Fig 2.

Absence of infectious PreXMRV-1 and PreXMRV-2 virus. (A) Western blotting of cell lysates and virus from 293T cells transfected with XMRV (VP62), PreXMRV-1, or PreXMRV-2. MLV unprocessed Gag and p30 capsid were detected in XMRV-transfected cells using the monoclonal MLV anti-capsid antibody (α-CA) but not in PreXMRV-1- or PreXMRV-2-transfected 293T cells (top). a and b, two independent transfections. α-Tubulin was used as a loading control. Viral supernatants were concentrated 100-fold and analyzed at the indicated dilutions. (B) Relative env RNA copy numbers normalized to GAPDH copy numbers for XMRV, PreXMRV-1, and PreXMRV-2. Quantitative PCR using a universal env primer/probe set able to detect VP62, PreXMRV-1, and PreXMRV-2 was performed after conversion of total cytoplasmic RNA to cDNA using random hexamer primers. (C) Monitoring the presence of RCRs using DIG cells. During reverse transcription, the F portion of gfp is used for minus-strand transfer, resulting in reconstitution of a functional gfp to generate GFP-positive cells; furthermore, the retroviral vector containing the reconstituted gfp can spread through the culture in the presence of an RCR. Ψ, MLV packaging signal. (D) PreXMRV-1 and PreXMRV-2 are noninfectious. Supernatant harvested from 293T cells transfected with XMRV, PreXMRV-1, or PreXMRV-2 was used to infect target DIG cells. A GFP-positive D17 cell line, A3GFP11, was used as a positive control for FACS analysis (52).