due to the bacterial toxicity of many viral sequences, resulting in unwanted mutations within the viral genome. Here, we describe a novel in vitro workflow that leverages gene synthesis and replication cycle reaction to produce a supercoiled infectious clone plasmid that is easy to distribute and manipulate. We developed two infectious clones as proof of concept: a low passage dengue virus serotype 2 isolate (PUO-218) and the USA-WA1/2020 strain of SARS-CoV-2, which replicated similarly to their respective parental viruses. Furthermore, we generated a medically relevant mutant of SARS-CoV-2, Spike D614G. Results indicate that our workflow is a viable method to generate and manipulate infectious clones for viruses that are notoriously difficult for traditional bacterial-based cloning methods.

Keywords: Reverse genetics, SARS-CoV-2, dengue virus, infectious clones, mutagenesis

1. Introduction

The development of reverse genetics systems for emerging viruses is critical in responding to global health threats, as it enables studies to dissect the mechanisms by which these viruses cause disease (Aubry et al., 2015). The infectious cDNA clone allows researchers to explore many different aspects of virology, including viral kinetics (Chuong et al., 2019; Bates et al., 2020), evolution (Atieh et al., 2018; Marano and Weger-Lucarelli, 2023), tropism (Kümmerer et al., 2012; Weger-Lucarelli et al., 2015; Ayers et al., 2021), and vaccinology (Mire et al., 2012; Furuyama et al., 2022; Weger-Lucarelli et al., 2023). The traditional approach to produce infectious clones involves generating fragments from viral RNA by RT-PCR and then cloning these fragments into bacterial plasmids (Weger-Lucarelli et al., 2017). However, traditional cloning approaches can be challenging due to viral genomic instability in bacteria, especially with flaviviruses (Pu et al., 2011), alphaviruses (Steel et al., 2011), and coronaviruses (Scobey et al., 2013). The proposed mechanism for this instability is cryptic bacterial promoters in the viral genome which drive the production of toxic viral genes (Johansen, 1996; Li et al., 2011; Pu et al., 2011). In response to the plasmids’ toxicity, a selective pressure is imposed on the bacteria to propagate viral genomes containing deletions, mutations, and recombination events that mitigate the toxicity (Li et al., 2011).

Several approaches have attempted to mitigate the effects of host-specific toxicity on infectious clone stability. One approach involves maintaining the viral genome across several plasmids, combining the genomic fragments in vitro, and then using that product for direct transfection for CMV-based clones or in vitro transcription followed by transfection of RNA for bacteriophage promoter-based clones (Scobey et al., 2013; Cockrell et al., 2017; Ayers et al., 2021). This approach is effective but requires maintaining up to 7 plasmids, making these systems cumbersome. Several groups have developed reverse genetics systems in which the products of circular polymerase extension cloning/reaction (CPEC/CPER), Gibson assembly, or infectious subgenomic amplicons (ISAs) are directly transfected into permissive cells or used to produce infectious RNA using in vitro transcription (Edmonds et al., 2013; Aubry et al., 2014; Mohamed Ali et al., 2018; Torii et al., 2021). While these approaches remove the need for bacteria because the assembly occurs molecularly or within permissive eukaryotic cells, a single plasmid containing the full-length cDNA clone cannot be recovered, making generating mutant viruses more challenging.

Other attempts have been made to reduce the toxicity of full-length clones in bacteria. One approach was to insert introns into the viral genome to disrupt the open reading frame (ORF) (Johansen, 1996; López-Moya and García, 2000; Ulper et al., 2008). Another approach is introducing synonymous mutations into sequences believed to contain cryptic bacterial promoters to prevent transcription by bacterial polymerases (Pu et al., 2011). Others involved altering the plasmid architecture, like using low copy number bacterial artificial chromosomes (Yun et al., 2003; Usme-Ciro et al., 2014), heavily repressed promoters (Mishin et al., 2001; Cai et al., 2005), or using yeast-based systems (Polo et al., 1997; Rihn et al., 2021). Nonetheless, these systems still do not entirely remove the toxicity of viral genomes (Aubry et al., 2015).

