Zinc-finger antiviral protein inhibits XMRV infection

Abstract

Background: The zinc-finger antiviral protein (ZAP) is a host factor that specifically inhibits the replication of certain viruses, including Moloney murine leukemia virus (MoMLV), HIV-1, and certain alphaviruses and filoviruses. ZAP binds to specific viral mRNAs and recruits cellular mRNA degradation machinery to degrade the target RNA. The common features of ZAP-responsive RNA sequences remain elusive and thus whether a virus is susceptible to ZAP can only be determined experimentally. Xenotropic murine leukemia virus-related virus (XMRV) is a recently identified γ-retrovirus that was originally thought to be involved in prostate cancer and chronic fatigue syndrome but recently proved to be a laboratory artefact. Nonetheless, XMRV as a new retrovirus has been extensively studied. Since XMRV and MoMLV share only 67.9% sequence identity in the 3'UTRs, which is the target sequence of ZAP in MoMLV, whether XMRV is susceptible to ZAP remains to be determined.

Findings: We constructed an XMRV-luc vector, in which the coding sequences of Gag-Pol and part of Env were replaced with luciferase-coding sequence. Overexpression of ZAP potently inhibited the expression of XMRV-luc in a ZAP expression-level-dependent manner, while downregulation of endogenous ZAP rendered cells more sensitive to infection. Furthermore, ZAP inhibited the spreading of replication-competent XMRV. Consistent with the previously reported mechanisms by which ZAP inhibits viral infection, ZAP significantly inhibited the accumulation of XMRV-luc mRNA in the cytoplasm. The ZAP-responsive element in XMRV mRNA was mapped to the 3'UTR.

Conclusions: ZAP inhibits XMRV replication by preventing the accumulation of viral mRNA in the cytoplasm. Documentation of ZAP inhibiting XMRV helps to broaden the spectrum of ZAP's antiviral activity. Comparison of the target sequences of ZAP in XMRV and MoMLV helps to better understand the features of ZAP-responsive elements.

Figure 1. Overexpression of hZAP inhibits XMRV infection.

(A) Schematic structure of XMRV-luc vector. The coding sequences of Gag-Pol and part of Envelope were replaced with luciferase-coding sequence to generate pXMRV-luc. (B) Overexpression of hZAP inhibits XMRV-luc infection. 293TRex cells expressing hZAP-v1-myc and hZAP-v2-myc upon tetracycline induction were infected with VSV-G pseudotyped XMRV-luc. Cells were equally divided into two dishes at 6 h postinfection, with one mock treated and the other treated with tetracycline. Cells were lysed and luciferase activity was measured at 48 h postinfection (upper panel). The luciferase activity in the absence of ZAP was set as 100. Data presented are means ± SD of three independent experiments. The expression of hZAP was confirmed by Western blotting (lower panel). (C) ZAP inhibits XMRV-luc in an expression-level-dependent manner. 293TREx-hZAP-v2 cells were infected with XMRV-luc. At 6 h postinfection the cells were equally split and tetracycline was added to the concentrations indicated. Cells were lysed and luciferase activity was measured at 48 h postinfection. Fold inhibition was calculated as the luciferase activity in mock treated cells divided by the luciferase activity in the tetracycline treated cells (upper panel). Data presented are means ± SD of three independent experiments. The expression levels of hZAP-v2 were measured by Western blotting (lower panel). (D) ZAP inhibits XMRV replication. 293Trex-hZAP-v2 Cells were infected with XMRV produced in 293T cells. At 8 h postinfection, cells were mock treated or treated with doxycycline to induce hZAP-v2 expression. Samples were taken every day and subjected to RT assays.

Figure 2. Downregulation of endogenous hZAP enhances XMRV-luc expression.

HOS cells were transfected with control siRNA (Ctrl) or siRNAs directed against hZAP (ZAPi-1 and ZAPi-2), followed by infection with XMRV-luc for 5 h. At 48 h postinfection, cells were lysed. (A) Endogenous hZAP mRNA levels were measured by real-time PCR. (B) Luciferase activity was measured and presented as relative light units (RLU). Data presented are means ± SD of three measurements.

Figure 3. Expression of hZAP prevents the accumulation of XMRV-luc mRNA.

293TRex-hZAP-v2 cells harbouring XMRV-luc provirus were mock treated or treated with 1μg/ml tetracycline for 48 h to induce ZAP expression. (A) Cells were lysed and luciferase activity was measured (upper panel). Data presented are means ± SD of three independent experiments. The expression of hZAP was confirmed by Western blotting (lower panel). (B) Cytoplasmic RNA was extracted and subjected to Northern blotting to detect the mRNA indicated (upper panel). Expression of hZAP was confirmed by Western blotting (lower panel).

Figure 4. Downregulation of RIG-I does not affect ZAP's antiviral activity against XMRV-luc.

(A) Control shRNA and shRNAs against RIG-I (Ri446 and Ri583) were stably expressed in 293TRex-hZAP-v2 cells. RIG-I mRNA levels were measured by real-time PCR and normalized to that of GAPDH. Data presented are means ± SE of three parallel experiments. (B) Cells were transfected with pGl3-IFNβ-luc and pRL-TK. At 48 h posttransfection, cells were transfected with poly (I:C). Luciferase activity was assayed 12 h later. (C) Cells were infected with XMRV-luc, mock treated or treated with tetracycline for 48 h to induce ZAP expression, and luciferase activities were measured. Fold inhibition was calculated as the luciferase activity in mock treated cells divided by that in tetracycline treated cells. Data presented are means ± SE of three parallel experiments.

Figure 5. ZAP targets the 3′LTR of XMRV.

(A) Schematic structures of HR'-CMV-luc vectors. (B) 293TRex-hZAP-v2 cells were infected with the vectors indicated. At 3 h postinfection, cells were mock treated or treated with 1 μg/ml tetracycline to induce ZAP expression. At 48 h postinfection the cells were lysed and luciferase activity was measured. Fold inhibition was calculated as luciferase activity in mock treated cells divided by luciferase activity in tetracycline treated cells (up panel). Data presented are means ± SD of three independent experiments. The expression of hZAP-v2 was confirmed by Western blotting (lower panel).

Figure 6. Mapping of ZRE in the 3′ LTR of XMRV.

(A) Schematic structures of the truncation constructs of 3′UTR. (B) Analysis of the sensitivity of the 3′UTR truncation mutants to ZAP. Fold inhibition was measured as described in the legend to Figure 5B. Data presented are means ± SD of three independent experiments.