Transactivation of human parvovirus B19 gene expression in endothelial cells by adenoviral helper functions

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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.

Section: Discussionmentioning

confidence: 99%

Antibody-Mediated Enhancement of Parvovirus B19 Uptake into Endothelial Cells Mediated by a Receptor for Complement Factor C1q

Kietzell

Pozzuto

Heilbronn

et al. 2014

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“…In contrast to the studies performed in other nonpermissive cells, we did not observe a clear NS1-mediated transactivation of the p6 promoter in 293 cells (data not shown). Although this may due to the high basal activity of the p6 promoter caused by constitutive expression of the E1A protein in 293 cells (40), we have also been unable to score a significant NS1-induced transactivation of the p6 promoter in endothelial cells, despite a low basal p6 activity (41). Thus, NS1 may not generally act as a positive regulator of B19V structural gene expression but may also exhibit negative effects under conditions, where B19V genome replication is blocked.…”

Section: Discussionmentioning

confidence: 78%

“…The plasmid DNA was digested with SalI to obtain the linearized double-stranded B19V DNA full-length genomic fragment used for transfections. The NS1-negative B19V clone pB19-M20dNS1 has been described in detail before (41). It contains an insertion of 4 bp in the amino-terminal part of the NS1 gene, leading to a frameshift and a truncated nonfunctional protein of 213 amino acids.…”

Section: Methodsmentioning

confidence: 99%

“…Plasmid pTR-p6-Luc harboring the luciferase reporter gene under the control of the B19V p6 promoter has been described elsewhere (41). The pCATCH-NS1 plasmid for expression of an amino-terminally Flagtagged NS1 protein under the control of the human cytomegalovirus (HCMV) early promoter/enhancer was cloned by amplification of the complete NS1 ORF from pB19-M20 with oligonucleotides generating EcoRI and XbaI restriction sites, respectively, and insertion via these sites into eukaryotic expression pCATCH (20).…”

Section: Methodsmentioning

confidence: 99%

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Roles of E4orf6 and VA I RNA in Adenovirus-Mediated Stimulation of Human Parvovirus B19 DNA Replication and Structural Gene Expression

Winter

Kietzell

Heilbronn

et al. 2012

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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.

“…RUNX-1 colocalizes adjacent to nuclear sites of viral transcription; however, the effect of this transcription factor was not examined. Moreover, a new function of E4orf6 was recently unveiled, as the protein seems to be involved in transactivation of parvovirus B19 replication in endothelial cells (63). Besides E1A expression, E4orf6 expression enhances the activity of the parvovirus p6 promoter.…”

Section: Discussionmentioning

confidence: 99%

Functional Cooperation between Human Adenovirus Type 5 Early Region 4, Open Reading Frame 6 Protein, and Cellular Homeobox Protein HoxB7

Müller

Schreiner

Schmid

et al. 2012

cHuman adenovirus type 5 (HAdV5) E4orf6 (early region 4 open reading frame 6 protein) is a multifunctional early viral protein promoting efficient replication and progeny production. E4orf6 complexes with E1B-55K to assemble cellular proteins into a functional E3 ubiquitin ligase complex that not only mediates proteasomal degradation of host cell substrates but also facilitates export of viral late mRNA to promote efficient viral protein expression and host cell shutoff. Recent findings defined the role of E4orf6 in RNA splicing independent of E1B-55K binding. To reveal further functions of the early viral protein in infected cells, we used a yeast two-hybrid system and identified the homeobox transcription factor HoxB7 as a novel E4orf6-associated protein.Using a HoxB7 knockdown cell line, we observed a positive role of HoxB7 in adenoviral replication. Our experiments demonstrate that the absence of HoxB7 leads to inefficient viral progeny production, as HAdV5 gene expression is highly regulated by HoxB7-mediated activation of various adenoviral promoters. We have thus identified a novel role of E4orf6 in HAdV5 gene transcription via regulation of homeobox protein-dependent modulation of viral promoter activity. Human adenovirus type 5 (HAdV5) E4orf6 (early region 4 open reading frame 6 protein) is a multifunctional protein which promotes efficient viral replication and plays a major role in adenoviral transformation processes. During infection, this viral factor assembles an SCF-like E3 ubiquitin ligase complex based on the cellular proteins elongins B and C, cullin 5, and Rbx1 (4, 66; reviewed in references 74 and 83). In cooperation with E1B-55K (early region 1B 55K protein), this viral RING-type ligase ubiquitinates cellular substrates prior to proteasomal degradation. So far, p53, DNA ligase IV, Mre11, integrin ␣3, and BLM (Blooms helicase) have been identified as targets of this HAdV5 ligase complex (4,18,64,83).E1B-55K was also identified as a viral interaction partner of the transcription factor Daxx (76,79,89) and is required for proteasomal degradation of Daxx during HAdV5 infection (75, 76). Remarkably, in contrast to the cellular targets of HAdV5 E3 ubiquitin ligases mentioned above, E4orf6 is dispensable for Daxx removal. In reverse, the HAdV12 E4orf6 protein was shown to target TopBP1, a protein involved in the DNA damage response and cell cycle checkpoint control (70, 71). In this case, HAdV12 E1B-55K is apparently not relevant for the cullin2-E4orf6-dependent proteasomal degradation of TopBP1 (5). In addition, it was shown that formation of the HAdV5 E3 ubiquitin ligase complex is necessary for E4orf6-and E1B-55K-mediated export of viral late mRNA out of the nucleus into the cytoplasm (7, 88). This preferential export of viral transcripts over cellular mRNA is important for efficient viral protein expression and host cell shutoff.However, different studies show that during the course of infection, only up to 50% of E4orf6 is associated with E1B-55K (16,60,80). Therefore, we were interested in ad...