Susceptibility of xenotropic murine leukemia virus-related virus (XMRV) to retroviral restriction factors

Abstract

Xenotropic murine leukemia virus-related virus (XMRV) is a recently discovered gammaretrovirus that has been linked to prostate cancer and chronic fatigue syndrome. This virus is therefore an important potential human pathogen and, as such, it is essential to understand its host cell tropism. Intriguingly, infectious virus has been recovered from patient-derived peripheral blood mononuclear cells. These cells express several antiviral restriction factors that are capable of inhibiting the replication of a wide range of retroviruses, including other gamma retroviruses. This raises the possibility that, similar to HIV, XMRV may have acquired resistance to restriction. We therefore investigated the susceptibility of XMRV to a panel of different restriction factors. We found that both human APOBEC3 and tetherin proteins are able to block XMRV replication. Expression of human TRIM5alpha, however, had no effect on viral infectivity. There was no evidence that XMRV expressed countermeasures to overcome restriction. In addition, the virus was inhibited by factors from nonhuman species, including mouse Apobec3, tetherin, and Fv1 proteins. These results have important implications for predicting the natural target cells for XMRV replication, for relating infection to viral pathogenicity and pathology, and for the design of model systems with which to study XMRV-related diseases.

Fig. 1.

XMRV infection in a panel of cell lines. LacZ-encoding XMRV either expressing the XMRV envelope or pseudotyped with the G protein of VSV (white and black bars respectively), were produced in 293T cells by transient transfection. Virus titers were measured by RT-ELISA and equivalent amounts of virus were used to challenge the cell lines indicated. Productive infection was measured after 48 h as the induction of β-galactosidase activity, monitored using a chemiluminescent substrate. Results are given in counts per second.

Fig. 2.

Sensitivity of XMRV to human and mouse APOBEC3 proteins. LacZ-encoding XMRV or Mo-MLV viruses were produced in 293T cells transiently transfected with 0.5 μg HA-tagged APOBEC-expressing plasmid or empty vector. (A) Virus titers were measured by RT-ELISA and equivalent amounts of virus were used to challenge D17 cells. Productive infection was measured after 48 h as the induction of β-galactosidase activity, monitored using a chemiluminescent substrate. Results are given in counts per second and are representative of at least two independent experiments. (B) Whole-cell lysates were prepared from the 293T cells used above and expression of APOBEC proteins was confirmed by immunoblot analysis using a high-affinity anti-HA monoclonal antibody.

Fig. 3.

Sensitivity of XMRV to human and monkey tetherins. (A) 293T cells were transiently transfected with XMRV proviral DNA in combination with a retroviral vector that encodes an eGFP reporter, increasing plasmid doses of a human, Rhesus, and AGM tetherin-expression vector, and either empty vector or HIV-1 Vpu. Viral supernatants were harvested 48 h posttransfection and used to infect 293T cells. After 24 h, the number of GFP-positive cells was analyzed by flow cytometry. (B and C) HeLa cells that constitutively express human tetherin were transfected with XMRV/CNCG, HIV-1 NL4.3 (HIV-1wt), or a Vpu-defective HIV-1 NL4.3 (HIV-1Vpu-) with or without Vpu or XMRV env expression in trans, as indicated. Forty-eight hours posttransfection, the viral supernatants were harvested and processed as in A.

Fig. 4.

Sensitivity of XMRV to Fv1 and primate TRIM5 proteins. (A) Table indicating the positions of amino acids in capsid that differ between XMRV, N- and B-tropic MLV. (B) FACS profiles of CrFK cells that were transduced with retroviral vectors expressing Fv1ⁿ or Fv1^b and EYFP before infecting with XMRV and N- or B-tropic MLV carrying the EGFP marker. The percentages of infected Fv1-negative versus Fv1-positive cells are shown above each panel. (C) Ratios of infected restriction factor-positive cells to restriction factor-negative cells. Ratios that are less than 0.3 are taken to represent restriction (shaded black); ratios greater than 0.7 represent absence of restriction (not shaded). Numbers between 0.3 and 0.7 (shaded gray) are taken to represent partial restriction.