Fig. 5 in Successful transcription but not translation or assembly of Solenopsis invicta virus 3 in a baculovirus-driven expression system

Fig. 5. Confirmation of heterologous expression of SINV-3 transcript by amplification of the 3' (A) and 5' termini (B) from RNA templates purified from SINV- 3-transfected Sf21 cells. Regions amplified are illustrated in the genome diagram between (A) and (B). (A) Three plaque preparations (AcSINV-3 CiC, AcSINV-3 CiD, and AcSINV-3 DiD) were separated by centrifugation into soluble and pelleted fractions, treated with DNase I, reverse transcribed, and the 3' end of the genome amplified by PCR. Lane assignments were as follows: 1 = mass marker (bp); 2, 6 = AcSINV-3 CiC; 3, 7 = AcSINV-3 CiD; 4, 8 = AcSINV-3 DiD; 5, 9 = mock infection; 10 = positive control (wild-type virus); 11 = negative control; 12 = non-template control. (B) AcSINV-3 plaque preparations (CiC and DiD) evaluated by PCR of the 5' end of the genome (RNA preparations). Lane assignments were as follows: 1 = mass marker (bp); 2, 3 = DNase treated, reverse transcribed; 4, 5 = without DNase treatment, reverse transcribed; 6, 7 = DNase treated, without reverse transcription; 8, 9 = without DNase treatment, without reverse transcription; 10 = positive control; 11 = negative control; 12 = non-template control. (C) PCR amplification of the entire SINV-3 genome from DNA preparations of AcSINV-3 CiC (lane 2) and AcSINV-3 DiD (lane 3). Lane 1, molecular markers; lane 4, non-template control. (D) Western blot to evaluate translation of the SINV-3 transcript by detection of viral capsid protein 2 (VP2). Lane assignments were as follows: 1 = AcSINV-3 CiC (4 dpi); 2 = AcSINV-3 CiC (5 dpi); 3 = AcSINV-3 CiD (4 dpi); 4 = AcSINV-3 DiD (5 dpi); 5 = mock infection (negative control); 6 = positive control (purified wild-type SINV-3; kDa).