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 Surface-Exposed Adeno-Associated Virus Vp1-NLS Capsid Fusion Protein Rescues Infectivity of Noninfectious Wild-Type Vp2/Vp3 and Vp3-Only Capsids but Not That of Fivefold Pore Mutant Virions

2007 ◽  
Vol 81 (15) ◽  
pp. 7833-7843 ◽  
Author(s):  
Joshua C. Grieger ◽  
Jarrod S. Johnson ◽  
Brittney Gurda-Whitaker ◽  
Mavis Agbandje-McKenna ◽  
R. Jude Samulski
Keyword(s):  
Conformational Changes ◽  
Structural Changes ◽  
Wild Type ◽  
Dot Blot ◽  
Adeno Associated Virus ◽  
N Terminus ◽  
Localization Signal ◽  
Significant Effort

ABSTRACT Over the past 2 decades, significant effort has been dedicated to the development of adeno-associated virus (AAV) as a vector for human gene therapy. However, understanding of the virus with respect to the functional domains of the capsid remains incomplete. In this study, the goal was to further examine the role of the unique Vp1 N terminus, the N terminus plus the recently identified nuclear localization signal (NLS) (J. C. Grieger, S. Snowdy, and R. J. Samulski, J. Virol 80:5199-5210, 2006), and the virion pore at the fivefold axis in infection. We generated two Vp1 fusion proteins (Vp1 and Vp1NLS) linked to the 8-kDa chemokine domain of rat fractalkine (FKN) for the purpose of surface exposure upon assembly of the virion, as previously described (K. H. Warrington, Jr., O. S. Gorbatyuk, J. K. Harrison, S. R. Opie, S. Zolotukhin, and N. Muzyczka, J. Virol 78:6595-6609, 2004). The unique Vp1 N termini were found to be exposed on the surfaces of these capsids and maintained their phospholipase A2 (PLA2) activity, as determined by native dot blot Western and PLA2 assays, respectively. Incorporation of the fusions into AAV type 2 capsids lacking a wild-type Vp1, i.e., Vp2/Vp3 and Vp3 capsid only, increased infectivity by 3- to 5-fold (Vp1FKN) and 10- to 100-fold (Vp1NLSFKN), respectively. However, the surface-exposed fusions did not restore infectivity to AAV virions containing mutations at a conserved leucine (Leu336Ala, Leu336Cys, or Leu336Trp) located at the base of the fivefold pore. EM analyses suggest that Leu336 may play a role in global structural changes to the virion directly impacting downstream conformational changes essential for infectivity and not only have local effects within the pore, as previously suggested.

scholarly journals The Role of Electrostatic Interactions on Klentaq1 Insight for Domain Separation

2012 ◽  
Vol 6 ◽  
pp. BBI.S9390 ◽  
Author(s):  
Santi Nurbaiti ◽  
Muhamad A. Martoprawiro ◽  
Akhmaloka ◽  
Rukman Hertadi
Keyword(s):  
Protein Stability ◽  
Electrostatic Interactions ◽  
Thermal Adaptation ◽  
Atomic Level ◽  
Salt Bridges ◽  
Wild Type ◽  
Interdomain Interactions ◽  
The Relationship ◽  
Domain Separation

We investigated the relationship between the thermostability of Klentaq1 and factors stabilizing interdomain interactions. When thermal adaptation of Klentaq1 was analyzed at the atomic level, the protein was stable at 300 and 350 K. It gradually unfolded at 373 K and almost spontaneously unfolded at 400 K. Domain separation was induced by disrupting electrostatic interactions in two salt bridges formed by Lys354-Glu445 and Asp371-Arg435 on the interface domain. The role of these interactions in protein stability was evaluated by comparing free energy solvation (ΔΔGsolv) between wild type and mutants. Substitution of Asp371 by Glu or Asn, and also Glu445 by Asn resulted in a positive value of ΔΔGsolv, suggesting that mutations destabilized the protein structure. Nevertheless, substitution of Glu445 by Asp gave a negative value to ΔΔGsolv reflecting increasing protein stability. Our results demonstrate that interactions at the interface domains of Klentaq1 are essential factors correlated with the Klentaq1 thermostability.

