Inhibition of xenotropic murine leukemia virus-related virus by APOBEC3 proteins and antiviral drugs

Abstract

Xenotropic murine leukemia virus-related virus (XMRV), a gammaretrovirus, has been isolated from human prostate cancer tissue and from activated CD4(+) T cells and B cells of patients with chronic fatigue syndrome, suggesting an association between XMRV infection and these two diseases. Since APOBEC3G (A3G) and APOBEC3F (A3F), which are potent inhibitors of murine leukemia virus and Vif-deficient human immunodeficiency virus type 1 (HIV-1), are expressed in human CD4(+) T cells and B cells, we sought to determine how XMRV evades suppression of replication by APOBEC3 proteins. We found that expression of A3G, A3F, or murine A3 in virus-producing cells resulted in their virion incorporation, inhibition of XMRV replication, and G-to-A hypermutation of the viral DNA with all three APOBEC3 proteins. Quantitation of A3G and A3F mRNAs indicated that, compared to the human T-cell lines CEM and H9, prostate cell lines LNCaP and DU145 exhibited 50% lower A3F mRNA levels, whereas A3G expression in 22Rv1, LNCaP, and DU145 cells was nearly undetectable. XMRV proviral genomes in LNCaP and DU145 cells were hypermutated at low frequency with mutation patterns consistent with A3F activity. XMRV proviral genomes were extensively hypermutated upon replication in A3G/A3F-positive T cells (CEM and H9), but not in A3G/A3F-negative cells (CEM-SS). We also observed that XMRV replication was susceptible to the nucleoside reverse transcriptase (RT) inhibitors zidovudine (AZT) and tenofovir and the integrase inhibitor raltegravir. In summary, the establishment of XMRV infection in patients may be dependent on infection of A3G/A3F-deficient cells, and cells expressing low levels of A3G/A3F, such as prostate cancer cells, may be ideal producers of infectious XMRV. Furthermore, the anti-HIV-1 drugs AZT, tenofovir, and raltegravir may be useful for treatment of XMRV infection.

FIG. 1.

Effects of A3G, A3F, and mA3 proteins on XMRV infection in single-replication-cycle assay. (A and D) Schematic outline of the experimental design, along with the proviral plasmids used in the experiment. XMRV was produced upon transfection of XMRV infectious molecular clone VP62 with (A) and without (D) MSCV-IRES-Luc reporter plasmids, in the presence or absence of A3G, A3F, or mA3 in 293T cells. The viruses produced were used to infect LNCaP prostate carcinoma cells, and the relative infectivity of the virus was measured by determining firefly luciferase activity in infected cells (A) or by PCR amplifying and sequencing the proviral DNA (D). CMV, cytomegalovirus promoter; R, repeat; U₅, unique 5′; GAG-PRO-POL, GAG-protease-polymerase polyprotein gene; ENV, envelope gene; U₃, unique 3′; LTF, long terminal repeat; Ψ, packaging signal; IRES, internal ribosomal entry site; Luc, luciferase gene. (B) VP62, the MSCV-IRES-Luc plasmid, and different amounts of A3G, A3F, and mA3 expression plasmids were transfected in 293T cells; 48 h posttransfection, the viruses were used to infect LNCaP cells in triplicate. The infected LNCaP cells were lysed 48 h posttransfection, and the luciferase activity in the cell lysates was determined. The luciferase activity in the vector control was set to 100%. The asterisks indicate that luciferase activity was less than 1% of that of the vector control. The error bars show the standard deviations of the luciferase activity observed in three independent experiments. (C) Western blot analysis of the incorporation of A3G, A3F, or mA3 protein in XMRV virions. Virus particles were produced in 293T cells by transient transfection of VP62 and MSCV-IRES-Luc in the presence of either 0.5, 1, or 2 μg of A3G expression plasmid; 1, 2, or 4 μg of either A3F or mA3 expression plasmid; or 4 μg of an empty-vector plasmid. The viruses from the supernatant were collected 48 h posttransfection, filtered, and concentrated by ultracentrifugation. At the same time point, the transfected cells were lysed. Equal amounts of cell lysate and equal volumes of concentrated virus were analyzed by immunoblotting them with anti-MLV capsid, anti-FLAG, or anti-HA antibodies. To ensure that equivalent aliquots were loaded onto the gels, the same lysates were analyzed using anti-tubulin antibody. (E) The extent of G-to-A hypermutation was analyzed in the presence of 0.5 μg A3G, 1 μg A3F, and 4 μg mA3. A schematic overview of the XMRV genome is shown, and the region sequenced (nt 2465 to 3660) is indicated with a red bar. The Hypermut (http://www.hir.lanl.gov/content/sequence/HYPERMUT/hypermut.html) color code was used to indicate the mutations: GG to AG in red, GA to AA in cyan, GC to AC in green, GT to AT in magenta, gaps in yellow, and all other mutations in black. In total, 2 of 11 clones were hypermutated in the presence of A3G, 5 of 22 with A3F, and 6 of 21 with mA3. (F) Total numbers of mutations and their occurrence in different dinucleotide contexts. The numbers in parentheses indicate percentages of total G-to-A mutations.

FIG. 2.

Rare hypermutation in the XMRV 22Rv1 provirus. (A) Mutations in proportion to the XMRV genome that were found while sequencing XMRV-22Rv1. In total, 20 clones each of fragments 1 and 2 were sequenced completely. One clone of fragment 1 (clone 1.1) and 2 clones of fragment 2 (clones 2.1 and 2.2) were found to be hypermutated. The Hypermut color code is described in the legend to Fig. 1. (B) The total number and the sequence context of mutations are shown for each clone. Most of the mutations occurred in the GA dinucleotide context.

FIG. 3.

A3G and A3F mRNA levels and A3G protein expression in prostate cancer and T-cell lines. (A) The A3G and A3F mRNA copy numbers were determined by real-time RT-PCR assays and normalized to PBGD. The reactions were prepared in duplicate, and the average and standard error of 3 independent extractions are shown. Copy numbers below the detection limit of 100 copies are marked with asterisks. (B) Equal amounts of protein (5 μg) from the prostate cancer cell lines 22Rv1, LNCaP, and DU145 and the human T-cell lines CEM, CEM-SS, and H9 were analyzed by Western blotting to detect A3G and α-tubulin, which served as a loading control.

FIG. 4.

XMRV hypermutation in prostate cancer and T-cell lines. Mutations found in 22Rv1 (A), LNCaP (B), DU145 (C), CEM-SS (D), CEM (E), and H9 (F) cells in comparison to the XMRV-22Rv1 consensus sequence are marked with vertical bars at their positions. The Hypermut color code is described in the legend to Fig. 1. Below each set of sequences, a detailed table with the total numbers of single-nucleotide mutations is shown.

FIG. 5.

Single-replication-cycle drug susceptibility assays. Phenotypic drug susceptibility testing for AZT (A), 3TC (B), ddI (C), d4T (D), ABC (E), TDF (F), raltegravir (G), and foscarnet (H) was performed with HIV-1 (pNLuc + VSV-G) (open circles) and XMRV (VP62+MSCV-IRES-Luc) (filled circles) reporter viruses in LNCaP cells. The intersections of the vertical lines with the drug concentration axis show the IC₅₀ for each curve. Representative graphs are shown. (I) The mean IC₅₀s and standard deviations were calculated using SIGMAPLOT 8.0 as described in Materials and Methods.