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.

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Insertion of completely complementary microRNA (miR) target sites (miRTS) into a transgene has been shown to be a valuable approach to specifically repress transgene expression in non-targeted tissues. miR-122TS have been successfully used to silence transgene expression in the liver following systemic application of cardiotropic adeno-associated virus (AAV) 9 vectors. For miR-206-mediated skeletal muscle-specific silencing of miR-206TS-bearing AAV9 vectors, however, we found this approach failed due to the expression of another member (miR-1) of the same miR family in heart tissue, the intended target. We introduced single-nucleotide substitutions into the miR-206TS and searched for those which prevented miR-1-mediated cardiac repression. Several mutated miR-206TS (m206TS), in particular m206TS-3G, were resistant to miR-1, but remained fully sensitive to miR-206. All these variants had mismatches in the seed region of the miR/m206TS duplex in common. Furthermore, we found that some m206TS, containing mismatches within the seed region or within the 3' portion of the miR-206, even enhanced the miR-206- mediated transgene repression. In vivo expression of m206TS-3G- and miR-122TS-containing transgene of systemically applied AAV9 vectors was strongly repressed in both skeletal muscle and the liver but remained high in the heart. Thus, site-directed mutagenesis of miRTS provides a new strategy to differentiate transgene de-targeting of related miRs.