The 5′ Untranslated Region of Human Bocavirus Capsid Transcripts Regulates Viral mRNA Biogenesis and Alternative Translation

The capsid mRNA transcripts of human bocavirus 1 (HBoV1) can be generated by alternative splicing from the mRNA precursor transcribed from the P5 promoter. However, the alternative translation regulation mechanism of capsid mRNA transcripts is largely unknown. Here we report that the polycistronic capsid mRNA transcripts encode VP1, VP2, and VP3 in vitro and in vivo. The 5′ untranslated regions (UTRs) of capsid mRNA transcripts, which consist of exons, affected not only the abundance of mRNA but also the translation pattern of capsid proteins. Further study showed that exons 2 and 3 were critical for the abundance of mRNA, while exon 4 regulated capsid translation. Alternative translation of capsid mRNA involved a leaky scan mechanism. Mutating the upstream ATGs (uATGs) located in exon 4 resulted in more mRNA transcripts polyadenylated at the proximal polyadenylation [(pA)p] site, leading to increased capsid mRNA transcripts. Moreover, uATG mutations induced more VP1 expression, while VP3 expression was decreased, which resulted in less progeny virus production. Our data show that the 5′ UTR of HBoV1 plays a critical role in the modulation of mRNA abundance, alternative RNA processing, alternative translation, and progeny virus production. IMPORTANCE Alternative translation of HBoV1 capsid mRNAs is vital for the viral life cycle, as capsid proteins perform essential functions in genome packaging, assembly, and antigenicity. The 5′ untranslated regions (UTRs) of capsid mRNAs are generated by alternative splicing, and they contain different exons. Our study shows that the 5′ UTR not only modulates mRNA abundance but also regulates capsid expression. Two upstream ATGs (uATGs) that were upstream of the capsid translation initiation site in the 5′ UTR were found to affect viral capsid mRNA polyadenylation, alternative translation, and progeny virus production. The results reveal that uATGs play an important role in the viral life cycle and represent a new layer to regulate HBoV1 RNA processing, which could be a target for gene therapy.