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 Separate Basic Region Motifs within the Adeno-Associated Virus Capsid Proteins Are Essential for Infectivity and Assembly

2006 ◽  
Vol 80 (11) ◽  
pp. 5199-5210 ◽  
Author(s):  
Joshua C. Grieger ◽  
Stephen Snowdy ◽  
Richard J. Samulski
Keyword(s):  
Nuclear Periphery ◽  
Capsid Proteins ◽  
Nuclear Localization Sequence ◽  
Mutant Form ◽  
Wild Type ◽  
Nuclear Targeting ◽  
Adeno Associated Virus ◽  
Gene Therapy Vector ◽  
Virion Assembly ◽  
Transfected Cells

ABSTRACT Adeno-associated virus (AAV) is gaining momentum as a gene therapy vector for human applications. However, there remain impediments to the development of this virus as a vector. One of these is the incomplete understanding of the biology of the virus, including nuclear targeting of the incoming virion during initial infection, as well as assembly of progeny virions from structural components in the nucleus. Toward this end, we have identified four basic regions (BR) on the AAV2 capsid that represent possible nuclear localization sequence (NLS) motifs. Mutagenesis of BR1 (120QAKKRVL126) and BR2 (140PGKKRPV146) had minor effects on viral infectivity (∼4- and ∼10-fold, respectively), whereas BR3 (166PARKRLN172) and BR4 (307RPKRLN312) were found to be essential for infectivity and virion assembly, respectively. Mutagenesis of BR3, which is located in Vp1 and Vp2 capsid proteins, does not interfere with viral production or trafficking of intact AAV capsids to the nuclear periphery but does inhibit transfer of encapsidated DNA into the nucleus. Substitution of the canine parvovirus NLS rescued the BR3 mutant to wild-type (wt) levels, supporting the role of an AAV NLS motif. In addition, rAAV2 containing a mutant form of BR3 in Vp1 and a wt BR3 in Vp2 was found to be infectious, suggesting that the function of BR3 is redundant between Vp1 and Vp2 and that Vp2 may play a role in infectivity. Mutagenesis of BR4 was found to inhibit virion assembly in the nucleus of transfected cells. This affect was not completely due to the inefficient nuclear import of capsid subunits based on Western blot analysis. In fact, aberrant capsid foci were observed in the cytoplasm of transfected cells, compared to the wild type, suggesting a defect in early viral assembly or trafficking. Using three-dimensional structural analysis, the lysine- and arginine-to-asparagine change disrupts hydrogen bonding between these basic residues and adjacent beta strand glutamine residues that may prevent assembly of intact virions. Taken together, these data support that the BR4 domain is essential for virion assembly. Each BR was also found to be conserved in serotypes 1 to 11, suggesting that these regions are significant and function similarly in each serotype. This study establishes the importance of two BR motifs on the AAV2 capsid that are essential for infectivity and virion assembly.

scholarly journals A Zinc Ion Controls Assembly and Stability of the Major Capsid Protein of Rotavirus

2003 ◽  
Vol 77 (6) ◽  
pp. 3595-3601 ◽  
Author(s):  
Inge Erk ◽  
Jean-Claude Huet ◽  
Mariela Duarte ◽  
Stéphane Duquerroy ◽  
Felix Rey ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Major Capsid Protein ◽  
Chelating Agent ◽  
Zinc Ion ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
High Concentration ◽  
Mutant Proteins

