Bettina Bundgaard

Human herpesvirus-6B induces expression of the human endogenous retrovirus K18-encoded superantigen.

BACKGROUND The superantigen, encoded by the envelope gene (env) of the human endogenous retrovirus (HERV)-K18, may be involved in autoimmunity. Its expression is transactivated in B cells during infection with Epstein-Barr virus and in peripheral blood mononuclear cells (PBMCs) that are treated with IFN-alpha. OBJECTIVES We investigated whether HHV-6B infection was able to induce the expression of HERV-K18 env. STUDY DESIGN The expression of HERV-K18 env gene was measured by real-time quantitative PCR in HHV-6B-infected PBMC. RESULTS Infection of PBMCs with HHV-6B resulted in a rapid and dose-dependent induction of HERV-K18 env gene expression, predominantly in monocytes. Induction was dependent on the interaction of glycoprotein H with CD46, but did not require viral transcription or DNA synthesis. Cycloheximide inhibited both the induction and basal expression of HERV-K18 env, indicating that de novo synthesis of proteins was necessary. CONCLUSIONS HHV-6B induced transcriptional activation of the endogenous superantigen HERV-K18 independently of virus replication, which may have consequences for the development of autoimmunity.

Human Herpesvirus 6B Induces Cell Cycle Arrest Concomitant with p53 Phosphorylation and Accumulation in T Cells

ABSTRACT 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.

A Central Role for CK1 in Catalyzing Phosphorylation of the p53 Transactivation Domain at Serine 20 after HHV-6B Viral Infection

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 Ser20 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 Ser20 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 Ser20 site phosphorylation of p53 in T-cells, we sought to identify the kinase responsible for this virus-induced p53 modification. The p53 Ser20 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 Ser20 site kinase activity. Immunodepletion of CK1 but not VRK1 removed the kinase activity from the peak fraction, and bacterially expressed CK1 exhibited Ser20 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 Ser20 site p53 kinases. Low levels of the CK1 inhibitor D4476 selectively inhibited HHV-6B-induced Ser20 site phosphorylation of p53. However, x-ray-induced Ser20 site phosphorylation of p53 was not blocked by D4476. These data highlight a central role for CK1 as the Ser20 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 Ser20.

Synergy between Vitamin D3 and Toll-Like Receptor Agonists Regulates Human Dendritic Cell Response during Maturation

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.

Induction of Cell-Cell Fusion from Without by Human Herpesvirus 6B

ABSTRACT Human herpesvirus (HHV) 6A induce fusion from without (FFWO), whereas HHV-6B is believed to be ineffective in this process. Here, we demonstrate that HHV-6B induces rapid fusion in both epithelial cells and lymphocytes. The fusion was identified 1 h postinfection, could be inhibited by antibodies to HHV-6B gH and to the cellular receptor CD46, and was dependent on virus titer but independent of de novo protein synthesis and UV inactivation of the virus. Comparisons indicate that HHV-6A is only 10-fold more effective in inducing FFWO than HHV-6B. These data demonstrate that HHV-6B can induce FFWO in epithelial cells and lymphocytes.

Human herpesvirus 6B inhibits cell proliferation by a p53-independent pathway

BACKGROUND Various forms of cellular stress can activate the tumour suppressor protein p53, an important regulator of cell cycle arrest, apoptosis, and cellular senescence. Cells infected by human herpesvirus 6B (HHV-6B) accumulate aberrant amounts of p53. OBJECTIVES The aim of this study was to investigate the role of p53 accumulation in the HHV-6B-induced cell cycle arrest. STUDY DESIGN The role of p53 was studied using the p53 inhibitor pifithrin-a, and cells genetically deficient in functional p53 by homologous recombination. RESULTS In response to HHV-6B infection, epithelial cells were arrested in the G1/S phase of the cell cycle concomitant with an aberrant accumulation of p53. However, the known p53-induced mediator of cell cycle arrest, p21, was not upregulated. Approximately 90% of the cells expressed HHV-6B p41, indicative of viral infection. The presence of pifithrin-a, a p53 inhibitor, did not reverse the HHV-6B-induced cell cycle block. In support of this, HHV-6B infection of p53(-/-) cells induced a cell cycle block before S-phase with kinetics similar to or faster than that observed by infection in wt cells. CONCLUSIONS HHV-6B infection inhibited host cell proliferation concomitantly with p53 accumulation, but importantly the block in cell cycle occurred by a pathway independent of p53.

Restriction of human herpesvirus 6B replication by p53

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-alpha, 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.

U20 Is Responsible for Human Herpesvirus 6B Inhibition of Tumor Necrosis Factor Receptor-Dependent Signaling and Apoptosis

ABSTRACT 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-α), 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 IκBα 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, IκBα Ser-32 phosphorylation, and NF-κB 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.

Altered frequency of T regulatory cells is associated with disability status in relapsing- remitting multiple sclerosis patients

A perturbed immunoregulation may be part of the pathogenesis of relapsing-remitting multiple sclerosis (RRMS). In this study, we demonstrate a dichotomy within the frequency of Treg among newly diagnosed RRMS patients but not in healthy controls. A group of RRMS patients was characterized by a significantly lower percentage of Treg cells than that of their matched, healthy controls, and this was inversely correlated with their score on the expanded disability status scale (EDSS). Since the EDSS reflected severity of the last attack, this study demonstrates a correlation between low frequency of Treg and severity of clinical disease in RRMS.

The DR6 protein from human herpesvirus-6B induces p53-independent cell cycle arrest in G2/M.

HHV-6B infection inhibits cell proliferation in G2/M, but no protein has so far been recognized to exert this function. Here we identify the protein product of direct repeat 6, DR6, as an inhibitor of G2/M cell-cycle progression. Transfection of DR6 reduced the total number of cells compared with mock-transfected cells. Lentiviral transduction of DR6 inhibited host cell DNA synthesis in a p53-independent manner, and this inhibition was DR6 dose-dependent. A deletion of 66 amino acids from the N-terminal part of DR6 prevented efficient nuclear translocation and the ability to inhibit DNA synthesis. DR6-induced accumulation of cells in G2/M was accompanied by an enhanced expression of cyclin B1 that accumulated predominantly in the cytoplasm. Pull-down of cyclin B1 brought down pCdk1 with the inactivating phosphorylation at Tyr15. Together, DR6 delays cell cycle with an accumulation of cells in G2/M and thus might be involved in HHV-6B-induced cell-cycle arrest.