The Experts below are selected from a list of 165 Experts worldwide ranked by ideXlab platform
David J. Pintel - One of the best experts on this subject based on the ideXlab platform.
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The Human Bocavirus 1 NP1 Protein Is a Multifunctional Regulator of Viral RNA Processing.
Journal of virology, 2018Co-Authors: Yanming Dong, Olufemi O. Fasina, David J. PintelAbstract:Human bocavirus 1 (HBoV1) encodes a genus-specific protein, NP1, which regulates viral alternative pre-mRNA processing. Similar to NP1 of the related bocavirus minute virus of canine (MVC), HBoV1 NP1 suppressed cleavage and polyadenylation of RNAs at the viral internal polyadenylation site (pA)p. HBoV1 (pA)p is a complex region. It contains 5 significant cleavage and polyadenylation sites, and NP1 was found to regulate only the three of these sites that are governed by canonical AAUAAA hexamer signals. HBoV1 NP1 also facilitated splicing of the upstream intron adjacent to (pA)p. Alternative polyadenylation and splicing of the upstream intron were independent of each other, functioned efficiently within an isolated transcription unit, and were responsive independent of NP1. Characterization of HBoV1 NP1 generalizes its function within the genus Bocaparvovirus, uncovers important differences, and provides important comparisons with MVC NP1 for mechanistic and evolutionary considerations.IMPORTANCE The Parvovirinae are small nonenveloped icosahedral viruses that are important pathogens in many animal species, including humans. The NP1 protein of human bocavirus 1 (HBoV1), similar to NP1 of the bocavirus minute virus of canine (MVC), regulates viral alternative RNA processing by both suppressing polyadenylation at an internal site, (pA)p, and facilitating splicing of an upstream adjacent intron. These effects allow both extension into the capsid gene and splicing of the viral pre-mRNA that correctly registers the capsid gene open reading frame. Characterization of HBoV1 NP1 generalizes this central mode of parvovirus gene regulation to another member of the bocavirus genus and uncovers both important similarities and differences in function compared to MVC NP1 that will be important for future comparative studies.
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Minute Virus of Canines NP1 Protein Governs the Expression of a Subset of Essential Nonstructural Proteins via Its Role in RNA Processing
Journal of virology, 2017Co-Authors: Olufemi O. Fasina, Stephanie Stupps, Wanda Figueroa-cuilan, David J. PintelAbstract:Parvoviruses use a variety of means to control the expression of their compact genomes. The Bocaparvovirus minute virus of canines (MVC) encodes a small, genus-specific protein, NP1, which governs access to the viral capsid gene via its role in alternative polyadenylation and alternative splicing of the single MVC pre-mRNA. In addition to NP1, MVC encodes five additional nonstructural proteins (NS) that share an initiation codon at the left end of the genome and which are individually encoded by alternative multiply spliced mRNAs. We found that three of these proteins were encoded by mRNAs that excise the NP1-regulated MVC intron immediately upstream of the internal polyadenylation site, (pA)p, and that generation of these proteins was thus regulated by NP1. Splicing of their progenitor mRNAs joined the amino termini of these proteins to the NP1 open reading frame, and splice site mutations that prevented their expression inhibited virus replication in a host cell-dependent manner. Thus, in addition to controlling capsid gene access, NP1 also controls the expression of three of the five identified NS proteins via its role in governing MVC pre-mRNA splicing. IMPORTANCE The Parvovirinae are small nonenveloped icosahedral viruses that are important pathogens in many animal species, including humans. Minute virus of canine (MVC) is an autonomous parvovirus in the genus Bocaparvovirus. It has a single promoter that generates a single pre-mRNA. NP1, a small genus-specific MVC protein, participates in the processing of this pre-mRNA and so controls capsid gene access via its role in alternative internal polyadenylation and splicing. We show that NP1 also controls the expression of three of the five identified NS proteins via its role in governing MVC pre-mRNA splicing. These NS proteins together are required for virus replication in a host cell-dependent manner.