ABSTRACT The recent determination of the crystal structure of VP6, the major capsid protein of rotavirus, revealed a trimer containing a central zinc ion coordinated by histidine 153 from each of the three subunits. The role of the zinc ion in the functions of VP6 was investigated by site-directed mutagenesis. The mutation of histidine 153 into a serine (H153S and H153S/S339H) did not prevent the formation of VP6 trimers. At pH <7.0, about the pK of histidine, wild-type and mutated VP6 proteins display similar properties, giving rise to identical tubular and spherical assemblies. However, at pH >7.0, histidine 153 mutant proteins did not assemble into the characteristic 45-nm-diameter tubes, in contrast to wild-type VP6. These observations showed that under conditions in which histidine residues are not charged, the properties of VP6 depended on the presence of the centrally coordinated zinc atom in the trimer. Indeed, wild-type VP6 depleted of the zinc ion by a high concentration (100 mM) of a metal-chelating agent behaved like the H153 mutant proteins. The susceptibility of wild-type VP6 to proteases is greatly increased in the absence of zinc. NH2-terminal sequencing of the proteolytic fragments showed that they all contained the β-sheet-rich VP6 head domain, which appeared to be less sensitive to protease activity than the α-helical basal domain. Finally, the mutant proteins assembled well on cores, as demonstrated by both electron microscopy and rescue of transcriptase activity. Zinc is thus not necessary for the transcription activity. All of these observations suggest that, in solution, VP6 trimers present a structural flexibility that is controlled by the presence of a zinc ion.

scholarly journals A Heterologous Coiled Coil Can Substitute for Helix I of the Sindbis Virus Capsid Protein

2003 ◽  
Vol 77 (15) ◽  
pp. 8345-8353 ◽  
Author(s):  
Rushika Perera ◽  
Chanakha Navaratnarajah ◽  
Richard J. Kuhn
Keyword(s):  
Capsid Protein ◽  
Sindbis Virus ◽  
Coiled Coil ◽  
Wild Type ◽  
Transcription Activator ◽  
Particle Assembly ◽  
Virus Capsid ◽  
Assembly Pathway ◽  
Trimer Formation ◽  
Virus Capsid Protein

ABSTRACT Alphavirus core assembly proceeds along an assembly pathway involving a dimeric assembly intermediate. Several regions of the alphavirus capsid protein have been implicated in promoting and stabilizing this dimerization, including a putative heptad repeat sequence named helix I. This sequence, which spans residues 38 to 55 of the Sindbis virus capsid protein, was implicated in stabilizing dimeric contacts initiated through the C-terminal two-thirds of the capsid protein and nucleic acid. The studies presented here demonstrate that helix I can be functionally replaced by the corresponding sequence of a related alphavirus, western equine encephalitis virus, and also by an unrelated sequence from the yeast transcription activator, GCN4, that was previously shown to form a dimeric coiled coil. Replacing helix I with the entire leucine zipper domain of GCN4 (residues 250 to 281) produced a virus with the wild-type phenotype as determined by plaque assay and one-step growth analysis. However, replacement of helix I with a GCN4 sequence that favored trimer formation produced a virus that exhibited ∼40-fold reduction in virus replication compared to the wild-type Sindbis virus. Changing residues within the Sindbis virus helix I sequence to favor trimer formation also produced a virus with reduced replication. Peptides corresponding to helix I inhibited core-like particle assembly in vitro. On the basis of these studies, it is proposed that helix I favors capsid protein-capsid protein interactions through the formation of dimeric coiled-coil interactions and may stabilize assembly intermediates in the alphavirus nucleocapsid core assembly pathway.

scholarly journals The Chemokine CCL2 Is Required for Control of Murine Gastric Salmonella enterica Infection

2005 ◽  
Vol 73 (10) ◽  
pp. 6514-6522 ◽  
Author(s):  
R. William DePaolo ◽  
Rashida Lathan ◽  
Barrett J. Rollins ◽  
William J. Karpus
Keyword(s):  
Typhoid Fever ◽  
Salmonella Enterica ◽  
Critical Factor ◽  
Wild Type ◽  
Necrosis Factor Alpha ◽  
Factor Alpha ◽  
Vitro Analysis ◽  
In Vitro Analysis

