Fig. 1

Lack of Gag-Pro-Pol polyprotein processing in virions derived from the original ENTV-1 molecular clone. a The ENTV-1 genome was amplified from genomic DNA isolated from an ENA tumor (ENTV-1NA4) in two overlapping fragments. The CMV promoter was fused to the 5’ ENTV-1 genome at the 5’ border of the R region in an intermediate fragment by overlap extension PCR and was introduced into the 5’ genome fragment via the BsrGI restriction site. The fragments were ligated together at the MmeI restriction site and the ENTV-1 molecular clone was generated by ligation into the pLG338/30 plasmid. b Singly spliced transcripts coding for the envelope protein were detected in RNA extracted from HEK 293T cells transfected with the ENTV-1 or JSRV molecular clone and the resulting PCR products are shown. A schematic outlining this strategy and the location of the primers (arrows) used in the RT-PCR is shown above. M indicates the lanes containing a 100 bp DNA ladder (Life Technologies). c Immunoblot analysis of capsid protein in the lysates of concentrated virus particles derived from the indicated molecular clones after transfection of HEK 293T cells. M indicates the lane containing the molecular weight marker and lanes labelled ECa contain bacterially expressed and purified ENTV-1 capsid protein. The black arrows indicate unprocessed Gag at 78 kDa and the red arrows indicate processed capsid at 26 kDa. The dashes on the right hand side demarcate unprocessed forms of Gag