Fig. 4

Alanine point mutations of conserved N-terminal Gag residues abolish particle budding, infectivity and Gag processing. a Alignment of FV Gag sequences using ClustalW2 Multiple Sequence Alignment and identification of highly conserved residues. Sequences were obtained from SwissProt (FFV Gag, accession number O56860; EFV Gag, accession number Q9J4C8; BFV Gag, accession number O41893; SloEFV Gag from endogenous sloth FV [56]; PFV Gag, accession number P14349; SFVcpz Gag from chimpanzee, accession number Q87039; SFVora from orangutan, accession number P27400; SFVmac Gag from macaque, accession number Q00071; SFVagm from African green monkey, accession number M74895). Highly conserved residues are marked in bold face letters and asterisks below the alignment. The proposed FFV Gag cytoplasmic targeting and retention signal (CTRS) is marked by a frame. Alanine point mutations of FFV Gag (L10A, Q11A, Q12A, L13A, Y14A, H25A, G26A, D27A, I28A, I29A, R32A, G36A, W38A, G39A, R43A, L51A and D53A, in red) were constructed using the proviral vector pCF-7 and the Gag expression clone pBC-Gag-oRPE. b 293T cells were transfected in 10 cm dishes with 8 µg of plasmid encoding for proviral Gag mutants (L10A, Q11A, Q12A, L13A, Y14A, H25A, G26A, D27A, I28A, I29A, R32A, G36A, W38A, G39A, R43A, L51A and D53A, lanes 1–10 and 13–19, respectively), pCF-7 (lanes 11 and 20) or pcDNA (lanes 12 and 21). Cell lysates and VLPs in supernatants were analyzed by SDS-PAGE. Positions of Gag p48 and p52, full-length Env precursor gp130^Env, mature processed Env gp48™, Pol precursor p127^Pol and PR-RT-RN domain p65^Pol are marked. c Two days p.t., infectivity was assessed by titration onto FeFab cells. Mean relative titers of three independent titration experiments are shown. Error bars represent the standard deviation