Claudia Filippone

Molecular and Functional Analyses of a Human Parvovirus B19 Infectious Clone Demonstrates Essential Roles for NS1, VP1, and the 11-Kilodalton Protein in Virus Replication and Infectivity

ABSTRACT 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.

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.

VP1u phospholipase activity is critical for infectivity of full-length parvovirus B19 genomic clones.

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.

Functional analysis and quantitative determination of the expression profile of human parvovirus B19.

Comprehension of the pathogenetic potential of human parvovirus B19 requires the definition of the complete spectrum of cellular tropism and a functional analysis of the viral genome in infected cells. In this study, we carried out a systematic functional analysis of B19 virus genome in the course of infection of susceptible bone marrow mononuclear cells and myeloblastoid UT7/EpoS1 cells, in terms of dynamics of nucleic acid synthesis. A PCR array was designed and a comprehensive analysis was performed by quantitative PCR and RT-PCR, yielding extended information on the presence and abundance of the diverse classes of viral nucleic acids, on the temporal regulation of genome expression and on its relationship with the cell cycle. The analysis performed indicate that the synthesis of viral nucleic acids is correlated to the progression through the S phase of the cell cycle, that an extended pattern of transcriptional activity occurs throughout the course of infection, with a maximal rate of transcription preceding the onset of S-phase dependent replication of the viral genome, and that utilization of transcript processing signals is relatively constant throughout the course of infection. The information obtained led to the definition of a unified model of functional and expression profiling of parvovirus B19 genome.

HepG2 hepatocellular carcinoma cells are a non-permissive system for B19 virus infection

Parvovirus B19 has been associated with liver dysfunction and has been considered a potential aetiological agent of fulminant hepatitis and hepatitis-associated aplastic anaemia. The possible effects of B19 virus infection on the liver have been investigated using HepG2 hepatocellular carcinoma cells as a model system, but the reported results are inconsistent. To investigate this relationship further, this study followed the course of B19 virus infection of HepG2 cells in terms of viral DNA, RNA and protein production by quantitative PCR, RT-PCR and immunofluorescence assays. The data showed that B19 virus is able to bind and possibly enter HepG2 cells, but that viral genome replication or transcription is not supported and that viral proteins are not produced. As far as HepG2 cells can be considered a representative model system, any possible pathogenic role of B19 virus on the liver cannot be ascribed to infection or to a direct cytopathic effect on hepatocytes.