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

Comparison of the nucleotide coding and amino acid sequences of the feline A3C genes

Pairwise comparison of the domestic cat A3 cDNA to the predicted A3Ca, A3Cb and A3Cc genomic coding sequences and the predicted amino acid sequences. Amino acid sequence alignment of A3C cDNA and the predicted proteins for A3C genes. Highlighted in yellow are amino acid residues different between the A3Cs based on the genomic sequence, whereas amino acid sites displaying non-synonymous amino acid substitutions are boxed in blue and red for A3Cb and A3Cc, respectively, as identified by SNP genotyping of eight domestic cat breeds for exons 2-4 of A3Ca, A3Cb and A3Cc (for more details see Table 4 in Additional data file 2). Arrows indicate exonic junctions. Below the alignments, variant amino acids are boxed in red for A3Cc (for example, W65R) and blue for A3Cb with respect to the breed from which they were identified: Turkish van (VAN), Egyptian mau (MAU), Sphynx (SPH), Birman (BIR) and Japanese bobtail (BOB). A dash indicates the amino acid is identical to genomic sequence. Numbers adjacent to breed identifiers refer to alleles 1 and 2 identified by clonal sequence analysis of the PCR products that are in phase for exons 3 and 4, but not for exon 2 (1/2). The residue corresponding to functionally significant amino acid replacement identified in human A3G (D128K) is highlighted by an asterisk (see text).<p><b>Copyright information:</b></p><p>Taken from "Functions, structure, and read-through alternative splicing of feline APOBEC3 genes"</p><p>http://genomebiology.com/2008/9/3/R48</p><p>Genome Biology 2008;9(3):R48-R48.</p><p>Published online 3 Mar 2008</p><p>PMCID:PMC2397500.</p><p></p

Cat A3 proteins selectively inhibit the infectivity of different retroviruses

A3 expression in the transfected 293T cells was detected by immunoblotting with anti-HA monoclonal antibody. Wild-type (wt) or ΔFFV wild type (b), wild-type FIV-Luc, Δvif FIV-Luc + Vif expression plasmid (vif.V5) and ΔFIV luciferase reporter vector particles (c), FeLV/GFP (d), and ΔSIVagm luciferase viruses (e) were produced in 293T cells in the presence or absence of the indicated APOBEC3s.<p><b>Copyright information:</b></p><p>Taken from "Functions, structure, and read-through alternative splicing of feline APOBEC3 genes"</p><p>http://genomebiology.com/2008/9/3/R48</p><p>Genome Biology 2008;9(3):R48-R48.</p><p>Published online 3 Mar 2008</p><p>PMCID:PMC2397500.</p><p></p

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

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

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