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NP1 Protein of the Bocaparvovirus Minute Virus of Canines Controls Access to the Viral Capsid Genes via Its Role in RNA Processing
Journal of virology, 2015Co-Authors: Olufemi O. Fasina, Yanming Dong, David J. PintelAbstract:ABSTRACT Minute virus of canines (MVC) is an autonomous parvovirus in the genus Bocaparvovirus. It has a single promoter that generates a single pre-mRNA processed via alternative splicing and alternative polyadenylation to produce at least 8 mRNA transcripts. MVC contains two polyadenylation sites, one at the right-hand end of the genome, (pA)d, and another complex site, (pA)p, within the capsid-coding region. During viral infection, the mRNAs must extend through (pA)p and undergo additional splicing of the immediately upstream 3D∕3A intron to access the capsid gene. MVC NP1 is a 22-kDa nuclear phosphoprotein unique to the genus Bocaparvovirus of the Parvovirinae which we have shown governs suppression of (pA)p independently of viral genome replication. We show here that in addition to suppression of (pA)p, NP1 is also required for the excision of the MVC 3D∕3A intron, independently of its effect on alternative polyadenylation. Mutations of the arginine∕serine (SR) di-repeats within the intrinsically disordered amino terminus of NP1 are required for splicing of the capsid transcript but not suppression of polyadenylation at (pA)p. 3′-end processing of MVC mRNAs at (pA)p is critical for viral genome replication and the optimal expression of NP1 and NS1. Thus, a finely tuned balance between (pA)p suppression and usage is necessary for efficient virus replication. NP1 is the first parvovirus protein implicated in RNA processing. Its characterization reveals another way that parvoviruses govern access to their capsid protein genes, namely, at the RNA level, by regulating the essential splicing of an intron and the suppression of an internal polyadenylation site. IMPORTANCE The Parvovirinae are small nonenveloped icosahedral viruses that are important pathogens in many animal species, including humans. Although parvoviruses have only subtle early-to-late expression shifts, they all regulate access to their capsid genes. Minute virus of canines (MVC) is an autonomous parvovirus in the genus Bocaparvovirus. It has a single promoter generating a single pre-mRNA which is processed via alternative splicing and alternative polyadenylation to generate at least 8 mRNA transcripts. MVC contains two polyadenylation sites, one at the right-hand end of the genome, (pA)d, and another, (pA)p, within the capsid-coding region. It had not been clear how the potent internal polyadenylation motif is suppressed to allow processing, export, and accumulation of the spliced capsid protein-encoding mRNAs. We show here that MVC NP1, the first parvovirus protein to be implicated in RNA processing, governs access to the MVC capsid gene by facilitating splicing and suppressing internal polyadenylation of MVC pre-mRNAs.
Jan Gorm Lisby - One of the best experts on this subject based on the ideXlab platform.
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Multicenter evaluation of the QIAstat Respiratory Panel—A new rapid highly multiplexed PCR based assay for diagnosis of acute respiratory tract infections
PLoS ONE, 2020Co-Authors: Marijo Parčina, Uffe Vest Schneider, Benoit Visseaux, Robert Jozić, Irene Hannet, Jan Gorm LisbyAbstract:Acute respiratory tract infections (ARTI), including the common cold, pharyngitis, sinusitis, otitis media, bronchiolitis and pneumonia are the most common diagnoses among patients seeking medical care in western countries, and account for most antibiotic prescriptions. While a confirmed and fast ARTI diagnosis is key for antibiotic prescribing, empiric antimicrobial treatment remains common, because viral symptoms are often clinically similar and difficult to distinguish from those caused by bacteria. As a result, inappropriate antibiotic prescriptions are high and in certain settings likely higher than the commonly estimated 30%. The QIAstat Respiratory Panel® assay (QIAstat RP) is a multiplexed in vitro diagnostics test for the rapid simultaneous detection of 21 pathogens directly from respiratory samples, including human mastadenovirus A-G, primate Bocaparvovirus 1+2, human coronavirus (HKU1, NL63, OC43, 229E), human metapneumovirus A/B, rhinovirus/enterovirus, influenza A virus (no subtype, subtype H1, H1N1/2009, H3), influenza B virus, human respirovirus 1+3, human orthorubulavirus 2+4, human orthopneumovirus, Bordetella pertussis, Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella pneumophila. We describe the first multicenter study of 445 respiratory samples, collected through the 2016-2017 and 2018 respiratory seasons, with performance compared against BioFire FilmArray RP v1.7 and discrepancy testing by Seegene Allplex RP. The QIAstat RP demonstrated a positive percentage of agreement of 98.0% (95% CI: 96.0-99.1%) and a negative percentage agreement of 99.8% (95% CI: 99.6-99.9%). With use of this comprehensive and rapid test, improved patient outcomes and antimicrobial stewardship may potentially be achieved.
