mA3 influences FV-specific neutralizing antibody responses in highly resistant (Fv2^r/r) mice. (A–B) mA3^+/+ and mA3^−/− B6 mice (>16 weeks old) were infected with 5000 SFFU of FV. A, mA3 influences early viremic control at 7 dpi; B, mA3 is required for FV-specific neutralizing antibody production 28 dpi. (C) mA3 influences neutralizing antibody responses in 129/Ola mice. mA3^+/+ and mA3^−/− 129/Ola mice were crossed with mA3^+/+ and mA3^−/− B6 mice to generate F₁ offspring. FV-specific neutralizing antibody titers (IC₇₅) at 28 dpi with 5000 SFFU of FV are shown. Additional information is found in the Supplementary Text. Statistical analysis was performed using a two-tailed Student’s t test.

The Rfv3 genetic restriction is mediated by mA3 in vivo. (A–C) mA3 confers the Rfv3 phenotype in low-recovery H-2^b/d mice. Congenic mA3⁺ and mA3⁻ (B6 × BALB/c) F₁ mice were infected with 140 spleen focus-forming units (SFFU) of FV. A, mA3 is required for viremic control at an early timepoint (7 dpi); B, mA3 inactivation compromises Rfv3-associated survival from FV-induced disease; C, mA3 influences FV-specific IgG production 14 dpi. FV binding IgG was measured by flow cytometry using FV antigen-expressing FBL-3 cells. (D) mA3 confers the Rfv3 phenotype in high-recovery H-2^b/b mice. Congenic (A.BY × BALB/c)F₁ mice were infected with 1400 SFFU of FV. Mean 28 dpi neutralizing antibody (NAb) titers (75% inhibitory concentration, IC₇₅) are significantly lower in mA3 ⁻ F₁ mice. Open circles indicate individual mice data, gray bars indicate means and dashed lines refer to the assay detection limit. Statistical analyses were performed with two-tailed Student’s t test.

Molecular basis of Rfv3^s susceptibility. (A) Aberrant mA3 exon 2 splicing in Rfv3^s/s mice. If the wild-type mA3 open reading frame (ORF1) is used, frameshift-induced translational termination will result in a nonfunctional peptide. However, two start sites in an alternative reading frame (ORF3) may be utilized to translate a mutant mA3 protein with a novel N-terminus and a 56 amino acid deletion. (B) Decreased Exon2⁺, but not total mA3 transcripts, in Rfv3^s/s mice. Quantitative RT-PCR of total (left) and Exon2⁺ (right) mA3 splenocyte transcripts in Rfv3^r/r and Rfv3^s/s mouse strains were performed. Amplification levels were normalized to beta-actin (left) or total mA3 (right). (C) Decreased antiviral activity of the mA3 Δexon2 relative to the mA3 Δexon5 spliceoform. An FV molecular clone was co-transfected in NIH3T3 cells with FLAG-tagged mA3 constructs. The infectivity of harvested virions were assayed in Mus dunni cells and normalized against reverse transcriptase activity. Co-transfections with FLAG vector alone were set at 0% inhibition (not shown). Expression of mA3 was assessed by anti-FLAG immunoblotting. Error bars correspond to SD from triplicate experiments.