The Experts below are selected from a list of 252 Experts worldwide ranked by ideXlab platform
Kevin E. Brown - One of the best experts on this subject based on the ideXlab platform.
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Adeno-associated virus (AAV)-3-based vectors transduce haematopoietic cells not susceptible to transduction with AAV-2-based vectors
Journal of General Virology, 2000Co-Authors: Atsushi Handa, Hiroaki Mizukami, Jianming Qiu, Shin-ichi Muramatsu, Kevin E. BrownAbstract:Although adeno-associated virus (AAV)-2 has a broad tissue-host range and can transduce a wide variety of tissue types, some cells, such as erythro-megakaryoblastoid cells, are non-permissive and appear to lack the AAV-2 receptor. However, limited studies have been reported with the related Dependovirus AAV-3. We have previously cloned this virus, characterized its genome and produced an infectious clone. In this study, the gene for green fluorescent protein (GFP) was inserted into AAV-2- and AAV-3-based plasmids and recombinant viruses were produced. These viruses were then used to transduce haematopoietic cells and the transduction efficiencies were compared. In contrast to recombinant (r) AAV-2, rAAV-3 successfully transduced erythroid and megakaryoblastoid cells, although rAAV-2 was superior in transduction of lymphocyte-derived cell lines. Recently, it was reported that heparan sulphate can act as a receptor of AAV-2. The infectivity of rAAV-2 and rAAV-3 was tested with mutant cell lines of Chinese hamster ovary cells that were defective for heparin or heparan sulphate expression on the cell surface. There was no correlation between the ability of rAAV-2 or rAAV-3 to infect cells and the cell surface expression of heparan sulphate and, although heparin blocked both rAAV-2 and rAAV-3 transduction, the ID50 of rAAV-3 was higher than that of rAAV-2. In addition, virus-binding overlay assays indicated that AAV-2 and AAV-3 bound different membrane proteins. These results suggest not only that there are different cellular receptors for AAV-2 and AAV-3, but that rAAV-3 vectors may be preferred for transduction of some haematopoietic cell types.
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The interaction of heparin sulfate and adeno-associated virus 2.
Virology, 2000Co-Authors: Jianming Qiu, Atsushi Handa, Martha Kirby, Kevin E. BrownAbstract:Abstract Recently heparan sulfate was proposed as the host cell receptor for the Dependovirus, adeno-associated virus type 2 (AAV2). We show that although heparan sulfate on the cell surface may contribute to the binding of AAV2 to permissive cells, the amount of heparan sulfate on the cell surface as determined by flow cytometry using four different monoclonal antibodies does not correlate with AAV2 binding to cells or recombinant AAV2 transduction efficiency. Experiments with either mutant CHO cells or cells treated with chlorate to remove sulfate groups showed that sulfation was not absolutely required for infection or binding: in the absence of cell surface sulfation, recombinant AAV2 was still able to be transduced in previously permissive cells. Heparin is commonly used as a substitute in studies of the interaction between heparan sulfate and ligand, and we demonstrate that the binding affinity of AAV2/heparin is low, with a K d value of ∼2.0 nM. A study of the direct interaction between AAV2 and artificial glycosaminoglycans showed that a high degree of sulfation on heparin was critical for the ability to bind AAV2 and compete rAAV2 transduction and that both O- and N-sulfate groups are required. Overall, our data suggest that, as has been shown for other viruses, the presence of a high-affinity AAV2 receptor mediates AAV2 infection in addition to the low-affinity heparan sulfate binding.
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GOOSE PARVOVIRUS : AN AUTONOMOUS MEMBER OF THE Dependovirus GENUS ?
Virology, 1995Co-Authors: Kevin E. Brown, Spencer W. Green, Neal S. YoungAbstract:Goose parvovirus is the etiological agent of Derzsy's disease, a fatal hepatitis of young geese. The virus infects geese and Muscovy ducks and can be propagated in the laboratory in primary embryonic goose fibroblasts. To date the virus has only been classified by morphological, biochemical, and culture characteristics as an autonomous parvovirus. We now report the cloning and partial sequencing of 3434 nucleotides of the viral genome. Three overlapping clones were obtained, encoding regions in the nonstructural and capsid coding region. The nucleotide sequence show little homology to other autonomous parvoviruses but 55% homology to the Dependovirus AAV2. The homology to AAV2 was also confirmed at the amino acid level (nonstructural protein 55%, capsid coding region 51%). DNA cross hybridization studies indicate an even closer similarity of goose parvovirus to the yet unsequenced human Dependoviruses AAV1 and AAV3 than to AAV2. These findings suggest that goose parvovirus may be genetically related to the Dependovirus genus rather than to the other autonomous parvoviruses.
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goose parvovirus an autonomous member of the Dependovirus genus
Virology, 1995Co-Authors: Kevin E. Brown, Spencer W. Green, Neal S. YoungAbstract:Goose parvovirus is the etiological agent of Derzsy's disease, a fatal hepatitis of young geese. The virus infects geese and Muscovy ducks and can be propagated in the laboratory in primary embryonic goose fibroblasts. To date the virus has only been classified by morphological, biochemical, and culture characteristics as an autonomous parvovirus. We now report the cloning and partial sequencing of 3434 nucleotides of the viral genome. Three overlapping clones were obtained, encoding regions in the nonstructural and capsid coding region. The nucleotide sequence show little homology to other autonomous parvoviruses but 55% homology to the Dependovirus AAV2. The homology to AAV2 was also confirmed at the amino acid level (nonstructural protein 55%, capsid coding region 51%). DNA cross hybridization studies indicate an even closer similarity of goose parvovirus to the yet unsequenced human Dependoviruses AAV1 and AAV3 than to AAV2. These findings suggest that goose parvovirus may be genetically related to the Dependovirus genus rather than to the other autonomous parvoviruses.
