scholarly journals Mutational Analysis of Narrow Pores at the Fivefold Symmetry Axes of Adeno-Associated Virus Type 2 Capsids Reveals a Dual Role in Genome Packaging and Activation of Phospholipase A2 Activity

2005 ◽  
Vol 79 (4) ◽  
pp. 2528-2540 ◽  
Author(s):  
Svenja Bleker ◽  
Florian Sonntag ◽  
Jürgen A. Kleinschmidt
Keyword(s):  
Phospholipase A2 ◽  
Virus Type ◽  
Mutational Analysis ◽  
Pla2 Activity ◽  
Genome Packaging ◽  
Adeno Associated Virus ◽  
Conserved Residues ◽  
Axes Of Symmetry ◽  
Phospholipase A2 Activity

ABSTRACT Adeno-associated virus type 2 (AAV2) capsids show 12 pores at the fivefold axes of symmetry. We mutated amino acids which constitute these pores to investigate possible functions of these structures within the AAV2 life cycle. Mutants with alterations in conserved residues were impaired mainly in genome packaging or infectivity, whereas few mutants were affected in capsid assembly. The packaging phenotype was characterized by increased capsid-per-genome ratios. Analysis of capsid-associated DNA versus encapsidated DNA revealed that this observation was due to reduced and not partial DNA encapsidation. Most mutants with impaired infectivity showed a decreased capability to expose their VP1 N termini. As a consequence, the activation of phospholipase A2 (PLA2) activity, which is essential for efficient infection, was affected on intact capsids. In a few mutants, the exposure of VP1 N termini and the development of PLA2 activity were associated with enhanced capsid instability, which is obviously also deleterious for virus infection. Therefore, PLA2 activity seems to be required on intact capsids for efficient infection. In conclusion, these results suggest that the pores at the fivefold axes function not only as portals for AAV2 single-stranded DNA packaging but also as channels for presentation of the PLA2 domain on AAV2 virions during infection.

scholarly journals Impact of Capsid Conformation and Rep-Capsid Interactions on Adeno-Associated Virus Type 2 Genome Packaging

2006 ◽  
Vol 80 (2) ◽  
pp. 810-820 ◽  
Author(s):  
Svenja Bleker ◽  
Michael Pawlita ◽  
Jürgen A. Kleinschmidt
Keyword(s):  
Amino Acid ◽  
Capsid Protein ◽  
Amino Acid Exchange ◽  
Genome Packaging ◽  
Adeno Associated Virus ◽  
Rep Proteins ◽  
Axes Of Symmetry ◽  
Conserved Amino Acids ◽  
Acid Exchange

ABSTRACT Single-stranded genomes of adeno-associated virus (AAV) are packaged into preformed capsids. It has been proposed that packaging is initiated by interaction of genome-bound Rep proteins to the capsid, thereby targeting the genome to the portal of encapsidation. Here we describe a panel of mutants with amino acid exchanges in the pores at the fivefold axes of symmetry on AAV2 capsids with reduced packaging and reduced Rep-capsid interaction. Mutation of two threonines at the rim of the fivefold pore nearly completely abolished Rep-capsid interaction and packaging. This suggests a Rep-binding site at the highly conserved amino acids at or close to the pores formed by the capsid protein pentamers. A different mutant (P. Wu, W. Xiao, T. Conlon, J. Hughes, M. Agbandje-McKenna, T. Ferkol, T. Flotte, and N. Muzyczka, J. Virol. 74:8635-8647, 2000) with an amino acid exchange at the interface of capsid protein pentamers led to a complete block of DNA encapsidation. Analysis of the capsid conformation of this mutant revealed that the pores at the fivefold axes were occupied by VP1/VP2 N termini, thereby preventing DNA introduction into the capsid. Nevertheless, the corresponding capsids had more Rep proteins bound than wild-type AAV, showing that correct Rep interaction with the capsid depends on a defined capsid conformation. Both mutant types together support the conclusion that the pores at the fivefold symmetry axes are involved in genome packaging and that capsid conformation-dependent Rep-capsid interactions play an essential role in the packaging process.

scholarly journals Mutational Analysis of Adeno-Associated Virus Type 2 Rep68 Protein Endonuclease Activity on Partially Single-Stranded Substrates

2000 ◽  
Vol 74 (6) ◽  
pp. 2936-2942 ◽  
Author(s):  
Michael D. Davis ◽  
Jianwen Wu ◽  
Roland A. Owens
Keyword(s):  
Amino Acids ◽  
Virus Type ◽  
Active Site ◽  
Mutational Analysis ◽  
Covalent Attachment ◽  
Endonuclease Activity ◽  
Adeno Associated Virus ◽  
Mutant Proteins ◽  
Endonucleolytic Cleavage