An alternative idea is to shift from ameliorating the host issue to removing the host from the system entirely. Having previously demonstrated the ability to rescue and manipulate infectious clones using rolling circle amplification (RCA), an in vitro process (Weger-Lucarelli et al., 2018; Bates et al., 2020; Marano et al., 2020; Kang et al., 2021), we sought to use an emerging technology called replication-cycle reaction (RCR) (Su’Etsugu et al., 2017; Nara and Su’Etsugu, 2021) to develop an in vitro pipeline to produce novel infectious clones. The RCR system works by inserting an origin of replication C sequence (OriC) into a plasmid. This plasmid can then be amplified using a reconstituted mixture of 25 polypeptides and 14 proteins critical for chromosomal replication in E.coli bacteria (Su’Etsugu et al., 2017). The RCR reaction begins with DnaA binding to the OriC sequence along with several initiation factors (Su’Etsugu et al., 2017). The replication fork is then progressed using DnaB and DnaC helicase, gryases, DnaG primase, and the DNA polymerase III holoenzyme (Pol III HE) complex (Su’Etsugu et al., 2017). The Okazaki fragments are repaired using DNA polymerase I, RNAse H, and a DNA ligase (Su’Etsugu et al., 2017). The daughter plasmids are deconcatenated using topoisomerase IV (TOPO IV), TOPO III, and RecQ helicase (Su’Etsugu et al., 2017). Finally, the DNA gyrase introduces a negative supercoil structure to the products, priming them for another round of replication (Su’Etsugu et al., 2017). The original authors demonstrate that the resulting products of an RCR reaction were supercoiled using gel electrophoresis (Su’Etsugu et al., 2017).

This process selectively amplifies products that contain the OriC sequence and does so with high fidelity, with an error rate of ~10−8 per base per replication cycle (Su’Etsugu et al., 2017). This approach removes the need for a living host and the risk of selection due to sequence-specific toxicity. Leveraging RCR, we developed a novel workflow to generate and manipulate viral infectious clones (Figure 1). Briefly, this process uses the high efficiency and rapidly decreasing costs of chemical gene synthesis (Cockrell et al., 2017; Rihn et al., 2021), high fidelity PCR, in vitro assembly, and in vitro amplification. By starting with clonal gene fragments, the resulting product is a clonal supercoiled plasmid population that can be manipulated identically to a bacterial-derived plasmid, and from which virus can be rescued.

Figure 1. Novel in vitro infectious clone generation workflow.

Figure 1.

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To produce a new clone, the viral genome is chemically synthesized and cloned into plasmids. The plasmids are used as a template for PCR, and the amplicons are assembled into a backbone containing the origin of replication C using a modified form of Gibson assembly. The assembled plasmid is then amplified using an OriC – mediated replication-cycle reaction (RCR), which involves the reconstituted 14 proteins and 25 polypeptides necessary for E. coli chromosomal replication. The resulting supercoiled product is then transfected into susceptible cells to produce infectious virus. The clone and/or viral stock is then assessed by deep sequencing and growth kinetics to ensure matching sequences and behaviors with the parental virus.

In this paper, we generated infectious clones of two medically relevant viruses using the above-described workflow. We selected dengue virus serotype 2 (DENV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since they are two viruses of significant public health concern and are known to be difficult to manipulate in bacteria. Based on sequencing and viral kinetics, we concluded that our innovative workflow could effectively generate recombinant viruses with similar replication kinetics to the parental virus. Furthermore, we used our SARS-CoV-2 USA-WA1/2020 clone to generate a Spike D614G mutant virus. This workflow represents a paradigm shift in developing and manipulating infectious clones for emerging pathogens, as it leverages the host-free nature of previously described methods, such as CPER and Gibson assembly, while still producing recoverable plasmids like those from bacterial cloning approaches.

