Despite its strong host tropism for erythroid progenitor cells, human parvovirus B19 (B19V) can also infect a variety of additional cell types. Acute and chronic inflammatory cardiomyopathies have been associated with a high prevalence of B19V DNA in endothelial cells of the myocardium. To elucidate the mechanisms of B19V uptake into endothelium, we first analyzed the surface expression of the well-characterized primary B19V receptor P antigen and the putative coreceptors ␣ 5 ␤ 1 integrins and Ku80 antigen on primary and permanent endothelial cells. The receptor expression pattern and also the primary attachment levels were similar to those in the UT7/Epo-S1 cell line regarded as functional for B19V entry, but internalization of the virus was strongly reduced. As an alternative B19V uptake mechanism in endothelial cells, we demonstrated antibody-dependent enhancement (ADE), with up to a 4,000-fold increase in B19V uptake in the presence of B19V-specific human antibodies. ADE was mediated almost exclusively at the level of virus internalization, with efficient B19V translocation to the nucleus. In contrast to monocytes, where ADE of B19V has been described previously, enhancement does not rely on interaction of the virus-antibody complexes with Fc receptors (FcRs), but rather, involves an alternative mechanism mediated by the heat-sensitive complement factor C1q and its receptor, CD93. Our results suggest that ADE represents the predominant mechanism of endothelial B19V infection, and it is tempting to speculate that it may play a role in the pathogenicity of cardiac B19V infection. IMPORTANCEBoth efficient entry and productive infection of human parvovirus B19 (B19V) seem to be limited to erythroid progenitor cells. However, in vivo, the viral DNA can also be detected in additional cell types, such as endothelial cells of the myocardium, where its presence has been associated with acute and chronic inflammatory cardiomyopathies. In this study, we demonstrated that uptake of B19V into endothelial cells most probably does not rely on the classical receptor-mediated route via the primary B19V receptor P antigen and coreceptors, such as ␣ 5 ␤ 1 integrins, but rather on antibody-dependent mechanisms. Since the strong antibody-dependent enhancement (ADE) of B19V entry requires the CD93 surface protein, it very likely involves bridging of the B19V-antibody complexes to this receptor by the complement factor C1q, leading to enhanced endocytosis of the virus.

Despite its very narrow tropism for erythroid progenitor cells, human parvovirus B19 (B19V) has recently been shown to replicate and form infectious progeny virus in 293 cells in the presence of early adenoviral functions provided either by infection with adenovirus type 5 or by addition of the pHelper plasmid encoding the E2a, E4orf6, and VA RNA functions. In the present study we dissected the individual influence of these functions on B19V genome replication and expression of structural proteins VP1 and VP2. We show that, in the presence of the constitutively expressed E1A and E1B, E4orf6 alone is able to promote B19V DNA replication, resulting in a concomitant increase in VP expression levels. The stimulatory effects of E4orf6 require the integrity of the BC box motifs, which target cellular proteins such as p53 and the Mre11 DNA repair complex for proteosomal degradation through formation of an E3 ubiquitin ligase complex with E1B. VA RNA also strongly induces VP expression but, in contrast to E4orf6, in a replication-independent manner. This stimulation could be attributed exclusively to the VA I RNA transcript and does not involve major activating effects at the level of the B19V p6 promoter, but the nucleotide residues required for the welldefined pathway of VA I RNA mediated stimulation of translation through functional inactivation of protein kinase R. These data show that the cellular pathways regulating B19V replication may be very similar to those governing the productive cycle of the helper-dependent parvoviruses, the adeno-associated viruses.

Human parvovirus B19 (B19V) DNA is highly prevalent in endothelial cells lining up intramyocardial arterioles and postcapillary venules of patients with chronic myocarditis and cardiomyopathies. We addressed the question of a possible stimulation of B19V gene expression in endothelial cells by infection with adenoviruses. Adenovirus infection led to a strong augmentation of B19V structural and nonstructural proteins in individual endothelial cells infected with B19V or transfected with an infectious B19V genome. Transactivation was mostly mediated at the level of transcription and not due to adenovirus-mediated induction of second-strand synthesis from the single-stranded parvoviral genome. The main adenoviral functions required were E1A and E4orf6, which displayed synergistic effects. Furthermore, a limited B19V genome replication could be demonstrated in endothelial cells and adenovirus infection induced the appearance of putative dimeric replication intermediates. Thus the almost complete block in B19V gene expression seen in endothelial cells can be abrogated by infection with other viruses.

A coupled luminescent method (CLM) based on glyceraldehyde-3-phosphate dehydrogenase released from injured target cells was used to evaluate the cytotoxicity of antigen-specific HLA class I-restricted CTLs. In contrast to established methods, CLM does not require the pretreatment of target cells with radioactive or toxic labeling substances. CTLs from healthy HLA-A2 positive donors were stimulated by autologous dendritic cells (DCs) pulsed with HLA-A2 restricted HCMV-pp65 nonamer peptides. HLA-A2 positive T2 cells or autologous monocytes pulsed with HCMV-pp65 nonamer peptide served as target cells. Lysis was detected only in HCMV-pp65-pulsed target cells incubated with CTLs from seropositive donors stimulated by HCMV-pp65-pulsed DCs. After 3 days, stimulation 38% of T2 cells and 17% of monocytes were lysed at an effector to target ratio of 8:1. In conclusion, CLM represents a highly sensitive, fast, material-saving and non-toxic/non-radioactive method for the measurement of antigen-specific CTL cytotoxic activity.

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