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Cloning and sequencing of the simian parvovirus genome.
Virology, 1995Co-Authors: Kevin E. Brown, Spencer W. Green, M. Gerard O'sullivan, Neal S. YoungAbstract:We recently reported the identification of a novel simian parvovirus in cynomolgus monkeys with severe anemia. We now describe the cloning and sequencing of 4986 nucleotides of the viral DNA. Like the human parvovirus B19, simian parvovirus encapsidates both positive and negative single-stranded DNA. The positive strand contains two large open reading frames, with the left open reading frame encoding the nonstructural protein(s) and the right reading frame encoding the two capsid proteins. Simian parvovirus has little homology with the autonomous parvoviruses or the Dependovirus AAV-2 but 50% overall homology with parvovirus B19 DNA. At the amino acid level there was 70% homology with B19 capsid proteins and 50% homology with B19 nonstructural protein. Based on this genetic similarity and with the known tropism of the virus for cynomolgus erythroid precursors, we suggest that this new virus should be classified as a new member of the Erythrovirus genus of the Parvoviridae.
Jianming Qiu - One of the best experts on this subject based on the ideXlab platform.
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Structure of the NS1 Protein N-Terminal Origin Recognition/Nickase Domain from the Emerging Human Bocavirus
Journal of virology, 2013Co-Authors: Sunil Kumar Tewary, Jianming Qiu, Haiyan Zhao, Weiran Shen, Liang TangAbstract:Human bocavirus is a newly identified, globally prevalent, parvovirus that is associated with respiratory infection in infants and young children. Parvoviruses encode a large nonstructural protein 1 (NS1) that is essential for replication of the viral single-stranded DNA genome and DNA packaging and may play versatile roles in virus-host interactions. Here, we report the structure of the human bocavirus NS1 N-terminal domain, the first for any autonomous parvovirus. The structure shows an overall fold that is canonical to the histidine-hydrophobic-histidine superfamily of nucleases, which integrates two distinct DNA-binding sites: (i) a positively charged region mediated by a surface hairpin (residues 190 to 198) that is responsible for recognition of the viral origin of replication of the double-stranded DNA nature and (ii) the nickase active site that binds to the single-stranded DNA substrate for site-specific cleavage. The structure reveals an acidic-residue-rich subdomain that is present in bocavirus NS1 proteins but not in the NS1 orthologs in erythrovirus or Dependovirus, which may mediate bocavirus-specific interaction with DNA or potential host factors. These results provide insights into recognition of the origin of replication and nicking of DNA during bocavirus genome replication. Mapping of variable amino acid residues of NS1s from four human bocavirus species onto the structure shows a scattered pattern, but the origin recognition site and the nuclease active site are invariable, suggesting potential targets for antivirals against this clade of highly diverse human viruses.
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Processing of adeno-associated virus RNA.
Frontiers in bioscience : a journal and virtual library, 2008Co-Authors: Jianming Qiu, David PintelAbstract:Adeno-associated viruses (AAVs), members of the Dependovirus genus of the subfamily Parvovirinae, family Parvoviridae, have single-stranded DNA genomes that replicate via a double-stranded DNA intermediate which serves as the transcription template. They exhibit a highly compact, overlapping genetic organization which is surprisingly variable amongst various members within the group. Additionally, recent work has shown them to be excellent models for understanding potential interactions between alternative splicing and alternative polyadenylation, the export of unspliced RNAs, and transactivating proteins that influence RNA processing co-transcriptionally via interaction with the transcription template.
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The Expression Strategy of Goose Parvovirus Exhibits Features of both the Dependovirus and Parvovirus Genera
Journal of virology, 2005Co-Authors: Jianming Qiu, Zoltán Zádori, Fang Cheng, Yuko Yoto, David J. PintelAbstract:The RNA transcription profile of the goose parvovirus (GPV) was determined, and it is a surprising hybrid of features of the Parvovirus and Dependovirus genera of the Parvovirinae subfamily of the Parvoviridae. Similar to the Dependovirus adeno-associated virus type 5, RNAs transcribed from the GPV upstream P9 promoter, which encode the viral nonstructural proteins, were polyadenylated at a high efficiency at a polyadenylation site [(pA)p] located within an intron in the center of the genome. Efficient usage of (pA)p required a downstream element that overlaps with the polypyrimidine tract of the A2 3' splice site of the central intron. An upstream element required for efficient use of (pA)p was also identified. RNAs transcribed from the P42 promoter, presumed to encode the viral capsid proteins, primarily extended through (pA)p and were polyadenylated at a site, (pA)d, located at the right end of the genome and ultimately spliced at a high efficiency. No promoter analogous to the Dependovirus P19 promoter was detected; however, similar to minute virus of mice and other members of the Parvovirus genus, a significant portion of pre-mRNAs generated from the P9 promoter were additionally spliced within the putative GPV Rep1 coding region and likely encode an additional, smaller, nonstructural protein. Also similar to members of the Parvovirus genus, detectable activity of the GPV P42 promoter was highly dependent on transactivation by the GPV Rep1 protein in a manner dependent on binding to a cis-element located in the P42 promoter.
