Influence of RT inhibitors on XMRV infection. (A) LNCaP cells were infected with GFP-carrying XMRV in the presence of 30 nM of AZT, 3TC, Tenofovir, D4T, Efavirenz, Nevirapine and 118-D-24. As a control, equivalent amount of DMSO was used. Three days after infection, GFP-positive cell populations were analyzed by FACS. (B) 293T cells were maintained in the presence of 30 nM of the indicated antiretroviral compounds for 72 hours, and cell viability was assessed by MTT assay. (C) LNCaP cells were infected with 20 μl of GFP-carrying XMRV in the presence of 6, 30 and 150 nM of AZT or equivalent amount of DMSO. Three days after infection, viral infectivity was determined by FACS. (D) LNCaP cells were treated with 6, 30 and 150 nM of AZT for three days, and subjected to MTT assay. (E) LNCaP cells were infected with benzonase-treated XMRV in the presence of 30 nM of AZT or equivalent amounts of DMSO. Total cellular DNA was isolated at 8 and 24 hr post infection. The copy numbers of viral reverse-transcripts were determined by ABI Real Time PCR system.

(A) Influence of AZT treatment on XMRV replication. LNCaP cells were infected with XMRV stock for a week, and then co-cultured with uninfected LNCaP cells (day 0). The XMRV-infected LNCaP cells were maintained in the presence of 30 nM AZT or DMSO. Culture supernatants were collected every two days. Cells were passaged on days 7 and 13. On day 13, AZT treated cells were divided into two culture bottles; one was maintained in 30 nM AZT and the other in DMSO. Similarly, DMSO-treated cells were divided into two bottles and treated with DMSO or 30 nM AZT for additional eight days. (B) Alignment of portions of HIV-1 and XMRV RT amino acid sequences. Amino acid sequence alignments of various RNA-dependent DNA polymerases including HIV-1, XMRV, MLV, PERV and hTERT. The HIV-1 motif D amino acid residues were underlined. Codons associated with resistance of HIV-1 to AZT (T215Y, K220Q) are indicated by arrows. (C) Alignment of portions of HIV-1 and XMRV RT amino acid sequences. Identical residues are indicated with an asterisk. The HIV-1 polymerase active-site YMDD and the LPQG motif are underlined. Codons associated with resistance of HIV-1 to multi-nucleoside analogs (Q151M), Tenofovir (K65R), D4T (V75I), Efavirenz (K103N), Nevirapine (V106A), 3TC (K65R/M184V) and AZT (T215Y, K220Q) are indicated by arrows.

No evidence of anti-XMRV activity in HIV protease inhibitors. (A) LNCaP, 293T and NIH3T3 cells were infected with GFP-carrying XMRV. Two days after infection, GFP-positive cell populations were analyzed by flow cytometry. (B) GFP-carrying XMRV was produced in the presence of 30 nM of Ritonavir, Indinavir and Saquinavir, or equivalent amount of DMSO (Control). Influence of drug treatment on viral infectivity was determined by infecting 293T cells with the GFP-carrying XMRV produced in the presence of antiviral compounds. (C) 293T and LNCaP cells were treated with 30 nM of a PI for 72 hours, and cell viability was determined by MTT assay and shown as the average of absorbance (630 nm) of triplicated experiments with standard deviation. (D) GFP-carrying XMRV was produced in 293T cells in the presence of increasing amounts of Ritonavir and the resulting viral titers were determined by FACS. Equivalent amounts of DMSO were used as controls. (E) 293T and LNCaP cells were treated with 6, 30 and 150 nM of Ritonavir for three days, and subjected to MTT assay. (F) 293T cells were transfected with a XMRV proviral DNA plasmid, treated with various concentrations (6, 30, 150 nM) of Ritonavir or equivalent amount of DMSO. Cell lysates were analyzed to detect XMRV precursor Gag (Pr-Gag) and proteolytically cleaved mature capsid protein (CA). As a loading control, β-actin was detected by anti-β-actin antibody. (G) 293T cells were transfected with a HIV-1 provirus plasmid, pNL4-3. HIV-1-producer cells were treated with 6 nM of Ritonavir, and cell lysates were analyzed for HIV-1 precursor Gag (Pr-gag) and mature capsid (CA) proteins.