Xuefeng Deng

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.

Parvovirus B19 Infection of Human Primary Erythroid Progenitor Cells Triggers ATR-Chk1 Signaling, Which Promotes B19 Virus Replication

ABSTRACT Human parvovirus B19 (B19V) infection is restricted to erythroid progenitor cells of the human bone marrow. Although the mechanism by which the B19V genome replicates in these cells has not been studied in great detail, accumulating evidence has implicated involvement of the cellular DNA damage machinery in this process. Here, we report that, in ex vivo-expanded human erythroid progenitor cells, B19V infection induces a broad range of DNA damage responses by triggering phosphorylation of all the upstream kinases of each of three repair pathways: ATM (ataxia-telangiectasi mutated), ATR (ATM and Rad3 related), and DNA-PKcs (DNA-dependent protein kinase catalytic subunit). We found that phosphorylated ATM, ATR, and DNA-PKcs, and also their downstream substrates and components (Chk2, Chk1, and Ku70/Ku80 complex, respectively), localized within the B19V replication center. Notably, inhibition of kinase phosphorylation (through treatment with either kinase-specific inhibitors or kinase-specific shRNAs) revealed requirements for signaling of ATR and DNA-PKcs, but not ATM, in virus replication. Inhibition of the ATR substrate Chk1 led to similar levels of decreased virus replication, indicating that signaling via the ATR-Chk1 pathway is critical to B19V replication. Notably, the cell cycle arrest characteristic of B19V infection was not rescued by interference with the activity of any of the three repair pathway kinases.

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

ABSTRACT 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.

Human bocavirus 1 infects commercially available primary human airway epithelium cultures productively.

Abstract Human bocavirus 1 (HBoV1), a human parvovirus, belongs to the genus Bocavirus of the Parvoviridae family. It causes wheezing in young children with acute respiratory tract infections. HBoV1 has been shown to infect polarized human airway epithelium (HAE) made in house, and induces airway epithelial damage. In this study, two commercially available HAE cultures, EpiAirway and MucilAir HAE, were examined for HBoV1 infection. Both HAE cultures support fully productive HBoV1 infection. Infected EpiAirway and MucilAir HAE cultures showed loss of cilia, disruption of the tight junction barrier, and a significant decrease in transepithelial electrical resistance. Notably, HBoV1 persistent infection was demonstrated by maintaining HBoV1-infected EpiAirway HAE for as long as 50 days. After 2 days post-infection, progeny virus was produced consistently daily at a level of over 2×108 viral genome copies per culture (0.6cm2). This study is the first to use commercial sources of HAE cultures for HBoV1 infection. The availability of these cultures will enable a wide range of laboratories to study HBoV1 infection.

Human Parvovirus B19 Infection Causes Cell Cycle Arrest of Human Erythroid Progenitors at Late S Phase That Favors Viral DNA Replication

ABSTRACT 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 (M20mTAD2) 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 M20mTAD2 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.

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.

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

ABSTRACT 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.

SMC1-Mediated Intra-S-Phase Arrest Facilitates Bocavirus DNA Replication

ABSTRACT 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.

The human parvovirus B19 non-structural protein 1 N-terminal domain specifically binds to the origin of replication in the viral DNA

The non-structural protein 1 (NS1) of human parvovirus B19 plays a critical role in viral DNA replication. Previous studies identified the origin of replication in the viral DNA, which contains four DNA elements, namely NSBE1 to NSBE4, that are required for optimal viral replication (Guan et al., 2009). Here we have demonstrated in vitro that the NS1 N-terminal domain (NS1N) binds to the origin of replication in a sequence-specific, length-dependent manner that requires NSBE1 and NSBE2, while NSBE3 and NSBE4 are dispensable. Mutagenesis analysis has identified nucleotides in NSBE1 and NSBE2 that are critical for NS1N binding. These results suggest that NS1 binds to the NSBE1-NSBE2 region in the origin of replication, while NSBE3 and NSBE4 may provide binding sites for potential cellular factors. Such a specialized nucleoprotein complex may enable NS1 to nick the terminal resolution site and separate DNA strands during replication.

Internal polyadenylation of parvoviral precursor mRNA limits progeny virus production

Aleutian Mink Disease Virus (AMDV) is the only virus in the genus Amdovirus of family Parvoviridae. In adult mink, AMDV causes a persistent infection associated with severe dysfunction of the immune system. Cleavage of AMDV capsid proteins has been previously shown to play a role in regulating progeny virus production (Fang Cheng et al., J. Virol. 84:2687-2696, 2010). The present study shows that AMDV has evolved a second strategy to limit expression of capsid proteins by preventing processing of the full-length capsid protein-encoding mRNA transcripts. Characterization of the cis-elements of the proximal polyadenylation site [(pA)p] in the infectious clone of AMDV revealed that polyadenylation at the (pA)p site is controlled by an upstream element (USE) of 200 nts in length, the AAUAAA signal, and a downstream element (DSE) of 40 nts. A decrease in polyadenylation at the (pA)p site, either by mutating the AAUAAA signal or the DSE, which does not affect the encoding of amino acids in the infectious clone, increased the expression of capsid protein VP1/VP2 and thereby increased progeny virus production approximately 2-3-fold. This increase was accompanied by enhanced replication of the AMDV genome. Thus, this study reveals correlations among internal polyadenylation, capsid production, viral DNA replication and progeny virus production of AMDV, indicating that internal polyadenylation is a limiting step for parvovirus replication and progeny virus production.