Extraocular muscle volume index at the orbital apex with optic neuritis: a combined parameter for diagnosis of dysthyroid optic neuropathy.

OBJECTIVES: To evaluate the diagnostic performance of the extraocular muscle volume index at the orbital apex (AMI) and the signal intensity ratio (SIR) of the optic nerve in dysthyroid optic neuropathy (DON). METHODS: Clinical data and magnetic resonance imaging were collected retrospectively from 63 Graves' ophthalmopathy patients, including 24 patients with DON and 39 without DON. The volume of these structures was obtained by reconstructing their orbital fat and extraocular muscles. The SIR of the optic nerve and axial length of eyeball were also measured. The posterior 3/5 of the retrobulbar space volume was used as the orbital apex to compare parameters in patients with or without DON. Area under the receiver operating characteristic curve (AUC) analysis was used to select the morphological and inflammatory parameters with the highest diagnostic value. A logistic regression was performed to identify the risk factors of DON. RESULTS: One hundred twenty-six orbits (35 with DON and 91 without DON) were analyzed. Most of the parameters in DON patients were significantly higher than in non-DON patients. However, the SIR 3 mm behind the eyeball of the optic nerve and AMI had the highest diagnostic value in these parameters and are independent risk factors of DON by stepwise multivariate logistic regression analysis. Combining AMI and SIR had a higher diagnostic value than a single index. CONCLUSIONS: Combining AMI with SIR 3 mm behind the eyeball's orbital nerve can be a potential parameter for diagnosing DON. CLINICAL RELEVANCE STATEMENT: The present study provided a quantitative index based on morphological and signal changes to assess the DON, allowing clinicians and radiologists to monitor DON patients timely. KEY POINTS: The extraocular muscle volume index at the orbital apex (AMI) has excellent diagnostic performance for dysthyroid optic neuropathy. A signal intensity ratio (SIR) of 3 mm behind the eyeball has a higher AUC compared to other slices. Combining AMI and SIR has a higher diagnostic value than a single index.

The N-terminal 5-68 amino acids domain of the minor capsid protein VP1 of human parvovirus B19 enters human erythroid progenitors and inhibits B19 infection.

Parvovirus B19 (B19V) infection causes diseases in humans ranging from the mild erythema infectiosum to severe hematological disorders. The unique region of the minor structural protein VP1 (VP1u) of 227 amino acids harbors strong neutralizing epitopes which elicit dominant immune responses in patients. Recent studies have shown that the VP1u selectively binds to and enters B19V permissive cells through an unknown cellular proteinaceous receptor. In the present study, we demonstrated that purified recombinant VP1u effectively inhibits B19V infection of ex vivo expanded primary human erythroid progenitors. Furthermore, we identified the amino acid sequence 5-68 of the VP1 (VP1u5-68aa) is sufficient to confer the inhibition of B19V infection at a level similar to that of the full-length VP1u. In silico structure prediction suggests that the VP1u5-68aa contains three α-helices. Importantly, we found that the inhibition capability of the minimal domain VP1u5-68aa is independent of its dimerization but is likely dependent on the structure of the three predicated α-helices. As VP1u5-68aa outcompetes the full-length VP1u in entering cells, we believe that VP1u5-68aa functions as a receptor-binding ligand during virus entry. Finally, we determined the effective inhibition potency of VP1u5-68aa in B19V infection of human erythroid progenitors, which has a half maximal effective concentration (EC50) of 67 nM, suggesting an anti-viral peptide candidate to combat B19V infection.IMPORTANCEHuman parvovirus B19 infection causes severe hematological disorders, including transient aplastic crisis, pure red cell aplasia, and hydrops fetalis. A productive B19 infection is highly restricted to human erythroid progenitors in human bone marrow and fetal liver. In the current study, we identified that the N-terminal 5-68 amino acids domain of the minor viral capsid protein VP1 enters ex vivo expanded human erythroid progenitors, which is nearly 5 times more efficient than the full-length VP1 unique region (1-227aa). Importantly, purified recombinant 5-68aa of the VP1 has a high efficiency in inhibition of parvovirus B19 infection of human erythroid progenitors, which has a half maximal effective concentration (EC50) of 67 nM and a low cytotoxicity. The N-terminal 5-68 amino acids holds the potential as an effective antiviral of parvovirus B19 caused hematological disorders, as well as a carrier to deliver proteins to human erythroid progenitors.

