MMTV infection is restricted by cell-intrinsic and virion-packaged mA3. (A) Western blotting of cell extracts from mA3-transfected TRH3 cells probed with anti-HA or anti-β-actin antisera. The first two panels are the cells infected with the mA3⁻ virions (ΔEnv), and the second two panels are the cells infected with the mA3⁺ virions [(ΔEnv(+mA3)]. (B) Western blotting of MMTV virions used in the study. The one-hit ΔEnv virus was made by transient transfection of 293T cells, with V5-tagged mA3 (+) or pcDNA3.1 (−). The pGR102ES virus was produced from CGRES6 cells transiently transfected with an expression vector containing HA-tagged mA3 (+) or pcDNA3.1 (−). Blots were probed with anti-V5, stripped, and reprobed with goat anti-MMTV antisera (top) or probed with anti-HA and reprobed with goat anti-MMTV Gag (p27) (bottom). (C) TRH3 cells were transfected with mA3 (mA3⁺) or pcDNA3.1 (mA3⁻) and then infected with ΔEnv or ΔEnv(+mA3) virions. Infection was analyzed by RT-qPCR; the relative infection levels are shown. Each bar represents the average of three wells. P values of ≤0.001 are significant for all comparisons [mA3⁺ for ΔEnv versus mA3⁻ for ΔEnv, mA3⁺ for MMTVΔEnv(+mA3) versus mA3⁻ for ΔEnv(+mA3), mA3⁺ for ΔEnv versus mA3⁺ for ΔEnv(+mA3), mA3⁻ for ΔEnv versus mA3⁻ for ΔEnv(+mA3)]. This experiment was performed three times with similar results. (D) TRH3 cells were transfected with mA3 (mA3⁺) or pcDNA3.1 (mA3⁻) and then infected with GR102ES virus with or without packaged mA3. FACS analysis was performed to determine the percentage of GFP⁺ cells. Each bar represents the average of three wells. *, P ≤ 0.01; **, P ≤ 0.001; ***, P ≤ 0.03. This experiment was performed twice with similar results. All error bars represent standard deviations. The statistical significance of differences between groups was tested using paired two-tailed Student's t test. α, anti-.

Cell-intrinsic mA3 restricts HIV-1 infection in murine DCs. (A) Virion-packaged mA3 was analyzed by Western blotting, using anti-HA antibody and the Odyssey infrared imaging system. Symbols: −, virions made with empty vector; +, virions made with mA3 expression vector. (B and C) NIH 3T3 cells (B) or DCs from mA3^+/+ and mA3^−/− mice (C) were infected with VSV-G-pseudotyped lentiviral constructs with or without packaged mA3 for 48 h (NIH 3T3 cells) or 24 h (DCs). The number of GFP⁺ colonies (B) or FACS analysis (C) to determine the percentage of GFP⁺ cells was used to measure infection levels. Bars represent relative infection levels normalized to viral RNA levels present in the infecting particles. For DC infection, filled bars represent mA3^+/+ DCs, and open bars represent mA3^−/− DCs. DCs pretreated with AZT showed no infection. P values of ≤0.001 are significant for all comparisons. This experiment was performed twice with similar results. All error bars represent standard deviations. The statistical significance of differences between groups was tested using paired two-tailed Student's t test.

IFN-α treatment results in increased mA3 expression and decreased MMTV infection. (A and B) BMDCs from C57BL/6 (mA3^+/+) and mA3^−/− mice were grown in the presence of increasing amounts of 0, 10, or 100 U/ml IFN-α for 24 h, and then RNA was isolated and analyzed by RT-qPCR for mA3 expression (A) or the cells were infected with GR102ES virus for 24 h and DNA was isolated and analyzed for RT-qPCR for viral DNA (B). *, P ≤ 0.001; **, P ≤ 0.005; ***, P ≤ 0.01. P values are significant for comparisons of mA3^+/+ and mA3^−/− cells at each IFN-α concentration. All error bars represent standard deviations. The statistical significance of differences between groups was tested using paired two-tailed Student's t test.