2. Materials and Methods

2.1. Cells and viruses

Vero (CCL-81), BHK-21 (CCL-10; herein called BHK-21), and Calu-3 (HTB-55) cells were obtained from American Type Culture Collection. Vero E6 cells expressing transmembrane protease, serine 2, and human angiotensin-converting enzyme 2 (VeroE6 hACE2-TMPRSS2) cells were obtained from BEI (Catalog No. NR-54970). HEK293A cells were kindly provided by Dr. Jamie Smyth from the Fralin Biomedical Research Institute. Vero and BHK-21 cells were cultured at 37°C with 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal bovine serum (FBS), 1% nonessential amino acids, and 0.1% gentamicin. For VeroE6 hACE2-TMPRSS2 cells, the media described above was supplemented with puromycin to a 0.01 mg/mL final concentration. Dengue virus serotype 2 (DENV2) strain PUO-218 (GenBank: ON398847) was obtained from the Centers for Disease Control and Prevention (Lot: TC00838). SARS-CoV-2 USA-WA1/2020 strain (GenBank: MN985325.1) was acquired from BEI resources (NR-52281; Lot: 70034262). Before testing, both parental viruses were passaged once in Vero (DENV2) and VeroE6 hACE2-TMPRSS2 (SARS-CoV-2).

2.2. Construction of novel infectious clones using the OriC Workflow

Infectious clones were designed in silico using SnapGene 6.0.2 software (GSL Biotech). One (DENV2) or two (SARS-CoV-2) restriction enzyme sites were ablated by synonymous mutation to allow downstream differentiation between the parental and clone-derived virus. Fragments with 40 bp overlaps were designed for clonal gene synthesis by Twist Bioscience (DENV2) or Bio Basic Inc. (SARS-CoV-2).

These plasmids and a vector containing the OriC cassette were then used as templates for PCRs using the SuperFi II Master Mix (Invitrogen; Catalog number: 12368010). The bands were then gel purified using the Macherey-Nagel NucleoSpin Gel and PCR Clean-up kit (Item Number 740609). Fragments were quantified by Qubit before assembly and then mixed in equimolar ratios to a final concentration of 8 ng/μL. The DNA was then assembled using the OriCiro 2x Recombination Assembly mix and incubated for 30 minutes at 42°C, followed by a 65°C step for 2 minutes to eliminate misassembled products. The amplification mixture was prepared according to the kit’s protocol. Following priming at 33°C for 15 minutes, the assembly product was mixed with the amplification mix, incubated for 6 hours at 33°C and then held at 12°C. The next day, the product was supercoiled by diluting the product two-fold in 1x amplification buffer according to the manufacturer’s protocol and incubating at 33°C for 30 minutes. The supercoiled product was then tested by restriction digestion to confirm proper assembly. The remaining supercoiled product was then supplemented with a final concentration of 20 mM EDTA.

2.3. Constructing a novel DENV clone using Circular Polymerase Extension Reaction (CPER)

To generate the above-described DENV2 clone using CPER, we modified previously described approaches (Amarilla et al., 2021; Tamura et al., 2022). PCR fragments were generated as described above. The resulting five fragments were then pooled at an equimolar ratio of 0.1 pmol each. We then assembled the pooled fragments with PrimeSTAR GXL Premix (Catalog number: R051A) using the following cycling conditions: 98°C for 2 minutes followed by 20 cycles of 10s at 98°C, 15s at 55°C, and 12 min at 68°C, and a final extension at 68°C for 12 min.

2.4. Rescue of infectious clones

For the DENV2 infectious clone, viral rescue was performed similarly as previously described (Marano et al., 2020). Briefly, we used the EquiPhi29 DNA Polymerase (Thermo Scientific; Catalog number: A39390) to amplify the RCR product by RCA and then used HEK293A cells for transfection. The virus, representing the passage zero (p0) stock, was harvested seven days post-transfection and titered by plaque assay on Vero cells. We then passaged the p0 stock once in Vero cells at a multiplicity of infection (MOI) of 0.01 to produce the p1 stock, which was used for downstream testing. For the SARS-CoV-2 infectious clone, due to the large size of the plasmid, supercoiled plasmid obtained by RCR was co-transfected with pUC19 (Søndergaard et al., 2020) into BHK-21 cells. The supernatant was collected two- and three-days post-transfection, pooled, and used to blindly infect VeroE6 hACE2-TMPRSS2 (Rihn et al., 2021) to produce a p1 stock for downstream testing. Infectious virus was titered by plaque assay on VeroE6 hACE2-TMPRSS2 cells.