Phosphorylated STAT5 directly facilitates parvovirus B19 DNA replication in human erythroid progenitors through interaction with the MCM complex.

Productive infection of human parvovirus B19 (B19V) exhibits high tropism for burst forming unit erythroid (BFU-E) and colony forming unit erythroid (CFU-E) progenitor cells in human bone marrow and fetal liver. This exclusive restriction of the virus replication to human erythroid progenitor cells is partly due to the intracellular factors that are essential for viral DNA replication, including erythropoietin signaling. Efficient B19V replication also requires hypoxic conditions, which upregulate the signal transducer and activator of transcription 5 (STAT5) pathway, and phosphorylated STAT5 is essential for virus replication. In this study, our results revealed direct involvement of STAT5 in B19V DNA replication. Consensus STAT5-binding elements were identified adjacent to the NS1-binding element within the minimal origins of viral DNA replication in the B19V genome. Phosphorylated STAT5 specifically interacted with viral DNA replication origins both in vivo and in vitro, and was actively recruited within the viral DNA replication centers. Notably, STAT5 interacted with minichromosome maintenance (MCM) complex, suggesting that STAT5 directly facilitates viral DNA replication by recruiting the helicase complex of the cellular DNA replication machinery to viral DNA replication centers. The FDA-approved drug pimozide dephosphorylates STAT5, and it inhibited B19V replication in ex vivo expanded human erythroid progenitors. Our results demonstrated that pimozide could be a promising antiviral drug for treatment of B19V-related diseases.

Parvovirus B19 NS1 protein induces cell cycle arrest at G2-phase by activating the ATR-CDC25C-CDK1 pathway.

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.

Selective inhibition of cyclooxygenase-2 protects porcine aortic endothelial cells from human antibody-mediated complement-dependent cytotoxicity.

BACKGROUND: Cyclooxygenase-2 (COX-2) is an inducible enzyme with catalytic activity for biosynthesis of prostaglandins which are the key mediators of inflammation. COX-2 is also the therapeutic target for widely used non-steroidal anti-inflammatory drugs (NSAIDs). However, the involvement of COX-2 in xenotransplantation (eg, pig-to-non-human primate) remains poorly recognized. METHODS: We investigated the mechanisms that regulate COX-2 expression and the effects of COX-2 on porcine aortic endothelial cell (PAEC) viability using in vitro pig-to-primate xenotransplantation model and in vivo pig-to-mouse cellular transplant model. Regulation of COX-2 expression was assessed by real-time quantitative polymerase chain reaction (qPCR) and Western blotting. The effects of inhibition or downregulation of COX-2 on PAEC viability were assessed by propidium iodide (PI)-Annexin V staining and Cell Counting Kit-8 assay. RESULTS: Human serum triggered robust COX-2 expression in PAECs in a dose- and time-dependent manner. Induction of COX-2 expression by human serum was partially through activation of both canonical and non-canonical nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κb) signaling and increasing intracellular calcium. Cytokines like tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1ß), IL-17, were able to induce COX-2 expression. Selective inhibition of COX-2 by celecoxib dramatically decreased PAEC death in vitro and in vivo as defined by propidium iodide (PI)-Annexin V staining. Consistently, downregulation of COX-2 expression by NF-κb inhibitors or calcium chelator BAPTA decreased human serum-induced PAEC death as well. Silencing of COX-2 expression by small interfering RNA (siRNA) protected PAEC viability when transplanted under kidney capsule of C57BL/6 mice. CONCLUSIONS: Taken together, our data suggest that COX-2 is highly induced in PAECs by xenogenic serum and associated with human antibody-mediated complement-dependent cytotoxicity. COX-2 might be a potential therapeutic target to improve xenotransplantation.

