Nonstructural Protein NP1 of Human Bocavirus 1 Plays a Critical Role in the Expression of Viral Capsid Proteins

Abstract

A novel chimeric parvoviral vector, rAAV2/HBoV1, in which the recombinant adeno-associated virus 2 (rAAV2) genome is pseudopackaged by the human bocavirus 1 (HBoV1) capsid, has been shown to be highly efficient in gene delivery to human airway epithelia (Z. Yan et al., Mol Ther 21:2181-2194, 2013, http://dx.doi.org/10.1038/mt.2013.92). In this vector production system, we used an HBoV1 packaging plasmid, pHBoV1NSCap, that harbors HBoV1 nonstructural protein (NS) and capsid protein (Cap) genes. In order to simplify this packaging plasmid, we investigated the involvement of the HBoV1 NS proteins in capsid protein expression. We found that NP1, a small NS protein encoded by the middle open reading frame, is required for the expression of the viral capsid proteins (VP1, VP2, and VP3). We also found that the other NS proteins (NS1, NS2, NS3, and NS4) are not required for the expression of VP proteins. We performed systematic analyses of the HBoV1 mRNAs transcribed from the pHBoV1NSCap packaging plasmid and its derivatives in HEK 293 cells. Mechanistically, we found that NP1 is required for both the splicing and the read-through of the proximal polyadenylation site of the HBoV1 precursor mRNA, essential functions for the maturation of capsid protein-encoding mRNA. Thus, our study provides a unique example of how a small viral nonstructural protein facilitates the multifaceted regulation of capsid gene expression. IMPORTANCEA novel chimeric parvoviral vector, rAAV2/HBoV1, expressing a full-length cystic fibrosis transmembrane conductance regulator (CFTR) gene, is capable of correcting CFTR-dependent chloride transport in cystic fibrosis human airway epithelium. Previously, an HBoV1 nonstructural and capsid protein-expressing plasmid, pHBoV1NSCap, was used to package the rAAV2/HBoV1 vector, but yields remained low. In this study, we demonstrated that the nonstructural protein NP1 is required for the expression of capsid proteins. However, we found that the other four nonstructural proteins (NS1 to -4) are not required for expression of capsid proteins. By mutating the cis elements that function as internal polyadenylation signals in the capsid protein-expressing mRNA, we constructed a simple HBoV1 capsid protein-expressing gene that expresses capsid proteins as efficiently as pHBoV1NSCap does, and at similar ratios, but independently of NP1. Our study provides a foundation to develop a better packaging system for rAAV2/HBoV1 vector production. Human bocavirus 1 (HBoV1), first identified in 2005 (1), belongs to the genus Bocaparvovirus in the subfamily Parvovirinae of the family Parvoviridae (2). The genus Bocaparvovirus consists of three groups of viruses, namely, HBoV1 to -4, bovine parvovirus 1 (BPV1), and minute virus of canines (MVC/CnMV) (3). HBoV1 causes respiratory tract infections in young children worldwide (4-11). In vitro, the virus infects only polarized (welldifferentiated) human airway epithelium cultured at an air-liquid interface (HAE-ALI) (12-15). We have constructed an infectious clone of HBoV1...

Parvovirus Expresses a Small Noncoding RNA That Plays an Essential Role in Virus Replication

“…1B and C) but not with those that end at or before nt 5200 (26), and that it is present only in total RNA and not in mRNA samples (34), we speculated that this transcript was encoded by HBoV1 sequence encompassed by the fragment from nt 5124 to 5395 and not a spliced form of the viral polyadenylated mRNA. To identify the exact ends of the BocaSR, we performed both 5= and 3= rapid amplification of cDNA ends (RACE) (Fig.…”

“…1A, in which a negative-sense genome (ϪssDNA) of HBoV1 is diagrammed. We also analyzed VP- encoding mRNA transcripts in total RNA isolated from pCMVNS*Cap-transfected HEK293 cells, using a Cap2 probe spanning nt 3921 to 5200 and an NSCap2 probe spanning nt 542 to 5124 (26). These analyses revealed that transfection with the HBoV1 duplex genome produces NS-, NP1-, and VP-encoding mRNAs (26,34) (Fig.…”

“…NP1, which is comprised of 219 aa, has a molecular mass of 25 kDa. It plays important roles not only in replication of the viral DNA (21,23) but also in processing of the viral mRNA transcripts (25,26). NP1 is required for the splicing of viral mRNAs, as well as for read-through from the proximal polyadenylation site (26).…”

DNA Damage Signaling Is Required for Replication of Human Bocavirus 1 DNA in Dividing HEK293 Cells

“…NP1 plays important roles not only in the replication of bocaparvovirus DNA (22,30) but also in the processing of the viral pre-mRNA (31)(32)(33). It is essential to generate the mRNA that encodes viral capsid proteins VP1, VP2, and VP3 (33). HBoV1 NP1 colocalizes within the autonomous parvovirus-associated replication (APAR) bodies and complements the function of the minute virus of mice (MVM) NS2 protein in viral DNA replication during the early phase of infection (34).…”

“…NP1 plays important roles not only in the replication of bocaparvovirus DNA (22,30) but also in the processing of the viral pre-mRNA (31)(32)(33). It is essential to generate the mRNA that encodes viral capsid proteins VP1, VP2, and VP3 (33).…”