scholarly journals Addition of Six-His-Tagged Peptide to the C Terminus of Adeno-Associated Virus VP3 Does Not Affect Viral Tropism or Production

2002 ◽  
Vol 76 (23) ◽  
pp. 12023-12031 ◽  
Author(s):  
Huang-Ge Zhang ◽  
Jinfu Xie ◽  
Igor Dmitriev ◽  
Elena Kashentseva ◽  
David T. Curiel ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Affinity Purification ◽  
High Titer ◽  
Plasmid Vector ◽  
Nitrilotriacetic Acid ◽  
Start Codon ◽  
Wild Type ◽  
Adeno Associated Virus ◽  
Viral Tropism ◽  
His Tag

ABSTRACT Production of large quantities of recombinant adeno-associated virus (AAV) is difficult and not cost-effective. To overcome this problem, we have explored the feasibility of creating a recombinant AAV encoding a 6×His tag on the VP3 capsid protein. We generated a plasmid vector containing a six-His (6×His)-tagged AAV VP3. A second plasmid vector was generated that contained the full-length AAV capsid capable of producing VP1 and VP2, but not VP3 due to a mutation at position 2809 that encodes the start codon for VP3. These plasmids, necessary for production of AAV, were transfected into 293 cells to generate a 6×His-tagged VP3mutant recombinant AAV. The 6×His-tagged VP3 did not affect the formation of AAV virus, and the physical properties of the 6×His-modified AAV were equivalent to those of wild-type particles. The 6×His-tagged AAV did not affect the production titer of recombinant AAV and could be used to purify the recombinant AAV using an Ni-nitrilotriacetic acid column. Addition of the 6×His tag did not alter the viral tropism compared to wild-type AAV. These observations demonstrate the feasibility of producing high-titer AAV containing a 6×His-tagged AAV VP3 capsid protein and to utilize the 6×His-tagged VP3 capsid to achieve high-affinity purification of this recombinant AAV.

scholarly journals Relevance of Assembly-Activating Protein for Adeno-associated Virus Vector Production and Capsid Protein Stability in Mammalian and Insect Cells

2017 ◽  
Vol 91 (20) ◽  
Author(s):  
Stefanie Grosse ◽  
Magalie Penaud-Budloo ◽  
Anne-Kathrin Herrmann ◽  
Kathleen Börner ◽  
Julia Fakhiri ◽  
...  
Keyword(s):  
Protein Stability ◽  
Capsid Protein ◽  
Insect Cells ◽  
Critical Factor ◽  
Capsid Proteins ◽  
Wild Type ◽  
Particle Assembly ◽  
Adeno Associated Virus ◽  
Recombinant Vectors

ABSTRACT The discovery that adeno-associated virus 2 (AAV2) encodes an eighth protein, called assembly-activating protein (AAP), transformed our understanding of wild-type AAV biology. Concurrently, it raised questions about the role of AAP during production of recombinant vectors based on natural or molecularly engineered AAV capsids. Here, we show that AAP is indeed essential for generation of functional recombinant AAV2 vectors in both mammalian and insect cell-based vector production systems. Surprisingly, we observed that AAV2 capsid proteins VP1 to -3 are unstable in the absence of AAP2, likely due to rapid proteasomal degradation. Inhibition of the proteasome led to an increase of intracellular VP1 to -3 but neither triggered assembly of functional capsids nor promoted nuclear localization of the capsid proteins. Together, this underscores the crucial and unique role of AAP in the AAV life cycle, where it rapidly chaperones capsid assembly, thus preventing degradation of free capsid proteins. An expanded analysis comprising nine alternative AAV serotypes (1, 3 to 9, and rh10) showed that vector production always depends on the presence of AAP, with the exceptions of AAV4 and AAV5, which exhibited AAP-independent, albeit low-level, particle assembly. Interestingly, AAPs from all 10 serotypes could cross-complement AAP-depleted helper plasmids during vector production, despite there being distinct intracellular AAP localization patterns. These were most pronounced for AAP4 and AAP5, congruent with their inability to rescue an AAV2/AAP2 knockout. We conclude that AAP is key for assembly of genuine capsids from at least 10 different AAV serotypes, which has implications for vectors derived from wild-type or synthetic AAV capsids. IMPORTANCE Assembly of adeno-associated virus 2 (AAV2) is regulated by the assembly-activating protein (AAP), whose open reading frame overlaps with that of the viral capsid proteins. As the majority of evidence was obtained using virus-like particles composed solely of the major capsid protein VP3, AAP's role in and relevance for assembly of genuine AAV capsids have remained largely unclear. Thus, we established a trans-complementation assay permitting assessment of AAP functionality during production of recombinant vectors based on complete AAV capsids and derived from any serotype. We find that AAP is indeed a critical factor not only for AAV2, but also for generation of vectors derived from nine other AAV serotypes. Moreover, we identify a new role of AAP in maintaining capsid protein stability in mammalian and insect cells. Thereby, our study expands our current understanding of AAV/AAP biology, and it concomitantly provides insights into the importance of AAP for AAV vector production.

