Figure 1.
Schematic representation of the full length and domains of CiRIG-I.
The full-length CiRIG-I consists of two CARDs, a DExD/H helicase and a RD. To investigate the functions of pivotal domains of CiRIG-I, a series of plasmids were constructed, including the full-length CiRIG-I (1–947, 947 aa); CiRIG-I-ΔCARDs (249–947, 697 aa); CiRIG-I-ΔRD (1–758, 758 aa); CiRIG-I-CARDs-RD (1–253 and 756–947, 445 aa); CiRIG-I-CARDs (1–254, 254 aa); CiRIG-I-RD (755–947, 193 aa). For detailed representation: aa (amino acids), CARDs (2–91, 100–187 aa), helicase domain (272–749 aa), RD (820–942 aa). The scale was shown at the bottom left corner.
Figure 2.
Illustration of pCMV-EGFP-CMV-SV40 plasmid.
It contains additional CMV promoter and SV40 polyA transcription termination sequence. It holds kanamycin (Kan) or neomycin (Neo) selection sequence, restriction enzyme sites (EcoRI and BamHI), and the skeleton component of original pCMV-EGFP plasmid.
Figure 3.
Virus titer test and antiviral activity assay of CiRIG-I.
(A) Virus titer was checked in transfected cells, including pCMV (control) and six representative RIG-I variants by plaque assay. Fresh CIK cells were seeded in 96-well plates, and they were inoculated with 2-fold serial dilution of transfected cell supernatants (harvested at 12 and 48 h) in duplicate. At 72 h post infection, the cells were observed and the viral titer was calculated. The results represented two individual experiments, and error bars indicated the standard deviation (SD). (B) In antiviral activity assay, the steadily transfected cells were seeded into a 96-well plate, then infected with 2-fold-diluted GCRV in duplicate. At 60 h post infection, cells were fixed with 10% paraformaldehyde for 10 min at room temperature, and then stained with 0.05% (wt/vol) crystal violet for 30 min. Washed with water and drained, and the plates were photographed under a light box.
Figure 4.
The mRNA expressions of several downstream genes of CiRIG-I post GCRV infection in seven stable transgenic cells.
These genes included CiIPS-1 (A), CiIFN-I (B) and CiMx2 (C). Seven constructs include pCMV (control), pRIG-I, pΔCARDs, pΔRD, pCARDs-RD, pCARDs and pRD. The mRNA expressions were measured at 0, 2, 24 and 48 h post GCRV challenge. The EF1α gene was used as an internal control to normalize the cDNA template. Error bars indicated SD. Asterisk (*) was marked significant difference (P<0.05) between experimental group and control group. Detailed values were listed at the bottom of the figure.
Figure 5.
The relative virus quantities in pΔCARDs transfected cells post GCRV infection.
They were measured at 2, 24 and 48 h post stimulation. The GCRV quantities in pΔCARDs tranfected cells were relative to those in pCMV transfected cells. Error bars indicated SD. Asterisks (*) indicated significant differences between the pCMV and pΔCARDs transfected cells at indicated time points.
Figure 6.
The mRNA expression patterns of several downstream genes of CiRIG-I post poly(I:C) stimulation in seven stable transgenic cells.
These genes included CiIPS-1 (A), CiIFN-I (B) and CiMx2 (C). The mRNA expressions were measured at 0, 2, 24 and 72 h post stimulation and the concentration of poly(I:C) was 5 µg/ml. Other captions were the same as Fig. 4.
Figure 7.
The mRNA expression profiles of several downstream genes of CiRIG-I after LPS challenge in seven stable transgenic cells.
These genes included CiIPS-1 (A), CiIFN-I (B) and CiMx2 (C). The concentration of LPS was 10 µg/ml. The mRNA expression was measured at 0, 2, 24 and 72 h. Other captions were referenced as Fig. 4.
Figure 8.
The mRNA expression patterns of several downstream genes of CiRIG-I after PGN stimulation in seven stable transgenic cells.
These genes included CiIPS-1 (A), CiIFN-I (B) and CiMx2 (C). The ultimate concentration of PGN was 10 µg/ml. The mRNA expression was measured at 0, 2, 24 and 72 h. Other captions were the same as Fig. 4.
Table 1.
Primers used for the construction of vectors and qRT-PCR analyses.
Table 2.
The abbreviation of constructed vectors.