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.

中文翻译：

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正如三个异源重组基因组克隆所揭示的那样，在人类博卡病毒2的环状基因组中保留非编码区的所有二级结构对于DNA复制和病毒装配很重要。

所有人类博卡病毒（HBoV）基因型的附加体结构均已破译，包括HBoV2的环状基因组（HBoV2-C1）。为了识别圆形HBoV2基因组的作用，将三个不同的线性化HBoV2-C1基因组克隆到pBlueScript SKII（+）中，以获得pBlueScript HBoV2 5043-5042（保留所有二级结构），pBlueScript-HBoV2 5075-5074（保留发夹编号2）以及5'末端结构）和pBlueScript-HBoV2 5220-5219（仅在5'-基因组末端保留5'末端结构）。重组质粒分别转染了HEK293细胞，通过实时PCR测定，表明在转染后72 h，pBlueScript HBoV2 5043-5042转染的HEK293细胞中积累了更多的HBoV2 DNA。然而，如通过点印迹杂交和RNAscope所确定，pBlueScript-HBoV2 5075-5074转录的mRNA比其他构建体要多。在三个HBoV2基因组克隆之间未观察到NS1-70蛋白表达的显着差异。但是，电子显微镜显示HBoV2病毒颗粒仅存在于pBlueScript HBoV2 5043-5042转染的HEK293细胞中。通过在HEK293转染的细胞中使用三个异源重组HBoV2基因组克隆，只有具有完整二级结构的基因组才能产生病毒颗粒，这表明将这些结构保留在环状基因组中对于HBoV2 DNA复制和病毒组装很重要。电子显微镜显示，HBoV2病毒颗粒仅存在于pBlueScript HBoV2 5043-5042转染的HEK293细胞中。通过在HEK293转染的细胞中使用三个异源重组HBoV2基因组克隆，只有具有完整二级结构的基因组才能产生病毒颗粒，这表明将这些结构保留在环状基因组中对于HBoV2 DNA复制和病毒组装很重要。电子显微镜显示，HBoV2病毒颗粒仅存在于pBlueScript HBoV2 5043-5042转染的HEK293细胞中。通过在HEK293转染的细胞中使用三个异源重组HBoV2基因组克隆，只有具有完整二级结构的基因组才能产生病毒颗粒，这表明将这些结构保留在环状基因组中对于HBoV2 DNA复制和病毒组装很重要。