Keeping All Secondary Structures of the Non-coding Region in the Circular Genome of Human Bocavirus 2 is Important for DNA Replication and Virus Assembly, As Revealed by Three Hetero-recombinant Genomic Clones

The episomal structures of all human bocavirus (HBoV) genotypes have been deciphered, including the circular genome of HBoV2 (HBoV2-C1). To discern the role of the circular HBoV2 genome, three distinct linearized HBoV2-C1 genomes were cloned into pBlueScript SKII(+) to obtain pBlueScript HBoV2 5043-5042 (retaining all secondary structures), pBlueScript-HBoV2 5075-5074 (retaining hairpin number 2 and the 5' terminal structure), and pBlueScript-HBoV2 5220-5219 (retaining only the 5' terminal structure at the 5' -genome end). The recombinant plasmids were separately transfected HEK293 cells, revealing that more HBoV2 DNA had accumulated in the pBlueScript HBoV2 5043-5042-transfected HEK293 cells at 72 h post-transfection, as determined by real-time PCR. However, more mRNA was transcribed by pBlueScript-HBoV2 5075-5074 than by the other constructs, as determined by dot-blot hybridization and RNAscope. No significant differences in NS1-70 protein expression were observed among the three HBoV2 genomic clones. However, electron microscopy showed that HBoV2 virus particles were only present in the pBlueScript HBoV2 5043-5042-transfected HEK293 cells. By using three hetero-recombinant HBoV2 genomic clones in HEK293 transfected cells, only the genome with intact secondary structures produced virus particles, suggesting that retaining these structures in a circular genome is important for HBoV2 DNA replication and virus assembly.