Experiment Overview
Our exeperiment consists of three parts: 1. Single Round Infectious Particle (SRIP) production 2. SRIP detecting cell production 3. SRIP infection
In this part, we will give an overview of the construction of the system. There are two types of cells we will create. The first is SRIP producing cells (hereafter referred to as “SRIP producing cells”). The second is a cell for detecting SRIP infection (hereafter referred to as “SRIP detecting cell”).
First, let's look at SRIP producing cells. This is based on Dr. Suzuki's paper [3], in which the sequences encoding the capsid, membrane, and envelope that make up the virus particle itself and the non-structural protein sequences encoding the proteins necessary for replication and other functions are inserted into separate plasmids. Some of the non-structural proteins are modified and replaced with appropriate sequences. These plasmids are co-transfected into HEK293T cells or C6/36 cells to create SRIP producing cells.
Next, we will discuss SRIP detecting cells. As described below, SRIP detecting cells are produced by constructing a genome using plasmids and synthetic DNA and co-transfection of the constructed plasmids into Vero cells or C6/36 cells.
Fig.1: overview of experiment
1. SRIP Production
Step 1. Construction
The viral genomes of Flaviviridae all consist of sequences encoding capsid, membrane, and envelope, which constitute the viral particles themselves, and non-structual protein sequences encoding proteins necessary for replication and other processes. These sequences are divided into structual protein and non-structual protein, and genes for C-Cre and autodegradable peptides are attached to the non-structual protein. Now, the particles produced do not contain the gene for the structural protein, but instead carry the gene for the C-Cre.
The backbone of the C-Cre particles is not dengue virus but yellow fever virus, as described in the paper by Dr. Suzuki [1]. prME is from DENV1-4 respectively, but C and NS1-5 are from yellow fever virus.
The process of creation was as follows. First, the Nano Luciferase portion of pCMV-YF-nluc rep provided by Dr. Suzuki is cut out using the restriction enzymes SnaBⅠ and XmaⅠ, and both ends are CIP treated. At this time, a portion of the promoter is also cut out, so the synthetic DNA contains the cut out promoter sequence, 5' UTR, dC, C-Cre, and the cut out FMDV2A sequence. The synthetic DNA is treated with restriction enzymes SnaBⅠ and XmaⅠ, and ligated with the restriction enzyme-treated plasmid that we received as an insert. The ligated product is transformed into E. coli DH5α and cultured. Extract the plasmid from the colony to obtain the plasmid you want.
SRIP producting construction protocol
Fig.2: SRIP producting construction
Step 2. HEK 293T culture cell
As shown in the figure, capsid, prME, and C-Cre and nonstructural protein sequences are transformed into HEK293T cells and C6/36 cells, respectively. HEK293T cell is a cell derived from human embryonic kidney expressing the SV40 largeT antigen. The transduced genome is transcribed and translated in HEK293T cells to produce particles. As mentioned again, the particles do not contain the genome encoding the structural protein, and no new particles are formed in the cells even if they infect other cells. protocol
Step3. SRIP Production
Cells incorporating the flavivirus genome synthesize their respective structural proteins and a genome that does not contain the structural protein sequence. These assemble to form particles that do not contain structural proteins and are released outside the cell. These particles are called "single-round infectious particles(SRIP)" because they do not allow new particles to be constructed in the cells they infect.
Fig.3: Concept of SRIP-producing cells
2. Production SRIP Detecting Cell
Step1. Construction
The cells for detection should behave differently when infected with SRIP than when uninfected with SRIP. The difference in behavior is expressed by the difference in the fluorescent proteins. The Cre-LoxP system was employed as the system used to switch between the two. In addition, as mentioned earlier, since Split-Cre is employed, the N-Cre sequence corresponding to the one put on SRIP is incorporated. When Cre recombinase and the Cre-LoxP system functions, the expressed fluorescent proteins must be altered; by placing the N-Cre, IRES, and EGFP sequences between the LoxP sequences and the mCherry behind them, the Cre- LoxP system was constructed so that when the LoxP system functions, the N-Cre, IRES, and EGFP sequences are cut out and mCherry is expressed.