2.5. Comparing rescue kinetics of a CPER-derived, RCR-derived, and RCA-derived DENV clone

To amplify the DENV RCR product by RCA, we used the EquiPhi29 DNA Polymerase The RCR, RCA, and CPER products were quantified before transfection using the Qubit 3.0 fluorometer (ThermoFisher). Transfections were performed as previously described (Marano et al., 2020) using 250 ng of unpurified DNA in 48-well plates of HEK293A cells using the jetOPTIMUS® transfection system (Polyplus; Catalog number: 101000051). Supernatant was collected every 24 hours and assessed by plaque assay.

2.6. Library Preparation and NGS Sequencing

For the DENV2 infectious clone, sequencing libraries were prepared from the RCA product using the sparQ DNA Frag & Library Prep Kit from Quantabio (Cat. 95194–024). After assessing the library size by Tapestation, the samples were sequenced using 150 bp paired-end reads on the Illumina Novaseq 6000. To analyze the resulting data, a previously described workflow was used. Briefly, paired-end reads were trimmed to a quality score of 30 using BBDuk (Bushnell, 2014) before being mapped to the DENV reference sequence using the Burrows-Wheeler aligner (BWA) (Rodgers et al., 2017), and variants occurring at >25% were identified using LoFreq (Li and Durbin, 2009). The consensus sequence of the clone was then produced using Genome Analysis Toolkit (GATK) (McKenna et al., 2010) and aligned to the reference sequence.

For the SARS-CoV-2 infectious clone and SARS-CoV-2 Spike D614G mutant, viral RNA was sequenced using the plexWell ARTIC protocol V4.1 and the plexWell 384 Library Prep Kit protocol. Briefly, cDNA was prepared from RNA samples using SuperScript IV Reverse Transcriptase (Invitrogen, Waltham, MA). PCR amplification was performed using Q5 Polymerase (NEB, Ipswitch, MA) and primers from ARTIC nCoV-2019 Amplicon Panel, V4.1 (Integrated DNA Technologies, Coralville, IA). Amplicons were quantified using a Qubit 2.0 fluorometer (Thermo/Fisher) and then barcoded and pooled using the plexWell™ 384 Library Preparation Kit (seqWell, Beverly, MA). Pooled libraries were amplified using a Kappa HiFi Hot Start Ready Mix kit (Roche, CA). Libraries were sequenced using the 300-cycle MiSeq Sequencing Kit (Illumina, San Diego, CA). To analyze the data, paired-end reads were trimmed to a quality score of 30 using BBDuk (Bushnell, 2014) and aligned to the appropriate reference sequence using BWA (Rodgers et al., 2017). Primer sequences were trimmed using iVar (Grubaugh et al., 2019). Variants were identified using samtools (Danecek et al., 2021), and variants occurring at ≥25% were called using iVar.

2.7. Comparing viral kinetics of clone-derived and parental viruses.

Vero (DENV2) or VeroE6 hACE2-TMPRSS2 (SARS-CoV-2) cells were plated to an 80–90% confluency in 24-well plates. Viral stocks were diluted in Roswell Park Memorial Institute medium (RPMI 1640) with 25 mM HEPES and 1% FBS. Cells were infected at an MOI of 0.01 plaque-forming units (PFU) per cell and incubated at 37°C for one hour. After the adsorption period, the cells were washed in phosphate-buffered saline (PBS), and then fresh media was added. The supernatant was harvested every 24 hours until 50–75% CPE was observed. Harvested samples were stored at −80°C until titration by plaque assay. Viral RNA was used to generate cDNA for both viruses to confirm the clone identity using the Maxima H- RT kit (Catalog number: EP0752). The resulting cDNA was used to amplify the region of the genome containing the ablated restriction site. The amplicons were then digested with either EcoRI (DENV2) or HindIII (SARS-CoV-2) and tested by gel electrophoresis to observe the banding differences between parental- and clone-derived viruses.