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Adeno-associated virus (AAV)-3-based vectors transduce haematopoietic cells not susceptible to transduction with AAV-2-based vectors
Journal of General Virology, 2000Co-Authors: Atsushi Handa, Hiroaki Mizukami, Jianming Qiu, Shin-ichi Muramatsu, Kevin E. BrownAbstract:Although adeno-associated virus (AAV)-2 has a broad tissue-host range and can transduce a wide variety of tissue types, some cells, such as erythro-megakaryoblastoid cells, are non-permissive and appear to lack the AAV-2 receptor. However, limited studies have been reported with the related Dependovirus AAV-3. We have previously cloned this virus, characterized its genome and produced an infectious clone. In this study, the gene for green fluorescent protein (GFP) was inserted into AAV-2- and AAV-3-based plasmids and recombinant viruses were produced. These viruses were then used to transduce haematopoietic cells and the transduction efficiencies were compared. In contrast to recombinant (r) AAV-2, rAAV-3 successfully transduced erythroid and megakaryoblastoid cells, although rAAV-2 was superior in transduction of lymphocyte-derived cell lines. Recently, it was reported that heparan sulphate can act as a receptor of AAV-2. The infectivity of rAAV-2 and rAAV-3 was tested with mutant cell lines of Chinese hamster ovary cells that were defective for heparin or heparan sulphate expression on the cell surface. There was no correlation between the ability of rAAV-2 or rAAV-3 to infect cells and the cell surface expression of heparan sulphate and, although heparin blocked both rAAV-2 and rAAV-3 transduction, the ID50 of rAAV-3 was higher than that of rAAV-2. In addition, virus-binding overlay assays indicated that AAV-2 and AAV-3 bound different membrane proteins. These results suggest not only that there are different cellular receptors for AAV-2 and AAV-3, but that rAAV-3 vectors may be preferred for transduction of some haematopoietic cell types.
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The interaction of heparin sulfate and adeno-associated virus 2.
Virology, 2000Co-Authors: Jianming Qiu, Atsushi Handa, Martha Kirby, Kevin E. BrownAbstract:Abstract Recently heparan sulfate was proposed as the host cell receptor for the Dependovirus, adeno-associated virus type 2 (AAV2). We show that although heparan sulfate on the cell surface may contribute to the binding of AAV2 to permissive cells, the amount of heparan sulfate on the cell surface as determined by flow cytometry using four different monoclonal antibodies does not correlate with AAV2 binding to cells or recombinant AAV2 transduction efficiency. Experiments with either mutant CHO cells or cells treated with chlorate to remove sulfate groups showed that sulfation was not absolutely required for infection or binding: in the absence of cell surface sulfation, recombinant AAV2 was still able to be transduced in previously permissive cells. Heparin is commonly used as a substitute in studies of the interaction between heparan sulfate and ligand, and we demonstrate that the binding affinity of AAV2/heparin is low, with a K d value of ∼2.0 nM. A study of the direct interaction between AAV2 and artificial glycosaminoglycans showed that a high degree of sulfation on heparin was critical for the ability to bind AAV2 and compete rAAV2 transduction and that both O- and N-sulfate groups are required. Overall, our data suggest that, as has been shown for other viruses, the presence of a high-affinity AAV2 receptor mediates AAV2 infection in addition to the low-affinity heparan sulfate binding.
John A. Chiorini - One of the best experts on this subject based on the ideXlab platform.
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Structure of Adeno-Associated Virus Type 4
Journal of virology, 2005Co-Authors: Eric Padron, Valorie D. Bowman, Nikola Kaludov, Lakshmanan Govindasamy, Hazel C. Levy, Phillip Nick, Robert Mckenna, Nicholas Muzyczka, John A. Chiorini, Timothy S. BakerAbstract:Adeno-associated virus (AAV) is a member of the Parvoviridae, belonging to the Dependovirus genus. Currently, several distinct isolates of AAV are in development for use in human gene therapy applications due to their ability to transduce different target cells. The need to manipulate AAV capsids for specific tissue delivery has generated interest in understanding their capsid structures. The structure of AAV type 4 (AAV4), one of the most antigenically distinct serotypes, was determined to 13-A resolution by cryo-electron microscopy and image reconstruction. A pseudoatomic model was built for the AAV4 capsid by use of a structure-based sequence alignment of its major capsid protein, VP3, with that of AAV2, to which AAV4 is 58% identical and constrained by its reconstructed density envelope. The model showed variations in the surface loops that may account for the differences in receptor binding and antigenicity between AAV2 and AAV4. The AAV4 capsid surface topology also shows an unpredicted structural similarity to that of Aleutian mink disease virus and human parvovirus B19, autonomous members of the genus, despite limited sequence homology.
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Structure of Adeno-Associated Virus Type 4
2004Co-Authors: Eric Padron, Nikola Kaludov, Lakshmanan Govindasamy, Phillip Nick, Robert Mckenna, Nicholas Muzyczka, John A. Chiorini, Valorie Bowman, Hazel Levy, Timothy S. BakerAbstract:Adeno-associated virus (AAV) is a member of the Parvoviridae, belonging to the Dependovirus genus. Currently, several distinct isolates of AAV are in development for use in human gene therapy applications due to their ability to transduce different target cells. The need to manipulate AAV capsids for specific tissue delivery has generated interest in understanding their capsid structures. The structure of AAV type 4 (AAV4), one of the most antigenically distinct serotypes, was determined to 13-Å resolution by cryo-electron microscopy and image reconstruction. A pseudoatomic model was built for the AAV4 capsid by use of a structure-based sequence alignment of its major capsid protein, VP3, with that of AAV2, to which AAV4 is 58 % identical and constrained by its reconstructed density envelope. The model showed variations in the surface loops that may account for the differences in receptor binding and antigenicity between AAV2 and AAV4. The AAV4 capsid surface topology also shows an unpredicted structural similarity to that of Aleutian mink disease virus and human parvovirus B19, autonomous members of the genus, despite limited sequence homology. Adeno-associated virus (AAV) is a member of the Parvoviridae family (45). AAV virions have a T�1 icosahedral capsid consisting of 60 copies of three related proteins, VP1, VP2, and VP3, at an estimated ratio of 1:1:8, which surrounds a singlestrande
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Cloning of an avian adeno-associated virus (AAAV) and generation of recombinant AAAV particles.
