In Vitro Modeling of Human Bocavirus 1 Infection of Polarized Primary Human Airway Epithelia

et al. 2017

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Human bocavirus 1 (HBoV1), an emerging human-pathogenic respiratory virus, is a member of the genus Bocaparvovirus of the Parvoviridae family. In human airway epithelium air-liquid interface (HAE-ALI) cultures, HBoV1 infection initiates a DNA damage response (DDR), activating all three phosphatidylinositol 3-kinaserelated kinases (PI3KKs): ATM, ATR, and DNA-PKcs. In this context, activation of PI3KKs is a requirement for amplification of the HBoV1 genome (X. Deng, Z. Yan, F. Cheng, J. F. Engelhardt, and J. Qiu, PLoS Pathog, 12:e1005399, 2016, https://doi.org/ 10.1371/journal.ppat.1005399), and HBoV1 replicates only in terminally differentiated, nondividing cells. This report builds on the previous discovery that the replication of HBoV1 DNA can also occur in dividing HEK293 cells, demonstrating that such replication is likewise dependent on a DDR. Transfection of HEK293 cells with the duplex DNA genome of HBoV1 induces hallmarks of DDR, including phosphorylation of H2AX and RPA32, as well as activation of all three PI3KKs. The large viral nonstructural protein NS1 is sufficient to induce the DDR and the activation of the three PI3KKs. Pharmacological inhibition or knockdown of any one of the PI3KKs significantly decreases both the replication of HBoV1 DNA and the downstream production of progeny virions. The DDR induced by the HBoV1 NS1 protein does not cause obvious damage to cellular DNA or arrest of the cell cycle. Notably, key DNA replication factors and major DNA repair DNA polymerases (polymerase [Pol ] and polymerase [Pol ]) are recruited to the viral DNA replication centers and facilitate HBoV1 DNA replication. Our study provides the first evidence of the DDR-dependent parvovirus DNA replication that occurs in dividing cells and is independent of cell cycle arrest.IMPORTANCE The parvovirus human bocavirus 1 (HBoV1) is an emerging respiratory virus that causes lower respiratory tract infections in young children worldwide. HEK293 cells are the only dividing cells tested that fully support the replication of the duplex genome of this virus and allow the production of progeny virions. In this study, we demonstrate that HBoV1 induces a DDR that plays significant roles in the replication of the viral DNA and the production of progeny virions in HEK293 cells. We also show that both cellular DNA replication factors and DNA repair DNA polymerases colocalize within centers of viral DNA replication and that Pol and Pol play an important role in HBoV1 DNA replication. Whereas the DDR that leads to the replication of the DNA of other parvoviruses is facilitated by the cell cycle, the DDR triggered by HBoV1 DNA replication or NS1 is not. HBoV1 is the first parvovirus whose NS1 has been shown to be able to activate all three PI3KKs (ATM, ATR, and DNA-PKcs).

Section: Importancementioning

confidence: 99%

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

Deng

Peng

et al. 2017

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“…Severe and deadly cases associated with high viral load, anti-HBoV1 IgM antibody detection, or increased IgG antibody production have been documented (7)(8)(9). In vitro, HBoV1 infects polarized primary human airway epithelium cultured at an airliquid interface (HAE-ALI) (10) and causes damage to the airway epithelium (11)(12)(13). Currently, no specific treatments for HBoV1 infection are available for hospitalized infants.…”

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confidence: 99%

Identification and Functional Analysis of Novel Nonstructural Proteins of Human Bocavirus 1

Shen

Deng

et al. 2015

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102

Human bocavirus 1 (HBoV1) is a single-stranded DNA parvovirus that causes lower respiratory tract infections in young children worldwide. In this study, we identified novel splice acceptor and donor sites, namely, A1= and D1=, in the large nonstructural protein (NS1)-encoding region of the HBoV1 precursor mRNA. The novel small NS proteins (NS2, NS3, and NS4) were confirmed to be expressed following transfection of an HBoV1 infectious proviral plasmid and viral infection of polarized human airway epithelium cultured at an air-liquid interface (HAE-ALI). We constructed mutant pIHBoV1 infectious plasmids which harbor silent mutations (sm) smA1= and smD1= at the A1= and D1= splice sites, respectively. The mutant infectious plasmids maintained production of HBoV1 progeny virions at levels less than five times lower than that of the wild-type plasmid. Importantly, the smA1= mutant virus that does not express NS3 and NS4 replicated in HAE-ALI as effectively as the wild-type virus; however, the smD1= mutant virus that does not express NS2 and NS4 underwent an abortive infection in HAE-ALI. Thus, our study identified three novel NS proteins, NS2, NS3, and NS4, and suggests an important function of the NS2 protein in HBoV1 replication in HAE-ALI. IMPORTANCEHuman bocavirus 1 infection causes respiratory diseases, including acute wheezing in infants, of which life-threatening cases have been reported. In vitro, human bocavirus 1 infects polarized human bronchial airway epithelium cultured at an air-liquid interface that mimics the environment of human lower respiratory airways. Viral nonstructural proteins are often important for virus replication and pathogenesis in infected tissues or cells. In this report, we identified three new nonstructural proteins of human bocavirus 1 that are expressed during infection of polarized human bronchial airway epithelium. Among them, we proved that one nonstructural protein is critical to the replication of the virus in polarized human bronchial airway epithelium. The creation of nonreplicating infectious HBoV1 mutants may have particular utility in vaccine development for this virus. Human bocavirus 1 (HBoV1) belongs to genus Bocaparvovirus of the Parvoviridae family (1, 2). HBoV1 causes lower respiratory tract infections, especially in infants less than 2 years old (3-7). Severe and deadly cases associated with high viral load, anti-HBoV1 IgM antibody detection, or increased IgG antibody production have been documented (7-9). In vitro, HBoV1 infects polarized primary human airway epithelium cultured at an airliquid interface (HAE-ALI) (10) and causes damage to the airway epithelium (11-13). Currently, no specific treatments for HBoV1 infection are available for hospitalized infants.The RNA transcription profiles of three species in the genus Bocaparvovirus, i.e., canine minute virus (CnMV/MVC) (14), bovine parvovirus type 1 (BPV1) (15), and HBoV1 (10), have been studied during virus infection. These species share similarities in their gene expression, with distinguishing featur...

mentioning

confidence: 99%

“…We have constructed an infectious clone of HBoV1 (pIHBoV1). Transfecting pIHBoV1 in human embryonic kidney 293 (HEK 293) cells results in efficient replication of the HBoV1 genome and production of HBoV1 virions, which are infectious in HAE-ALI (13,14).The transcription profile of HBoV1 has been studied in transfection of both HBoV1 nonreplicating and replicating doublestranded DNA (dsDNA) forms of the viral genome in HEK 293 cells (16,17), as well as during HBoV1 infection of HAE-ALI (12, 17). All the viral mRNA transcripts are generated from both alternative splicing and alternative polyadenylation of one HBoV1 precursor mRNA (pre-mRNA), which is transcribed from the P5 promoter (Fig.…”

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confidence: 99%

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

Cheng

Shen

et al. 2016

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100

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...