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

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: Dengue virus; Infectious clones; Mutagenesis; Reverse genetics; SARS-CoV-2.

Figure 1.. Novel in vitro infectious clone generation workflow.

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.

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

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.

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

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.

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

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).