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Parvovirus B19 (B19V) is pathogenic for humans and has an extreme tropism for human erythroid progenitors. We report cell type-specific expression of the B19V capsid genes (VP1 and VP2) and greatly increased B19V capsid protein production in nonpermissive cells by codon optimization. Codon usage limitation, rather than promoter type and the 3' untranslated region of the capsid genes, appears to be a key factor in capsid protein production in nonpermissive cells. Moreover, B19 virus-like particles were successfully generated in nonpermissive cells by transient transfection of a plasmid carrying both codon-optimized VP1 and VP2 genes.

Human parvovirus B19 (B19V) is the only human pathogenic parvovirus. It causes a wide spectrum of human diseases, including fifth disease (erythema infectiosum) in children and pure red cell aplasia in immunocompromised patients. B19V is highly erythrotropic and preferentially replicates in erythroid progenitor cells (EPCs). Current understanding of how B19V interacts with cellular factors to regulate disease progression is limited, due to a lack of permissive cell lines and animal models. Here, we employed a recently developed primary human CD36(+) EPC culture system that is highly permissive for B19V infection to identify cellular factors that lead to cell cycle arrest after B19V infection. We found that B19V exploited the E2F family of transcription factors by downregulating activating E2Fs (E2F1 to E2F3a) and upregulating repressive E2Fs (E2F4 to E2F8) in the primary CD36(+) EPCs. B19V nonstructural protein 1 (NS1) was a key viral factor responsible for altering E2F1-E2F5 expression, but not E2F6-E2F8 expression. Interaction between NS1 and E2F4 or E2F5 enhanced the nuclear import of these repressive E2Fs and induced stable G₂ arrest. NS1-induced G₂ arrest was independent of p53 activation and increased viral replication. Downstream E2F4/E2F5 targets, which are potentially involved in the progression from G₂ into M phase and erythroid differentiation, were identified by microarray analysis. These findings provide new insight into the molecular pathogenesis of B19V in highly permissive erythroid progenitors.

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.

The pre-mRNA processing strategy of the B19 virus is unique among parvoviruses. B19 virus-generated pre-mRNAs are transcribed from a single promoter and are extensively processed by alternative splicing and alternative polyadenylation to generate 12 transcripts. Blockage of the production of full-length B19 virus transcripts at the internal polyadenylation site [(pA)p] was previously reported to be a limiting step in B19 virus permissiveness. We show here that in the absence of genome replication, internal polyadenylation of B19 virus RNAs at (pA)p is favored in cells which are both permissive and nonpermissive for B19 viral replication. Replication of the B19 virus genome, however, introduced either by viral infection or by transfection of an infectious clone into permissive cells or forced by heterologous replication systems in nonpermissive cells, enhanced readthrough of (pA)p and the polyadenylation of B19 virus transcripts at the distal site [(pA)d]. Therefore, replication of the genome facilitates the generation of sufficient full-length transcripts that encode the viral capsid proteins and the essential 11-kDa nonstructural protein. Furthermore, we show that polyadenylation of B19 viral RNA at (pA)p likely competes with splicing at the second intron. Thus, we conclude that replication of the B19 virus genome is the primary limiting step governing B19 virus tropism.

Three full-length genomic clones (pB19-M20, pB19-FL and pB19-HG1) of parvovirus B19 were produced in different laboratories. pB19-M20 was shown to produce infectious virus. To determine the differences in infectivity, all three plasmids were tested by transfection and infection assays. All three clones were similar in viral DNA replication, RNA transcription, and viral capsid protein production. However, only pB19-M20 and pB19-HG1 produced infectious virus. Comparison of viral sequences showed no significant differences in ITR or NS regions. In the capsid region, there was a nucleotide sequence difference conferring an amino acid substitution (E176K) in the phospholipase A2-like motif of the VP1-unique (VP1u) region. The recombinant VP1u with the E176K mutation had no catalytic activity as compared with the wild-type. When this mutation was introduced into pB19-M20, infectivity was significantly attenuated, confirming the critical role of this motif. Investigation of the original serum from which pB19-FL was cloned confirmed that the phospholipase mutation was present in the native B19 virus.

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.

In an attempt to experimentally define the roles of viral proteins encoded by the B19 genome in the viral life cycle, we utilized the B19 infectious clone constructed in our previous study to create two groups of B19 mutant genomes: (i) null mutants, in which either a translational initiation codon for each of these viral genes was substituted by a translational termination codon or a termination codon was inserted into the open reading frame by a frameshift; and (ii) a deletion mutant, in which half of the hairpin sequence was deleted at both the 5' and the 3' termini. The impact of these mutations on viral infectivity, DNA replication, capsid protein production, and distribution was systematically examined. Null mutants of the NS and VP1 proteins or deletion of the terminal hairpin sequence completely abolished the viral infectivity, whereas blocking expression of the 7.5-kDa protein or the putative protein X had no effect on infectivity in vitro. Blocking expression of the proline-rich 11-kDa protein significantly reduced B19 viral infectivity, and protein studies suggested that the expression of the 11-kDa protein was critical for VP2 capsid production and trafficking in infected cells. These findings suggest a previously unrecognized role for the 11-kDa protein, and together the results enhance our understanding of the key features of the B19 viral genome and proteins.

Human parvovirus B19 has a nonenveloped, icosahedral capsid packaging a linear single-stranded DNA genome of 5.6 kb with long inverted terminal repeats (ITR) at both the 5' and 3' end. Previous attempts to construct a full-length B19 clone were unsuccessful due to deletions in the ITR sequences. We cloned the complete parvovirus B19 genome with intact ITRs from an aplastic crisis patient. Sequence analysis of the complete viral genome indicated that both 5' and 3' ITRs have two sequence configurations and several base changes within the ITRs compared to previous published sequences. After transfection of the plasmid into permissive cells, spliced and non-spliced viral transcripts and viral capsid proteins could be detected. Southern blot analysis of the DNA purified from the plasmid-transfected cells confirmed parvovirus B19 DNA replication. Production of infectious virus by the B19 plasmid was shown by inoculation of cell lysate derived from transfected cells into fresh cells. Together, these results indicate the first successful production of an infectious clone for parvovirus B19 virus.