scholarly journals High-Titer, Wild-Type Free Recombinant Adeno-Associated Virus Vector Production Using Intron-Containing Helper Plasmids

2000 ◽  
Vol 74 (24) ◽  
pp. 11456-11463 ◽  
Author(s):  
Lei Cao ◽  
Yuhong Liu ◽  
Matthew J. During ◽  
Weidong Xiao
Keyword(s):  
Gene Expression ◽  
High Titer ◽  
Infectious Clone ◽  
Wild Type ◽  
Helper Plasmid ◽  
Intron Insertion ◽  
Adeno Associated Virus ◽  
Vector Systems ◽  
Simplex Virus ◽  
High Level

ABSTRACT Recombinant adeno-associated virus (rAAV) is capable of directing long-term, high-level transgene expression without destructive cell-mediated immune responses. However, traditional packaging methods for rAAV vectors are generally inefficient and contaminated with replication-competent AAV (rcAAV) particles. Although wild-type AAV is not associated with any known human diseases, contaminating rcAAV particles may affect rAAV gene expression and are an uncontrolled variable in many AAV gene transfer studies. In the current study, a novel strategy was designed to both optimize AAV rep gene expression and increase vector yield, as well as simultaneously to diminish the potential of generating rcAAV particles from the helper plasmid. The strategy is based on the insertion of an additional intron in the AAV genome. In the AAV infectious clone, the intron insertion had no effects on the properties of Rep proteins expressed. Normal levels of both Rep and Cap proteins were expressed, and the replication of the AAV genome was not impaired. However, the generation of infectious rcAAV particles using intronized AAV helper was greatly diminished, which was due to the oversized AAV genome caused by the insertion of the artificial introns. Moreover, the rAAV packaging was significantly improved with the appropriate choice of intron and insertion position. The intron is another element that can regulate therep and cap gene expression from the helper plasmid. This study provides for a novel AAV packaging system which is highly versatile and efficient. It can not only be combined with other AAV packaging systems, including rep-containing cell lines and herpes simplex virus hybrid packaging methods, but also be used in other vector systems as well.

scholarly journals Proteomics of HCV virions reveals an essential role for the nucleoporin Nup98 in virus morphogenesis

2016 ◽  
Vol 113 (9) ◽  
pp. 2484-2489 ◽  
Author(s):  
Marion Lussignol ◽  
Martina Kopp ◽  
Kelly Molloy ◽  
Gema Vizcay-Barrena ◽  
Roland A. Fleck ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Culture Media ◽  
Nonstructural Protein ◽  
Affinity Purification ◽  
High Titer ◽  
Functional Characterization ◽  
Buoyant Density ◽  
Protein Composition ◽  
Cellular Proteins ◽  
Enveloped Virus

Hepatitis C virus (HCV) is a unique enveloped virus that assembles as a hybrid lipoviral particle by tightly interacting with host lipoproteins. As a result, HCV virions display a characteristic low buoyant density and a deceiving coat, with host-derived apolipoproteins masking viral epitopes. We previously described methods to produce high-titer preparations of HCV particles with tagged envelope glycoproteins that enabled ultrastructural analysis of affinity-purified virions. Here, we performed proteomics studies of HCV isolated from culture media of infected hepatoma cells to define viral and host-encoded proteins associated with mature virions. Using two different affinity purification protocols, we detected four viral and 46 human cellular proteins specifically copurifying with extracellular HCV virions. We determined the C terminus of the mature capsid protein and reproducibly detected low levels of the viral nonstructural protein, NS3. Functional characterization of virion-associated host factors by RNAi identified cellular proteins with either proviral or antiviral roles. In particular, we discovered a novel interaction between HCV capsid protein and the nucleoporin Nup98 at cytosolic lipid droplets that is important for HCV propagation. These results provide the first comprehensive view to our knowledge of the protein composition of HCV and new insights into the complex virus–host interactions underlying HCV infection.

scholarly journals Transcriptional Analysis of the Adeno-Associated Virus Integration Site

2009 ◽  
Vol 83 (23) ◽  
pp. 12512-12525 ◽  
Author(s):  
Nathalie Dutheil ◽  
Els Henckaerts ◽  
Erik Kohlbrenner ◽  
R. Michael Linden
Keyword(s):  
Transcriptional Activity ◽  
Integration Site ◽  
Regulatory Elements ◽  
Transcriptional Analysis ◽  
Start Codon ◽  
Adeno Associated Virus ◽  
Site Specific ◽  
Complex Interactions ◽  
Site Specific Integration ◽  
Virus Integration

