The tumor suppressor protein p53 is activated by distinct cellular stresses including radiation, hypoxia, type I interferon, and DNA/RNA virus infection. The transactivation domain of p53 contains a phosphorylation site at Ser 20 whose modification stabilizes the binding of the transcriptional co-activator p300 and whose mutation in murine transgenics induces B-cell lymphoma. Although the checkpoint kinase CHK2 is implicated in promoting Ser 20 site phosphorylation after irradiation, the enzyme that triggers this phosphorylation after DNA viral infection is undefined. Using human herpesvirus 6B (HHV-6B) as a virus that induces Ser 20 site phosphorylation of p53 in T-cells, we sought to identify the kinase responsible for this virus-induced p53 modification. The p53 Ser 20 kinase was fractionated and purified using cation, anion, and dye-ligand exchange chromatography. Mass spectrometry identified casein kinase 1 (CK1) and vaccinia-related kinase 1 (VRK1) as enzymes that coeluted with virus-induced Ser 20 site kinase activity. Immunodepletion of CK1 but not VRK1 removed the kinase activity from the peak fraction, and bacterially expressed CK1 exhibited Ser 20 site kinase activity equivalent to that of the virus-induced native CK1. CK1 modified p53 in a docking-dependent manner, which is similar to other known Ser 20 site p53 kinases. Low levels of the CK1 inhibitor D4476 selectively inhibited HHV-6B-induced Ser 20 site phosphorylation of p53. However, x-ray-induced Ser 20 site phosphorylation of p53 was not blocked by D4476. These data highlight a central role for CK1 as the Ser 20 site kinase for p53 in DNA virus-infected cells but also suggest that distinct stresses may selectively trigger different protein kinases to modify the transactivation domain of p53 at Ser 20 .The tumor suppressor protein p53 is a key player in the survival or death decision that cells face after exposure to a variety of metabolic and genotoxic stresses (1). The transient accumulation and activation of p53 in response to various cellular stresses enables the protein to modulate the expression of numerous genes involved in cell cycle arrest, DNA repair, and/or apoptosis. The initiation of either transient cell cycle arrest and damage repair or apoptosis is dependent on the cell and damage type, the severity of damage, and the cellular microenvironment. Phosphorylation and acetylation events that control interactions between the transcription factor p53 and its negative regulators (Mdm2, COP1, and Pirh2) or coactivators (p300) are ultimately involved in modulating p53-dependent gene expression in response to cellular stress (2). In particular, phosphorylation at Thr 18 within the N-terminal conserved BOX-I domain of p53 blocks the binding of Mdm2, whereas phosphorylation at Ser 20 , also within the BOX-I domain, enables the binding of p300 (3-5). Thus, phosphorylation in this transactivation domain serves to stimulate rather than inhibit p53 function. In addition, phosphorylation at Ser 392 within the C terminus of p53 stimulates the seq...

We studied the interactions between human herpesvirus 6B (HHV-6B) and its host cell. Productive infections of T-cell lines led to G1/S- and G2/M-phase arrest in the cell cycle concomitant with an increased level and enhanced DNA-binding activity of p53. More than 70% of HHV-6B-infected cells did not bind annexin V, indicating that the majority of cells were not undergoing apoptosis. HHV-6B infection induced Ser20 and Ser15 phosphorylation on p53, and the latter was inhibited by caffeine, an ataxia telangiectasia mutated kinase inhibitor. Thus, a productive HHV-6B infection suppresses T-cell proliferation concomitant with the phosphorylation and accumulation of p53