2.8. Generating mutant viruses and comparing fitness by competition assay.

To generate the mutant SARS-CoV-2 Spike D614G virus, mutagenic primers were designed bearing the desired point mutation. The SARS-CoV-2 infectious clone served as a template for PCRs. It was serially diluted to the lowest concentration that still resulted in amplification, which was performed to reduce the likelihood of downstream WT virus contamination. The mutants were then constructed and rescued as described above. To confirm the mutation, viral stocks were first confirmed by Sanger sequencing, followed by whole genome NGS, as described above. After confirmation, the mutant virus was mixed with the wild-type (WT) virus at a 1:1 PFU ratio. The virus mix was then used to infect VeroE6 hACE2-TMPRSS2 cells at an MOI of 0.01. Viral RNA was extracted from the inoculum and viral supernatant at 1-day post-infection (dpi), which was used to generate cDNA using the Maxima H- RT kit. The resulting cDNA was used to amplify the region of the genome containing the mutation of interest. The amplicon was then submitted for Sanger sequencing, and the relative ratios of wild-type and mutant virus were determined using EditR (Kluesner et al., 2018). Relative fitness of the mutant compared to the wild-type virus was defined as W = F(t)/F(0), where F(x) represents the relative proportion of mutant virus (either at baseline or 1 dpi) (Liu et al., 2021).

2.9. Statistical Analysis

Statistical analysis was performed in Prism 8 (GraphPad, San Diego, CA, USA). A 2-way ANOVA test was performed with a Dunnett’s correction for multiple comparisons to compare construction methods. A one-way ANOVA was performed with a Tukey correction for multiple comparisons to compare peak titers. A 2-way ANOVA test was performed with a Šidák correction for multiple comparisons for the viral kinetics assays. After a Shapiro-Wilk test confirmed normality, a one-sample t-test was used for the competition assays.

3. Results

3.1. Generating a Medically Relevant DENV2 Infectious Clone

Cryptic bacterial promoters within the DENV genome make developing infectious clones extremely challenging (Li et al., 2011; Pu et al., 2011). Using a host-free approach, we sought to develop a full-length DENV serotype 2 infectious clone. We selected DENV2 strain PUO-218, a low passage virus isolated in Thailand in 1980 (Gruenberg et al., 1988), which is also a component of Sanofi’s Dengvaxia vaccine (Guirakhoo et al., 2001). To generate the clone, the viral genome was synthesized in four clonal fragments (Figure 2A). We created a synonymous mutation that ablated an EcoRI site within the NS3 sequence to allow for differentiation between the clone and the parental virus. The four synthesized fragments and a donor plasmid containing the CMV promoter, hepatitis delta virus ribozyme (HDVr) sequence, and the OriC sequence were templates for high-fidelity PCR. The CMV promoter allows for direct recovery of infectious virus through cellular RNA polymerase II while the HDVr ensures an authentic 3′ UTR sequence; the OriC sequence is required for RCR amplification. The purified amplicons were then assembled into a circular plasmid using a modified Gibson assembly. We then amplified the circular molecule containing the OriC sequence by RCR to produce a supercoiled plasmid containing the full viral genome, as per the protocol established in Su’Etsugu et al., 2017 (Su’Etsugu et al., 2017). Simultaneously, we generated an identical DENV clone using a CPER approach (Tamura et al., 2022). We then directly compared the viral replication kinetics following transfections for the DENV clones generated by CPER, RCR, and RCA-amplified RCR products to benchmark our workflow (Figure 2B). The RCA reaction was included as it provides a convenient means to produce large quantities of DNA (Marano et al., 2020) in place of multiple RCR reactions. We observed that transfections using RCR produced significantly higher titers from 3 to 5 days post-transfection than those using CPER products (P <.0001). Using RCA product for transfections, we also observed increased viral production compared to CPER (3- and 4-days post-transfection, P <.0001). Transfections using RCA produced a higher peak titer than RCR or CPER (Supplemental Figure 1). These data demonstrate that our workflow is comparable to CPER, with significantly faster peak titers, suggesting a higher transfection efficiency.

