The Genome of Human Parvovirus B19 Can Replicate in Nonpermissive Cells with the Help of Adenovirus Genes and Produces Infectious Virus

Abstract

Human parvovirus B19 (B19V) is a member of the genus Erythrovirus in the family Parvoviridae. In vitro, autonomous B19V replication is limited to human erythroid progenitor cells and in a small number of erythropoietin-dependent human megakaryoblastoid and erythroid leukemic cell lines. Here we report that the failure of B19V DNA replication in nonpermissive 293 cells can be overcome by adenovirus infection. More specifically, the replication of B19V DNA in the 293 cells and the production of infectious progeny virus were made possible by the presence of the adenovirus E2a, E4orf6, and VA RNA genes that emerged during the transfection of the pHelper plasmid. Using this replication system, we identified the terminal resolution site and the nonstructural protein 1 (NS1) binding site on the right terminal palindrome of the viral genome, which is composed of a minimal origin of replication spanning 67 nucleotides. Plasmids or DNA fragments containing an NS1 expression cassette and this minimal origin were able to replicate in both pHelper-transfected 293 cells and B19V-semipermissive UT7/Epo-S1 cells. Our results have important implications for our understanding of native B19V infection.

Novel adenovirus-based helper system to support production of recombinant parvovirus

“…In agreement with these results, adenovirus 2 facilitated DNA replication of the closely related parvovirus MVM, 27 whereas pXX6 adenoviral vector, enabled the erythrovirus B19 to replicate its DNA and produce infectious progeny particles in non-permissive HEK 293 cells. 25 This led us to speculate that one or several gene products of pXX6 may directly modulate parvoviral DNA replication or set up favorable cellular conditions for that. In support of an active role of adenovirus genes in parvoviral DNA replication, it has been shown that E4(orf6) participates in the conversion of the singlestranded AAV-2 viral genome into the double-stranded replicative forms.…”

“…The adenovirus genes E2a, E4(orf6) and VA RNA were found to boost human erythrovirus B19 replication and production in HEK 293 cells, an otherwise non-permissive cell line for B19 production. 25 Furthermore, parvovirus H-1PV failed to initiate replication in secondary cultures of normal human embryonic lung cells, unless these cells are co-infected with adenovirus serotype 12. 26 In addition, adenovirus serotype 2 induced an increase in parvovirus MVMp DNA replication in HeLa cells, associated with re-localization of MVMp DNA from host nucleoli to adenovirus replication factories.…”

Internal Polyadenylation of the Parvovirus B19 Precursor mRNA Is Regulated by Alternative Splicing

“…AY386330) (19). The parent plasmid C1NS1(Ϫ), which contains the SV40 replication origin and produces a prematurely terminated NS1 gene has been described previously (14,17,20). The parent plasmid pB19-N8 was described previously (19 -21).…”

“…Cells were transfected with 2 g of DNA per 60-mm plate using Lipofectamine TM and PLUS TM reagent (Invitrogen) according to the manufacturer's instructions. UT7/Epo-S1 cells, which are permissive to B19V infection (20,25,26), were cultured in DMEM with 10% FCS and 2 units/ml of Epo (Epogen; Amgen, Thousand Oaks, CA) at 37°C in 5% CO 2 . Plasmids B19-N8, B19-N8A2-1U2AF, and B19-N8A2-1/ 2KO were digested with XhoI/EcoRI to recover the excised B19V DNA fragments that were used for electroporation.…”

“…Plasmids B19-N8, B19-N8A2-1U2AF, and B19-N8A2-1/ 2KO were digested with XhoI/EcoRI to recover the excised B19V DNA fragments that were used for electroporation. UT7/ Epo-S1 cells were electroporated with 2 g of linearized B19V DNA per 2 ϫ 10 6 cells with an Amaxa Nucleofector device (Lonza) as described previously (20). Primary human erythroid progenitor cells (CD36 ϩ EPCs) were generated; large numbers of CD36…”