ABSTRACT Salmonella enterica is a gram-negative intracellular pathogen that can cause a variety of diseases ranging from gastroenteritis to typhoid fever. The Typhimurium serotype causes gastroenteritis in humans; however, infection of mice results in an enteric fever that resembles human typhoid fever and has been used as a model for typhoid fever. The present study examined the role of the chemokine CCL2 in the control of Salmonella infection. Upon infection with salmonellae, mucosal expression of CCL2 is rapidly up-regulated, followed by systemic expression in the spleen. CCL2−/− mice became moribund earlier and had a higher rate of mortality compared to wild-type C57BL/6 mice. Moreover, CCL2−/− mice had significantly higher levels of bacteria in the liver compared to wild-type controls. Mucosal and serum interleukin-6 and tumor necrosis factor alpha levels were elevated in CCL2−/− mice compared to wild-type mice. In vitro analysis demonstrated that CCL2−/− macrophages infected with salmonellae resulted in dysregulated cytokine production compared to macrophages derived from wild-type mice. These data are the first to directly demonstrate CCL2 as a critical factor for immune responses and survival following S. enterica infection.

scholarly journals The Role of WWP1-Gag Interaction and Gag Ubiquitination in Assembly and Release of Human T-Cell Leukemia Virus Type 1

2007 ◽  
Vol 81 (18) ◽  
pp. 9769-9777 ◽  
Author(s):  
Gisela Heidecker ◽  
Patricia A. Lloyd ◽  
Ferri Soheilian ◽  
Kunio Nagashima ◽  
David Derse
Keyword(s):  
Leukemia Virus ◽  
Cell Leukemia ◽  
Wild Type ◽  
Particle Assembly ◽  
Cell Leukemia Virus Type ◽  
Hect Domain ◽  
Human T Cell ◽  
T Cell Leukemia Virus

ABSTRACT The PPPY motif in the matrix (MA) domain of human T-cell leukemia virus type 1 (HTLV-1) Gag associates with WWP1, a member of the HECT domain containing family of E3 ubiquitin ligases. Mutation of the PPPY motif arrests particle assembly at an early stage and abolishes ubiquitination of MA. Similar effects are seen when Gag is expressed in the presence of a truncated form of WWP1 that lacks the catalytically active HECT domain (C2WW). To understand the role of ubiquitination in budding, we mutated the four lysines in MA to arginines and identified lysine 74 as the unique site of ubiquitination. Virus-like particles produced by the K74R mutant did not contain ubiquitinated MA and showed a fourfold reduction in the release of infectious particles. Furthermore, the K74R mutation rendered assembly hypersensitive to C2WW inhibition; K74R Gag budding was inhibited at significantly lower levels of expression of C2WW compared with wild-type Gag. This finding indicates that the interaction between Gag and WWP1 is required for functions other than Gag ubiquitination. Additionally, we show that the PPPY− mutant Gag exerts a strong dominant-negative effect on the budding of wild-type Gag, further supporting the importance of recruitment of WWP1 to achieve particle assembly.

scholarly journals Alphavirus Nucleocapsid Protein Contains a Putative Coiled Coil α-Helix Important for Core Assembly

2001 ◽  
Vol 75 (1) ◽  
pp. 1-10 ◽  
Author(s):  
Rushika Perera ◽  
Katherine E. Owen ◽  
Timothy L. Tellinghuisen ◽  
Alexander E. Gorbalenya ◽  
Richard J. Kuhn
Keyword(s):  
Amino Acid ◽  
Capsid Protein ◽  
Coiled Coil ◽  
Terminal Region ◽  
Wild Type Virus ◽  
Wild Type ◽  
Particle Assembly ◽  
In Vitro Assembly ◽  
Α Helix

