The pathogenic mechanism by which parvovirus B19 may induce inflammatory cardiomyopathy (iCMP) is complex but is known to involve inflammatory processes, possibly including activation of JAK/STAT signaling. The nonstructural B19 protein NS1 acts as a transactivator triggering signaling cascades that eventually lead to activation of interleukin 6 (IL-6). We examined the impact of NS1 on modulation of STAT signaling in human endothelial cells (HMEC-1). The NS1 sequences were identified from B19 DNA isolated from the myocardia of patients with fatal iCMP. B19 infection as well as NS1 overexpression in HMEC-1 cells produced a significant upregulation in the phosphorylation of both tyrosine 705 and serine 727 STAT3 (P < 0.05). The increased STAT3 phosphorylation was accompanied by dimerization, nuclear translocation, and DNA binding of pSTAT3. In contrast, NS1 expression did not result in increased STAT1 activation. Notably, the expression levels of the negative regulators of STAT activation, SOCS1 and SOCS3, were not altered by NS1. However, the level of PIAS3 was upregulated in NS1-expressing HMEC-1 cells. Analysis of the transcriptional activation of target genes revealed that NS1-induced STAT3 signaling was associated with upregulation of genes involved in immune response (e.g., the IFNAR1 and IL-2 genes) and downregulation of genes associated with viral defense (e.g., the OAS1 and TYK2 genes). Our results demonstrate that B19 NS1 modulates the STAT/PIAS pathway. The NS1-induced upregulation of STAT3/PIAS3 in the absence of STAT1 phosphorylation and the lack of SOCS1/SOCS3 activation may contribute to the mechanisms by which B19 evades the immune response and establishes persistent infection in human endothelial cells. Thus, NS1 may play a critical role in the mechanism of viral pathogenesis in B19-associated iCMP.Human parvovirus B19 is emerging as an important pathogenic agent in the etiology of inflammatory cardiomyopathy (iCMP). Recent studies have indicated an association between infection with B19 and acute myocarditis in both children and adults (5, 44). However, the role of B19 infection in the development of chronic iCMP patients is still unclear. We have recently demonstrated that endothelial cells but not cardiac myocytes are B19-specific target cells in patients with B19-associated myocarditis (21). Furthermore, B19 could be detected frequently in patients with unexplained isolated diastolic dysfunction (2, 49).The B19 single-stranded DNA genome of 5,600 base pairs contains two open reading frames encoding the nonstructural protein NS1 and two structural capsid proteins, VP1 and VP2, by a combination of alternative splicing (15). A functional phospholipase A2-like activity has been demonstrated recently in the VP1 region which is involved in intracellular Ca 2ϩ regulation (28). In addition, three small viral proteins of unknown function have been described previously (39,55).The main function of NS1 includes transactivation of the viral P6 promoter, which is important for viral replication in a proces...

The expression of many cellular genes is modulated by DNA methylation and histone acetylation. These processes can influence malignant cell transformation and are also responsible for the silencing of DNA constructs introduced into mammalian cells for therapeutic or research purposes. As a better understanding of these biological processes may contribute to the development of novel cancer treatments and to study the complex mechanisms regulating gene silencing, we established a cellular system suitable to dissect the mechanisms regulating DNA methylation and histone acetylation. For this purpose, we stably transfected the neuroblastoma cell line U87 with a cytomegalovirus promoter-driven reporter gene construct whose expression was analyzed following treatment with the DNA methylation inhibitor 5'-aza-2'-deoxycytidine or histone deacetylation inhibitor trichostatin A. Both substances reactivated the silenced cytomegalovirus promoter, but with different reaction kinetics. Furthermore, whereas the kinetics of reactivation by trichostatin A did not substantially change over the time range considered (5 days), reactivation induced by 5'-aza-2'-deoxycytidine showed profound differences between day 1 and longer time points. We showed that this effect is related to the down-regulation of DNA replication by 5'-aza-2'-deoxycytidine. Finally, we have shown that the simultaneous administration of trichostatin A and 5'-aza-2'-deoxycytidine results in reactivation of the CMV promoter according to a cooperative, not synergistic or additive, mechanism. In conclusion, our cellular system should represent a powerful tool to investigate the complex mechanisms regulating gene silencing and to identify new anticancer drugs.

Recent reports demonstrated an association of human parvovirus B19 with inflammatory cardiomyopathy (iCMP), which is accompanied by endothelial dysfunction. As intracellular Ca 2؉ activity is a key regulator of cell function and participates in mechanisms leading to endothelial dysfunction, the present experiments explored the effects of the B19 capsid proteins VP1 and VP2. A secreted phospholipase A2 (PLA2)-like activity has been located in the VP1 unique region of the B19 minor capsid protein. As PLA2 has recently been shown to activate the store-operated or capacitative Ca 2؉ channel I CRAC , we analyzed the impact of the viral PLA2 motif on Ca 2؉ entry. We cloned the VP1 and VP2 genes isolated from a patient suffering from fatal B19 iCMP into eukaryotic expression vectors. We also generated a B19 replication-competent plasmid to demonstrate PLA2 activity under the control of the complete B19 genome. After the transfection of human endothelial cells (HMEC-1), cytosolic Ca Human parvovirus B19, a nonenveloped virus of about 22 to 24 nm in diameter, is a member of the genus Erythrovirus within the family of Parvoviridae (35). B19 infection occurs frequently in humans, and this is documented by the high prevalence of specific immunoglobulin G (IgG) antibodies in young children (5% to 15%), adults (60%), and seniors older than 69 years (85%) (15). B19 is the causative agent of erythema infectiosum (fifth disease), hydrops fetalis, and transient aplastic anemia (1,86). Several studies disclosed an association between B19 and a variety of diseases (43,48,63,80), such as arthritis (56, 77), vasculitic syndromes (19, 24, 31, 80), hepatitis (27,36,38,75,85), and neurological disorders (2, 85). Specifically, B19 infections have been observed to be associated with acute and chronic myocarditis (13,16,30,44,53,(58)(59)(60)71). Moreover, the development of endothelial and isolated left ventricle diastolic dysfunction has been associated with B19 infection (81). During pregnancy, parvovirus B19 infection may cause maternal and fetal myocarditis, congenital abnormalities, stillbirth, and abortion (10, 21, 39, 61). The particularly severe course of the antenatal disease may relate to the preference of B19 for proliferating tissues (79).The cellular receptor for B19 infection has been regarded as a blood group P antigen based on the failure of B19 infection in a patient with a hereditary P antigen defect (12). The P antigen is necessary for B19 binding but not sufficient for virus entry into cells. In this regard, the ␣5␤1 integrin and the recently identified Ku80 autoantigen act as cellular coreceptors for human parvovirus B19 infection (57, 82). Therefore, target cells of B19 are mainly erythroid progenitor cells expressing high levels of P antigen as well as the coreceptors ␣5␤1 integrin and Ku80 autoantigen. However, nonerythroid cell lineages, such as fetal myocytes, follicular dendritic cells, and endothelial cells can be infected by B19 (12,28,29,57,82). We have recently localized B19 genomes in endothelial cells of my...

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