Journal of virology, 2003Co-Authors: Ioannis Bossis, John A. ChioriniAbstract:Recent studies have proposed that adeno-associated viruses (AAVs) are not evolutionarily linked to other mammalian autonomous parvoviruses but are more closely linked to the autonomous parvoviruses of birds. To better understand the relationship between primate and avian AAVs (AAAVs), we cloned and sequenced the genome of an AAAV (ATCC VR-865) and generated recombinant AAAV particles. The genome of AAAV is 4,694 nucleotides in length and has organization similar to that of other AAVs. The entire genome of AAAV displays 56 to 65% identity at the nucleotide level with the other known AAVs. The AAAV genome has inverted terminal repeats of 142 nucleotides, with the first 122 forming the characteristic T-shaped palindromic structure. The putative Rep-binding element consists of a tandem (GAGY)4 repeat, and the putative terminal resolution site (trs), CCGGT/CG, contains a single nucleotide substitution relative to the AAV2 trs. The Rep open reading frame of AAAV displays 50 to 54% identity at the amino acid level with the other AAVs, with most of the diversity clustered at the carboxyl and amino termini. Comparison of the capsid proteins of AAAV and the primate Dependoviruses indicate that divergent regions are localized to surface-exposed loops. Despite these sequence differences, we were able to produce recombinant AAAV particles carrying a lacZ reporter gene by cotransfection in 293T cells and were able to examine transduction efficiency in both chicken primary cells and several cell lines. Our findings indicate that AAAV is the most divergent AAV described to date but maintains all the characteristics unique to the genera of Dependovirus.
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Adeno-Associated Virus Type 2 Rep78 Inhibition of PKA and PRKX: Fine Mapping and Analysis of Mechanism
Journal of virology, 2002Co-Authors: Michael Schmidt, John A. Chiorini, Sandra Afione, Robert M KotinAbstract:Hormones and neurotransmitters utilize cyclic AMP (cAMP) as a second messenger in signal transduction pathways to regulate cell growth and division, differentiation, gene expression, and metabolism. Adenoassociated virus type 2 (AAV-2) nonstructural protein Rep78 inhibits members of the cAMP signal transduction pathway, the protein kinases PKA and PRKX. We mapped the kinase binding and inhibition domain of Rep78 for PRKX to amino acids (aa) 526 to 561 and that for PKA to aa 526 to 621. These polypeptides were as potent as full-length Rep78 in kinase inhibition, which suggests that the kinase-inhibitory domain is entirely contained in these Rep peptides. Steady-state kinetic analysis of Rep78-mediated inhibition of PKA and PRKX showed that Rep78 appears to increase the Km value of the peptide kinase substrate, while the maximal velocity of the reaction was unaffected. This indicates that Rep78 acts as a competitive inhibitor with respect to the peptide kinase substrate. We detected homology between a cellular pseudosubstrate inhibitor of PKA, the protein kinase inhibitor PKI, and the PRKX and PKA inhibition domains of Rep78. Due to this homology and the competitive inhibition mechanism of Rep78, we propose that Rep78 inhibits PKA and PRKX kinase activity by pseudosubstrate inhibition. Adeno-associated virus type 2 (AAV-2) is a member of the Parvoviridae family and is assigned to the genus Dependovirus. Productive infection requires coinfection with a helper virus such as adenovirus or herpesvirus (2, 36). The AAV virion consists of a nonenveloped, icosahedral capsid harboring a linear, single-stranded DNA genome of ca. 4.7 kilonucleotides (knt). The coding regions containing the genes for the nonstructural proteins (rep) and capsid proteins (cap) are flanked by 145-nt inverted terminal repeats (ITR) that function in cis as the replication origin. Expression of the rep open reading
Robert M Kotin - One of the best experts on this subject based on the ideXlab platform.
