Ex Vivo-Generated CD36 ⁺ Erythroid Progenitors Are Highly Permissive to Human Parvovirus B19 Replication

Abstract

The pathogenic parvovirus B19 (B19V) has an extreme tropism for human erythroid progenitor cells. In vitro, only a few erythroid leukemic cell lines (JK-1 and KU812Ep6) or megakaryoblastoid cell lines (UT7/Epo and UT7/Epo-S1) with erythroid characteristics support B19V replication, but these cells are only semipermissive. By using recent advances in generating large numbers of human erythroid progenitor cells (EPCs) ex vivo from hematopoietic stem cells (HSCs), we produced a pure population of CD36 ؉ EPCs expanded and differentiated from CD34 ؉ HSCs and assessed the CD36 ؉ EPCs for their permissiveness to B19V infection. Over more than 3 weeks, cells grown in serum-free medium expanded more than 800,000-fold, and 87 to 96% of the CD36 ؉ EPCs were positive for globoside, the cellular receptor for B19V. Immunofluorescence (IF) staining showed that about 77% of the CD36 ؉ EPCs were positive for B19V infection, while about 9% of UT7/Epo-S1 cells were B19V positive. Viral DNA detected by real-time PCR increased by more than 3 logs in CD36 ؉ EPCs; the increase was 1 log in UT7/Epo-S1 cells. Due to the extensive permissivity of CD36 ؉ EPCs, we significantly improved the sensitivity of detection of infectious B19V by real-time reverse transcription-PCR and IF staining 100-and 1,000-fold, respectively, which is greater than the sensitivity of UT7/Epo-S1 cell-based methods. This is the first description of an ex vivo method to produce large numbers of EPCs that are highly permissive to B19V infection and replication, offering a cellular system that mimics in vivo infection with this pathogenic human virus.

Human parvovirus B19 causes cell cycle arrest of human erythroid progenitors via deregulation of the E2F family of transcription factors

“…Due to its restricted tropism for EPCs, there are no fully permissive cell lines for B19V propagation. Here, we employed the primary CD36 + EPC-based culture system, which generates fully permissive cells for B19V propagation (36). Upon B19V infection or NS1 transduction, CD36 + EPCs displayed G 2 arrest in a time-dependent manner.…”

“…Human CD36 + EPCs were generated as described in our previous study (36). 293T cells were maintained in DMEM containing 10% fetal bovine serum and antibiotics.…”

“…To quantitatively evaluate B19V infection, we performed real-time PCR as described previously (36). To confirm changes in putative E2F4 target genes induced by B19V NS1, we performed SYBR green-based real-time PCR using the RT² qPCR Primer Assay kit (SuperArray Bioscience) following the manufacturer's instructions.…”

“…B19V infection results in a rapid arrest of cell proliferation in the primary CD36 + EPCs (36). Given the important roles of E2Fs in cell proliferation and cell cycle progression, we investigated effects of E2F transcription factors on B19V-induced arrest of cell proliferation and cell cycling in CD36 + EPCs.…”

“…Recently, we developed a cell culture system that allows differentiation and expansion of CD34 + HSCs into CD36 + EPCs, which are highly permissive to B19V infection and replication, providing a cellular system that mimics in vivo infection of this pathogenic human virus (36). B19V infection results in a rapid arrest of cell proliferation in the primary CD36 + EPCs (36).…”