Apobec3 encodes Rfv3, a gene influencing neutralizing antibody control of retrovirus infection

Abstract

Recovery from Friend virus 3 (Rfv3) is a single autosomal gene encoding a resistance trait that influences retroviral neutralizing antibody responses and viremia. Despite extensive research for 30 years, the molecular identity of Rfv3 has remained elusive. Here, we demonstrate that Rfv3 is encoded by Apobec3. Apobec3 maps to the same chromosome region as Rfv3 and has broad inhibitory activity against retroviruses, including HIV. Not only did genetic inactivation of Apobec3 convert Rfv3-resistant mice to a susceptible phenotype, but Apobec3 was also found to be naturally disabled by aberrant messenger RNA splicing in Rfv3-susceptible strains. The link between Apobec3 and neutralizing antibody responses highlights an Apobec3-dependent mechanism of host protection that might extend to HIV and other human retroviral infections.

Fig. 1

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.

Fig. 1

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.

Fig. 1

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.

Fig. 1

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.

Fig. 2

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.

Fig. 2

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.

Fig. 2

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.

Fig. 3

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.

Fig. 3

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.

Fig. 3

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.