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

Human bocavirus 1 (HBoV1) belongs to the species Primate bocaparvovirus of the genus Bocaparvovirus of the Parvoviridae family. HBoV1 causes acute respiratory tract infections in young children and has a selective tropism for the apical surface of well-differentiated human airway epithelia (HAE). In this study, we identified an additional HBoV1 gene, bocavirus-transcribed small noncoding RNA (BocaSR), within the 3' noncoding region (nucleotides [nt] 5199 to 5338) of the viral genome of positive sense. BocaSR is transcribed by RNA polymerase III (Pol III) from an intragenic promoter at levels similar to that of the capsid protein-coding mRNA and is essential for replication of the viral DNA in both transfected HEK293 and infected HAE cells. Mechanistically, we showed that BocaSR regulates the expression of HBoV1-encoded nonstructural proteins NS1, NS2, NS3, and NP1 but not NS4. BocaSR is similar to the adenovirus-associated type I (VAI) RNA in terms of both nucleotide sequence and secondary structure but differs from it in that its regulation of viral protein expression is independent of RNA-activated protein kinase (PKR) regulation. Notably, BocaSR accumulates in the viral DNA replication centers within the nucleus and likely plays a direct role in replication of the viral DNA. Our findings reveal BocaSR to be a novel viral noncoding RNA that coordinates the expression of viral proteins and regulates replication of viral DNA within the nucleus. Thus, BocaSR may be a target for antiviral therapies for HBoV and may also have utility in the production of recombinant HBoV vectors.IMPORTANCE Human bocavirus 1 (HBoV1) is pathogenic to humans, causing acute respiratory tract infections in young children. In this study, we identified a novel HBoV1 gene that lies in the 3' noncoding region of the viral positive-sense genome and is transcribed by RNA polymerase III into a noncoding RNA of 140 nt. This bocavirus-transcribed small RNA (BocaSR) diverges from both adenovirus-associated (VA) RNAs and Epstein-Barr virus-encoded small RNAs (EBERs) with respect to RNA sequence, representing a third species of this kind of Pol III-dependent viral noncoding RNA and the first noncoding RNA identified in autonomous parvoviruses. Unlike the VA RNAs, BocaSR localizes to the viral DNA replication centers of the nucleus and is essential for expression of viral nonstructural proteins independent of RNA-activated protein kinase R and replication of HBoV1 genomes. The identification of BocaSR and its role in virus DNA replication reveals potential avenues for developing antiviral therapies.

Human Bocavirus 1 Is a Novel Helper for Adeno-associated Virus Replication.

Human bocavirus 1 (HBoV1) is an autonomous parvovirus that infects well-differentiated primary human airway epithelia (HAE) in vitro In human embryonic kidney HEK293 cells, the transfection of a duplex HBoV1 genome initiates viral DNA replication and produces progeny virions that are infectious in HAE. HBoV1 takes advantage of signaling pathways in the DNA damage response for efficient genome amplification in both well-differentiated (nondividing) HAE and dividing HEK293 cells. On the other hand, adeno-associated virus 2 (AAV2) is a helper-dependent dependoparvovirus, and productive AAV2 replication requires coinfection with a helper virus (e.g., adenovirus or herpesvirus) or treatment with genotoxic agents. Here, we report that HBoV1 is a novel helper virus for AAV2 replication. Coinfection by HBoV1 and AAV2 rescued AAV2 replication in HAE cells. The helper function of HBoV1 for AAV2 is not limited to HAE cells but also includes HEK293 and HeLa cells. Importantly, the helper function of HBoV1 for AAV2 relies on neither HBoV1 replication nor the DNA damage response. Following transfection of HEK293 cells, the minimal requirements for the replication of the AAV2 duplex DNA genome and the production of progeny virions included the HBoV1 NP1 and NS4 proteins and a newly identified viral long noncoding RNA (BocaSR). However, following infection of HEK293 and HeLa cells with AAV2 virions, HBoV1 NS2 (but not NS4), NP1, and BocaSR were required for AAV2 DNA replication and progeny virion formation. These new methods for packaging the AAV2 genome may be useful for generating recombinant AAV-packaging cell lines and the directed evolution of AAV capsids.IMPORTANCE We first report that an autonomous parvovirus, HBoV1, helps the replication of a dependoparvovirus, AAV2, in differentiated human airway epithelia. We identified the minimal sets of HBoV1 genes required to facilitate the replication of the AAV2 duplex genome and for AAV2 infection. Notably, together with the expression of the NP1 and BocaSR genes, HBoV1 NS2 is required for the productive infection of HEK293 and HeLa cells by AAV2, whereas NS4 is sufficient for viral DNA replication of an AAV2 duplex genome. The identification of HBoV1 as a helper virus for AAV2 replication has implications for the improvement of recombinant AAV production in HEK293 cells and cell types that do not express the adenovirus E1 gene as well as for the rescue of wild-type AAV genomes from tissues during directed evolution in the absence of wild-type adenovirus. A further understanding of the mechanism underlying HBoV1 helper-dependent AAV2 replication may also provide insights into its functions in HBoV1 replication.