Figure 2. Generation and Characterization of a dengue virus (DENV) Infectious Clone.

Figure 2.

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A) The DENV2 PUO-218 strain genome was synthesized in four clonal fragments. The four resulting plasmids and a donor plasmid containing the necessary components for expression and replication were used as templates for PCRs. The amplicons from these reactions were then assembled and amplified by RCR to produce a supercoiled infectious clone. B) Comparison of the rescue kinetics of DENV2 PUO-218 clone generated by replication cycle reaction (RCR), amplified by rolling circle amplification (RCA), and generated by circular polymerase extension reaction (CPER). Statistical analysis was performed using a 2-way ANOVA with a Šidák correction for multiple comparisons with all values compared to the CPER titers (**** P< .0001). C) Growth curve of DENV2 PUO-218 isolate and infectious clone in Vero cells. Data represent two biological replicates, each containing three technical replicates, and the error bars represent the standard deviation. No significant difference was detected at any time by a 2-way ANOVA with a Šidák correction for multiple comparisons. D) A representative gel image of an EcoRI digest of the genetically marked region of the infectious clone. The 100bp DNA ladder from NEB (#N3231) was used for reference, with the first sample lane containing the viral isolate and the second containing the clone.

3.2. Comparison of DENV2 Clone-derived Virus to the Parental Virus

We next compared the growth of the parental DENV2 PUO-218 isolate to the infectious clone-derived virus in Vero cells to ensure similar growth kinetics. Vero cells were selected as they are highly susceptible to DENV (Martínez-Barragán and del Angel, 2001). We found no significant differences in viral titer between the parental and the clone on any day (Figure 2C). To confirm the identity of each virus, we performed RT-PCR on the viral stocks and digested the amplicons with EcoRI. We observed the expected two bands at 400 bp and 732 bp for the parental virus and a single band in the clone-derived virus at roughly 1.1 kb (Figure 2D). Finally, next-generation sequencing revealed no variants above 25% of the population within the clone-derived virus, indicating no unwanted mutations were generated during the cloning or viral rescue process. These data indicate that the DENV2 clone’s sequence and in vitro replication match the parental virus.

3.3. Generating a SARS-CoV-2 Infectious Clone

We next sought to produce an infectious clone of an early isolate of SARS-CoV-2, USA-WA1/2020 strain. The SARS-CoV-2 genome was synthesized as four clonal fragments (Figure 3A). Two HindIII sites within Orf1a and Spike were ablated by introducing synonymous mutations during the design phase to distinguish between parental- and clone-derived viruses. As with the DENV2 clone, the clonal fragments and a donor plasmid were used as templates for PCR, and those amplicons were assembled and then amplified by RCR to produce a supercoiled infectious clone plasmid. The RCR product was then transfected into BHK-21 cells, and we used the supernatant for a blind passage in VeroE6 hACE2-TMPRSS2, as previously described (Rihn et al., 2021)

Figure 3. Generation and Characterization of a SARS-CoV-2 Infectious Clone.

Figure 3.

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A) To produce the SARS-CoV-2 USA-WA1/2020 infectious clone, the viral genome was synthesized in four clonal fragments. The four resulting plasmids, along with a donor plasmid that contained the necessary components for expression and replication, were used as templates for PCRs. The amplicons from these reactions were then assembled and amplified by RCR to produce a supercoiled infectious clone. B) Growth curve of SARS-CoV-2 USA-WA1/2020 strain isolate and the infectious clone in VeroE6 hACE2-TMPRSS2 cells. Data represent two biological replicates, each consisting of three technical replicates. Statistical analysis was performed using a 2-way ANOVA test with a Šidák correction for multiple comparisons (** P = .003 *** P = .0005). C) A representative gel image of a HindIII digest of the genetically marked region of the infectious clone. The 1kb Plus DNA Ladder from NEB (#N3200) was used for reference, with the first sample lane containing the viral isolate and the second containing the clone.