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Multicenter evaluation of the QIAstat Respiratory Panel-A new rapid highly multiplexed PCR based assay for diagnosis of acute respiratory tract infections.
PloS one, 2020Co-Authors: Marijo Parčina, Uffe Vest Schneider, Benoit Visseaux, Robert Jozić, Irene Hannet, Jan Gorm LisbyAbstract:Acute respiratory tract infections (ARTI), including the common cold, pharyngitis, sinusitis, otitis media, bronchiolitis and pneumonia are the most common diagnoses among patients seeking medical care in western countries, and account for most antibiotic prescriptions. While a confirmed and fast ARTI diagnosis is key for antibiotic prescribing, empiric antimicrobial treatment remains common, because viral symptoms are often clinically similar and difficult to distinguish from those caused by bacteria. As a result, inappropriate antibiotic prescriptions are high and in certain settings likely higher than the commonly estimated 30%. The QIAstat Respiratory Panel® assay (QIAstat RP) is a multiplexed in vitro diagnostics test for the rapid simultaneous detection of 21 pathogens directly from respiratory samples, including human mastadenovirus A-G, primate Bocaparvovirus 1+2, human coronavirus (HKU1, NL63, OC43, 229E), human metapneumovirus A/B, rhinovirus/enterovirus, influenza A virus (no subtype, subtype H1, H1N1/2009, H3), influenza B virus, human respirovirus 1+3, human orthorubulavirus 2+4, human orthopneumovirus, Bordetella pertussis, Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella pneumophila. We describe the first multicenter study of 445 respiratory samples, collected through the 2016–2017 and 2018 respiratory seasons, with performance compared against BioFire FilmArray RP v1.7 and discrepancy testing by Seegene Allplex RP. The QIAstat RP demonstrated a positive percentage of agreement of 98.0% (95% CI: 96.0–99.1%) and a negative percentage agreement of 99.8% (95% CI: 99.6–99.9%). With use of this comprehensive and rapid test, improved patient outcomes and antimicrobial stewardship may potentially be achieved.
Richard A. Hesse - One of the best experts on this subject based on the ideXlab platform.
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First identification of porcine parvovirus 6 in North America by viral metagenomic sequencing of serum from pigs infected with porcine reproductive and respiratory syndrome virus
Virology Journal, 2015Co-Authors: Erin E. Schirtzinger, Andrew W. Suddith, Benjamin M. Hause, Richard A. HesseAbstract:Background Currently, eight species in four genera of parvovirus have been described that infect swine. These include ungulate protoparvovirus 1 (classical porcine parvovirus, PPV), ungulate tetraparvovirus 2 (PPV3), ungulate tetraparvovirus 3 (which includes PPV2, porcine hokovirus, porcine partetravirus and porcine PARV4), ungulate copiparvovirus 2 (which includes PPV4 and PPV5), ungulate Bocaparvovirus 2 (which includes porcine bocavirus 1, 2 and 6), ungulate Bocaparvovirus 3 (porcine bocavirus 5), ungulate Bocaparvovirus 4 (porcine bocavirus 7) and ungulate Bocaparvovirus 5 (porcine bocavirus 3, 4–1 and 4–2). PPV6, the most recently described porcine parvovirus, was first identified in China in late 2014 in aborted pig fetuses. Prevalence of PPV6 in China was found to be similar in finishing age pigs from farms with and without evidence of swine reproductive failure. Methods Porcine parvovirus 6 (PPV6) was detected by sequence-independent single primer amplification (SISPA) and confirmed by overlapping and real-time PCR in the serum of porcine reproductive and respiratory virus (PRRSv) positive samples. Results Seven nearly complete genomes of PPV6 were identified in PRRSv genotype 2 positive serum samples submitted to state veterinary diagnostic laboratories in 2014. Further testing using overlapping and real-time PCR determined PPV6 to be present in 13.2 % of the serums tested. Additionally, PPV6 was present in samples from all of the geographic locations sampled encompassing nine states in the United States and one state in Mexico. The presence of PPV6 in serum indicates that the PPV6 infection is disseminated and not localized to a specific tissue type. Alignments of the near full length genomes, NS1, and capsid genes identified one of the five PPV6 isolates from China (98.6–99.5 % identity with the North American strains) to be the North American strains nearest relative. Conclusions These results are the first to report the presence of PPV6 in North America and demonstrate that the virus is found in multiple geographic areas in the United States and in Mexico. The overall prevalence of PPV6 in PRRSv viremic animals is relatively low. Further, all of the PPV6 genomes found in North America are most closely related to a PPV6 strain first identified in 2014 in healthy pigs from the Tianjin province of China.