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scalable generation of high titer recombinant adeno associated virus type 5 in insect cells
Journal of Virology, 2006Co-Authors: Masashi Urabe, Takayo Nakakura, Yoko Obara, Hiroaki Mizukami, Akihiro Kume, Robert M Kotin, Keiya OzawaAbstract:Recombinant adeno-associated virus (rAAV) is being developed as a gene transfer vector. rAAV based on serotype 2 (rAAV2) successfully transduces nondividing cells, including muscle, liver, and brain cells (29). Conventional rAAV production requires packaging of rAAV DNA into type 2 capsids by transient transfection of HEK293 cells with two or three plasmids: an AAV helper plasmid encoding rep and cap genes devoid of inverted terminal repeat (ITR) sequences, a vector plasmid harboring the therapeutic gene between ITRs, and an adenovirus helper plasmid expressing E2A, virus-associated (VA) RNA, and E4orf6. Transient cotransfection is the major limitation for scale-up of rAAV production. Since rAAV can be purified using column chromatography, which can result in highly purified rAAV while eliminating other contaminating viruses, some efforts were made to develop rAAV production systems by using recombinant mammalian viruses such as adenovirus (10) or herpes virus (4) which do not rely on the plasmid transfection and therefore may be amenable to scale-up production. Recombinant baculoviruses based on the Autographa californica nuclear polyhedrosis virus are widely employed for production of heterologous proteins in cultured insect cells. The highly active, late A. californica nuclear polyhedrosis virus promoters, such as polyhedrin and p10 promoters, regulate the expression of heterologous proteins, resulting in large amounts of foreign proteins. Insect cells may be grown in suspension cultures in volumes ranging from shake flasks of sizes from, e.g., 50 to 400 ml, up to commercial-size bioreactors, e.g., 1,000 liters and larger. Recently, we described a highly scalable and efficient method for packaging rAAV2 in insect cells by use of baculovirus expression vectors (31). The ease of scale-up production is perhaps the most attractive feature of this production system. Infection of insect cells in suspension culture with recombinant baculoviruses eliminates the transfection process. Standard downstream processing to recover rAAV, such as tangential flow filtration and column chromatography, is readily applied. In addition to vectors derived from serotype 2, other serotypes, utilizing different cell surface receptors, constitute a vector set from which an appropriate vector can be selected for a specific application. AAV5 is the most divergent Dependovirus characterized (2), and type 5 AAV vectors have desirable properties that differ from other serotype vectors. AAV5 utilizes different receptors from other serotypes (14, 30), and rAAV5 has demonstrated different tropism from AAV2 (5), thus making it worthwhile to establish a method to produce rAAV5 in insect cells. AAV is a member of the family Parvoviridae. The genome of AAV is a linear, single-stranded DNA of 4.7 kb in length. The ITRs flank the unique coding sequences for the nonstructural replication initiator proteins, Rep, and the structural capsid proteins, VP. The ITRs serve as origins of DNA replication and may also function as the packaging signal. Type 2 Rep78 is generated by the p5 promoter, while Rep68 is translated from spliced mRNA from the p5 promoter. The small Rep polypeptides Rep52 and Rep40 are expressed by the p19 promoter with nonspliced or spliced mRNA. The p40 promoter regulates expression of capsid proteins VP1, VP2, and VP3. Alternate usage of two splice sites and translation of VP2 at a non-AUG codon results in a stoichiometry of 1:1:10 of VP1, VP2, and VP3. Both p5 proteins Rep78 and Rep68 are AAV origin binding proteins, and the presence of either is required for AAV DNA replication and processing replicative intermediates of the virus DNA (13). Also, either Rep52 or Rep40 is necessary for packaging the single-stranded, linear virion genome into preformed empty capsids (17). The transcriptional map of type 5 AAV differs from that of type 2; the p7 promoter or p19 promoter transcribes mRNA for Rep78 or Rep52. Type 5 AAV does not encode the spliced form of Rep polypeptides Rep68 and Rep40 (25). Structural protein VP1 is a minor constituent in the AAV capsid. But the VP1-unique portion of approximately 140 amino acid residues is highly conserved among different serotypes and has a phospholipase A2 motif. The YXGGX and HDXXY motifs (where X is any amino acid residue) in phospholipase A2 indicate the catalytic site and Ca2+ binding loop, respectively (see Fig. Fig.3A).3A). Enzymes classified into the secretory phospholipase A2 family hydrolyze the ester bond at the 2-acyl ester position of glycerophospholipids in the presence of Ca2+ and are involved in many aspects of cellular pathways, such as lipid membrane metabolism and signal transduction pathways (1, 21). The VP1-unique portion of parvovirus is required for transfer of the virus from late endosomes to the nucleus (36). A mutant virus lacking the VP1-unique portion or the activity of phospholipase is not processed properly, and thus no virus or vector genes are expressed. FIG. 3. (A) Chimeric VP genes constructed. The portions derived from type 2 are indicated in gray, while those from type 5 are in white. The common portions are indicated in black. The phospholipase A2 motifs are shaded. The YXGGX and HDXXY motifs (where X is ... In the present study, we describe a rAAV5 production system based on recombinant baculovirus and insect cells. In order to achieve high production levels of rAAV5 particles, we replaced a portion of the VP1 polypeptide with the corresponding portion of type 2. The VP1 substitution did not alter the tropism of rAAV5, which behaved indistinguishably from rAAV5 with wild-type VP1. In an attempt to improve the yields of rAAV5 particles, we used type 1 Rep52 instead of type 5, which resulted in the production of more than 5 × 104 vector genomes (vg) per insect cell.
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517. Type 1 Rep52 Is Superior to Authentic Rep52 for Producing Recombinant Adeno-Associated Virus Type 5 in Insect Cells
Molecular Therapy, 2005Co-Authors: Masashi Urabe, Takayo Nakakura, Hiroaki Mizukami, Akihiro Kume, Robert M Kotin, Keiya OzawaAbstract:Top of pageAbstract The recently described method for producing recombinant adeno-associated virus (rAAV) type 2 in insect cells facilitates scale-up by using suspension cell cultures and baculovirus expression vectors (Hum Gene Ther 13:1935-1943, 2002). AAV5 is one of the most divergent Dependovirus characterized and has been shown to utilize different receptors than AAV2 for example, and has demonstrated different tissue tropism from other serotype rAAVs. In addition to vectors derived from serotype 2, other serotypes constitute a vector set from which an optimal one can be selected for specific applications. Thus, we established a method to generate rAAV5 in insect cells. The current process requires triple infection with recombinant baculoviruses that provide the following: 1. AAV structural proteins that form the virus capsid (VP 1, 2, 3); 2. two of the AAV non-structural proteins for replication and encapsidation (Rep 78 and Rep 52); and 3. the AAV vector DNA which contain the gene of interest flanked by the AAV origins of replication (ITRs). Compared to other rAAV serotypes produced in insect cells, the rAAV5 yield per cell was substantially lower and we tested some modifications of the system. The initial Rep baculovirus drove type 5 Rep72 expression with a truncated promoter for the immediate-early 1 gene of Orgyia pseudotsugata nuclear polyhedrosis virus. The titers of the Rep baculovirus were relatively lower than others. We constructed Rep baculoviruses with a series of truncated p10 promoters for Rep78 and selected one that could produce rAAV5 at a high titer and propagate well. Rep52 or small Rep protein packages AAV genome into preformed empty capsids. We examined Rep52 of other serotypes, 1, 2, 3, 4 for generation of rAAV5 particles. The titer of rAAV5-GFP produced with type 1, 2, 3, or 4 small Rep was 56,000|[plusmn]|3,200, 41,000|[plusmn]|18,900, 42,000|[plusmn]|7,300, or 39,000|[plusmn]|3,500 particles per Sf9 cell while rAAV5-GFP produced by authentic Rep52 was 13,500|[plusmn]|3,200. The rAAV5-GFP produced with either serotype small Rep has an equal ratio of nucleic acid to capsid protein assessed by real-time PCR quantification and silver staining of purified rAAV5 particles. Also, rAAV5-GFP produced with either combination of large and small Rep proteins, transduced the simian Cos cell line with similar efficiency. The analysis by cesium density gradient of insect-cell lysates indicated that approximately 50% of all capsids contained the vector genome. These results indicate that heteroserotypic small Rep polypeptide is able to substitute for AAV5 small Rep and package AAV vector genome with type 5 in Sf9 cells and the new Rep baculovirus expressing type 5 Rep78 under the control of a truncated p10 promoter and type 1 Rep52 will contribute to the development of more efficient production of rAAV5 in insect cells.
