A Novel Chimeric Adenoassociated Virus 2/Human Bocavirus 1 Parvovirus Vector Efficiently Transduces Human Airway Epithelia

Yan, Ziying; Keiser, Nicholas W.; Song, Yi; Deng, Xuefeng; Cheng, Fang; Qiu, Jianming; Engelhardt, John F.

doi:10.1038/mt.2013.92

Cited by 68 publications

(109 citation statements)

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“…capsid protein (VP*) whose size is between those of VP1 and "VP2" in HBoV1-infected human airway epithelium and HBoV1 plasmid-transfected HEK 293 cells (17), as well as in the purified rAAV2/HBoV1 vector (19). However, we did not know whether this VP* band was a cleaved protein of VP1 or a novel capsid protein translated from a noncanonical initiation site.…”

Section: Resultsmentioning

confidence: 80%

“…However, in the current vector production system, the vector is packaged using the packaging plasmid pHBoV1NSCap, which carries an HBoV1 nonreplicating dsDNA genome (containing the P5 promoter and NS and capsid protein [Cap] genes). The efficiency of the vector production is on average 10 times lower than that of the rAAV2 vector packaged by the AAV2 capsid (19). We hypothesize that this lower efficiency is likely due to the unnecessary expression of the HBoV1 NS gene from the packaging plasmid, pHBoV1NSCap.…”

mentioning

confidence: 83%

“…The parent plasmid pHBoV1NSCap has been used to package the rAAV2/HBoV1 vector, which contains an incomplete HBoV1 genome (nucleotides [nt] 97 to 5395) that lacks the intact left-and right-end hairpins (19). All the other plasmids based on the pHBoV1NSCap (12,16,17).…”

Section: Methodsmentioning

confidence: 99%

“…This cross-genus packaging generates an HBoV1 capsidpseudotyped rAAV2 vector (rAAV2/HBoV1) (19). The chimeric vector can deliver a full-length cystic fibrosis transmembrane con-ductance regulator (CFTR) gene with a strong promoter to cystic fibrosis (CF) HAE, with demonstrated efficacy in correcting CFTR-dependent chloride transport (19).…”

mentioning

confidence: 99%

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

Zou

Cheng

Shen

et al. 2016

J Virol

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100

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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...

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Section: Resultsmentioning

confidence: 80%

mentioning

confidence: 83%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Zou

Cheng

Shen

et al. 2016

J Virol

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100

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“…BPV, in particular, has been shown to bind to N-and Olinked sialic acid moieties, which may serve as primary receptors for cell entry (22,23). Cell-based studies with human bocavirus 1 (HBoV1) virions predict that primary receptors and coreceptors on the apical membrane of human airway epithelial cells influence efficient virion infection and cellular transduction (24). Information on Bocaparvovirus capsid structure will provide a platform to begin the elucidation of sites on the capsid that serve as important determinants of host range, tissue tropism, pathogenicity, and bocaparvovirus-related disease emergence.…”