ABSTRACT The alphavirus nucleocapsid core is formed through the energetic contributions of multiple noncovalent interactions mediated by the capsid protein. This protein consists of a poorly conserved N-terminal region of unknown function and a C-terminal conserved autoprotease domain with a major role in virion formation. In this study, an 18-amino-acid conserved region, predicted to fold into an α-helix (helix I) and embedded in a low-complexity sequence enriched with basic and Pro residues, has been identified in the N-terminal region of the alphavirus capsid proteins. In Sindbis virus, helix I spans residues 38 to 55 and contains three conserved leucine residues, L38, L45, and L52, conforming to the heptad amino acid organization evident in leucine zipper proteins. Helix I consists of an N-terminally truncated heptad and two complete heptad repeats with β-branched residues and conserved leucine residues occupying the a andd positions of the helix, respectively. Complete or partial deletion of helix I, or single-site substitutions at the conserved leucine residues (L45 and L52), caused a significant decrease in virus replication. The mutant viruses were more sensitive to elevated temperature than wild-type virus. These mutant viruses also failed to accumulate cores in the cytoplasm of infected cells, although they did not have defects in protein translation or processing. Analysis of these mutants using an in vitro assembly system indicated that the majority were defective in core particle assembly. Furthermore, mutant proteins showed a trans-dominant negative phenotype in in vitro assembly reactions involving mutant and wild-type proteins. We propose that helix I plays a central role in the assembly of nucleocapsid cores through coiled coil interactions. These interactions may stabilize subviral intermediates formed through the interactions of the C-terminal domain of the capsid protein and the genomic RNA and contribute to the stability of the virion.

scholarly journals Adeno-Associated Virus Type 2 VP2 Capsid Protein Is Nonessential and Can Tolerate Large Peptide Insertions at Its N Terminus

2004 ◽  
Vol 78 (12) ◽  
pp. 6595-6609 ◽  
Author(s):  
Kenneth H. Warrington ◽  
Oleg S. Gorbatyuk ◽  
Jeffrey K. Harrison ◽  
Shaun R. Opie ◽  
Sergei Zolotukhin ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Virus Type ◽  
Fluorescent Protein ◽  
Amino Acid Sequences ◽  
Peptide Ligands ◽  
Full Length ◽  
Capsid Proteins ◽  
Adeno Associated Virus ◽  
Gene Therapy Vector

ABSTRACT Direct insertion of amino acid sequences into the adeno-associated virus type 2 (AAV) capsid open reading frame (cap ORF) is one strategy currently being developed for retargeting this prototypical gene therapy vector. While this approach has successfully resulted in the formation of AAV particles that have expanded or retargeted viral tropism, the inserted sequences have been relatively short, linear receptor binding ligands. Since many receptor-ligand interactions involve nonlinear, conformation-dependent binding domains, we investigated the insertion of full-length peptides into the AAV cap ORF. To minimize disruption of critical VP3 structural domains, we confined the insertions to residue 138 within the VP1-VP2 overlap, which has been shown to be on the surface of the particle following insertion of smaller epitopes. The insertion of coding sequences for the 8-kDa chemokine binding domain of rat fractalkine (CX3CL1), the 18-kDa human hormone leptin, and the 30-kDa green fluorescent protein (GFP) after residue 138 failed to lead to formation of particles due to the loss of VP3 expression. To test the ability to complement these insertions with the missing capsid proteins in trans, we designed a system for producing AAV vectors in which expression of one capsid protein is isolated and combined with the remaining two capsid proteins expressed separately. Such an approach allows for genetic modification of a specific capsid protein across its entire coding sequence leaving the remaining capsid proteins unaffected. An examination of particle formation from the individual components of the system revealed that genome-containing particles formed as long as the VP3 capsid protein was present and demonstrated that the VP2 capsid protein is nonessential for viral infectivity. Viable particles composed of all three capsid proteins were obtained from the capsid complementation groups regardless of which capsid proteins were supplied separately in trans. Significant overexpression of VP2 resulted in the formation of particles with altered capsid protein stoichiometry. The key finding was that by using this system we successfully obtained nearly wild-type levels of recombinant AAV-like particles with large ligands inserted after residue 138 in VP1 and VP2 or in VP2 exclusively. While insertions at residue 138 in VP1 significantly decreased infectivity, insertions at residue 138 that were exclusively in VP2 had a minimal effect on viral assembly or infectivity. Finally, insertion of GFP into VP1 and VP2 resulted in a particle whose trafficking could be temporally monitored by using confocal microscopy. Thus, we have demonstrated a method that can be used to insert large (up to 30-kDa) peptide ligands into the AAV particle. This system allows greater flexibility than current approaches in genetically manipulating the composition of the AAV particle and, in particular, may allow vector retargeting to alternative receptors requiring interaction with full-length conformation-dependent peptide ligands.

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