Human Parvovirus Infection of Human Airway Epithelia Induces Pyroptotic Cell Death by Inhibiting Apoptosis.

Human bocavirus 1 (HBoV1) is a human parvovirus that causes acute respiratory tract infections in young children. In this study, we confirmed that, when polarized/well-differentiated human airway epithelia are infected with HBoV1 in vitro, they develop damage characterized by barrier function disruption and cell hypotrophy. Cell death mechanism analyses indicated that the infection induced pyroptotic cell death characterized by caspase-1 activation. Unlike infections with other parvoviruses, HBoV1 infection did not activate the apoptotic or necroptotic cell death pathway. When the NLRP3-ASC-caspase-1 inflammasome-induced pathway was inhibited by short hairpin RNA (shRNA), HBoV1-induced cell death dropped significantly; thus, NLRP3 mediated by ASC appears to be the pattern recognition receptor driving HBoV1 infection-induced pyroptosis. HBoV1 infection induced steady increases in the expression of interleukin 1α (IL-1α) and IL-18. HBoV1 infection was also associated with the marked expression of the antiapoptotic genes BIRC5 and IFI6 When the expression of BIRC5 and/or IFI6 was inhibited by shRNA, the infected cells underwent apoptosis rather than pyroptosis, as indicated by increased cleaved caspase-3 levels and the absence of caspase-1. BIRC5 and/or IFI6 gene inhibition also significantly reduced HBoV1 replication. Thus, HBoV1 infection of human airway epithelial cells activates antiapoptotic proteins that suppress apoptosis and promote pyroptosis. This response may have evolved to confer a replicative advantage, thus allowing HBoV1 to establish a persistent airway epithelial infection. This is the first report of pyroptosis in airway epithelia infected by a respiratory virus.IMPORTANCE Microbial infection of immune cells often induces pyroptosis, which is mediated by a cytosolic protein complex called the inflammasome that senses microbial pathogens and then activates the proinflammatory cytokines IL-1 and IL-18. While virus-infected airway epithelia often activate NLRP3 inflammasomes, studies to date suggest that these viruses kill the airway epithelial cells via the apoptotic or necrotic pathway; involvement of the pyroptosis pathway has not been reported previously. Here, we show for the first time that virus infection of human airway epithelia can also induce pyroptosis. Human bocavirus 1 (HBoV1), a human parvovirus, causes lower respiratory tract infections in young children. This study indicates that HBoV1 kills airway epithelial cells by activating genes that suppress apoptosis and thereby promote pyroptosis. This strategy appears to promote HBoV1 replication and may have evolved to allow HBoV1 to establish persistent infection of human airway epithelia.

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

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-kinase-related 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 η [Pol η] and polymerase κ [Pol κ]) 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 η and Pol κ 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).

Adeno-associated Virus (AAV) Serotypes Have Distinctive Interactions with Domains of the Cellular AAV Receptor.

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.

Replication of an Autonomous Human Parvovirus in Non-dividing Human Airway Epithelium Is Facilitated through the DNA Damage and Repair Pathways.

Human bocavirus 1 (HBoV1) belongs to the genus Bocaparvovirus of the Parvoviridae family, and is an emerging human pathogenic respiratory virus. In vitro, HBoV1 infects well-differentiated/polarized primary human airway epithelium (HAE) cultured at an air-liquid interface (HAE-ALI). Although it is well known that autonomous parvovirus replication depends on the S phase of the host cells, we demonstrate here that the HBoV1 genome amplifies efficiently in mitotically quiescent airway epithelial cells of HAE-ALI cultures. Analysis of HBoV1 DNA in infected HAE-ALI revealed that HBoV1 amplifies its ssDNA genome following a typical parvovirus rolling-hairpin DNA replication mechanism. Notably, HBoV1 infection of HAE-ALI initiates a DNA damage response (DDR) with activation of all three phosphatidylinositol 3-kinase-related kinases (PI3KKs). We found that the activation of the three PI3KKs is required for HBoV1 genome amplification; and, more importantly, we identified that two Y-family DNA polymerases, Pol η and Pol κ, are involved in HBoV1 genome amplification. Overall, we have provided an example of de novo DNA synthesis (genome amplification) of an autonomous parvovirus in non-dividing cells, which is dependent on the cellular DNA damage and repair pathways.