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First identification of porcine parvovirus 6 in North America by viral metagenomic sequencing of serum from pigs infected with porcine reproductive and respiratory syndrome virus
Virology journal, 2015Co-Authors: Erin E. Schirtzinger, Andrew W. Suddith, Benjamin M. Hause, Richard A. HesseAbstract:Currently, eight species in four genera of parvovirus have been described that infect swine. These include ungulate protoparvovirus 1 (classical porcine parvovirus, PPV), ungulate tetraparvovirus 2 (PPV3), ungulate tetraparvovirus 3 (which includes PPV2, porcine hokovirus, porcine partetravirus and porcine PARV4), ungulate copiparvovirus 2 (which includes PPV4 and PPV5), ungulate Bocaparvovirus 2 (which includes porcine bocavirus 1, 2 and 6), ungulate Bocaparvovirus 3 (porcine bocavirus 5), ungulate Bocaparvovirus 4 (porcine bocavirus 7) and ungulate Bocaparvovirus 5 (porcine bocavirus 3, 4–1 and 4–2). PPV6, the most recently described porcine parvovirus, was first identified in China in late 2014 in aborted pig fetuses. Prevalence of PPV6 in China was found to be similar in finishing age pigs from farms with and without evidence of swine reproductive failure. Porcine parvovirus 6 (PPV6) was detected by sequence-independent single primer amplification (SISPA) and confirmed by overlapping and real-time PCR in the serum of porcine reproductive and respiratory virus (PRRSv) positive samples. Seven nearly complete genomes of PPV6 were identified in PRRSv genotype 2 positive serum samples submitted to state veterinary diagnostic laboratories in 2014. Further testing using overlapping and real-time PCR determined PPV6 to be present in 13.2 % of the serums tested. Additionally, PPV6 was present in samples from all of the geographic locations sampled encompassing nine states in the United States and one state in Mexico. The presence of PPV6 in serum indicates that the PPV6 infection is disseminated and not localized to a specific tissue type. Alignments of the near full length genomes, NS1, and capsid genes identified one of the five PPV6 isolates from China (98.6–99.5 % identity with the North American strains) to be the North American strains nearest relative. These results are the first to report the presence of PPV6 in North America and demonstrate that the virus is found in multiple geographic areas in the United States and in Mexico. The overall prevalence of PPV6 in PRRSv viremic animals is relatively low. Further, all of the PPV6 genomes found in North America are most closely related to a PPV6 strain first identified in 2014 in healthy pigs from the Tianjin province of China.
Olufemi O. Fasina - One of the best experts on this subject based on the ideXlab platform.
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The Human Bocavirus 1 NP1 Protein Is a Multifunctional Regulator of Viral RNA Processing.
Journal of virology, 2018Co-Authors: Yanming Dong, Olufemi O. Fasina, David J. PintelAbstract:Human bocavirus 1 (HBoV1) encodes a genus-specific protein, NP1, which regulates viral alternative pre-mRNA processing. Similar to NP1 of the related bocavirus minute virus of canine (MVC), HBoV1 NP1 suppressed cleavage and polyadenylation of RNAs at the viral internal polyadenylation site (pA)p. HBoV1 (pA)p is a complex region. It contains 5 significant cleavage and polyadenylation sites, and NP1 was found to regulate only the three of these sites that are governed by canonical AAUAAA hexamer signals. HBoV1 NP1 also facilitated splicing of the upstream intron adjacent to (pA)p. Alternative polyadenylation and splicing of the upstream intron were independent of each other, functioned efficiently within an isolated transcription unit, and were responsive independent of NP1. Characterization of HBoV1 NP1 generalizes its function within the genus Bocaparvovirus, uncovers important differences, and provides important comparisons with MVC NP1 for mechanistic and evolutionary considerations.IMPORTANCE The Parvovirinae are small nonenveloped icosahedral viruses that are important pathogens in many animal species, including humans. The NP1 protein of human bocavirus 1 (HBoV1), similar to NP1 of the bocavirus minute virus of canine (MVC), regulates viral alternative RNA processing by both suppressing polyadenylation at an internal site, (pA)p, and facilitating splicing of an upstream adjacent intron. These effects allow both extension into the capsid gene and splicing of the viral pre-mRNA that correctly registers the capsid gene open reading frame. Characterization of HBoV1 NP1 generalizes this central mode of parvovirus gene regulation to another member of the bocavirus genus and uncovers both important similarities and differences in function compared to MVC NP1 that will be important for future comparative studies.
