The case of Parvovirus B19
Text by Giorgio Gallinella
Parvovirus B19 (B19V) is a human pathogenic virus, characterized by a selective tropism for erythroid progenitor cells (EPCs) in the bone marrow, and responsible for a wide range of clinical manifestations. To study its lifecycle, a central issue is the availability of genomic clones possessing full competence and ability to generate infectious virus. In our study, we established a new model genetic system for B19V by a synthetic approach. We designed a reference genome sequence, generated a corresponding artificial construct, and setup an efficient strategy to generate infectious virus, via transfection in UT7/EpoS1 cells followed by amplification in EPCs. The synthetic genome was able to generate virus with biological properties paralleling those of native virus, and we showed that its infectious activity was dependent on the preservation of self-complementarity and sequence heterogeneity within the terminal regions, that serve as origins of replication.
Our basic idea was to design and construct a virus of defined genome sequence, chosen as a consensus, possibly ancestral sequence. A synthetic approach was easily followed, but a main obstacle was cloning of the inverted terminal regions of B19V genome (ITR), palindromic hairpins that due to sequence heterogeneity can be present in different combinations of isomers, called flip/flop. These regions are rather unstable when cloned in plasmid vectors. Was all of the ITRs necessary to maintain their potential as origins of replication? Was flip/flop heterogeneity of some relevance? To answer these questions, we constructed and compared a collection of inserts, differing in the extension and isomer combination of the ITRs.
Then, we had to find cells that could be efficiently transfected and support viral replication. EPCs are permissive, but their transfection is critical, with a very variable outcome. The myeloblastoid UT7/EpoS1 cell line can be transfected with constant even if low efficiency, can support B19V DNA replication but the yield of infectious virus is minimal. The key point of our work was a prolonged time course experiment following transfection of UT7/EpoS1 cells, when we realized that infectious virus could eventually be released from cells, and that this virus could be maintained and amplified in subsequent passages in EPCs. Thus, we could compare the activity of our collection of inserts, and gain insight into the requirements of the ITRs to ensure replication of the virus. As a final output, we generated a fully competent synthetic virus that can constitute a reference tool for investigation of the structural and functional characteristics of B19V.
Top: schematic diagram of B19V genome indicating the extension of inserts relative to the two inverted terminal regions (ITR) and the internal region (IR).
Center: flowchart of the experimental scheme, from the cloned B19V DNA inserts to transfection in UT7/EpoS1 cells to two successive rounds of infection in EPCs, each time with supernatant obtained from the previous step and progressive amplification of the viral yield.
Bottom: histograms, showing for each group of inserts the amounts of viral DNA detected in transfected UT7/EpoS1 cells (DNA within cells or benzonase-resistant DNA in cell culture supernatants at 6 days post-transfection), and infected EPCs (input DNA at 2 hpi, DNA within cells or in cell culture supernatants at 6 days post-infection).
Ability to generate infectious virus was observed for the inserts extending beyond the dyad axis of symmetry within terminal regions (CK inserts), irrespective of the terminal isomer combination, and also for inserts extending up to the site of dyad symmetry (CH inserts), in this case provided that the two truncated terminal regions preserve sequence heterogeneity (CH01 and CH10, but not CH00 and CH11). Further truncation (CI and CJ inserts) also led to loss of functional competence.
Introducing the Parvo Group
Pictured from left to right, Francesca Bonvicini, Gloria Bua, Giorgio Gallinella, Elisabetta Manaresi and Ilaria Conti. Department of Pharmacy and Biotechnology, University of Bologna, Italy.
About the research
Elisabetta Manaresi, Ilaria Conti, Gloria Bua, Francesca Bonvicini, Giorgio Gallinella
Virology, Volume 508, August 2017, Pages 54–62