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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 tyrosine705 and serine727 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. [ABSTRACT FROM AUTHOR]

Recent reports demonstrated an association of human parvovirus B19 with inflammatory cardiomyopathy (iCMP), which is accompanied by endothelial dysfunction. As intracellular Ca2+ 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 Ca2+ channel ICRAC, we analyzed the impact of the viral PLA2 motif on Ca2+ 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 Ca2+ activity was determined by utilizing Fura-2 fluorescence. VP1 and VP2 expression did not significantly modify basal cytosolic Ca2+ activity or the decline of cytosolic Ca2+ activity following the removal of extracellular Ca2+. However, expression of VP1 and of the full-length B19 clone, but not of VP2, significantly accelerated the increase of cytosolic Ca2+ activity following the readdition of extracellular Ca2+ in the presence of thapsigargin, indicating an activation of ICRAC. The effect of VP1 was mimicked by the PLA2 product lysophosphatidylcholine and abolished by an inactivating mutation of the PLA2-encoding region of the VP1 gene. Our observations point to the activation of Ca2+ entry by VP1 PLA2 activity, an effect likely participating in the pathophysiology of B19 infection. [ABSTRACT FROM AUTHOR]

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(2+) activity was determined by utilizing Fura-2 fluorescence. VP1 and VP2 expression did not significantly modify basal cytosolic Ca(2+) activity or the decline of cytosolic Ca(2+) activity following the removal of extracellular Ca(2+). However, expression of VP1 and of the full-length B19 clone, but not of VP2, significantly accelerated the increase of cytosolic Ca(2+) activity following the readdition of extracellular Ca(2+) in the presence of thapsigargin, indicating an activation of I(CRAC.) The effect of VP1 was mimicked by the PLA2 product lysophosphatidylcholine and abolished by an inactivating mutation of the PLA2-encoding region of the VP1 gene. Our observations point to the activation of Ca(2+) entry by VP1 PLA2 activity, an effect likely participating in the pathophysiology of B19 infection.