Journal: Journal of Virology

Article Title: Genetic engineering strategy for generating a stable dsRNA virus vector using a virus-like codon-modified transgene

doi: 10.1128/jvi.00492-23

Figure Lengend Snippet: Improved stability of codon-modified NLuc gene in recombinant RV vectors. (A) GC content of SA11 NSP1, NLuc, and RvNLuc genes. (B) Expression of NLuc and RvNLuc by plasmid vectors. Expression plasmids pCAG-NLuc and pCAG-RvNLuc were transfected into 293T cells at the indicated amounts. At 16 hours post-transfection, cell lysate NLuc activities were examined. (C) Gene construction of NLuc- or RvNLuc-coding NSP1 gene. NLuc or RvNLuc genes were inserted into the NSP1 ORF. NLuc and RvNLuc were expressed as fusion proteins with 27 NSP1 N-terminal peptides. The latter region of NSP1 downstream of the NLuc/RvNLuc ORF was not expressed. White arrowhead: start codon, black arrowhead: stop codon. Dashed arrows indicate forward and reverse primers for detecting transgene insertion. (D–E) Emergence of transgene-deletion mutants after ten serial passages of reporter RVs expressing NLuc or RvNLuc in MA104 cells. (D) Electrophoresis of the dsRNA genome purified from passage 1 (p1) and p10 viruses was examined. The positions of NSP1-NLuc/RvNLuc genes and wild-type NSP1 genes are indicated by arrows. (E) PCR amplification of the partial NSP1 and NLuc/RvNLuc region using NSP1 specific primers shown in (C). (F) Western blot analysis of NSP1 expression in MA104 cells infected with rSA11 or rSA11-NLuc (p1 and p10) and rSA11- RvNLuc (p1 and p10). VP6 and β-actin were used as controls. (G–H) Viral replication and NLuc activity of rSA11, rSA11-NLuc, and rSA11-RvNLuc in MA104 cells.

Article Snippet: Cells Epithelial monkey kidney MA104 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Nacalai Tesque) supplemented with 5% fetal bovine serum (FBS) (Gibco).

Techniques: Modification, Recombinant, Expressing, Plasmid Preparation, Transfection, Electrophoresis, Purification, Amplification, Western Blot, Infection, Activity Assay

Journal: Journal of Virology

Article Title: Genetic engineering strategy for generating a stable dsRNA virus vector using a virus-like codon-modified transgene

doi: 10.1128/jvi.00492-23

Figure Lengend Snippet: Generation of stable RV vectors encoding codon-modified fluorescent proteins. (A) GC contents of SA11 NSP1, ZsG, RvZsG, AsR, and RvAsR genes. (B) Construction of the NSP1 gene segment encoding fluorescent proteins. Reporter genes were inserted within NSP1 genes as described in Fig. 1. Dashed arrows indicate forward and reverse primers for detecting the insertion of transgenes. (C–F) Emergence of deletion mutant transgenes after 10 serial passages of RV vectors. (C and E upper) Electropherotypes of dsRNA purified from rSA11, rSA11-ZsG, rSA11-RvZsG, rSA11-AsR, and rSA11-RvAsR. White arrows indicate deletion mutant genes. (C and E lower) PCR amplification using primers targeting NSP1 1–20 and 300–320 nucleotides. (D and F) Ratios of ZsG/RvZsG-positive cells per virus-infected cells. MA104 cells were infected with RV vectors at an MOI of 0.1 FFU/cell. At 16 hours post-infection, cells were fixed and virus-infected cells were visualized using rabbit anti-NSP4 serum and anti-rabbit IgG antibody-CF488 conjugate. Five replicates of each virus were examined. P values determined by the t-test are indicated. (G–I) Fluorescence intensities of ZsG and RvZsG. (G and H left) Fluorescent images of ZsG and RvZsG expressed by (G) RV vectors (rSA11-ZsG and rSA11-RvZsG) or (H) plasmid vectors (pCAG-ZsG and pCAG-RvZsG). In infected cells, viral NSP4 was visualized by immunofluorescence using rabbit-anti NSP4 antibody followed by goat antirabbit IgG-cf594 conjugate. Nuclei were stained with Hoechst blue. (G right) Fluorescent intensities of ZsG, RvZsG, and NSP4 were measured by Nikon C2 confocal microscopy. Fluorescent intensity ratios of ZsG and RvZsG to NSP4 are shown. n=50. (I) Replication kinetics of rSA11-ZsG and rSA11-RvZsG. p1: passage 1, p10: passage 10.

Article Snippet: Cells Epithelial monkey kidney MA104 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Nacalai Tesque) supplemented with 5% fetal bovine serum (FBS) (Gibco).

Techniques: Modification, Mutagenesis, Purification, Amplification, Virus, Infection, Fluorescence, Plasmid Preparation, Immunofluorescence, Staining, Confocal Microscopy

Journal: Journal of Virology

Article Title: Genetic engineering strategy for generating a stable dsRNA virus vector using a virus-like codon-modified transgene

doi: 10.1128/jvi.00492-23

Figure Lengend Snippet: Stability of partially codon-modified NLuc gene in RV vector. (A) Schematic showing generation of RV vectors carrying NLuc and RvNLuc chimeric transgenes. The chimeric NLuc genes, RvNLuc1-171, RvNLuc172-342, and RvNLuc343-516 were inserted within NSP1 gene segments and used to generate the RV vectors R1180, R1181, and R1182, respectively. (B) GC content of RV NSP1, NLuc, RV NLuc, and chimeric NLuc genes. (C) dsRNA profiles of R1180, R1181, and R1182 RV vectors after serial passages. Representative data of passage 1, 5, 7, and 10 samples are shown. White arrows indicate deletion mutant genes. (D) Twelve plaque clones obtained from R1180 (p10), R1181 (p10), and R1182 (p10) were amplified in MA104 cells for 72 hours and NLuc activities in the cell lysates were examined. (E) Schematic showing truncated NSP1-RvNLuc343-516 genes obtained from R1182 passage 10.

Article Snippet: Cells Epithelial monkey kidney MA104 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Nacalai Tesque) supplemented with 5% fetal bovine serum (FBS) (Gibco).

Techniques: Modification, Plasmid Preparation, Mutagenesis, Clone Assay, Amplification