Cell-intrinsic and virion-packaged mA3 restricts MMTV infection in vivo. Five mA3^+/+ and five mA3^−/− mice were infected with ΔEnv and ΔEnv(+mA3), and DCs from their draining lymph nodes were pooled and analyzed for infection. Infection was analyzed by RT-qPCR; the relative infection levels are shown. Filled bars, mA3^+/+ iDCs; open bars, mA3^−/− iDCs. P values of ≤0.001 are significant for all comparisons. Mice pretreated with AZT showed no infection. This experiment was performed twice with similar results. All error bars represent standard deviations. The statistical significance of differences between groups was tested using paired two-tailed Student's t test.

MMTV infection of BMDCs is restricted by cell-intrinsic and virus-packaged mA3 ex vivo. (A) BMDCs from mA3^+/+ and mA3^−/− mice were infected ex vivo with ΔEnv and ΔEnv(+mA3) viruses. Infection was analyzed by RT-qPCR; the relative infection levels are shown. Cells were also pretreated with AZT to block infection. Each bar represents the average of three independent BMDC cultures. P values of ≤0.001 are significant for all comparisons. (B) mA3^+/+ and mA3^−/− BMDCs infected with GR102ES virus. Infection was analyzed by FACS for GFP⁺ cells. Each bar represents the average of three independent cultures. P values of ≤0.001 are significant for all comparisons, except where marked with asterisks. *, P ≤ 0.02; **, P ≤ 0.003. This experiment was performed six times with similar results. All error bars represent standard deviations. The statistical significance of differences between groups was tested using paired two-tailed Student's t test.

MMTV more highly infects iDCs and mDCs from mA3^−/− mice than those from mA3^+/+ mice. (A) BMDCs were treated with LPS for 24 h and then stained with anti-CD40 or anti-CD86 and anti-CD11c antibodies and analyzed by FACS. A representative plot for mA3^+/+ BMDCs is shown. The table shows the percentage of CD40⁺ and CD86⁺ cells for triplicate samples of mA3^−/− and mA3^+/+ BMDCs. (B) RNA from the untreated or LPS-matured BMDCs was analyzed by reverse-transcribed RT-qPCR, using primers specific for mA3 exons 6 and 7. The relative levels normalized to those of GAPDH primers are shown. (C) BMDCs from mA3^+/+ and mA3^−/− mice were grown in the presence or absence of LPS for 24 h and then infected with ΔEnv or ΔEnv(+mA3). RT-qPCR analysis was carried out with DNA from immature (untreated) or matured (LPS-treated) BMDCs infected with ΔEnv mA3⁻ and mA3⁺ virions; the relative infection levels are shown. Each bar represents the average of three independent BMDC cultures. *, P ≤ 0.001; **, P ≤ 0.1. Cells were also pretreated with AZT to block infection. This experiment was performed three times with similar results. All error bars represent standard deviations. The statistical significance of differences between groups was tested using paired two-tailed Student's t test.

Cell-intrinsic mA3 inhibits ERT formation. TRH3 cells were transfected with mA3 (+A3) or with empty vector (−A3). (A) Twenty-four hours after transfection, aliquots of cells were taken, intracellularly stained for mA3, and analyzed by FACS, or extracts were made and analyzed by Western blotting (inset). (B to D) The mA3-transfected cells were infected with GR102ES, and at different time points (0, 2, 4, 6, and 24 h) after infection, aliquots of cells were taken for DNA isolation. RT-qPCR analyses were performed using primers that measure strong-stop DNA/ERT (B), LRT (C), or products from both LRT and integrated proviruses (D); all were normalized to those of GAPDH. The relative levels of PCR product are shown. Solid line, mA3⁺ cells; dashed line, mA3⁻ cells. All error bars represent standard deviations.

Increased mA3 expression in vivo results in reduced MMTV infection. Three mice of each genotype (mA3^−/−, mA3^+/+, and TLR4^−/−), were pretreated with 1 μg LPS and, 24 h later, inoculated subcutaneously with GR102ES virions. Twenty-four hours after infection, the mice were sacrificed, and DNA and RNA were isolated from DCs isolated from pooled lymph nodes. (A) Reverse-transcribed RT-qPCR analysis to measure mA3 RNA levels. (B) RT-qPCR analysis to detect integrated viral sequences. Open bars, untreated mice; filled bars, LPS-treated mice. *, P ≤ 0.1; **, P ≤ 0.01. This experiment was performed twice with similar results. All error bars represent standard deviations. The statistical significance of differences between groups was tested using paired two-tailed Student's t test.