Low serum level of epidermal growth factor in chronic ketamine users.

BACKGROUND: Growth factors play an important role in brain development. Whether epidermal growth factor (EGF) plays a role in the pathophysiology of ketamine related disorders is unexplored. In this study, we examined the serum levels of EGF in chronic ketamine users as compared with healthy controls. The possible correlation between serum EGF levels with the demographic, ketamine use characteristics and psychopathological symptoms were analyzed. METHODS: Sixty-seven chronic ketamine users and 40 healthy subjects were recruited. Serum EGF levels were measured by enzyme-linked immunosorbent assay. Psychopathological symptoms were assessed using Positive and Negative Syndrome Scale, Beck Depression Inventory and Beck Anxiety Inventory. RESULTS: The serum level of EGF in the chronic ketamine users was significantly lower than that of healthy subjects (22.34 ± 4.81 pg/ml vs. 87.10 ± 2.96 pg/ml, F = 15.169, p < 0.01). The serum EGF level was negatively correlated with the current average dose of ketamine consumption per day of use (p = 0.015), and positively associated with the Positive and Negative Syndrome Scale positive symptom score (p = .022). CONCLUSIONS: Serum level of EGF decreased in chronic ketamine users compared with healthy subjects, which may play a role in the pathophysiology of ketamine related disorders.

Analysis of cis and trans Requirements for DNA Replication at the Right-End Hairpin of the Human Bocavirus 1 Genome.

UNLABELLED: Parvoviruses are single-stranded DNA viruses that use the palindromic structures at the ends of the viral genome for their replication. The mechanism of parvovirus replication has been studied mostly in the dependoparvovirus adeno-associated virus 2 (AAV2) and the protoparvovirus minute virus of mice (MVM). Here, we used human bocavirus 1 (HBoV1) to understand the replication mechanism of bocaparvovirus. HBoV1 is pathogenic to humans, causing acute respiratory tract infections, especially in young children under 2 years old. By using the duplex replicative form of the HBoV1 genome in human embryonic kidney 293 (HEK293) cells, we identified the HBoV1 minimal replication origin at the right-end hairpin (OriR). Mutagenesis analyses confirmed the putative NS1 binding and nicking sites within the OriR. Of note, unlike the large nonstructural protein (Rep78/68 or NS1) of other parvoviruses, HBoV1 NS1 did not specifically bind OriR in vitro, indicating that other viral and cellular components or the oligomerization of NS1 is required for NS1 binding to the OriR. In vivo studies demonstrated that residues responsible for NS1 binding and nicking are within the origin-binding domain. Further analysis identified that the small nonstructural protein NP1 is required for HBoV1 DNA replication at OriR. NP1 and other viral nonstructural proteins (NS1 to NS4) colocalized within the viral DNA replication centers in both OriR-transfected cells and virus-infected cells, highlighting a direct involvement of NP1 in viral DNA replication at OriR. Overall, our study revealed the characteristics of HBoV1 DNA replication at OriR, suggesting novel characteristics of autonomous parvovirus DNA replication. IMPORTANCE: Human bocavirus 1 (HBoV1) causes acute respiratory tract infections in young children. The duplex HBoV1 genome replicates in HEK293 cells and produces progeny virions that are infectious in well-differentiated airway epithelial cells. A recombinant AAV2 vector pseudotyped with an HBoV1 capsid has been developed to efficiently deliver the cystic fibrosis transmembrane conductance regulator gene to human airway epithelia. Here, we identified both cis-acting elements and trans-acting proteins that are required for HBoV1 DNA replication at the right-end hairpin in HEK293 cells. We localized the minimal replication origin, which contains both NS1 nicking and binding sites, to a 46-nucleotide sequence in the right-end hairpin. The identification of these essential elements of HBoV1 DNA replication acting both in cis and in trans will provide guidance to develop antiviral strategies targeting viral DNA replication at the right-end hairpin and to design next-generation recombinant HBoV1 vectors, a promising tool for gene therapy of lung diseases.

