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. 2002 Mar;76(6):3023–3030. doi: 10.1128/JVI.76.6.3023-3030.2002

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Copyright © 2002, American Society for Microbiology.

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FIG. 1. — Expression of NLV capsid genes from VEE replicons. (A) Organization and transcription strategy of VEE replicons. In VEE replicons, the structural genes (capsid, E1, and PE2) are replaced with the NV capsid gene inserted into a polycloning site (PCS) under control of the subgenomic 26S promoter. Replicon transcripts coelectroporated into cells with the VEE defective packaging construct RNAs (VEE capsid and envelope glycoproteins) result in the packaging and release of infectious VEE VRPs that can be used as single-hit vaccine vectors in mammals (30, 41). Transfection will also result in the transient expression of high concentrations of rNV capsid protein which may self-assemble into NV VLPs. Upon infection of cells with VEE VRPs encoding the NV capsid gene, these single-hit replicon vectors will express high levels of the rNV capsid protein that self-assemble into ∼38-nm-diameter recombinant NLV particles which should be free of VEE VRPs. (B) Sequence of the NV major capsid gene. The NV capsid gene was cloned from stool samples of two human volunteers challenged with NV. The forward primer (NVCAPF1 [5′-GAT TTCCAGCAAGGTCATAC-3′]) and reverse primer (NVCAPR1 [5′-TTCGCCACCAACCTGTATGC-3′]) were designed to amplify the entire NV capsid region following reverse transcription-PCR (27, 46). The reaction was performed in a 50-μl reaction mixture with 5 μl of purified viral mRNA. Reverse transcription was performed at 50°C for 60 min, and then the temperature was elevated to 94°C for 2 min. Forty amplification cycles were performed, with one cycle consisting of 30 s at 94°C, annealing at 55°C for 1 min, and primer extension at 68°C for 2 min. While the NV1 sequence was identical to the published sequence (26, 27), the sequence of NV2 contained three amino acid changes. Chimeric constructs were assembled in which the N-terminal amino acid alterations in NV2 were joined in frame to the C terminus of NV1 (NV3) and in the opposite orientation (NV4). Using overlapping extension PCR, the NV capsid gene was fused to the VEE subgenomic promoter and inserted into the polycloning site of the VEE PVR21 plasmid vector and sequenced. (C) Expression of NV capsid proteins in cells from different mammals. Cells were infected with VRP-NV1 for 1 h at room temperature at a MOI of 5. At 18 h postinfection, the cultures were fixed and stained by FA staining for the presence of NV capsid proteins using NV antiserum from an infected volunteer using previously described techniques (5). Caco2 human colorectal adenocarcinoma cells, CRFK feline kidney cells, DBT murine astrocytoma cells, and swine testicular (ST) cells are shown.

FIG. 1. — Expression of NLV capsid genes from VEE replicons. (A) Organization and transcription strategy of VEE replicons. In VEE replicons, the structural genes (capsid, E1, and PE2) are replaced with the NV capsid gene inserted into a polycloning site (PCS) under control of the subgenomic 26S promoter. Replicon transcripts coelectroporated into cells with the VEE defective packaging construct RNAs (VEE capsid and envelope glycoproteins) result in the packaging and release of infectious VEE VRPs that can be used as single-hit vaccine vectors in mammals (30, 41). Transfection will also result in the transient expression of high concentrations of rNV capsid protein which may self-assemble into NV VLPs. Upon infection of cells with VEE VRPs encoding the NV capsid gene, these single-hit replicon vectors will express high levels of the rNV capsid protein that self-assemble into ∼38-nm-diameter recombinant NLV particles which should be free of VEE VRPs. (B) Sequence of the NV major capsid gene. The NV capsid gene was cloned from stool samples of two human volunteers challenged with NV. The forward primer (NVCAPF1 [5′-GAT TTCCAGCAAGGTCATAC-3′]) and reverse primer (NVCAPR1 [5′-TTCGCCACCAACCTGTATGC-3′]) were designed to amplify the entire NV capsid region following reverse transcription-PCR (27, 46). The reaction was performed in a 50-μl reaction mixture with 5 μl of purified viral mRNA. Reverse transcription was performed at 50°C for 60 min, and then the temperature was elevated to 94°C for 2 min. Forty amplification cycles were performed, with one cycle consisting of 30 s at 94°C, annealing at 55°C for 1 min, and primer extension at 68°C for 2 min. While the NV1 sequence was identical to the published sequence (26, 27), the sequence of NV2 contained three amino acid changes. Chimeric constructs were assembled in which the N-terminal amino acid alterations in NV2 were joined in frame to the C terminus of NV1 (NV3) and in the opposite orientation (NV4). Using overlapping extension PCR, the NV capsid gene was fused to the VEE subgenomic promoter and inserted into the polycloning site of the VEE PVR21 plasmid vector and sequenced. (C) Expression of NV capsid proteins in cells from different mammals. Cells were infected with VRP-NV1 for 1 h at room temperature at a MOI of 5. At 18 h postinfection, the cultures were fixed and stained by FA staining for the presence of NV capsid proteins using NV antiserum from an infected volunteer using previously described techniques (5). Caco2 human colorectal adenocarcinoma cells, CRFK feline kidney cells, DBT murine astrocytoma cells, and swine testicular (ST) cells are shown.