A) 293T cells were transfected with a plasmid encoding a Vif-deficient HIV-1 luciferase expression vector as well as plasmids expressing the HIV-1 Gag and Pol proteins, the VSV G glycoprotein and the indicated APOBEC3 protein. Virus supernatants were harvested at 48 h post-transfection and used to infect naïve 293T cells. Induced luciferase levels were assayed ~44 h later (upper panel). Data are given relative to cells transfected with a control plasmid expressing an irrelevant HA-tagged protein, β-arrestin, with SD indicated. The producer 293T cells were lysed and used for Western blots analyzing APOBEC3 protein expression (middle panel) or HIV-1 capsid expression (lower panel). B) Similar to panel A, except that the HIV-1-based plasmids were substituted with an MLV-based luciferase indicator vector and an MLV Gag-Pol expression plasmid. The lower panel was generated using an MLV capsid-specific goat antiserum. C) Similar to panel B, except that an XMRV Gag-Pol expression vector was substituted for the MLV Gag-Pol expression vector. As previously reported (Rodriguez and Goff, 2009), the XMRV capsid protein is recognized by the same goat polyclonal anti-MLV capsid antiserum.

In this experiment, the naturally XMRV-infected prostate cancer cell line 22RV1 was co-transfected with the indicated levels of the APOBEC3 expression plasmids pcDNA3/hA3G or pcDNA3/mA3, given in micrograms, and the retroviral luciferase expression vector pFB-Luc. At 40 h post-transfection, the media were changed and 8 h later harvested, filtered and used to infect naïve 293T cells. Induced luciferase levels were analyzed ~48 h later (upper panels). Data are shown relative to a blank vector control. Assays were performed in triplicate with SD indicated. The producer 22RV1 cells were also harvested and used for Western blots with an anti-HA monoclonal antibody (lower panels).

XMRV virions derived from the parental 22RV1 cell line (A) or from 22RV1 cells engineered to express hA3G (B), as described in Fig. 4A, were used to infect wildtype LNCaP cells. At 48 h post-infection, total DNA was harvested and a 450 bp segment of the XMRV gag gene isolated by PCR and cloned. Twenty-two clones were then sequenced from each infection (9,900 bp x 2) to reveal the mutations described in this figure.

This experiment was performed as described in Fig. 1 except that variable levels of the pcDNA3/hA3G and pcDNA3/mA3 expression vectors, shown in micrograms, were co-transfected into 293T cells. DNA levels were kept constant by varying the levels of a co-transfected blank pcDNA3 plasmid. Assays were performed in triplicate with SD indicated. The lower panels are APOBEC3 Western blots performed using extracts derived from the producer 293T cells.

A) This Western blot analyzes the level of hA3G expression in the hA3G-negative cell lines CEM-SS and 293T, the naturally hA3G-positive T-cell lines CEM and H9 and also in the parental prostate cancer cell lines 22RV1, DU145 and LNCaP. The polyclonal 22RV1/hA3G and LNCaP/hA3G cell lines were derived by lentiviral vector transduction. Endogenous β-actin was used as a loading control. B) The parental LNCaP cell line, and a transduced derivative expressing physiological levels of hA3G (panel A), were infected with XMRV virions produced by the wild type prostate cancer cell line 22RV1. At 2 days post-infection, the cells were split and supernatant RT levels monitored from day 3 to day 6 post-infection. RT activity is given in arbitrary units, average of three experiments with SD indicated.