3.4. Comparison of SARS-CoV-2 Clone-derived Virus to the Parental Virus

We next compared the growth of the parental and clone-derived virus in VeroE6 hACE2-TMPRSS2 since they are highly susceptible to SARS-CoV-2 (Matsuyama et al., 2020). We found that the clone-derived virus had reduced titers on days 1 (P = 0.0005; Figure 3B) and 2 (P = 0.003; Figure 3B) compared to the parental virus. RT-PCR confirmation was performed on the stocks, and the amplicons were digested with HindIII. We observed the expected bands at 1.1kb (representing two overlapping bands of equal size) for the parental virus and 2.2 kb for the clonal virus (Figure 3C). NGS was performed on the viral RNA to compare the clone-derived virus sequence to the parental virus, and the consensus sequences of both viruses were identical.

3.5. Generation and characterization of a medically relevant early pandemic mutant

We next sought to demonstrate that the new clones could serve as templates for further manipulation. To that end, using the SARS-CoV-2 infectious clone, we engineered a virus bearing a medically significant mutation, Spike D614G, hereafter called D614G, an early mutation in SARS-CoV-2 (Isabel et al., 2020). The mutant virus was generated by site-directed mutagenesis and assembled similarly to the parental infectious clone. Following rescue, we confirmed the mutation via NGS and detected no additional variants above the threshold.

We next compared D614G’s replication fitness to the WT virus using a competition assay. Briefly, the mutant was mixed at a 1:1 PFU ratio with WT SARS-CoV-2 infectious clone-derived virus. The mix was then used to infect VeroE6 hACE2-TMPRSS2. Viral RNA was extracted from the inoculum and the viral supernatant at 1 dpi and was used for RT-PCR to amplify the region containing the D614G mutation (Figure 4A). The relative fitness was assessed by comparing the starting and ending proportions for each virus (Figure 4B). As expected, the D614G mutant had significantly increased fitness (P = .0005), which aligns with previous findings regarding this mutation (Plante et al., 2021; Yang et al., 2021). These data demonstrate the successful engineering of a mutation using the RCR-based in vitro workflow.

Figure 4. Comparing Fitness of Early Pandemic SARS-CoV-2 Mutants Using a Novel Infectious Clone.

Figure 4.

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A) To assess the fitness of Spike D614G, wild-type (WT) and D614G were mixed at a 1:1 PFU ratio. After confirming the mix composition by Sanger sequencing and plaque assay, the mix was used to infect VeroE6 hACE2-TMPRSS2 cells. RNA was extracted from the viral supernatant at 1 dpi and used to synthesize cDNA downstream. B) Relative Fitness of SARS-CoV-2 USA-WA1/2020 wild-type infectious clone and a mutant bearing Spike D614G was calculated in VeroE6 hACE2-TMPRSS2 cells. Data represent two biological replicates, each consisting of three technical replicates, and the error bars represent the standard deviation from the mean. Statistical analysis was performed using a one-sample t-test with a null value of 1 (*** P = .0005).

4. Discussion

Infectious clones enable mechanistic studies to understand the impacts of mutations on viral biology and the generation of vaccines and diagnostic tools. However, state-of-the-art bacterial-based systems can be extremely cumbersome due to the toxicity of viral sequences (Pu et al., 2011; Steel et al., 2011; Scobey et al., 2013). To address these issues, we leveraged several emerging in vitro technologies, including gene synthesis and RCR, to develop an in vitro workflow to develop infectious clones (Fig 1). Using this system, we successfully produced infectious clones for DENV2 and SARS-CoV-2, two viruses of great medical importance.