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

UNLABELLED: 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. IMPORTANCE: Human 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 parvovirus B19 infection causes cell cycle arrest of human erythroid progenitors at late S phase that favors viral DNA replication.

Human parvovirus B19 (B19V) infection has a unique tropism to human erythroid progenitor cells (EPCs) in human bone marrow and the fetal liver. It has been reported that both B19V infection and expression of the large nonstructural protein NS1 arrested EPCs at a cell cycle status with a 4 N DNA content, which was previously claimed to be "G2/M arrest." However, a B19V mutant infectious DNA (M20(mTAD2)) replicated well in B19V-semipermissive UT7/Epo-S1 cells but did not induce G2/M arrest (S. Lou, Y. Luo, F. Cheng, Q. Huang, W. Shen, S. Kleiboeker, J. F. Tisdale, Z. Liu, and J. Qiu, J. Virol. 86:10748-10758, 2012). To further characterize cell cycle arrest during B19V infection of EPCs, we analyzed the cell cycle change using 5-bromo-2'-deoxyuridine (BrdU) pulse-labeling and DAPI (4',6-diamidino-2-phenylindole) staining, which precisely establishes the cell cycle pattern based on both cellular DNA replication and nuclear DNA content. We found that although both B19V NS1 transduction and infection immediately arrested cells at a status of 4 N DNA content, B19V-infected 4 N cells still incorporated BrdU, indicating active DNA synthesis. Notably, the BrdU incorporation was caused neither by viral DNA replication nor by cellular DNA repair that could be initiated by B19V infection-induced cellular DNA damage. Moreover, several S phase regulators were abundantly expressed and colocalized within the B19V replication centers. More importantly, replication of the B19V wild-type infectious DNA, as well as the M20(mTAD2) mutant, arrested cells at S phase. Taken together, our results confirmed that B19V infection triggers late S phase arrest, which presumably provides cellular S phase factors for viral DNA replication.

SMC1-mediated intra-S-phase arrest facilitates bocavirus DNA replication.

Activation of a host DNA damage response (DDR) is essential for DNA replication of minute virus of canines (MVC), a member of the genus Bocavirus of the Parvoviridae family; however, the mechanism by which DDR contributes to viral DNA replication is unknown. In the current study, we demonstrate that MVC infection triggers the intra-S-phase arrest to slow down host cellular DNA replication and to recruit cellular DNA replication factors for viral DNA replication. The intra-S-phase arrest is regulated by ATM (ataxia telangiectasia-mutated kinase) signaling in a p53-independent manner. Moreover, we demonstrate that SMC1 (structural maintenance of chromosomes 1) is the key regulator of the intra-S-phase arrest induced during infection. Either knockdown of SMC1 or complementation with a dominant negative SMC1 mutant blocks both the intra-S-phase arrest and viral DNA replication. Finally, we show that the intra-S-phase arrest induced during MVC infection was caused neither by damaged host cellular DNA nor by viral proteins but by replicating viral genomes physically associated with the DNA damage sensor, the Mre11-Rad50-Nbs1 (MRN) complex. In conclusion, the feedback loop between MVC DNA replication and the intra-S-phase arrest is mediated by ATM-SMC1 signaling and plays a critical role in MVC DNA replication. Thus, our findings unravel the mechanism underlying DDR signaling-facilitated MVC DNA replication and demonstrate a novel strategy of DNA virus-host interaction.

In vitro modeling of human bocavirus 1 infection of polarized primary human airway epithelia.

Human bocavirus 1 (HBoV1) is an emerging human-pathogenic respiratory virus. We characterized two important features of HBoV1 infection in polarized primary human airway epithelia (HAE). Apical HBoV1 infection of HAE at a low multiplicity of infection causes disruption of the tight junction barrier, loss of cilia, and epithelial cell hypertrophy, which are hallmarks of the airway epithelial damage caused by HBoV1 infection. HBoV1 also infects HAE from the basolateral surface productively, although less efficiently, and this also leads to the characteristic airway epithelial damage.