ABSTRACT The nonpathogenic human adeno-associated virus type 2 (AAV-2) has adopted a unique mechanism to site-specifically integrate its genome into the human MBS85 gene, which is embedded in AAVS1 on chromosome 19. The fact that AAV has evolved to integrate into this ubiquitously transcribed region and that the chromosomal motifs required for integration are located a few nucleotides upstream of the translation initiation start codon of MBS85 suggests that the transcriptional activity of MBS85 might influence site-specific integration and thus might be involved in the evolution of this mechanism. In order to begin addressing this question, we initiated the characterization of the human MBS85 promoter region and compared its transcriptional activity to that of the AAV-2 p5 promoter. Our results clearly indicate that AAVS1 is defined by a complex transcriptional environment and that the MBS85 promoter shares key regulatory elements with the viral p5 promoter. Furthermore, we provide evidence for bidirectional MBS85 promoter activity and demonstrate that the minimal motifs required for AAV site-specific integration are present in the 5′ untranslated region of the gene and play a posttranscriptional role in the regulation of MBS85 expression. These findings should provide a framework to further elucidate the complex interactions between the virus and its cellular host in this unique pathway to latency.

Induction of an Antitumoral Immune Response by Wild-Type Adeno-Associated Virus Type 2 in an In Vivo Model of Pancreatic Carcinoma

Pancreas ◽  
2007 ◽  
Vol 35 (1) ◽  
pp. 63-72 ◽  
Author(s):  
Sven Eisold ◽  
Jan Schmidt ◽  
Eduard Ryschich ◽  
Michael Gock ◽  
Ernst Klar ◽  
...  
Keyword(s):  
Immune Response ◽  
Pancreatic Carcinoma ◽  
Virus Type ◽  
In Vivo Model ◽  
Wild Type ◽  
Adeno Associated Virus

Influence of homologous phasins (PhaP) on PHA accumulation and regulation of their expression by the transcriptional repressor PhaR in Ralstonia eutropha H16

Microbiology ◽  
2005 ◽  
Vol 151 (3) ◽  
pp. 825-833 ◽  
Author(s):  
Markus Pötter ◽  
Helena Müller ◽  
Alexander Steinbüchel
Keyword(s):  
Ralstonia Eutropha ◽  
Current Model ◽  
Transcriptional Repressor ◽  
Start Codon ◽  
Wild Type ◽  
Mobility Shift ◽  
Intergenic Regions ◽  
Gel Mobility Shift ◽  
Dnasei Footprinting ◽  
Similar Growth

Phasins play an important role in the formation of poly(3-hydroxybutyrate) [poly(3HB)] granules and affect their size. Recently, three homologues of the phasin protein PhaP1 were identified in Ralstonia eutropha strain H16. The functions of PhaP2, PhaP3 and PhaP4 were examined by analysis of R. eutropha H16 deletion strains (ΔphaP1, ΔphaP2, ΔphaP3, ΔphaP4, ΔphaP12, ΔphaP123 and ΔphaP1234). When cells were grown under conditions permissive for poly(3HB) accumulation, the wild-type strain and all single-phasin negative mutants (ΔphaP2, ΔphaP3 and ΔphaP4), with the exception of ΔphaP1, showed similar growth and poly(3HB) accumulation behaviour, and also the size and number of the granules were identical. The single ΔphaP1 mutant and the ΔphaP12, ΔphaP123 and ΔphaP1234 mutants showed an almost identical growth behaviour; however, they accumulated poly(3HB) at a significantly lower level than wild-type and the single ΔphaP2, ΔphaP3 or ΔphaP4 mutants. Gel-mobility-shift assays and DNaseI footprinting experiments demonstrated the capability of the transcriptional repressor PhaR to bind to a DNA region +36 to +46 bp downstream of the phaP3 start codon. The protected sequence exhibited high similarity to the binding sites of PhaR upstream of phaP1, which were identified recently. In contrast, PhaR did not bind to the upstream or intergenic regions of phaP2 and phaP4, thus indicating that the expression of these two phasins is regulated in a different way. Our current model for the regulation of phasins in R. eutropha strain H16 was extended and confirmed.

scholarly journals Adeno-associated virus Rep proteins antagonize phosphatase PP1 to counteract KAP1 repression of the latent viral genome

2018 ◽  
Vol 115 (15) ◽  
pp. E3529-E3538 ◽  
Author(s):  
Sarah Smith-Moore ◽  
Stuart J. D. Neil ◽  
Cornel Fraefel ◽  
R. Michael Linden ◽  
Mathieu Bollen ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Helper Virus ◽  
Protein Phosphatase 1 ◽  
Wild Type ◽  
Fha Domain ◽  
Adeno Associated Virus ◽  
Histone 3 ◽  
Rep Proteins ◽  
Cellular Factors

