The Human Bocavirus 1 NP1 Protein Is a Multifunctional Regulator of Viral RNA Processing

Abstract

Human bocavirus 1 (HBoV1) encodes a genus-specific protein, NP1, which regulates viral alternative pre-mRNA processing. Similar to NP1 of the related bocavirus minute virus of canine (MVC), HBoV1 NP1 suppressed cleavage and polyadenylation of RNAs at the viral internal polyadenylation site (pA)p. HBoV1 (pA)p is a complex region. It contains 5 significant cleavage and polyadenylation sites, and NP1 was found to regulate only the three of these sites that are governed by canonical AAUAAA hexamer signals. HBoV1 NP1 also facilitated splicing of the upstream intron adjacent to (pA)p. Alternative polyadenylation and splicing of the upstream intron were independent of each other, functioned efficiently within an isolated transcription unit, and were responsive independent of NP1. Characterization of HBoV1 NP1 generalizes its function within the genus , uncovers important differences, and provides important comparisons with MVC NP1 for mechanistic and evolutionary considerations. The are small nonenveloped icosahedral viruses that are important pathogens in many animal species, including humans. The NP1 protein of human bocavirus 1 (HBoV1), similar to NP1 of the bocavirus minute virus of canine (MVC), regulates viral alternative RNA processing by both suppressing polyadenylation at an internal site, (pA)p, and facilitating splicing of an upstream adjacent intron. These effects allow both extension into the capsid gene and splicing of the viral pre-mRNA that correctly registers the capsid gene open reading frame. Characterization of HBoV1 NP1 generalizes this central mode of parvovirus gene regulation to another member of the bocavirus genus and uncovers both important similarities and differences in function compared to MVC NP1 that will be important for future comparative studies.

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

“…Development of a biotin proximity labeling assay to identify host proteins that interact with HBoV1 NP1. HBoV1 NP1 plays an important role in the production of capsid proteins through the regulation of viral pre-mRNA transcription and processing (19,22) and also in viral DNA replication (25). To identify the proteins associated with NP1 during HBoV1 replication, we used a proximity-dependent BioID assay (Fig.…”

“…Previously, we discovered that HBoV1 NP1 expression is essential to the maturation of viral mRNAs encoding VP. 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).…”

“…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).…”

Minute Virus of Canines NP1 Protein Interacts with the Cellular Factor CPSF6 To Regulate Viral Alternative RNA Processing

“…Unlike other parvoviruses, the bocaviruses control access to their capsid ORFs via alternative splicing and alternative polyadenylation (16,45,46). MVC and human bocavirus (HBoV) alternative pre-RNA processing is governed by a small nonstructural protein, NP1, that is unique to this genus, via interaction with the viral internal polyadenylation site (pA)p (45,46). Here, we further characterized the cis-acting signals within (pA)p and further examined the function of NP1 in conjunction with the cellular factor CPSF6.…”

“…Alternative polyadenylation and alternative splicing are critical RNA processing mechanisms used by parvoviruses (16,45,46,51,52) and other DNA viruses (21,53,54) to coordinate the temporal expression of viral proteins necessary for successful infection. A number of viral RNA binding proteins in addition to NP1, including herpesvirus simplex virus (HSV-1) ICP27 (18)(19)(20) and Kaposi sarcoma-associated herpesvirus (KSHV) ORF57 (21)(22)(23), regulate alternative polyadenylation and alternative spicing.…”