A Comprehensive RNA-seq Analysis of Human Bocavirus 1 Transcripts in Infected Human Airway Epithelium

Abstract

Human bocavirus 1 (HBoV1) infects well-differentiated (polarized) human airway epithelium (HAE) cultured at an air-liquid interface (ALI). In the present study, we applied next-generation RNA sequencing to investigate the genome-wide transcription profile of HBoV1, including viral mRNA and small RNA transcripts, in HBoV1-infected HAE cells. We identified novel transcription start and termination sites and confirmed the previously identified splicing events. Importantly, an additional proximal polyadenylation site (pA)p2 and a new distal polyadenylation site (pA)dREH lying on the right-hand hairpin (REH) of the HBoV1 genome were identified in processing viral pre-mRNA. Of note, all viral nonstructural proteins-encoding mRNA transcripts use both the proximal polyadenylation sites [(pA)p1 and (pA)p2] and distal polyadenylation sites [(pA)d1 and (pA)dREH] for termination. However, capsid proteins-encoding transcripts only use the distal polyadenylation sites. While the (pA)p1 and (pA)p2 sites were utilized at roughly equal efficiency for proximal polyadenylation of HBoV1 mRNA transcripts, the (pA)d1 site was more preferred for distal polyadenylation. Additionally, small RNA-seq analysis confirmed there is only one viral noncoding RNA (BocaSR) transcribed from nt 5199–5340 of the HBoV1 genome. Thus, our study provides a systematic and unbiased transcription profile, including both mRNA and small RNA transcripts, of HBoV1 in HBoV1-infected HAE-ALI cultures.

Cellular Cleavage and Polyadenylation Specificity Factor 6 (CPSF6) Mediates Nuclear Import of Human Bocavirus 1 NP1 Protein and Modulates Viral Capsid Protein Expression

“…How NP1 decreases the interaction between the CPSF complex and the (pA)p sites of the viral pre-mRNA warrants further investigations. The (pA)d REH site is also unique among the four HBoV1 pA sites, as it does not have a consensus CPSF-binding (AAUAAA) site (23,24), indicating that the building of the CPSF complex is different from that of a regular one, which may be less dependent on the binding of the CFIm complex to the UGUA signal upstream of the (pA)d REH site. Of note, we also observed an interaction of Fip1 with NP1 in a co-IP experiment.…”

“…It not only facilitates the effective splicing of viral pre-mRNA at the splice acceptor site upstream of the (pA)p sites but also assists the RNA Pol II complex to read through the (pA)p sites for the production of long viral mRNAs terminated at the distal polyadenylation [(pA)d] sites, which yields the VPencoding mRNAs; therefore, NP1 is crucial to capsid protein expression (19,22). A comprehensive analysis of viral mRNA transcripts during HBoV1 infection of HAE-ALI using transcriptome sequencing identified two (pA)p sites, (pA)p1 and (pA)p2, whose cleavage sites are nucleotide (nt) 3503 and nt 3341, respectively, and two (pA)d sites, (pA)d1 and (pA)d REH , which end viral mRNAs at nt 5171 and nt 5443, respectively (23). The two (pA)p sites were utilized at an equivalent efficiency in HBoV1-infected HAE-ALI as well as in HBoV1 RF dsDNA-transfected HEK293 cells (22,23).…”

“…A comprehensive analysis of viral mRNA transcripts during HBoV1 infection of HAE-ALI using transcriptome sequencing identified two (pA)p sites, (pA)p1 and (pA)p2, whose cleavage sites are nucleotide (nt) 3503 and nt 3341, respectively, and two (pA)d sites, (pA)d1 and (pA)d REH , which end viral mRNAs at nt 5171 and nt 5443, respectively (23). The two (pA)p sites were utilized at an equivalent efficiency in HBoV1-infected HAE-ALI as well as in HBoV1 RF dsDNA-transfected HEK293 cells (22,23). However, the (pA)d1 site was used more frequently than the (pA)d REH site during virus infection of HAE-ALI (23), but the opposite was found in viral RF dsDNA-transfected HEK293 cells (24).…”

“…The two (pA)p sites were utilized at an equivalent efficiency in HBoV1-infected HAE-ALI as well as in HBoV1 RF dsDNA-transfected HEK293 cells (22,23). However, the (pA)d1 site was used more frequently than the (pA)d REH site during virus infection of HAE-ALI (23), but the opposite was found in viral RF dsDNA-transfected HEK293 cells (24). In addition, HBoV1 NP1 is localized in viral DNA replication centers and is required for efficient viral DNA replication (13,25).…”

New Insights into Parvovirus Research

“…The transcription map of Bombyx mori bidensovirus has been thoroughly investigated and presented [11]. The transcriptome of Human Bocavirus 1 in polarised airway epithelial cells [12] has been analysed by comprehensive RNAseq, and, in this case, the use of NGS and combination of transcript mapping and quantitative analysis could yield a full insight into viral replication dynamics and expression. The aim now at hand by the application of next generation techniques is to obtain comprehensive paradigms to characterize a viral lifecycle and to interpret the effects of the virus within infected cells, possibly at single-cell level.…”