Adeno-associated virus (AAV) entry is determined by its interactions with specific surface glycans and a proteinaceous receptor(s). Adeno-associated virus receptor (AAVR) (also named KIAA0319L) is an essential cellular receptor required for the transduction of vectors derived from multiple AAV serotypes, including the evolutionarily distant serotypes AAV2 and AAV5. Here, we further biochemically characterize the AAV-AAVR interaction and define the domains within the ectodomain of AAVR that facilitate this interaction. By using a virus overlay assay, it was previously shown that the major AAV2 binding protein in membrane preparations of human cells corresponds to a glycoprotein with a molecular mass of 150 kDa. By establishing a purification procedure, performing further protein separation by two-dimensional electrophoresis, and utilizing mass spectrometry, we now show that this glycoprotein is identical to AAVR. While we find that AAVR is an N-linked glycosylated protein, this glycosylation is not a strict requirement for AAV2 binding or functional transduction. Using a combination of genetic complementation with deletion constructs and virus overlay assays with individual domains, we find that AAV2 functionally interacts predominantly with the second Ig-like polycystic kidney disease (PKD) repeat domain (PKD2) present in the ectodomain of AAVR. In contrast, AAV5 interacts primarily through the first, most membrane-distal, PKD domain (PKD1) of AAVR to promote transduction. Furthermore, other AAV serotypes, including AAV1 and -8, require a combination of PKD1 and PKD2 for optimal transduction. These results suggest that despite their shared dependence on AAVR as a critical entry receptor, different AAV serotypes have evolved distinctive interactions with the same receptor.IMPORTANCE Over the past decade, AAV vectors have emerged as leading gene delivery tools for therapeutic applications and biomedical research. However, fundamental aspects of the AAV life cycle, including how AAV interacts with host cellular factors to facilitate infection, are only partly understood. In particular, AAV receptors contribute significantly to AAV vector transduction efficiency and tropism. The recently identified AAV receptor (AAVR) is a key host receptor for multiple serotypes, including the most studied serotype, AAV2. AAVR binds directly to AAV2 particles and is rate limiting for viral transduction. Defining the AAV-AAVR interface in more detail is important to understand how AAV engages with its cellular receptor and how the receptor facilitates the entry process. Here, we further define AAV-AAVR interactions, genetically and biochemically, and show that different AAV serotypes have discrete interactions with the Ig-like PKD domains of AAVR. These findings reveal an unexpected divergence of AAVR engagement within these parvoviruses.

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...

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...

Human parvovirus B19 (B19V) infection of primary human erythroid progenitor cells (EPCs) arrests infected cells at both late S-phase and G2-phase, which contain 4N DNA. B19V infection induces a DNA damage response (DDR) that facilitates viral DNA replication but is dispensable for cell cycle arrest at G2-phase; however, a putative C-terminal transactivation domain (TAD2) within NS1 is responsible for G2-phase arrest. To fully understand the mechanism underlying B19V NS1-induced G2-phase arrest, we established two doxycycline-inducible B19V-permissive UT7/Epo-S1 cell lines that express NS1 or NS1mTAD2, and examined the function of the TAD2 domain during G2-phase arrest. The results confirm that the NS1 TAD2 domain plays a pivotal role in NS1-induced G2-phase arrest. Mechanistically, NS1 transactivated cellular gene expression through the TAD2 domain, which was itself responsible for ATR (ataxia-telangiectasia mutated and Rad3-related) activation. Activated ATR phosphorylated CDC25C at serine 216, which in turn inactivated the cyclin B/CDK1 complex without affecting nuclear import of the complex. Importantly, we found that the ATR-CHK1-CDC25C-CDK1 pathway was activated during B19V infection of EPCs, and that ATR activation played an important role in B19V infection-induced G2-phase arrest.

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).