Background
Herpes simplex virus 1 (HSV-1) is the archetypal member of the alphaherpesvirus subfamily with a large genome encoding over 80 viral proteins. UL41 is a HSV-1 tegument protein, and also a minor structural component of HSV-1 virions [
1,
2], and it can degrade host mRNAs by cutting them at preferred site [
3‐
5]. Upon HSV-1 infection, UL41 polypeptides enter the cell as components of infecting virions and contributes to an overall decrease in host protein synthesis [
6]. By shortening the half-life of mRNAs, UL41 helps to redirect the cell from host to viral gene expression, and facilitates the sequential expression of different classes of viral genes [
7]. Therefore, HSV-1 evades host responses to infection and diverts the resources of the cells to viral macromolecular synthesis.
The ZAP (zinc finger antiviral protein) was originally discovered in rat as an antiretroviral factor [
8]. The viruses that contain ZAP-responsive element (ZRE) in their viral mRNAs are sensitive to ZAP [
9]. Human ZAP (hZAP) exists in two isoforms, hZAPL and hZAPS respectively [
10]. Both hZAP isoforms have antiviral effects against several RNA viruses [
11,
12], while only ZAPS, not ZAPL was up-regulated under IFN treatment [
13,
14].
HSV-1 UL41 is an endoribonuclease with the substrate specificity of RNase A [
15]. ZAP could not inhibit HSV-1 infection [
16], and the molecular mechanism underneath is still illusive. In this study, we show for the first time that HSV-1 UL41 abrogate s hZAP’s antiviral activity by degrading hZAP mRNA.
Discussion
ZAP is an intrinsic host antiviral factor, which has been reported to not only inhibit the infection of a variety of RNA viruses [
8,
11,
12,
16,
19], but also several DNA viruses [
9,
14]. ZAP specifically inhibits the replication of many viruses by preventing the accumulation of viral mRNAs in the cytoplasm [
20]. However, previous study showed that HSV-1 could resist the antiviral effect of ZAP [
16]. In this study, we showed that hZAP does not suppress HSV-1 replication using the recombinant HSV-1 BAC-Luc, which behaved indistinguishably from the wild-type HSV-1 but could be easily quantified in vitro due to its luciferase activity [
17]. Furthermore, HSV-1 UL41 protein was demonstrated for the first time to degrade hZAP mRNA and dampen the antiviral activity of hZAP.
UL41 triggers degradation of host mRNAs and rapid shutoff of host cell protein synthesis [
21]. By shortening the half-life of mRNAs, UL41 helps redirect the cell from host to viral gene expression, and facilitates the sequential expression of different classes of viral genes [
7]. And three amino acid residues E192, D194, and D195 of UL41 are essential for the nuclease activity [
15]. UL41 mutants reportedly display a 5- to 10-fold reduction in virus yield in tissue culture infection [
1,
7,
22]. Previous studies demonstrated that UL41 could inhibit IFN-mediated antiviral response via multiple mechanisms [
23]. Moreover, UL41 was reported to reduce the expression of innate immune sensors, such as TLR2, TLR3, RIG-I and Mda-5 [
24]. and block IFN-γ signaling by reducing the expression of the IFN-gamma receptor alpha chain (IFNGR1) [
25]. Here, we found that UL41 could degrade hZAP mRNA, and that may facilitate the replication of HSV-1.
ZAP is an interferon-inducible gene and exhibits intrinsic antiviral activity [
26,
27]. Previous study demonstrated that MHV-68 infection up-regulates ZAP expression. ZAP inhibits the expression of MHV-68 ORF64, but MHV-68 RTA antagonizes the antiviral activity of ZAP [
28]. In this study, we demonstrate that HSV-1 infection down-regulates hZAP expression, and HSV-1 UL41 degrades hZAP mRNA. These findings will lead to a better understanding of the mechanisms employed by HSV-1 UL41 to dampen host antiviral signaling and develop the therapeutic interventions to modulate HSV-1 pathogenesis.
Conclusions
In summary, we have shown here for the first time that HSV-1 UL41 abrogate s the antiviral activity of hZAP by targeting its mRNA for degradation, and consequently inhibiting the expression of hZAP. These findings will lead to a better understanding of the mechanisms employed by HSV-1 UL41 to dampen host antiviral signaling and develop the therapeutic interventions to modulate HSV-1 pathogenesis.
Materials and methods
Cells, viruses and antibodies
HEK 293 T cells, Vero cells, 293Trex-hZAPL and 293Trex-hZAPS cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (Gibco-BRL) supplemented with 10 % fetal bovine serum (FBS) and 100 U/ml of penicillin and streptomycin. WT HSV-1 (strain F) and HSV-1-BAC-Luc (expressing firefly luciferase) were multiplied, titered and purified as previously described [
17]. The UL41-null mutant virus (R2621) was propagated and titered as described previously [
29].
Mouse anti-Myc and anti-Flag monoclonal antibodies (MAbs), Rabbit polyclonal anti-ZAPL and Rabbit polyclonal anti-ZAPS, Mouse anti-β-actin MAb were purchased from ABmart (Shanghai, China), Proteintech (Wuhan, China), Santa Cruz Biotechnology (Santa Cruz, CA), respectively. Rabbit polyclonal anti-ICP0, UL42 and UL46 were purchased from GL Biochem Ltd. (Shanghai, China), and Rabbit polyclonal anti-UL41 was kindly provided by Dr. Roizman. Tetracycline (Tet) was purchased from Biovision (San Francisco, CA).
Transfection, infection and dual-luciferase reporter (DLR) assay
HEK 293 T cells were transfected by standard calcium phosphate precipitation whereas hZAP-expressing cells were transfected using Lipofectamine 2000 (Invitrogen). VSV-G-pseudotyped NL4-3-luc virus were produced by transiently co-transfecting HEK 293 T cells with pVSV-G plasmid and pNL4-3-luc. 293Trex-hZAPS cells were co-transfected with reporter plasmid pNL4-3-Luc and internal control plasmid pRL-TK, with or without expression plasmids. At 6 h post-transfection, cells were mock treated or treated with Tetracycline (1000 ng/mL) for 36 h, and then luciferase assays were performed as previously described [
30].
RNA isolation, quantitative RT-PCR
Total RNA was extracted using Trizol (Invitrogen, California) according to the manufacturer’s manual. Samples were digested with DNase I and subjected to reverse transcription previously described [
30]. The cDNA was used as template for quantitative PCR (qPCR) to investigate the expression of hZAP. qPCR was performed according to the manufacturer’s instructions (SYBR Premix Ex Taq, Takara, Japan), and 18S rRNA was used as internal reference.
Statistical Analysis
The data were evaluated with the Student’s t-test and p < 0.05 were considered statistically significant.
Acknowledgement
We thank Dr. Guangxia Gao for providing hZAPL and hZAPS plasmids and 293Trex-hZAPL and 293Trex-hZAPS cells, and Dr. Bernard Roizman for providing the R2621 viruses. This work was supported by grants from the National Natural Science Foundation of China (81371795 and 81571974), Innovative Research Team in Soochow University (PCSIRT, IRT 1075) and the Priority Academic Program Development of Jiangsu Higher Education Institutions.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
CS and JZ conducted the experiments and wrote the manuscript. CZ provided overall supervision and financial support and edited the manuscript. All authors read and approved the final manuscript.