An important consideration when producing an infectious clone is that clone-derived and parental viruses replicate similarly and have the same amino acid sequence. Both clone-derived viruses matched the parental virus consensus sequence. For the DENV clone, the parental- and clone-derived growth kinetics were identical. In contrast, the clone-derived SARS-CoV-2 replicated to slightly lower levels compared to the WT virus, similar to what has been reported with other clones (Thi Nhu Thao et al., 2020; Xie et al., 2020). Evidence from other viruses has suggested that the loss of genetic diversity of a clone-derived virus can reduce viral fitness (Bordería et al., 2010; Fitzpatrick et al., 2010; Coffey et al., 2011), which could explain the observations with the SARS-CoV-2 clone. We further demonstrated that the infectious clone could be used for mutagenesis, as we generated the SARS-CoV-2 Spike D614G mutant (Isabel et al., 2020; Plante et al., 2021; Yang et al., 2021) and characterized it using a competition assay. We were able to recapitulate prior work demonstrating that SARS-CoV-2 Spike D614G has a replicative advantage compared to WT SARS-CoV-2 (Isabel et al., 2020; Plante et al., 2021; Yang et al., 2021).

The RCR and RCR/RCA products produced higher virus titers at earlier time points during viral rescue compared to CPER, which has previously been used to successfully produce recombinant viruses from various families. The delayed replication kinetics following transfection for the CPER product could be due to lower assembly efficiency compared to RCR. Furthermore, the RCR amplification step preferentially amplifies circular molecules, which would be expected to contain the full-length genome, while CPER reactions provide no such amplification. Future studies should compare the proportion of full-length viral genomes produced in CPER and RCR reactions. Additional studies should also compare transfection efficiency between the techniques since this may be critical for producing diverse viral libraries. These data suggested that RCR, RCR/RCA, and CPER are viable approaches to generate recombinant viruses.

Limitations of our study:

This work’s limitations include using only viruses from two viral families: Flaviviridae and Coronaviridae. However, these families likely represent the most challenging positive-sense RNA viruses to manipulate in bacteria; thus, we are confident that the workflow could be easily transferred to other positive-sense RNA viruses. Future studies involving negative-sense RNA viruses, multipartite viruses, and DNA viruses should be performed to expand the use of this technology. Another limitation is the genome size that was explored in this report. While coronaviruses have the largest RNA viral genomes (Almazán et al., 2000; Ogando et al., 2019), they are by no means the largest viral genomes, particularly when compared to giant aquatic viruses like Megavirus (Legendre et al., 2012) and Mimivirus (Colson et al., 2017). Given that the largest reported genome amplified with the OriC system was 1 Mb (Nara and Su’Etsugu, 2021), slightly below the genome size of some large aquatic viruses (Legendre et al., 2012; Colson et al., 2017), further characterizations may be needed for these giant viruses.

5. Conclusion

We demonstrated that replication cycle reaction (RCR) can be a critical tool for developing infectious clones. Specifically, we demonstrate that a DNA synthesis and RCR workflow was comparable to the current gold standard in in vitro clone construction, CPER. Further, we demonstrate that our workflow could be used to develop infectious clones of DENV2 and SARS-CoV-2 and medically relevant mutants of SARS-CoV-2. The results signal a foundational advancement in molecular virology and provide simple tools for many virologists to develop medically relevant infectious clones.

Supplementary Material

1

Highlights.

  • The development of infectious clones using bacteria is difficult due to toxicity.

  • Current methods to mitigate toxicity lack plasmid recoverability or are leaky.

  • An in vitro replication cycle reaction (RCR) method can mitigate toxicity.

  • Using an RCR method, we produced clones of dengue and SARS-CoV-2.

  • Using an RCR method, we generated a SARS-CoV-2 mutant.

Acknowledgments:

Figures 1, 2A, 3A, and 4A were generated using Biorender. The Molecular Diagnostics Laboratory performed viral RNA sequencing at the Fralin Biomedical Research Institute. The Virginia Tech Genomic Sequencing Center performed Sanger sequencing.

Funding:

This work was supported by seed grant funding from Virginia Tech’s Center for Emerging, Zoonotic, and Arthropod-borne Pathogens (CeZAP) and the Edward Via College of Osteopathic Medicine (VCOM) One Health seed funds awarded to J.W.L.

Conflicts of Interest:

J.M.M. has received financial support from OriCiro in travel reimbursement to present the above work. The other authors have no conflicts to report.

Footnotes

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Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data Availability Statement:

The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.

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Data Availability Statement

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