Abstract
Parvovirus B19 (B19V) is a human pathogenic virus of clinical relevance, characterized by a selective tropism for erythroid progenitor cells in bone marrow. Relevant information on viral characteristics and lifecycle can be obtained from experiments involving engineered genetic systems in appropriate in vitro cellular models. Previously, a B19V genome of defined consensus sequence was designed, synthesized and cloned in a complete and functional form, able to replicate and produce infectious viral particles in a producer/amplifier cell system. Based on such a system, we have now designed and produced a derived B19V minigenome, reduced to a replicon unit. The genome terminal regions were maintained in a form able to sustain viral replication, while the internal region was clipped to include only the left-side genetic set, containing the coding sequence for the functional NS1 protein. Following transfection in UT7/EpoS1 cells, this minigenome still proved competent for replication, transcription and production of NS1 protein. Further, the B19V minigenome was able to complement B19-derived, NS1-defective genomes, restoring their ability to express viral capsid proteins. The B19V genome was thus engineered to yield a two-component system, with complementing functions, providing a valuable tool for studying viral expression and genetics, suitable to further engineering for purposes of translational research.
Highlights
Accepted: 29 December 2021Within the family Parvoviridae [1], Parvovirus B19 (B19V) is a human pathogenic virus of clinical relevance, responsible for transient or persistent erythroid aplasia, infectious erythema, arthropathies, myocarditis and intrauterine infections, among others [2,3]
B19V has a marked tropism for erythroid progenitor cells (EPCs) in the bone marrow, both susceptible and permissive depending on their differentiation and proliferation state [4]
In the design of a B19V minigenome with potential replicative activity, the rational requirements were: (i) to preserve both terminal regions up to the sites of dyad symmetry, retaining the capacity of hairpin formation; and (ii) to preserve the internal region extending up to the pAp1 proximal cleavage-polyadenylation signal, as a gene cassette with potential for coding for the NS1 protein, while eliminating the genomic region coding for the viral capsid proteins
Summary
Within the family Parvoviridae [1], Parvovirus B19 (B19V) is a human pathogenic virus of clinical relevance, responsible for transient or persistent erythroid aplasia, infectious erythema, arthropathies, myocarditis and intrauterine infections, among others [2,3]. A reference genome of defined consensus sequence was designed, synthesized and cloned in a complete and functional form in a plasmid vector Such a genome was able to replicate and produce infectious viral particles in a producer/amplifier cell system, the myeloblastoid UT7/EpoS1cells, allowing generation and further propagation of virus in EPC cell cultures. The main objective of the present experiments was to create a genetic unit derived from the complete, competent cloned B19V genome, simplified to a potential replicon unit In this minigenome, the terminal regions were maintained in a form able to sustain viral replication, while the internal region was clipped to contain only the left-side genetic set. Viruses 2022, 14, 84 minigenome to complement defective forms of B19 genome rescuing production of viral capsid proteins and the possibility of producing transducing viral particles was tested
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