Fig. 3

Mutagenesis of the ENTV-1 protease to resemble the JSRV protease does not restore proteolytic processing. a ClustalW alignment of the predicted amino acid sequence of the protease from the ENTV-1 molecular clone with that of all 10 ENTV-1 isolates from North America (ENTV-1NA, see Table 1 [19] for accession numbers), ENTV-1OVC (accession number KC189895) [41], ENTV-2 (accession number NC 004994), JSRV (accession number AF105220) and an endogenous JSRV (enJS56A1, accession number AF153615). Dots indicate identical amino acids. The black arrow marks the catalytic amino acid, shown in red. The red box highlights the S199P mutation found only in the original ENTV-1 molecular clone. The blue boxes highlight amino acids where the ENTV-1 molecular clone is more similar to ENTV-1NA4, from which it was derived, and the three other isolates that were obtained from the same farm as ENTV-1NA4: ENTV-1NA2, ENTV-1NA3, and ENTV-1NA5. b Capsid protein immunoblot analysis of lysates from concentrated viruses derived from the ENTV-1 molecular clone containing the designated amino acid changes in the protease domain. M indicates the lane containing the molecular weight marker and lanes labelled as ECa contain bacterially expressed and purified ENTV-1 capsid protein. The black arrow indicates unprocessed Gag at 78 kDa and the red arrow indicates processed Capsid at 26 kDa