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Adeno-Associated Virus Type 2 Rep78 Inhibition of PKA and PRKX: Fine Mapping and Analysis of Mechanism
Journal of virology, 2002Co-Authors: Michael Schmidt, John A. Chiorini, Sandra Afione, Robert M KotinAbstract:Hormones and neurotransmitters utilize cyclic AMP (cAMP) as a second messenger in signal transduction pathways to regulate cell growth and division, differentiation, gene expression, and metabolism. Adenoassociated virus type 2 (AAV-2) nonstructural protein Rep78 inhibits members of the cAMP signal transduction pathway, the protein kinases PKA and PRKX. We mapped the kinase binding and inhibition domain of Rep78 for PRKX to amino acids (aa) 526 to 561 and that for PKA to aa 526 to 621. These polypeptides were as potent as full-length Rep78 in kinase inhibition, which suggests that the kinase-inhibitory domain is entirely contained in these Rep peptides. Steady-state kinetic analysis of Rep78-mediated inhibition of PKA and PRKX showed that Rep78 appears to increase the Km value of the peptide kinase substrate, while the maximal velocity of the reaction was unaffected. This indicates that Rep78 acts as a competitive inhibitor with respect to the peptide kinase substrate. We detected homology between a cellular pseudosubstrate inhibitor of PKA, the protein kinase inhibitor PKI, and the PRKX and PKA inhibition domains of Rep78. Due to this homology and the competitive inhibition mechanism of Rep78, we propose that Rep78 inhibits PKA and PRKX kinase activity by pseudosubstrate inhibition. Adeno-associated virus type 2 (AAV-2) is a member of the Parvoviridae family and is assigned to the genus Dependovirus. Productive infection requires coinfection with a helper virus such as adenovirus or herpesvirus (2, 36). The AAV virion consists of a nonenveloped, icosahedral capsid harboring a linear, single-stranded DNA genome of ca. 4.7 kilonucleotides (knt). The coding regions containing the genes for the nonstructural proteins (rep) and capsid proteins (cap) are flanked by 145-nt inverted terminal repeats (ITR) that function in cis as the replication origin. Expression of the rep open reading
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Recombinant Adeno-Associated Virus (r AAV) Vectors
Gene Therapy, 1999Co-Authors: M. Hallek, Robert M Kotin, C.-m. Wendtner, D. Michl, E.-l. WinnackerAbstract:Adeno-associated virus (AAV) is a single-stranded DNA Dependovirus of the family of Parvoviridae which has promising features as a vector for somatic gene therapy. Different recombinant (r) AAV vectors have been generated which seem to have some advantages in comparison with other vectors, like the ability to transduce terminally differentiated and non-dividing cells, the lack of any apparent pathogenicity, a low immunogenicity, a relatively high stability of transgene expression and the potential of targeted integration. Recent improvements of rAAV packaging now allow the generation of sufficient quantities of rAAV for clinical trials. Pre-clinical studies with rAAV are currently performed not only for the treatment of a variety of inherited, monogenic defects such as s-thalassemia, sickle cell anemia, Fanconi anemia, chronic granulomatous disease, Gaucher disease, metachromatic leukodystrophy, or cystic fibrosis, but also for acquired diseases like infection with HIV and non-Hodgkin’s lymphoma. The diversity of these studies indicates that rAAV might have a broad range of clinical applications. A first clinical trial with rAAV vectors has been started for cystic fibrosis. While several important issues including safety, tissue tropism and methods to achieve site-specific integration need further clarification, rAAV seems to have a sufficient number of advantages to be considered seriously as a future gene therapy vector.