mentioning

confidence: 99%

Structure of an Enteric Pathogen, Bovine Parvovirus

Kailasan

Halder

Gurda

et al. 2015

J Virol

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Bovine parvovirus (BPV), the causative agent of respiratory and gastrointestinal disease in cows, is the type member of the Bocaparvovirus genus of the Parvoviridae family. Toward efforts to obtain a template for the development of vaccines and small-molecule inhibitors for this pathogen, the structure of the BPV capsid, assembled from the major capsid viral protein 2 (VP2), was determined using X-ray crystallography as well as cryo-electron microscopy and three-dimensional image reconstruction (cryoreconstruction) to 3.2-and 8.8-Å resolutions, respectively. The VP2 region ordered in the crystal structure, from residues 39 to 536, conserves the parvoviral eight-stranded jellyroll motif and an ␣A helix. The BPV capsid displays common parvovirus features: a channel at and depressions surrounding the 5-fold axes and protrusions surrounding the 3-fold axes. However, rather than a depression centered at the 2-fold axes, a raised surface loop divides this feature in BPV. Additional observed density in the capsid interior in the cryo-reconstructed map, compared to the crystal structure, is interpreted as 10 additional N-terminal residues, residues 29 to 38, that radially extend the channel under the 5-fold axis, as observed for human bocavirus 1 (HBoV1). Surface loops of various lengths and conformations extend from the core jellyroll motif of VP2. These loops confer the unique surface topology of the BPV capsid, making it strikingly different from HBoV1 as well as the type members of other Parvovirinae genera for which structures have been determined. For the type members, regions structurally analogous to those decorating the BPV capsid surface serve as determinants of receptor recognition, tissue and host tropism, pathogenicity, and antigenicity. IMPORTANCEBovine parvovirus (BPV), identified in the 1960s in diarrheic calves, is the type member of the Bocaparvovirus genus of the nonenveloped, single-stranded DNA (ssDNA) Parvoviridae family. The recent isolation of human bocaparvoviruses from children with severe respiratory and gastrointestinal infections has generated interest in understanding the life cycle and pathogenesis of these emerging viruses. We have determined the high-resolution structure of the BPV capsid assembled from its predominant capsid protein VP2, known to be involved in a myriad of functions during host cell entry, pathogenesis, and antigenicity for other members of the Parvovirinae. Our results show the conservation of the core secondary structural elements and the location of the N-terminal residues for the known bocaparvovirus capsid structures. However, surface loops with high variability in sequence and conformation give BPV a unique capsid surface topology. Similar analogous regions in other Parvovirinae type members are important as determinants of receptor recognition, tissue and host tropism, pathogenicity, and antigenicity. B ovine parvovirus (BPV), the type member of the Bocaparvovirus genus of the Parvoviridae family, was discovered in 1961 in the gastrointestinal tract of di...

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Cystic Fibrosis: Gene Therapy

Sumner-Jones

Gill

2014

Encyclopedia of Life Sciences

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Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations of the CFTR gene, which encodes a member of the adenosine triphosphate‐binding cassette superfamily of transmembrane proteins. CFTR normally acts as a cyclic adenosine monophosphate‐activated chloride channel and as a regulator of other ion channels. The main cause of morbidity and mortality is the effect of CFTR dysfunction on the lung, which reduces life expectancy of CF patients (current median approximately 40 years according to UK and US national patient registries). There has been some progress in the identification of pharmacological agents that can correct the CFTR defect in patients carrying some classes of mutation, but with nearly 2000 different disease‐causing mutations reported, gene therapy remains the most likely option for the amelioration of lung disease in the majority of CF sufferers. The progress achieved and the hurdles identified as a result of the clinical trials of gene therapy vectors in CF patients are reviewed. Key Concepts: CF is a monogenic recessive disease with an incidence of 1:2500 in populations of Caucasian descent in particular, caused by mutations in the CFTR gene. It is a life‐limiting condition and disease management involves a heavy therapeutic burden with no definitive cure. Disruption of the CFTR gene leads to incorrect or insufficient transport of chloride ions across the epithelial cells of the body with a range of consequences in different organs, crucially inadequate clearance of bacteria from the lungs due to ineffective mucociliary clearance, resulting in progressive fibrosis and clogging of the lung with mucus, and eventually respiratory failure. Discovery of the genetic basis for CF quickly led to evaluation of gene therapy as a therapeutic option, with a range of viral and nonviral vectors. Although the vectors tested showed some efficacy in terms of correcting the fundamental chloride transport defect, no CF gene therapy trial has so far been able to demonstrate long‐term clinical improvement.

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A Novel Chimeric Adenoassociated Virus 2/Human Bocavirus 1 Parvovirus Vector Efficiently Transduces Human Airway Epithelia

Cited by 68 publications

References 48 publications

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

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

Structure of an Enteric Pathogen, Bovine Parvovirus

Cystic Fibrosis: Gene Therapy

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