MOESM2 of Replacement of feline foamy virus bet by feline immunodeficiency virus vif yields replicative virus with novel vaccine candidate potential

Additional file 2. Partial genome sequences from pCF7-Vif-4 and the stop mutations of the in vitro-selected FFV-Vif variants. The Trp codon and the downstream G residue (TGGG) ~ 130 bp upstream of the vif coding sequence are in bold face letters and underlined. In pCF7-Vif W/*1 (in blue), the mutation is from TGG to TGA and for mutant W/*2 (in green) the mutation is from TGGG to TAGA, with both mutations resulting in a Trp (W) to Stop (*) mutation (W/*) as indicated. The bet nucleotide sequence is in black, the linker sequence in pink with recognition sites for NheI (in brown) and SacII (in light violet). The vif gene is marked in blue with the authentic Met start codon in bold. The BettrVif fusion protein is highlighted in yellow with the amino acids color-coded as described above for the genes. The Met residue 14 amino acids upstream of the authentic vif start codon is highlighted in bold and underlining. The C-terminal amino acid sequence of tas is highlighted in red

MOESM4 of Replacement of feline foamy virus bet by feline immunodeficiency virus vif yields replicative virus with novel vaccine candidate potential

Additional file 4. Titers of pCF-7, pCF7-Vif-4 and engineered pCF7-Vif W/*1 and pCF7-Vif W/*2 variants. Plasmid pCF-7, pCF7-Vif-4, pCF7-Vif W/*1, and pCF7-Vif W/*2 and their engineered M/T and M+ variants were transfected into HEK 293T cells and 2 days post-transfection, cell-free supernatants were inoculated on CrFK cells and serially passaged every A 60 and B 84 h p.i. FFV titers were determined in duplicate using FeFAB reporter cells and are shown as bar diagrams for the different passages. Error bars represent the standard deviation

MOESM1 of Replacement of feline foamy virus bet by feline immunodeficiency virus vif yields replicative virus with novel vaccine candidate potential

Additional file 1. Rescue of Vif-deficient FIV and Bet-deficient FFV by FIV Vif and FFV Bet. A Vif-deficient FIV plasmid DNA was co-transfected with plasmids expressing FIV Vif or FFV Bet together with different feA3 restriction factors as given in the legend (left panel). Empty vector pcDNA3.1 served as control. Two days after transfection, cell-free supernatants were used to infect FIV reporter cells and luc activity induced by FIV infection was measured two days p.i. Titers are expressed as luc values of a representative experiment. B The Bet-deficient FFV genome pCF7-BBtr was co-transfected with plasmids expressing untagged and V5-tagged FFV Bet or two different amounts of FIV Vif expression plasmid together with the major FFV-restricting feA3Z2b-HA as shown below the bar diagram (right panel). Empty vector pcDNA3.1 served as control. Two days after transfection, cell-free supernatants were titrated in triplicate using FFV reporter cells as described in the “Methods” section and are expressed as focus-forming units (FFU) per ml inoculum of a representative experiment. Error bars represent the standard deviation

MOESM3 of Replacement of feline foamy virus bet by feline immunodeficiency virus vif yields replicative virus with novel vaccine candidate potential

Additional file 3. Mutations in Tas generated during the analysis of the upstream ATG do not affect Tas-mediated LTR transactivation. The LTR promoter-based luc reporter construct pFeFV-LTR-luc [73] was cotransfected into HEK 293T cells together with a CMV-IE-driven FFV Tas expression construct, the empty control pcDNA3.1 and proviral genomes pCF-7, pCF7-Vif-4, pCF7-Vif W/*1, and pCF7-Vif W/*2, and their engineered M/T and M+ variants. Two days post transfection, luc activity induced by FFV Tas expression was measured in duplicates. Data from a representative experiment normalized to co-expressed β-gal are expressed in a logarithmic bar diagram