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Here, we demonstrate that human herpesvirus 6B (HHV-6B) infection upregulates the tumour suppressor p53 and induces phosphorylation of p53 at Ser392. Interestingly, phosphorylation at the equivalent site has previously been shown to correlate with p53 tumour suppression in murine models. Although the signalling pathways leading to Ser392 phosphorylation are poorly understood, they seem to include casein kinase 2 (CK2), double-stranded RNA-activated protein kinase (PKR), p38 or cyclin-dependent kinase 9 (Cdk9). By using column chromatography and in vitro kinase assays, CK2 and p38, but not PKR or Cdk9, eluted in column fractions that phosphorylated p53 at Ser392. However, treatment of cells with neither the CK2 and Cdk9 inhibitor 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) nor p38 kinase inhibitors reduced HHV-6B-induced Ser392 phosphorylation significantly. Knockdown of the CK2β subunit or p38α by small interfering RNA had no effect on HHV-6B-induced phosphorylation of p53 at Ser392. Thus, HHV-6B induces p53 Ser392 phosphorylation by an atypical pathway independent of CK2 and p38 kinases, whereas mitogen-activated protein (MAP) kinase signalling pathways are involved in viral replication.

Human dendritic cells (DC) can be differentiated from blood monocytes in the presence of GM-CSF and IL-4 and matured by lipopolysaccharide (LPS). Vitamin D3 inhibits the maturation of human DC measured by changes in surface expression of HLA-DR, CD14, CD40, CD80, CD83, and CD86. We here examine the function of vitamin D3 during DC maturation. One of the earliest changes to LPS-induced maturation was an increase in CD83 expression. Vitamin D3 inhibited the increase in expression of HLA-DR, CD40, CD80, CD83, and CD86 and the decrease in expression of CD14, which was paralleled morphologically by vitamin D3-induced inhibition of dendritic cell differentiation. Vitamin D3 acted in synergy with the TLR agonists LPS and peptidoglycan (PGN) in inducing IL-6, IL-8, and IL-10, whereas vitamin D3 completely inhibited LPS-induced secretion of IL-12. The synergy occurred at concentrations where neither vitamin D3 nor the TLR agonists alone induced measurable cytokine secretion. Both LPS and PGN enhanced the level of the vitamin D3 receptor (VDR). Taken together, these data demonstrated that vitamin D3 and TLR agonists acted in synergy to alter secretion of cytokines from human DC in a direction that may provide an anti-inflammatory environment.

The immune system targets virus-infected cells by different means. One of the essential antiviral mechanisms is apoptosis induced by ligation of tumor necrosis factor receptor 1 (TNFR1). This receptor can be activated by tumor necrosis factor alpha (TNF-␣), which upon binding to TNFR1 induces the assembly of first an inflammatory and later a proapoptotic signaling complex. Here, we report that infection by human herpesvirus 6B (HHV-6B) inhibited poly(ADP-ribose) polymerase (PARP) cleavage, caspase 3 and 8 activation, and IB␣ Ser-32 phosphorylation downstream of TNFR1, indicating inhibition of both the inflammatory and apoptotic signaling pathways. We identified a hitherto uncharacterized viral protein, U20, as sufficient for mediating this inhibition. U20 was shown to locate to the cell membrane, and overexpression inhibited PARP cleavage, caspase 3 and 8 activation, IB␣ Ser-32 phosphorylation, and NF-B transcriptional activity. Moreover, small interfering RNA (siRNA) knockdown of U20 demonstrated that the protein is necessary for HHV-6B-mediated inhibition of TNFR signaling during infection. These results suggest an important novel function of U20 as a viral immune evasion protein during HHV-6B infection.

Human herpesvirus 6B (HHV-6B) induces significant accumulation of p53 in both the nucleus and cytoplasm during infection. Activation of p53 by DNA damage is known to induce either growth arrest or apoptosis; nevertheless, HHV-6B-infected cells are arrested in their cell cycle independently of p53, and only a minor fraction of the infected cells undergoes apoptosis. Using pifithrin-α, a p53 inhibitor, and p53-null cells, this study showed that infected epithelial cells accumulated viral transcripts and proteins to a significantly higher degree in the absence of active p53. Moreover, HHV-6B-induced cytopathic effects were greatly enhanced in the absence of p53. This suggests that, in epithelial cells, some of the functions of p53 leading to cell-cycle arrest and apoptosis are restrained by HHV-6B infection, whereas other cellular defences, causing inhibition of virus transcription, are partially retained.