Alleles and isoforms of mAPOBEC3 in FV-resistant C57BL/6 and -susceptible BALB/c mice. (A) The known genomic organization and splicing pattern of the mouse APOBEC3 gene is shown with short arrows indicating the positions of the PCR primers used. (B) Alignment of the amino acid sequences of mAPOBEC3 for the C57BL/6-derived exon 5-lacking isoform (mA3^bΔ5) (GenBank accession no. NM_030255), and BALB/c-derived full-length (mA3^d) [BC003314] and exon 5-lacking (mA3^dΔ5) isoforms (GenBank accession no. EDL04624). Open boxes show different amino acid residues, shaded boxes show the regions necessary for CDD activities, and the long horizontal lines indicate two CDDs as described in previous reports (15, 37). B10.A/SgSn mice showed amAPOBEC3 sequence that was completely identical to that of C57BL/6 mice, while A/WySn mice shared the mAPOBEC3 sequence with BALB/c mice.

Infectivities of F-MuLV virions produced from BALB/3T3 cells expressing mA3^bΔ5 mutants. (A and C) Cell lysates from the acutely infected transfectants were analyzed for the presence of APOBEC3 proteins. Immunoblot detection was performed with anti-FLAG and anti-actin Ab. (B and D) Mus dunni cells were infected with virus produced from the mAPOBEC3Δ5-expressing BALB/3T3 cells, and foci of gp70⁺-infected cells were enumerated (n = 3, mean ± standard deviation; *, P < 0.05; **, P < 0.005). All the experiments shown were performed with at least two representative clones of stable transfectants for each gene, and the results obtained with the independent clones were in agreement with the data shown here.

Expression of the different alleles and isoforms of mAPOBEC3 in FV-resistant and -susceptible mice and infectivities of F-MuLV virions produced from mAPOBEC3-expressing cells. (A) Comparisons of mAPOBEC3 mRNA expression levels between mouse strains, by Northern blotting. Female mice, 7 to 8 weeks old, were analyzed for endogenous mAPOBEC3 mRNA expression. mAPOBEC3 mRNA was detected in 5 μg total RNA extracted from the spleen and bone marrow of the indicated strains of mice. β-Actin was used as an internal control. The numbers shown below each lane indicate densitometric ratios of expression levels between mAPOBEC3 and β-actin messages, normalized to that in the spleen of C57BL/6 mice. (B) Levels of expression of mAPOBEC3 mRNA relative to GAPDH quantified by real-time PCR are shown. Means of three samples each are shown with bars indicating standard errors of the means. *, statistically significant differences from the expression levels in B10.A/SgSn mice, indicated by one-way ANOVA with Dunnett's posttest for multiple comparisons (P < 0.05). (C) Splicing variants of the APOBEC3 gene expressed in C57BL/6 and BALB/c mice. The known genomic organization and splicing pattern of the APOBEC3 gene along with the positions of the primers used are shown in Fig. 1. The primers a and b amplified the entire mAPOBEC3-coding region, while primers c and d encompassed exons 4 and 6. GAPDH was used as an internal control. (D) Expression of APOBEC3 mRNA in the BALB/3T3 cells stably transfected with each APOBEC3 gene was analyzed by RT-PCR. The same primers for mAPOBEC3 were used for samples in lanes 1 to 4. Samples in lanes 5 to 8 were amplified with each specific primer set. GAPDH was used as an internal control. Note the faint band of endogenous mA3^d cDNA in lane 1. (E) Flow cytometric analyses of the cell surface expression of F-MuLV gp70 on acutely infected stable transfectants are shown. Cells expressing the indicated genes were infected with F-MuLV at a multiplicity of infection of 2.0 and analyzed for surface gp70 expression with MAb 720 2 days later. (F and G) Proteins detected in cell lysate (F) and virus particles in the culture supernatant (G) from the infected BALB/3T3 cells expressing FLAG and FLAG-proteins are shown. Immunoblot detection was performed with the anti-FLAG, anti-gp70, anti-p30 or anti-actin Ab. (H and I) Infectivities of progeny F-MuLV produced from APOBEC3-expressing BALB/3T3 cells. Mus dunni cells were infected with the progeny virus produced from the indicated transfectants, and foci of infected cells were stained with anti-gp70 MAb for enumeration. The vertical axis in panel I shows F-MuLV infectivity as in panel H. The infectivities are shown as an equivalent of infectious virus per 1 ml of culture supernatant (n = 3, mean ± standard deviation; *, P < 0.05; †, P < 0.01; **, P < 0.005). The F-MuLV infectivity detected in the supernatant of hAPOBEC3G-expressing cells was drastically reduced, while only a moderate reduction in F-MuLV infectivity was observed when the indicator cells were inoculated with the supernatant from the hAPOBEC3F-expressing cells, consistent with the previous reports (1, 4, 13). All the experiments shown in panels C to I were performed with at least two representative clones of stable transfectants for each gene, and the results obtained with the independent clones were in agreement with the data shown.

Replication of F-MuLV in mAPOBEC3-deficient mice and the development of FV-induced disease in vivo. (A) Representative results for genotyping of the APOBEC3 alleles by PCR. (B) Expression of mAPOBEC3 mRNA in the bone marrow and spleen of the APOBEC3-deficient C57BL/6 mice analyzed by RT-PCR is shown. (C) C57BL/6 mice possessing either the APOBEC3^−/− (−/−) (n = 8), +/− (n = 10), or +/+ (n = 9) alleles were inoculated intravenously with 10⁵ FFU of purified F-MuLV. F-MuLV infectious centers were enumerated on Mus dunni cells with anti-gp70 MAb on PID 6. Short horizontal bars indicate the means. *, P < 0.02; **, P < 0.002. (D) Expression of APOBEC3 mRNA in mAPOBEC3-deficient CB6F₁ mice analyzed by RT-PCR. (E) CB6F₁ mice possessing either the APOBEC3^d/⁻ (d/−) (n = 10) or d/b (n = 10) alleles were inoculated intravenously with 10⁴ FFU of purified F-MuLV. F-MuLV infectious centers in the bone marrow were enumerated by coculturing Mus dunni cells and staining with anti-gp70 MAb on PID 6. Short horizontal bars indicate the means. The difference between the groups was analyzed by Student's t test: *, P < 1 × 10⁻⁷. (F and G) CB6F₁ mice possessing either the APOBEC3^d/⁻ (d/−) or d/b alleles were inoculated intravenously with 150 spleen FFU of FV complex. On PID 7, the cells prepared from the bone marrow were stained for TER119 and gp70 and were analyzed by flow cytometry (F). Five mice of each genotype were examined to calculate the means described in the text, and the dot graphs shown are of representative animals. On PID 21, hematocrit values were determined in the peripheral blood (G).