Adeno-associated virus (AAV) is a small human Dependovirus whose low immunogenicity and capacity for long-term persistence have led to its widespread use as vector for gene therapy. Despite great recent successes in AAV-based gene therapy, further improvements in vector technology may be hindered by an inadequate understanding of various aspects of basic AAV biology. AAV is unique in that its replication is largely dependent on a helper virus and cellular factors. In the absence of helper virus coinfection, wild-type AAV establishes latency through mechanisms that are not yet fully understood. Challenging the currently held model for AAV latency, we show here that the corepressor Krüppel-associated box domain-associated protein 1 (KAP1) binds the latent AAV2 genome at the rep ORF, leading to trimethylation of AAV2-associated histone 3 lysine 9 and that the inactivation of KAP1 repression is necessary for AAV2 reactivation and replication. We identify a viral mechanism for the counteraction of KAP1 in which interference with the KAP1 phosphatase protein phosphatase 1 (PP1) by the AAV2 Rep proteins mediates enhanced phosphorylation of KAP1-S824 and thus relief from KAP1 repression. Furthermore, we show that this phenomenon involves recruitment of the NIPP1 (nuclear inhibitor of PP1)–PP1α holoenzyme to KAP1 in a manner dependent upon the NIPP1 FHA domain, identifying NIPP1 as an interaction partner for KAP1 and shedding light on the mechanism through which PP1 regulates cellular KAP1 activity.

scholarly journals A novel murine model of fetal and neonatal alloimmune thrombocytopenia: response to intravenous IgG therapy

Blood ◽  
2006 ◽  
Vol 107 (7) ◽  
pp. 2976-2983 ◽  
Author(s):  
Heyu Ni ◽  
Pingguo Chen ◽  
Christopher M. Spring ◽  
Ebrahim Sayeh ◽  
John W. Semple ◽  
...  
Keyword(s):  
High Titer ◽  
Maternal Antibodies ◽  
Platelet Glycoprotein ◽  
Wild Type ◽  
Wild Type Male ◽  
Glycoprotein Iiia ◽  
Life Threatening ◽  
Β3 Integrin ◽  
Pathogenic Antibodies ◽  
Alloimmune Thrombocytopenia

AbstractFetal and neonatal alloimmune thrombo cytopenia (FNAITP) is a life-threatening bleeding disorder caused by maternal antibodies directed against fetal platelet antigens. The immunoreactive epitopes in FNAITP are primarily located in the extracellular regions of the platelet glycoprotein IIIa (β3 integrin). Here we have established a novel animal model of FNAITP using β3 integrin–deficient (β3-/-) mice. We demonstrated first that these mice are immunoresponsive to β3 integrin; β3-/- mice transfused with wild-type platelets generated specific anti–β3 antibodies which were able to induce thrombocytopenia in wild-type mice. Subsequently, β3-/- female mice (both naive and immunized) were bred with wild-type male mice to recapitulate the features of FNAITP. The titer of generated maternal antibodies correlated with the severity of FNAITP. High titer maternal anti–β3 anti-bodies caused severe fetal thrombocytopenia, intracranial hemorrhage, and even miscarriage. Furthermore, maternal administration of intravenous immunoglobulin G (IgG) ameliorated FNAITP and down-regulated pathogenic antibodies in both the maternal and fetal circulations.

scholarly journals Foamy Virus Integration

2004 ◽  
Vol 78 (5) ◽  
pp. 2472-2477 ◽  
Author(s):  
Thomas Juretzek ◽  
Teresa Holm ◽  
Kathleen Gärtner ◽  
Sylvia Kanzler ◽  
Dirk Lindemann ◽  
...  
Keyword(s):  
Infectious Virus ◽  
High Titer ◽  
Foamy Virus ◽  
Wild Type ◽  
Foamy Viruses ◽  
Unintegrated Dna ◽  
Particle Export ◽  
The Right ◽  
Virus Integration ◽  
Transcription And Replication

ABSTRACT It had been suggested that during integration of spumaretroviruses (foamy viruses) the right (U5) end of the cDNA is processed, while the left (U3) remains uncleaved. We confirmed this hypothesis by sequencing two-long terminal repeat (LTR) circle junctions of unintegrated DNA. Based on an infectious foamy virus molecular clone, a set of constructs harboring mutations at the 5′ end of the U3 region in the 3′ LTR was analyzed for particle export, reverse transcription, and replication. Following transient transfection some mutants were severely impaired in generating infectious virus, while others replicated almost like the wild type. The replication competence of the mutants was unrelated to the cleavability of the newly created U3 end. This became obvious with two mutants both belonging to the high-titer type. One mutant containing a dinucleotide artificially transferred from the right to the left end was trimmed upon integration, while another one with an unrelated dinucleotide in that place was not. The latter construct in particular showed that the canonical TG motif at the beginning of the provirus is not essential for foamy virus integration.

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