Infectivity of virions released from 3T3 or 3T3/mA3 cells infected with gGag⁺ or gGag⁻ MuLVs. Virions released from 3T3/mA3 cells infected with gGag⁺ or with gGag⁻ MuLVs were analyzed by immunoblotting for the presence of mA3 or gGag. (A) Immunoblot analysis of gGag⁺ virions, gGag⁻ virions, and a 3T3/mA3 cellular lysate for the presence of mA3 using a horseradish peroxidase (HRP)-conjugated anti-HA antibody (clone 3F10; Roche). The 3T3/mA3 cellular lysate was included to enable a size comparison of mA3 in the cells to those in the virions. The blot was also developed with an HRP-conjugated monoclonal antibody to p30 (MAb 18-7) (5) as a loading control. Exposure times for detecting mA3 were approximately 10-fold longer than those for p30. (B) Immunoblot analysis of gGag⁺ or gGag⁻ virions for the presence of gGag using an HRP-conjugated anti-gGag antibody (11). The blot was subsequently stripped and developed with an HRP-conjugated monoclonal antibody to p30 as a loading control. Exposure times for detection of gGag were approximately 20-fold longer than for p30. (C) 3T3 cells or 3T3/mA3 cells harboring the retroviral vector LAPSN were infected with gGag⁺ or gGag⁻ MuLVs. The cells were grown for 40 h, the medium was changed, and the virus was harvested after an 8-h period. Infectivity was assessed on uninfected M. dunni cells by alkaline phosphatase assays for newly transduced target cells and by focal immunofluorescence assays using a monoclonal antibody specifically reactive to the envelope proteins of the MuLVs. Infectivity titers, expressed as FFU, were normalized to the number of virions by assessing reverse transcriptase activity (RT). RT was expressed as the absorbance per ml at 405 nm. Statistical analysis was performed using the unpaired Student t test.

Effect of cellular mA3 on infection by gGag⁺ and gGag⁻ MuLVs. 3T3 and 3T3/mA3 cells were infected with mixtures of gGag⁺ and gGag⁻ viruses, each carrying a distinct retroviral vector encoding either alkaline phosphatase (LAPSN) or β-galactosidase (G1nβgSvNa), and assayed by scoring the number of foci of cells expressing the respective enzymes. The mixtures were adjusted to give equivalent titers of alkaline phosphatase and β-galactosidase on 3T3 cells. Infectivity was expressed as focus-forming units (FFU). Statistical analysis was performed using the unpaired Student t test.

Replication of gGag⁺ or gGag⁻ MuLVs in mA3 wild-type and knockout mice. (A) 129/Ola wild type (mA3^+/+) or 129/Ola mA3 knockout (mA3^−/−) mice were inoculated with gGag⁺ or gGag⁻ MuLVs. Three weeks after infection, the mice were sacrificed, sera were collected, and viruses were quantified by a focal immunofluorescence assay on M. dunni cells. Each point represents the level of viremia from an individual animal (n = 9 for gGag⁺ MuLV in mA3^+/+ mice, 9 for gGag⁺ MuLV in mA3^−/−mice, 7 for gGag⁻ MuLV in mA3^+/+ mice, and 14 for gGag⁻ MuLV in mA3^−/− mice). Statistical analysis was performed using the unpaired Student t test. (B) C57BL/6 wild-type mice (mA3^+/+) or C57BL/6 mA3 knockout mice (mA3^−/−) were inoculated with gGag⁺ or gGag⁻ MuLVs. Three weeks after infection, the mice were sacrificed, sera were collected, and viruses were quantified by focal immunofluorescence assays on M. dunni cells (n = 6 for the gGag⁺ MuLV in mA3^+/+ mice, 7 for the gGag⁺ MuLV in mA3^−/− mice, 9 for the gGag⁻ MuLV in mA3 ^+/+ mice, and 11 for the gGag⁻ MuLV in mA3^−/− mice). The Wilcoxon signed rank test was used to differentiate whole number integers from zero values. The unpaired Student t test was used to compare nonzero results.