New insights on thyroid hormone mediated regulation of herpesvirus infections

Abstract

Thyroid hormone (T₃) has been suggested to participate in the regulation of herpesvirus replication during reactivation. Clinical observations and in vivo experiments suggest that T₃ are involved in the suppression of herpes virus replication. In vitro, differentiated LNCaP cells, a human neuron-like cells, further resisted HSV-1 replication upon addition of T₃. Previous studies indicate that T₃ controlled the expression of several key viral genes via its nuclear receptors in differentiated LNCaP cells. Additional observation showed that differentiated LNCaP cells have active PI3K signaling and inhibitor LY294002 can reverse T₃-mediated repression of viral replication. Active PI3K signaling has been linked to HSV-1 latency in neurons. The hypothesis is that, in addition to repressing viral gene transcription at the nuclear level, T₃ may influence PI3K signaling to control HSV-1 replication in human neuron-like cells. We review the genomic and non-genomic regulatory roles of T₃ by examining the phosphoinositide 3-kinase (PI3K) pathway gene expression profile changes in differentiated LNCaP cells under the influence of hormone. The results indicated that 15 genes were down-regulated and 22 genes were up-regulated in T₃-treated differentiated LNCaP cells in comparison to undifferentiated state. Of all these genes, casein kinase 2 (CK2), a key component to enhance PI3K signaling pathway, was significantly increased upon T₃ treatment only while the cells were differentiated. Further studies revealed that CK2 inhibitors tetrabrominated cinnamic acid (TBCA) and 4, 5, 6, 7-tetrabromo-2H-benzotriazole (TBB) both reversed the T₃-mediated repression of viral replication. Together these observations suggested a new approach to understanding the roles of T₃ in the complicated regulation of HSV-1 replication during latency and reactivation.

Keywords: 2-Morpholin-4-yl-8-phenylchromen-4-one; 4,5,6,7-Tetrabromo-2H-benzotriazole; Casein kinase 2; Differentiation; Herpes simplex virus; Phosphoinositide 3-kinase; Tetrabrominated cinnamic acid; Thyroid hormone.

Fig. 1

A HSV-1 infectious viral particles (ivp) released from T₃ treated latently infected mouse TG explants. TGs from mice n = 10 latently infected with HSV-1 were explanted 30 days post infection. TG explants were separated into replicates of two treatment groups, +T₃ and −T₃, and were cultured for 8 days post explant. Media from each replicate was quantitatively tested daily for HSV-1 ivp via plaque assay. Two-way ANOVA with Holm-Sidak post hoc analysis suggests that statistically significant differences in ivp between +T₃ and −T₃ treatment at days 6, 7, and 8. Asterisk denotes p < 0.001. B PI3K Pathway is active in differentiated LNCaP cells with pAkt increase in differentiated cells. Western blot was performed using rabbit monoclonal IgG antibody against phospho-AKT pSer473 (ThermoSci, Cat#: OMA-03061) and mouse antibody AKT (Rockland, Cat#: 200-301-401) at a dilution of 1:1000 followed by the addition of conjugated secondary antibody for detection on extract from undifferentiated and differentiated LNCaP cells. C PI3K inhibitor reversed T₃-mediated repression HSV-1 viral replication from differentiated LNCaP cells treated with 100 nM T₃ and/or 20 µM LY294002 (Sigma Aldrich, cat#: L9908) was measured quantitatively by FLICIT assays [68]. In short, Vero cells were seeded on 384-well plates followed by exposure to media from EGFP HSV-1 infected cultures. The infected media samples were applied in serial dilutions in replicates and were incubated for 8–18 h when EGFP was observed. The numbers of total cells and infected cells were imaged and quantified by the BioTek Cytation3 fluorescent imaging station and Gen5 software then used to calculate the viral titer using an inverse Poisson’s equation as previously described. Two-way ANOVA with Holm-Sidak post hoc analysis suggests that statistically significant differences in fluorescently labeled infected cells per mL exists. a p < 0.018, b p < 0.004, c p < 0.012, d p < 0.035

Fig. 2

A Transcription profiles of genes involved in PI3K/Akt pathway measured by qRT-PCR arrays. Undifferentiated and 5-day differentiated LNCaP cells plated on poly-d-lysine coated T75 flasks were treated with and without 100 nM T₃ for 48 h. The total RNA was purified by TRIZOL and the cDNA was synthesized using RT2 first strand kit (QIAGEN, cat#: 330401). For the transcriptome heatmaps assessment, the cDNA was subjected to qRT-PCR array analyses via PI3K-AKT signaling pathway (SAB Target List) H96 (BIO-RAD, cat#: 100-34223). The protocol was described essentially by the manufactures based on the CFX Connect™ Real-Time PCR Detection System (BIO-RAD Cat# 1855200). Amplification was plotted and analyzed in triplicates using the BIO-RAD CFX manager software provided by the manufacturer. For each gene, the brightest red squares indicate at least a fourfold increase over the brightest blue square. A Showed the select genes from PI3K-AKT target list modulated significantly by T₃ treatment and differentiation. Akt, EIF, and mTOR genes regulated by T₃ and differentiation. B CK2 inhibitor TBB disrupts T₃ mediated reduction of viral replication. The viral replication was measured by T₃ removal assays [32] and FLICIT assays as shown in B with modification. TBB (Santa Cruz Bio, cat#: sc-202830) was added at 1 µM for CK2 inhibition. In short, differentiated cells were infected with HSV-1. At 48 hpi, infected cells were treated with (1) T₃, (2) T₃ washout, (3) T₃ plus TBB, or (4) T₃ washout plus TBB. The culture media were collected at 96 hpi and subjected to PLICIT assays. The results showed that infection with 100 nM of T₃ reduced viral replication and hormone washout reversed this reduction. The addition of TBB disrupted the T₃–mediated repression. FLICIT was reported previously [68] and described in the figure. Data in triplicates, two-way ANOVA with Holm-Sidak post hoc analysis suggests that statistically significant differences in fluorescently labeled infected cells exists; a, b, c, d, e p < 0.001. C TBCA reversed T₃-mediated suppression of viral replication in differentiated cells. LNCaP cells were infected under treatment of no T₃, with T₃, 110 nM TBCA (Millipore, cat#: 218710), or T₃ + TBCA followed by plaque assays to measure the release of infectious viruses. No suppression of viral replication was observed for undifferentiated cells under the influence of T₃ and/or TBCA when analyzed by ANOVA (data not shown). T₃ removal assays as described in A were used to investigate the effects of TBCA. At 48 hpi, infected cells were treated with (1) T₃, (2) T₃ washout, (3) T₃ plus TBCA, or (4) T₃ washout plus TBCA. It is shown that TBCA, similar to TBB, reversed the suppression of viral replication by T₃ as measured by viral plaque assay. Data in triplicates were analyzed by Two-way ANOVA with Holm-Sidak post hoc analysis suggests that statistically significant differences in pfu per mL exists; a p < 0.001, b p < 0.046, c p < 0.040