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Minute Virus of Canines NP1 Protein Governs the Expression of a Subset of Essential Nonstructural Proteins via Its Role in RNA Processing
Journal of virology, 2017Co-Authors: Olufemi O. Fasina, Stephanie Stupps, Wanda Figueroa-cuilan, David J. PintelAbstract:Parvoviruses use a variety of means to control the expression of their compact genomes. The Bocaparvovirus minute virus of canines (MVC) encodes a small, genus-specific protein, NP1, which governs access to the viral capsid gene via its role in alternative polyadenylation and alternative splicing of the single MVC pre-mRNA. In addition to NP1, MVC encodes five additional nonstructural proteins (NS) that share an initiation codon at the left end of the genome and which are individually encoded by alternative multiply spliced mRNAs. We found that three of these proteins were encoded by mRNAs that excise the NP1-regulated MVC intron immediately upstream of the internal polyadenylation site, (pA)p, and that generation of these proteins was thus regulated by NP1. Splicing of their progenitor mRNAs joined the amino termini of these proteins to the NP1 open reading frame, and splice site mutations that prevented their expression inhibited virus replication in a host cell-dependent manner. Thus, in addition to controlling capsid gene access, NP1 also controls the expression of three of the five identified NS proteins via its role in governing MVC pre-mRNA splicing. IMPORTANCE The Parvovirinae are small nonenveloped icosahedral viruses that are important pathogens in many animal species, including humans. Minute virus of canine (MVC) is an autonomous parvovirus in the genus Bocaparvovirus. It has a single promoter that generates a single pre-mRNA. NP1, a small genus-specific MVC protein, participates in the processing of this pre-mRNA and so controls capsid gene access via its role in alternative internal polyadenylation and splicing. We show that NP1 also controls the expression of three of the five identified NS proteins via its role in governing MVC pre-mRNA splicing. These NS proteins together are required for virus replication in a host cell-dependent manner.
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NP1 Protein of the Bocaparvovirus Minute Virus of Canines Controls Access to the Viral Capsid Genes via Its Role in RNA Processing
Journal of virology, 2015Co-Authors: Olufemi O. Fasina, Yanming Dong, David J. PintelAbstract:ABSTRACT Minute virus of canines (MVC) is an autonomous parvovirus in the genus Bocaparvovirus. It has a single promoter that generates a single pre-mRNA processed via alternative splicing and alternative polyadenylation to produce at least 8 mRNA transcripts. MVC contains two polyadenylation sites, one at the right-hand end of the genome, (pA)d, and another complex site, (pA)p, within the capsid-coding region. During viral infection, the mRNAs must extend through (pA)p and undergo additional splicing of the immediately upstream 3D∕3A intron to access the capsid gene. MVC NP1 is a 22-kDa nuclear phosphoprotein unique to the genus Bocaparvovirus of the Parvovirinae which we have shown governs suppression of (pA)p independently of viral genome replication. We show here that in addition to suppression of (pA)p, NP1 is also required for the excision of the MVC 3D∕3A intron, independently of its effect on alternative polyadenylation. Mutations of the arginine∕serine (SR) di-repeats within the intrinsically disordered amino terminus of NP1 are required for splicing of the capsid transcript but not suppression of polyadenylation at (pA)p. 3′-end processing of MVC mRNAs at (pA)p is critical for viral genome replication and the optimal expression of NP1 and NS1. Thus, a finely tuned balance between (pA)p suppression and usage is necessary for efficient virus replication. NP1 is the first parvovirus protein implicated in RNA processing. Its characterization reveals another way that parvoviruses govern access to their capsid protein genes, namely, at the RNA level, by regulating the essential splicing of an intron and the suppression of an internal polyadenylation site. IMPORTANCE The Parvovirinae are small nonenveloped icosahedral viruses that are important pathogens in many animal species, including humans. Although parvoviruses have only subtle early-to-late expression shifts, they all regulate access to their capsid genes. Minute virus of canines (MVC) is an autonomous parvovirus in the genus Bocaparvovirus. It has a single promoter generating a single pre-mRNA which is processed via alternative splicing and alternative polyadenylation to generate at least 8 mRNA transcripts. MVC contains two polyadenylation sites, one at the right-hand end of the genome, (pA)d, and another, (pA)p, within the capsid-coding region. It had not been clear how the potent internal polyadenylation motif is suppressed to allow processing, export, and accumulation of the spliced capsid protein-encoding mRNAs. We show here that MVC NP1, the first parvovirus protein to be implicated in RNA processing, governs access to the MVC capsid gene by facilitating splicing and suppressing internal polyadenylation of MVC pre-mRNAs.
