Zinc-Finger Antiviral Protein Inhibits XMRV Infection

<div><h3>Background</h3><p>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.</p> <h3>Findings</h3><p>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.</p> <h3>Conclusions</h3><p>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.</p> </div

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

<p>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).</p

Downregulation of endogenous hZAP enhances XMRV-luc expression.

<p>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.</p

Overexpression of hZAP inhibits XMRV infection.

<p>(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.</p

Mapping of ZRE in the 3′ LTR of XMRV.

<p> (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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039159#pone-0039159-g005" target="_blank">Figure 5B</a>. Data presented are means ± SD of three independent experiments.</p

ZAP targets the 3′LTR of XMRV.

<p>(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).</p

Table_1_Variations in olfactory function among bipolar disorder patients with different episodes and subtypes.pdf

PurposeMost studies on olfactory function in individuals with bipolar disorder (BD) have not distinguished between the different subtypes or between the acute phase (mania or depression) and euthymic state. In this study, we compared olfactory function among BD patients with different subtypes and episodes to explore the potential use of olfactory function as a biomarker for the early identification of BD.Patients and methodsThe study sample consisted of 117 BD patients who were hospitalized between April 2019 and June 2019, and 47 healthy volunteers as controls. The BD patients were divided into a bipolar I disorder (BD I) (n = 86) and bipolar II disorder (BD II) group (n = 31) according to the different subtypes, and divided into depressive BD (n = 36), manic BD (n = 44), or euthymic BD (n = 37) groups according to the types of episodes they experienced. We assessed olfactory sensitivity (OS) and olfactory identification (OI) via the Sniffin’ Sticks test and used the Hamilton Depression Rating Scale (HAMD) and Young Manic Rating Scale (YMRS) to evaluate BD characteristics among all subjects.ResultsCompared with controls, the participants with BD showed decreased OS and OI. We found statistically significant differences in OS and OI between the BD I group and controls, as well as differences in OS between the BD I and BD II group. Least-significant difference multiple comparisons revealed statistically significant differences in OS between the depressive BD group, manic BD group and controls and also between the manic BD and euthymic BD group. OI was positively correlated with the YMRS score in the BD I group and OS was negatively correlated with the HAMD score in the BD II group.ConclusionThis may be the first study to compare olfactory function in patients with BD I vs. BD II via pairwise comparisons. Our findings suggest that OS may have potential as a biomarker for distinguishing the different subtypes of BD and as a state-related biomarker for differentiating the acute phase from the euthymic state of BD. However, further prospective research is warranted.</p

Table_2_Variations in olfactory function among bipolar disorder patients with different episodes and subtypes.pdf

PurposeMost studies on olfactory function in individuals with bipolar disorder (BD) have not distinguished between the different subtypes or between the acute phase (mania or depression) and euthymic state. In this study, we compared olfactory function among BD patients with different subtypes and episodes to explore the potential use of olfactory function as a biomarker for the early identification of BD.Patients and methodsThe study sample consisted of 117 BD patients who were hospitalized between April 2019 and June 2019, and 47 healthy volunteers as controls. The BD patients were divided into a bipolar I disorder (BD I) (n = 86) and bipolar II disorder (BD II) group (n = 31) according to the different subtypes, and divided into depressive BD (n = 36), manic BD (n = 44), or euthymic BD (n = 37) groups according to the types of episodes they experienced. We assessed olfactory sensitivity (OS) and olfactory identification (OI) via the Sniffin’ Sticks test and used the Hamilton Depression Rating Scale (HAMD) and Young Manic Rating Scale (YMRS) to evaluate BD characteristics among all subjects.ResultsCompared with controls, the participants with BD showed decreased OS and OI. We found statistically significant differences in OS and OI between the BD I group and controls, as well as differences in OS between the BD I and BD II group. Least-significant difference multiple comparisons revealed statistically significant differences in OS between the depressive BD group, manic BD group and controls and also between the manic BD and euthymic BD group. OI was positively correlated with the YMRS score in the BD I group and OS was negatively correlated with the HAMD score in the BD II group.ConclusionThis may be the first study to compare olfactory function in patients with BD I vs. BD II via pairwise comparisons. Our findings suggest that OS may have potential as a biomarker for distinguishing the different subtypes of BD and as a state-related biomarker for differentiating the acute phase from the euthymic state of BD. However, further prospective research is warranted.</p

Involvement of Reactive Oxygen Species (ROS) in the IFN-γ induced senescence.

<p>(A) Melancytes were stimulated with indicated concentration of IFN-γ for 24 h. Generated ROS was detected with flow cytometer after labelled with the ROS sensor DCFH-DA. (B,C) Melanocytes were treated with or without 100 U/ml IFN-γ for 7 days in the presence of vehicle or 1 mM NAC. (B) Protein level of p21 was evaluated by Western blot. β-actin was probed as the loading control. (C) Percentages of SA-β-gal-positive cells were determined based on microscopic analysis. CON represents the cell culture without IFN-γ.</p

Interferon-γ Induces Senescence in Normal Human Melanocytes

<div><p>Background</p><p>Interferon-γ (IFN-γ) plays an important role in the proceedings of vitiligo through recruiting lymphocytes to the lesional skin. However, the potential effects of IFN-γ on skin melanocytes and the subsequent contribution to the vitiligo pathogenesis are still unclear.</p><p>Objective</p><p>To investigate the effects of IFN-γ on viability and cellular functions of melanocytes.</p><p>Methods</p><p>Primary human melanocytes were treated with IFN-γ. Cell viability, apoptosis, cell cycle melanin content and intracellular reactive oxygen species (ROS) level were measured. mRNA expression was examined by real-time PCR. The release of interleukin 6 (IL-6) and heat shock protein 70 (HSP-70) was monitored by ELISA. β-galactosidase staining was utilized to evaluate melanocyte senescence.</p><p>Results</p><p>Persistent IFN-γ treatment induced viability loss, apoptosis, cell cycle arrest and senescence in melanocytes. Melanocyte senescence was characterized as the changes in pigmentation and morphology, as well as the increase of β-galactosidase activity. Increase of p21^Cip1/Waf1 protein was evident in melanocytes after IFN-γ treatment. IFN-γ induction of senescence was attenuated by siRNAs against p21, Janus kinase 2 (JAK2) or signal transducer and activator of transcription 1 (STAT1), but not by JAK1 siRNA nor by p53 inhibitor pifithrin-α. IFN-γ treatment increased the accumulation of intracellular ROS in melanocytes, while ROS scavenger N-acetyl cysteine (NAC) effectively inhibited IFN-γ induced p21 expression and melanocyte senescence. IL-6 and HSP-70 release was significantly induced by IFN-γ treatment, which was largely inhibited by NAC. The increase of IL-6 and HSP-70 release could also be observed in senescent melanocytes.</p><p>Conclusion</p><p>IFN-γ can induce senescence in melanocytes and consequently enhance their immuno-competency, leading to a vitiligo-prone milieu.</p></div