PFV Gag mutants used in the study. (A) Schematic outline of the PFV Gag precursor protein. A solid arrow marks the primary processing site in p71Gag. The enlargement shows the protein sequences of the C-termini of wild type PFV Gag (wt) p68Gag subunit and the established GR box deletion mutant (ΔGR) as well as the mutant having 23 C-terminal arginine residues substituted by alanine (GR R/A). Amino acid residues introduced to or altered in comparison to the wild type sequence are indicated in red bold typeset. Differentially shaded boxes highlight C-terminal structural and functional domains. A, assembly domain; GRI-III, glycine-arginine-rich boxes I to III. The described minimal chromatin-binding sequence is underlined ,. The numbers indicate the amino acid position in the wt Gag protein. (B) Schematic outline of the PFV transfer vector puc2MD9 used. CAS: cis acting sequences I to III required for productive transduction. CMV: cytomegalovirus immediate early promoter, R: long terminal repeat (LTR) repeat region; U5: LTR unique 5′ region; ΔU3: enhancer – promoter deleted LTR unique 3’ region; partial coding sequences of PFV Gag, Pol and Env overlapping the CAS sequences are indicated by dashed boxes and marked with Δ. SFFV U3: spleen focus forming virus U3 enhancer promoter; EGFP: enhanced green fluorescent protein ORF; numbers indicate nucleotide positions in the HSRV2 RNA genome. Below the schematic outline the amplicons generated by the qPCR primer - probes sets (Table 1) specific for PFV pol or egfp ORFs are shown. Numbers indicate nucleotide position in the HSRV2 RNA genome or the egfp ORF.