Abstract
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.
Highlights
The capsid mRNA transcripts of human bocavirus 1 (HBoV1) can be generated by alternative splicing from the mRNA precursor transcribed from the P5 promoter
To investigate the cis elements that regulate capsid protein expression, the first 600 nucleotides of the VP3 open reading frame (ORF) and the VP1 unique region (VP1u) sequence with or without the 5= untranslated regions (UTRs) of the R6, R7, and R8 transcripts were inserted into a PCR-BluntII-TOPO vector that contained a 27-nt A tract to increase the in vitro translation efficiency (Fig. 2A)
Mutation of uATG1 or uATG2 and -3 in the infectious clone resulted in more RNA polyadenylation at thep site (Fig. 9, lane 2 versus lanes 3 and 4).Taken together, these results indicate that the upstream ATGs (uATGs) are essential cis elements in the HBoV1 5= UTR which are required for RNA processing and virus progeny production
Summary
The capsid mRNA transcripts of human bocavirus 1 (HBoV1) can be generated by alternative splicing from the mRNA precursor transcribed from the P5 promoter. The 5= untranslated regions (UTRs) of capsid mRNA transcripts, which consist of exons, affected the abundance of mRNA and the translation pattern of capsid proteins. 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. 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 detailed mechanism that regulates the alternative translation of capsid proteins is still not understood
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