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Adeno-Associated VirusDNA Replication InVitro: Activation bya Maltose Binding Protein/Rep 68Fusion Protein
1994Co-Authors: P Ward, Robert M Kotin, E Urcelay, Brian Safer, K. I. BernsAbstract:Theadeno-associated virus (AAV)nonstructural protein Rep68isrequired forviral DNAreplication. Anin vitro assayhasbeendeveloped inwhichaddition ofRep68toan extract fromuninfected HeLacells supports AAVDNAreplication. Inthis paper,we report characterization ofthereplication processwhenafusion ofthe maltose binding protein andRep68,expressed inEscherichia coli, was usedintheassay.Replication was observed whenthetemplate was either linear double-stranded AAVDNA or a plasmid construct containing intact AAVDNA.Whentherecombinant plasmid construct was usedasthetemplate, there was replication of pBR322DNA aswell as theAAVDNA;however, linear pBR322DNA was notreplicated. Whentheplasmid construct was thetemplate, replication appeared toinitiate on theintact plasmid andledtoseparation ofthe AAVsequencesfromthose ofthevector, a processwhichhasbeentermed rescue.There was no evidence that replication couldinitiate on theproducts ofrescue.Rep68can makea site-specific nick124nucleotides from the3'endofAAVDNA;thesite ofthenickhasbeencalled theterminal resolution site. Ourdataaremost consistent withinitiation occurring attheterminal resolution site andproceeding towardthe3'terminus. Whenthetemplate was theplasmid construct, either elongation continued pastthejunction intopBR322 sequencesorthenewlysynthesized sequencehairpinned, switched template strands, andreplicated theAAV DNA.Replication was linear for4h,during whichtime70%ofthemaximalsynthesis tookplace. Anadditional finding was thattheRepfusion could resolve AAVdimerlength duplex intermediates into monomer duplexes without DNA synthesis. Thehumanparvovirus adeno-associated virus type2(AAV) isclassified asa Dependovirus because oftherequirement for coinfection withhelper virus (either adenovirus [Ad]or herpesvirus) foroptimal replication incell culture (1,2,4,21). In theabsence ofhelper virus coinfection, theAAV genome integrates intothecellular genome toestablish a latent infection(1). Inseveral lines ofhumancells, integration hasbeen reported tobewithin adefined locus on chromosome 19q13.3qter(15-17, 27). Helper virus infection ofthelatently infected cell leads torescueandreplication oftheAAV genome,with virion production. We havereported an invitro assaywhich appearstobeamodelfortherescueandreplication ofAAV. Ithasmany oftheparameters determined forinvivoAAV rescueandreplication (10). Indeed, many oftheparameters of invivoreplication were determined after transfection of Ad-infected humancells withplasmid constructs thesame as or similar tothoseusedintheinvitro assay(11,25).In particular, theinvitro assayrequires an extract fromHeLa cells coinfected byAAV andAd;extracts fromuninfected cells orcells infected byAd orAAV alonedonotsupportreplication. Theinvitro assayalsohasspecific template requirements. TheAAV genome isa linear, single-stranded DNA (4,680 bases) with an inverted terminal repeat(ITR)of145bases (20, 28). Theterminal 125bases are an overall palindrome interrupted bytwosmaller, internal 21-base palindromes, one on either sideoftheoverall axisofsymmetry. Whenthepalindromicregion oftheitrisfolded on itself tomaximize
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scalable generation of high titer recombinant adeno associated virus type 5 in insect cells
Journal of Virology, 2006Co-Authors: Masashi Urabe, Takayo Nakakura, Yoko Obara, Hiroaki Mizukami, Akihiro Kume, Robert M Kotin, Keiya OzawaAbstract:Recombinant adeno-associated virus (rAAV) is being developed as a gene transfer vector. rAAV based on serotype 2 (rAAV2) successfully transduces nondividing cells, including muscle, liver, and brain cells (29). Conventional rAAV production requires packaging of rAAV DNA into type 2 capsids by transient transfection of HEK293 cells with two or three plasmids: an AAV helper plasmid encoding rep and cap genes devoid of inverted terminal repeat (ITR) sequences, a vector plasmid harboring the therapeutic gene between ITRs, and an adenovirus helper plasmid expressing E2A, virus-associated (VA) RNA, and E4orf6. Transient cotransfection is the major limitation for scale-up of rAAV production. Since rAAV can be purified using column chromatography, which can result in highly purified rAAV while eliminating other contaminating viruses, some efforts were made to develop rAAV production systems by using recombinant mammalian viruses such as adenovirus (10) or herpes virus (4) which do not rely on the plasmid transfection and therefore may be amenable to scale-up production. Recombinant baculoviruses based on the Autographa californica nuclear polyhedrosis virus are widely employed for production of heterologous proteins in cultured insect cells. The highly active, late A. californica nuclear polyhedrosis virus promoters, such as polyhedrin and p10 promoters, regulate the expression of heterologous proteins, resulting in large amounts of foreign proteins. Insect cells may be grown in suspension cultures in volumes ranging from shake flasks of sizes from, e.g., 50 to 400 ml, up to commercial-size bioreactors, e.g., 1,000 liters and larger. Recently, we described a highly scalable and efficient method for packaging rAAV2 in insect cells by use of baculovirus expression vectors (31). The ease of scale-up production is perhaps the most attractive feature of this production system. Infection of insect cells in suspension culture with recombinant baculoviruses eliminates the transfection process. Standard downstream processing to recover rAAV, such as tangential flow filtration and column chromatography, is readily applied. In addition to vectors derived from serotype 2, other serotypes, utilizing different cell surface receptors, constitute a vector set from which an appropriate vector can be selected for a specific application. AAV5 is the most divergent Dependovirus characterized (2), and type 5 AAV vectors have desirable properties that differ from other serotype vectors. AAV5 utilizes different receptors from other serotypes (14, 30), and rAAV5 has demonstrated different tropism from AAV2 (5), thus making it worthwhile to establish a method to