Fig. 1

a Top a plasmid encoding the full‐length replication‐competent clone Friend MLV was modified by adding a 2A peptide sequence (P2A) from picornaviruses, in frame with the C terminus of the envelope gene, followed by a Not1 restriction site and a stop codon. The vif gene was cloned in frame into the Not1 site to generate F-MLV‐2A‐vif. Bottom the Vif protein sequence of the NL4-3 molecular clone is shown. The single amino acid residue changes used for this study are highlighted in red at positions a11, a22, a40, and a143. b PCR of RNA isolated from viruses propagated in vitro using vif- and env-specific primers. c Western blot analysis of cell extracts from virus-producer cells. 293T cells were co-transfected with the indicated F-MLV-vif virus and 24 h post-transfection, equal amounts of cell lysates were analyzed by Western blot, using anti-Vif and anti-β-actin antisera. d Replication of F-MLV-Vif variants in vitro. Mus dunni cells were infected at an MOI = 0.1. Supernatants were collected 6 days post-infection and viral titers were measured by plaque assay. The data represent the average of 3 experiments, with the error bars showing standard deviations. e 2 day-old A3 KO mice were infected with 10⁴ PFU of each virus intraperitoneally. Splenocytes were isolated at 16 dpi and tenfold dilutions of these cells were co-cultured with Mus dunni cells for 3 days. Viral titers were assessed by plaque assay. The data represent the average of at least 5 mice per group, with the error bars showing standard deviations. ***P ≤ 0.001; NS not significant (one-way ANOVA)