Establishment of a reverse genetics system for studying human bocavirus in human airway epithelia.

Human bocavirus 1 (HBoV1) has been identified as one of the etiological agents of wheezing in young children with acute respiratory-tract infections. In this study, we have obtained the sequence of a full-length HBoV1 genome (including both termini) using viral DNA extracted from a nasopharyngeal aspirate of an infected patient, cloned the full-length HBoV1 genome, and demonstrated DNA replication, encapsidation of the ssDNA genome, and release of the HBoV1 virions from human embryonic kidney 293 cells. The HBoV1 virions generated from this cell line-based production system exhibits a typical icosahedral structure of approximately 26 nm in diameter, and is capable of productively infecting polarized primary human airway epithelia (HAE) from the apical surface. Infected HAE showed hallmarks of lung airway-tract injury, including disruption of the tight junction barrier, loss of cilia and epithelial cell hypertrophy. Notably, polarized HAE cultured from an immortalized airway epithelial cell line, CuFi-8 (originally derived from a cystic fibrosis patient), also supported productive infection of HBoV1. Thus, we have established a reverse genetics system and generated the first cell line-based culture system for the study of HBoV1 infection, which will significantly advance the study of HBoV1 replication and pathogenesis.

Migration-stimulating factor (MSF) is over-expressed in non-small cell lung cancer and promotes cell migration and invasion in A549 cells over-expressing MSF.

Migration-stimulating factor (MSF), an oncofetal truncated isoform of fibronectin, is a potent stimulator of cell invasion. However, its distribution and motogenic role in non-small cell lung cancer (NSCLC) have never been identified. In this study, real-time PCR and immunohistochemical staining (IHC) were performed to detect MSF mRNA and protein levels in tumor tissues and matched adjacent tumor-free tissues. Furthermore, to examine the effect of MSF on invasiveness, MSF was upregulated in A549 cells. The invasiveness and viability of A549 cells were then determined using a transwell migration assay and the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assays, respectively. The expression level of MSF in NSCLC tissue was markedly higher than in matched adjacent tumor-free tissue. Additionally, the level of MSF protein expression in stage III and IV NSCLC samples was higher than in stage I and II NSCLC samples. More importantly, we also demonstrated that migration and invasion of A549 cells increased substantially after upregulating MSF, although proliferation remained unchanged. Meanwhile, we found no correlation between increasing motility and invasiveness of MSF-overexpressing cells and expression levels and activities of matrix metalloprotease MMP-2 and MMP-9. Our current study shows that MSF plays a role in migration and invasion of A549 cells and suggests that MSF may be a potential biomarker of NSCLC progression.

A novel chimeric adenoassociated virus 2/human bocavirus 1 parvovirus vector efficiently transduces human airway epithelia.

Human bocavirus virus-1 (HBoV1), a newly discovered autonomous parvovirus with a 5,500 nt genome, efficiently infects human-polarized airway epithelia (HAE) from the apical membrane. We hypothesized that the larger genome and high airway tropism of HBoV1 would be ideal for creating a viral vector for lung gene therapy. To this end, we successfully generated recombinant HBoV1 (rHBoV1) from an open reading frames-disrupted rHBoV1 genome that efficiently transduces HAE from the apical surface. We next evaluated whether HBoV1 capsids could package oversized rAAV2 genomes. These studies created a rAAV2/HBoV1 chimeric virus (5.5 kb genome) capable of apically transducing HAE at 5.6- and 70-fold greater efficiency than rAAV1 or rAAV2 (4.7-kb genomes), respectively. Molecular studies demonstrated that viral uptake from the apical surface was significantly greater for rAAV2/HBoV1 than for rAAV2 or rAAV1, and that polarization of airway epithelial cells was required for HBoV1 capsid-mediated gene transfer. Furthermore, rAAV2/HBoV1-CFTR virus containing the full-length cystic fibrosis transmembrane conductance regulator (CFTR) gene coding sequence and the strong CBA promoter efficiently corrected CFTR-dependent chloride transport in cystic fibrosis (CF) HAE. In summary, using the combined advantages of AAV and HBoV1, we have developed a novel and promising viral vector for CF lung gene therapy and also potentially HBoV1 vaccine development.