Comparison of the effects of hA3G and mA3 on the infectivity of a luciferase vector, an hph vector, and MLV itself. 293T cells that had been stably transfected with pLXSH were transiently transfected as described in the legend to Fig. 1. The culture fluids from the transfectants were then assayed for pBABE-Luc infectivity, pLXSH infectivity, and MLV infectivity as described in Materials and Methods. Hygro, hygromycin.

Comparison of the effects of mA3 isoforms with and without exon 5 on the infectivity of an MLV-derived luciferase vector. Virions were produced as in the experiment shown in Fig. 1, with plasmids encoding mA3 lacking exon 5 (as in all of the other experiments described in this report), mA3 containing exon 5, or hA3G. The graph shows the luciferase-inducing activity divided by the virus particle concentration (as determined by RT activity following polyethylene glycol precipitation), so that the data represent the specific infectivity of the samples.

Subtilisin resistance of mA3 in MLV virions. MLV particles produced in the presence of hA3G or mA3 plasmids were subjected to subtilisin (SUB) digestion as described in Materials and Methods and then analyzed by immunoblotting for intact p30^CA (A), gp70^SU (B), or HA-tagged APO (C) protein. Lanes: 1 and 7, virions produced by cells expressing MLV and hA3G; 2 and 8, virions produced by cells expressing MLV and mA3; 3 and 9, virions produced by cells expressing MLV; 4 and 10, “virions” produced by cells expressing hA3G; 5 and 11, “virions” produced by cells expressing mA3. The samples in lanes 7 to 11 were digested with subtilisin before immunoblotting.

DNA synthesis by APO-inactivated MLV. 293T-mCAT1 cells were infected with the culture fluids that were assayed for infectivity in Fig. 2. They were lysed 24 h later and assayed by real-time PCR for hph DNA and 2-LTR circles as described in Materials and Methods. Values are corrected for differences in CCR5 DNA copy numbers.

Effects of hA3G and mA3 on the infectivity of an MLV-derived luciferase vector. 293T cells were transiently transfected with an infectious MLV clone, the pBABE-Luc plasmid, and different doses of expression plasmids for hA3G or mA3. 293T-mCAT1 cells were then infected with culture fluids from the transfectants, and lysates of these cells were assayed for luciferase activity. The error bars show the standard deviations of the luciferase assay results.

Comparison of the effects of hA3G and mA3 on the infectivity of HIV-1-derived and MLV-derived luciferase vectors. A, HIV-1 vector; B, MLV vector.

Comparison of the encapsidation of mA3 and hA3G in MLV virions. Virus particles were produced by transient transfection in the presence of either 3 or 10 μg of either mA3 or hA3G expression plasmid or in the presence of an empty vector (lane −). Virions were assayed for RT activity following precipitation from the culture fluids with polyethylene glycol. (A) Equal amounts of virus were loaded into the five lanes and analyzed by immunoblotting with antiserum against the HA epitope tag on the APO proteins. The cells were also lysed, and equal amounts of lysate (as determined by protein concentration) were analyzed by immunoblotting with the anti-HA antiserum. As an additional loading control, equal amounts of the lysates were also analyzed by immunoblotting with anti-β-actin antiserum. The sample in lane 1 was produced without an APOBEC expression plasmid; those in lanes 2 and 3 were produced with 3 and 10 μg of mA3 plasmid, respectively; and those in lanes 4 and 5 were produced with 3 and 10 μg of hA3G plasmid. In addition to autoradiography, the chemiluminescence of the immunoblots of the viral samples was measured on an Alpha Innotech ChemiImager 5500. Two different exposures were analyzed in this way, and the percent integrated density values of the viral samples (means ± standard deviations) were as follows: 3 μg mA3, 13.6 ± 1.2; 10 μg mA3, 32.1 ± 1.0; 3 μg hA3G, 11.4 ± 2.8; 10 μg hA3G, 43.0 ± 3.0. (B) The infectivity of the viruses shown in panel A was analyzed by luciferase assays. The graph shows the luciferase-inducing activity divided by the virus particle concentration, and thus, the data represent the specific infectivity of the samples.

Localization of APO proteins in the interior of MLV particles. MLV particles were prepared by transient transfection in the presence of 10 μg of hA3G (lanes 1 and 2), mA3 (lanes 3 and 4), or empty (lanes 5 and 6) expression plasmid. The virions in the culture fluids were fractionated as described in Materials and Methods, and the pellets were analyzed by immunoblotting against the HA epitope tag or p30^CA (top), gp70^SU (middle), or p15^MA (bottom). In lanes 1, 3, and 5, the virions were sedimented with no detergent, while in lanes 2, 4, and 6, Igepal was present in the 10% sucrose layer during centrifugation.

Effects of mutants of hA3G and mA3 on the infectivity of an MLV-derived luciferase vector. Mutant and wild-type (WT) APO proteins were assayed as described in the legend to Fig. 1, except that the luciferase activities were corrected for variations in the amount of virus, so that the graphs represent the specific infectivities of the virus preparations. Virus preparations were also analyzed by immunoblotting against the HA tag in the APO proteins. In panels C and D, lanes 1, 3, 5, and 7 are the samples transfected with 2.5 μg of APO plasmid; lanes 2, 4, 6, and 8 are the samples produced with 7.5 μg; and lane 9 is the sample produced with no APO.