produce rAAV5 in insect cells. AAV is a member of the family Parvoviridae. The genome of AAV is a linear, single-stranded DNA of 4.7 kb in length. The ITRs flank the unique coding sequences for the nonstructural replication initiator proteins, Rep, and the structural capsid proteins, VP. The ITRs serve as origins of DNA replication and may also function as the packaging signal. Type 2 Rep78 is generated by the p5 promoter, while Rep68 is translated from spliced mRNA from the p5 promoter. The small Rep polypeptides Rep52 and Rep40 are expressed by the p19 promoter with nonspliced or spliced mRNA. The p40 promoter regulates expression of capsid proteins VP1, VP2, and VP3. Alternate usage of two splice sites and translation of VP2 at a non-AUG codon results in a stoichiometry of 1:1:10 of VP1, VP2, and VP3. Both p5 proteins Rep78 and Rep68 are AAV origin binding proteins, and the presence of either is required for AAV DNA replication and processing replicative intermediates of the virus DNA (13). Also, either Rep52 or Rep40 is necessary for packaging the single-stranded, linear virion genome into preformed empty capsids (17). The transcriptional map of type 5 AAV differs from that of type 2; the p7 promoter or p19 promoter transcribes mRNA for Rep78 or Rep52. Type 5 AAV does not encode the spliced form of Rep polypeptides Rep68 and Rep40 (25). Structural protein VP1 is a minor constituent in the AAV capsid. But the VP1-unique portion of approximately 140 amino acid residues is highly conserved among different serotypes and has a phospholipase A2 motif. The YXGGX and HDXXY motifs (where X is any amino acid residue) in phospholipase A2 indicate the catalytic site and Ca2+ binding loop, respectively (see Fig. Fig.3A).3A). Enzymes classified into the secretory phospholipase A2 family hydrolyze the ester bond at the 2-acyl ester position of glycerophospholipids in the presence of Ca2+ and are involved in many aspects of cellular pathways, such as lipid membrane metabolism and signal transduction pathways (1, 21). The VP1-unique portion of parvovirus is required for transfer of the virus from late endosomes to the nucleus (36). A mutant virus lacking the VP1-unique portion or the activity of phospholipase is not processed properly, and thus no virus or vector genes are expressed. FIG. 3. (A) Chimeric VP genes constructed. The portions derived from type 2 are indicated in gray, while those from type 5 are in white. The common portions are indicated in black. The phospholipase A2 motifs are shaded. The YXGGX and HDXXY motifs (where X is ... In the present study, we describe a rAAV5 production system based on recombinant baculovirus and insect cells. In order to achieve high production levels of rAAV5 particles, we replaced a portion of the VP1 polypeptide with the corresponding portion of type 2. The VP1 substitution did not alter the tropism of rAAV5, which behaved indistinguishably from rAAV5 with wild-type VP1. In an attempt to improve the yields of rAAV5 particles, we used type 1 Rep52 instead of type 5, which resulted in the production of more than 5 × 104 vector genomes (vg) per insect cell.
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517. Type 1 Rep52 Is Superior to Authentic Rep52 for Producing Recombinant Adeno-Associated Virus Type 5 in Insect Cells
Molecular Therapy, 2005Co-Authors: Masashi Urabe, Takayo Nakakura, Hiroaki Mizukami, Akihiro Kume, Robert M Kotin, Keiya OzawaAbstract:Top of pageAbstract The recently described method for producing recombinant adeno-associated virus (rAAV) type 2 in insect cells facilitates scale-up by using suspension cell cultures and baculovirus expression vectors (Hum Gene Ther 13:1935-1943, 2002). AAV5 is one of the most divergent Dependovirus characterized and has been shown to utilize different receptors than AAV2 for example, and has demonstrated different tissue tropism from other serotype rAAVs. In addition to vectors derived from serotype 2, other serotypes constitute a vector set from which an optimal one can be selected for specific applications. Thus, we established a method to generate rAAV5 in insect cells. The current process requires triple infection with recombinant baculoviruses that provide the following: 1. AAV structural proteins that form the virus capsid (VP 1, 2, 3); 2. two of the AAV non-structural proteins for replication and encapsidation (Rep 78 and Rep 52); and 3. the AAV vector DNA which contain the gene of interest flanked by the AAV origins of replication (ITRs). Compared to other rAAV serotypes produced in insect cells, the rAAV5 yield per cell was substantially lower and we tested some modifications of the system. The initial Rep baculovirus drove type 5 Rep72 expression with a truncated promoter for the immediate-early 1 gene of Orgyia pseudotsugata nuclear polyhedrosis virus. The titers of the Rep baculovirus were relatively lower than others. We constructed Rep baculoviruses with a series of truncated p10 promoters for Rep78 and selected one that could produce rAAV5 at a high titer and propagate well. Rep52 or small Rep protein packages AAV genome into preformed empty capsids. We examined Rep52 of other serotypes, 1, 2, 3, 4 for generation of rAAV5 particles. The titer of rAAV5-GFP produced with type 1, 2, 3, or 4 small Rep was 56,000|[plusmn]|3,200, 41,000|[plusmn]|18,900, 42,000|[plusmn]|7,300, or 39,000|[plusmn]|3,500 particles per Sf9 cell while rAAV5-GFP produced by authentic Rep52 was 13,500|[plusmn]|3,200. The rAAV5-GFP produced with either serotype small Rep has an equal ratio of nucleic acid to capsid protein assessed by real-time PCR quantification and silver staining of purified rAAV5 particles. Also, rAAV5-GFP produced with either combination of large and small Rep proteins, transduced the simian Cos cell line with similar efficiency. The analysis by cesium density gradient of insect-cell lysates indicated that approximately 50% of all capsids contained the vector genome. These results indicate that heteroserotypic small Rep polypeptide is able to substitute for AAV5 small Rep and package AAV vector genome with type 5 in Sf9 cells and the new Rep baculovirus expressing type 5 Rep78 under the control of a truncated p10 promoter and type 1 Rep52 will contribute to the development of more efficient production of rAAV5 in insect cells.