The creation process is as follows. Prepare two synthetic DNAs, LoxP_N-Cre and LoxP_mCherry. Prepare two types of plasmids to be used, pIRES2-EGFP and pCAGGS.
First, pIRES2-EGFP is cut with restriction enzymes SmaⅠ and EcoRⅠ and CIP treated. Ligation of synthetic DNA LoxP_N-Cre treated with the same restriction enzymes is used to create a plasmid containing the sequence LoxP_N-Cre_IRES_EGFP. This is selected with E. coli DH5α, and colonies are taken to obtain the plasmid desired. Next, this plasmid is cut open and CIP treated with the restriction enzyme NotⅠ, which has a recognition sequence downstream of the EGFP sequence. Then, ligate the synthetic DNA LoxP_mCherry treated with the same restriction enzyme to create a plasmid containing the sequence. LoxP_N-Cre_IRES_EGFP_LoxP_mCherry, and select this with DH5α as before to obtain the plasmid you want.
Finally, treat this plasmid with restriction enzymes BglⅡ and EcoRⅠ to cut out the sequence of LoxP_N-Cre_IRES_EGFP_LoxP_mCherry. It was then treated with the same restriction enzymes, ligated into a CIP-treated plasmid called pCAGGS, and selected with DH5α. This plasmid is exactly what we wanted, and it will be used to transform cells to create the cells for infection detection.
SRIP detecting cell construction protocol
Step2. Production Detecting cell
The constructed plasmid with the sequence of LoxP_N-Cre_IRES_EGFP_LoxP_mCherry inserted into pCAGGS is transformed into Vero cells or C6/36 cells. In this process, the constructed plasmid is cleaved with a one-cutter restriction enzyme, linearized, and electroporated. At the same time, the plasmid carrying the neomycin resistance gene is linearized and electroporated, and the successfully transformed cells will also be resistant to neomycin. An antibiotic called G418 is used for this selection.
The constructed cells will express EGFP as long as they are not exposed to Cre.
protocol Fig.4: cleavage and transfection
3. SRIP Infection
The produced SRIP is mixed with the subject's sample (serum) and added to the prepared SRIP detecting cells. If there are neutralizing antibodies in the serum, SRIP cannot infect the SRIP detecting cells and the SRIP detecting cells remain green fluorescent, but if there are no neutralizing antibodies, SRIP infects the SRIP detecting cells, and the RNA in the particles is released into the cells. RNA in the particles is released into the cells. Since the RNA in the particles encodes C-Cre, it binds to N-Cre expressed in SRIP detecting cells and becomes Cre, allowing the Cre-LoxP system to function and mCherry to be expressed instead of EGFP.
Fig.5: SRIP is not infectious if there are neutralizing antibodies in the serum. If there are no neutralizing antibodies, SRIP is infectious. Fig.6: SRIP infecting Fig.7: Infected cell
References
[1] Yamanaka A, Matsuda M, Okabayashi T, Pitaksajjakul P, Ramasoota P, Saito K, Fukasawa M, Hanada K, Matsuura T, Muramatsu M, Shioda T, Suzuki R. Seroprevalence of Flavivirus Neutralizing Antibodies in Thailand by High-Throughput Neutralization Assay: Endemic Circulation of Zika Virus before 2012. mSphere. 2021 Aug 25;6(4):e0033921. doi: 10.1128/mSphere.00339-21. Epub 2021 Jul 14. PMID: 34259560; PMCID: PMC8386448
[2] Rajaee M, Ow DW. A new location to split Cre recombinase for protein fragment complementation. Plant Biotechnol J. 2017 Nov;15(11):1420-1428. doi: 10.1111/pbi.12726. Epub 2017 Apr 20. PMID: 28317293; PMCID: PMC5633763.
[3] Matsuda M, Yamanaka A, Yato K, Yoshii K, Watashi K, Aizaki H, Konishi E, Takasaki T, Kato T, Muramatsu M, Wakita T, Suzuki R. High-throughput neutralization assay for multiple flaviviruses based on single-round infectious particles using dengue virus type 1 reporter replicon. Sci Rep. 2018 Nov 9;8(1):16624. doi: 10.1038/s41598-018-34865-y. PMID: 30413742; PMCID: PMC6226426.