Herpes simplex virus 1 UL41 protein abrogates the antiviral activity of hZAP by degrading its mRNA

Abstract

Background: The zinc finger antiviral protein (ZAP) is a host restriction factor that inhibits the replication of various viruses by degradation of certain viral mRNA. However, previous study demonstrated that ectopic expression of rat ZAP did not suppress the replication of herpes simplex virus type 1 (HSV-1), an archetypal member of the alphaherpesvirus subfamily, and the molecular mechanism underneath is still illusive.

Results: Human ZAP (hZAP) does not suppress the replication of herpes simplex virus 1, and HSV-1 UL41 protein was identified as an antagonist of hZAP by degrading its mRNA. Infection of wild-type (WT), but not UL41-null mutant (R2621) virus, diminished the accumulation of hZAP to abrogate its antiviral activity. Moreover, ectopic expression of hZAP inhibited the replication of R2621 but not WT HSV-1.

Conclusion: HSV-1 UL41 was shown for the first time to evade the antiviral function of hZAP via its RNase activity.

Fig. 1

HSV-1 UL41 protein abrogated the antiviral activity of hZAP. a and b 293Trex-hZAPL cells and 293Trex-hZAPS cells were infected with HSV-1-BAC-Luc at an MOI of 1. Cells were treated or mock treated with tetracycline (100 ng/mL or 1000 ng/mL) 2 h post-infection to induce hZAP expression. The cells were lysed, and luciferase activities were measured at 36 h after infection. Fold inhibition was calculated as the ratio of the luciferase activity in mock-treated cells to that in tetracycline-treated cells. c 293Trex-hZAPS cells were transfected with 500 ng of NL4-3-Luc reporter plasmid, together with Renilla luciferase plasmid pRL-TK (50 ng) and pEYFP-N1 control vector or plasmids encoding the indicated viral proteins (1000 ng). At 6 h post-transfection, cells were treated with or without tetracycline (1000 ng/mL) to induce hZAPS expression and were incubated for additional 36 h, followed by cell lysis. The luciferase activity was determined by a dual-luciferase assay. Data shown are means ± SD from three separate experiments. (*P < 0.05)

Fig. 2

HSV-1 UL41 protein inhibits the expression of hZAP. a HEK 293 T cells were co-transfected with hZAPS-Myc plasmid along with increasing amounts of UL41-Flag plasmid. 24 h after transfection, cells were lysed and the samples were then subjected to WB analysis. The lower panel presented relative density analysis of hZAPS. b and c 293Trex-hZAPL cells and 293Trex-hZAPS cells were infected with WT HSV-1 or R2621 at an MOI of 0.1, 1 or 10, respectively. At 2 h post-infection, cells were mock treated or treated with tetracycline (1000 ng/mL). Cells were lysed and the samples were subjected to WB analysis 36 h post-infection. The relative density analysis of hZAPL and hZAPS were under the WB results, respectively. One representative of three independent experiments was shown

Fig. 3

HSV-1 UL41 protein promotes hZAP mRNA degradation. a 293Trex-hZAPL or 293Trex-hZAPS cells were transfected with pCMV-Flag control vector or increasing amount of UL41-Flag plasmid. At 6 h post-transfection, cells were mock treated or treated with tetracycline (1000 ng/mL) to induce hZAPL or hZAPS expression. Quantitative RT-PCR analysis was then performed to detect the mRNA level of hZAPL or hZAPS. b 293Trex-hZAPL cells or 293Trex-hZAPS cells were infected with WT HSV-1 or R2621 at an MOI of 1 or 10, respectively. At 2 h post-infection, cells were mock treated or treated with tetracycline (1000 ng/mL). Quantitative RT-PCR analysis was then performed to detect the mRNA level of hZAPL or hZAPS. One representative of three independent experiments was shown. (*P < 0.05, **P < 0.01)

Fig. 4

Expression of hZAP inhibits UL41-null mutant HSV-1 R2621 infection. a and b 293Trex-hZAPL cells or 293Trex-hZAPS cells were infected with WT HSV-1 or R2621 at an MOI of 1, respectively. At 2 h post-infection, cells were mock treated or treated with tetracycline (1000 ng/mL). At 36 h post-infection, cells were lysed and subjected to WB analysis with the indicated Ab. c and d 293Trex-hZAPL cells or 293Trex-hZAPS cells were infected with WT HSV-1. After 2 h post-infection, cells were treated with or without Tet (1000 ng/mL) to induce hZAPL or hZAPS expression. Viral growth curves were generated by traditional plaque assays at the indicated time points. e and f 293Trex-hZAPL cells or 293Trex-hZAPS cells were infected with R2621 virus. After 2 h post-infection, cells were treated with or without Tet (1000 ng/mL) to induce hZAPL or hZAPS expression. Viral growth curves were generated by traditional plaque assays at the indicated time points. One representative of three independent experiments was shown. (*P < 0.05)