John F. Engelhardt - One of the best experts on this subject based on the ideXlab platform.
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Cellular Cleavage and Polyadenylation Specificity Factor 6 (CPSF6) Mediates Nuclear Import of Human Bocavirus 1 NP1 Protein and Modulates Viral Capsid Protein Expression.
Journal of virology, 2020Co-Authors: Xiaomei Wang, Zekun Wang, Fang Cheng, Siyuan Hao, Jianke Wang, Kang Ning, Safder S. Ganaie, John F. EngelhardtAbstract:Human bocavirus 1 (HBoV1), which belongs to the genus Bocaparvovirus of the Parvoviridae family, causes acute respiratory tract infections in young children. In vitro, HBoV1 infects polarized primary human airway epithelium (HAE) cultured at an air-liquid interface (HAE-ALI). HBoV1 encodes a small nonstructural protein, nuclear protein 1 (NP1), that plays an essential role in the maturation of capsid protein (VP)-encoding mRNAs and viral DNA replication. In this study, we determined the broad interactome of NP1 using the proximity-dependent biotin identification (BioID) assay combined with mass spectrometry (MS). We confirmed that two host mRNA processing factors, DEAH-box helicase 15 (DHX15) and cleavage and polyadenylation specificity factor 6 (CPSF6; also known as CFIm68), a subunit of the cleavage factor Im complex (CFIm), interact with HBoV1 NP1 independently of any DNA or mRNAs. Knockdown of CPSF6 significantly decreased the expression of capsid protein but not that of DHX15. We further demonstrated that NP1 directly interacts with CPSF6 in vitro and colocalizes within the virus replication centers. Importantly, we revealed a novel role of CPSF6 in the nuclear import of NP1, in addition to the critical role of CPSF6 in NP1-facilitated maturation of VP-encoding mRNAs. Thus, our study suggests that CPSF6 interacts with NP1 to escort NP1 imported into the nucleus for its function in the modulation of viral mRNA processing and viral DNA replication.IMPORTANCE Human bocavirus 1 (HBoV1) is one of the significant pathogens causing acute respiratory tract infections in young children worldwide. HBoV1 encodes a small nonstructural protein (NP1) that plays an important role in the maturation of viral mRNAs encoding capsid proteins as well as in viral DNA replication. Here, we identified a critical host factor, CPSF6, that directly interacts with NP1, mediates the nuclear import of NP1, and plays a role in the maturation of capsid protein-encoding mRNAs in the nucleus. The identification of the direct interaction between viral NP1 and host CPSF6 provides new insights into the mechanism by which a viral small nonstructural protein facilitates the multiple regulation of viral gene expression and replication and reveals a novel target for potent antiviral drug development.
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Parvovirus Expresses a Small Noncoding RNA That Plays an Essential Role in Virus Replication.
Journal of virology, 2017Co-Authors: Zekun Wang, Xuefeng Deng, John F. Engelhardt, Weiran Shen, Fang Cheng, Ziying Yan, Jianming QiuAbstract: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.
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DNA Damage Signaling Is Required for Replication of Human Bocavirus 1 DNA in Dividing HEK293 Cells
Journal of virology, 2016Co-Authors: Xuefeng Deng, John F. Engelhardt, Weiran Shen, Ziying Yan, Wei Zou, Jianxin Peng, Kaiyu Liu, Jianming QiuAbstract:ABSTRACT 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).
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analysis of cis and trans requirements for dna replication at the right end hairpin of the human bocavirus 1 genome
Journal of Virology, 2016Co-Authors: Weiran Shen, Xuefeng Deng, John F. EngelhardtAbstract: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.
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Replication of an Autonomous Human Parvovirus in Non-dividing Human Airway Epithelium Is Facilitated through the DNA Damage and Repair Pathways.
PLoS pathogens, 2016Co-Authors: Xuefeng Deng, John F. Engelhardt, Fang Cheng, Ziying Yan, Jianming QiuAbstract: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.