ABSTRACT The endonuclease activity of the Rep68 and Rep78 proteins (Rep68/78) of adeno-associated virus type 2 (AAV) cuts at the terminal resolution site (trs) within the hairpin structure formed by the AAV inverted terminal repeats. Recent studies suggest that a DNA unwinding function of Rep68/78 may be required for endonuclease activity. We demonstrate that several mutant proteins which are endonuclease negative on a fully duplex hairpin substrate are endonuclease positive on a partially single-stranded hairpin substrate. Truncation analysis revealed that the endonuclease function is contained within the first 200 amino acids of Rep68/78. This endonucleolytic cleavage is believed to involve the covalent attachment of Rep68/78 to the trs via a phosphate-tyrosine linkage. A previous report (S. L. Walker, R. S. Wonderling, and R. A. Owens, J. Virol. 71:2722–2730, 1997) suggested that tyrosine 152 was part of the active site. We individually mutated each tyrosine within the first 200 amino acids of the Rep68 moiety of a maltose binding protein-Rep68/78 fusion protein to phenylalanine. Only mutation of tyrosine 156 resulted in a protein incapable of covalent attachment to a partially single-stranded hairpin substrate, suggesting that tyrosine 156 is part of the endonuclease active site.

scholarly journals The VP1 capsid protein of adeno-associated virus type 2 is carrying a phospholipase A2 domain required for virus infectivity

2002 ◽  
Vol 83 (5) ◽  
pp. 973-978 ◽  
Author(s):  
Anne Girod ◽  
Christiane E. Wobus ◽  
Zoltán Zádori ◽  
Martin Ried ◽  
Kristin Leike ◽  
...  
Keyword(s):  
Gene Expression ◽  
Phospholipase A2 ◽  
Capsid Protein ◽  
Virus Type ◽  
Early Gene ◽  
Virus Infectivity ◽  
Adeno Associated Virus ◽  
Unique Region ◽  
Early Gene Expression

The unique region of the VP1 protein of parvoviruses was proposed to contain a parvoviral phospholipase A2 (pvPLA2) motif. Here, PLA2 activity is shown in the unique region of adeno-associated virus type 2 (AAV-2) VP1 when expressed as an isolated domain in bacteria. Mutations in this region of the capsid protein strongly reduced the infectivity of mutant virions in comparison to wild-type AAV-2. This correlated with effects on the activity of PLA2. The mutations had no influence on capsid assembly, packaging of viral genomes into particles or binding to and entry into HeLa cells. However, a delayed onset and reduced amount of early gene expression, as measured by Rep immunofluorescence, was observed. These results suggest that pvPLA2 activity is required for a step following perinuclear accumulation of virions but prior to early gene expression.

Mutational analysis of the adeno-associated virus type 2 Rep68 protein helicase motifs.

1997 ◽  
Vol 71 (9) ◽  
pp. 6996-7004 ◽  
Author(s):  
S L Walker ◽  
R S Wonderling ◽  
R A Owens
Keyword(s):  
Virus Type ◽  
Mutational Analysis ◽  
Adeno Associated Virus

Biologically active Rep proteins of adeno-associated virus type 2 produced as fusion proteins in Escherichia coli.

1994 ◽  
Vol 68 (2) ◽  
pp. 797-804 ◽  
Author(s):  
J A Chiorini ◽  
M D Weitzman ◽  
R A Owens ◽  
E Urcelay ◽  
B Safer ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Virus Type ◽  
Fusion Proteins ◽  
Biologically Active ◽  
Adeno Associated Virus ◽  
Rep Proteins

scholarly journals Biodistribution of adeno‐associated virus type 2 carrying multi‐characteristic opsin in dogs following intravitreal injection

2021 ◽  
Vol 25 (18) ◽  
pp. 8676-8686
Author(s):  
Kissaou T. Tchedre ◽  
Subrata Batabyal ◽  
Melissa Galicia ◽  
Darryl Narcisse ◽  
Sourajit Mitra Mustafi ◽  
...  
Keyword(s):  
Intravitreal Injection ◽  
Virus Type ◽  
Adeno Associated Virus

scholarly journals Adeno-Associated Virus Type 2 and Hepatocellular Carcinoma?

2015 ◽  
Vol 26 (12) ◽  
pp. 779-781 ◽  
Author(s):  
Kenneth I. Berns ◽  
Barry J. Byrne ◽  
Terence R. Flotte ◽  
Guangping Gao ◽  
William W. Hauswirth ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Virus Type ◽  
Adeno Associated Virus

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

scholarly journals Cerebrospinal Fluid Secretory Ca2+-Dependent Phospholipase A2 Activity Is Increased in Alzheimer Disease

2009 ◽  
Vol 55 (12) ◽  
pp. 2171-2179 ◽  
Author(s):  
Sonia Chalbot ◽  
Henrik Zetterberg ◽  
Kaj Blennow ◽  
Tormod Fladby ◽  
Inge Grundke-Iqbal ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Alzheimer Disease ◽  
Phospholipase A2 ◽  
Microtiter Plate ◽  
Pla2 Activity ◽  
Activity Assay ◽  
Proinflammatory Mediators ◽  
Spla2 Activity ◽  
Phospholipase A2 Activity ◽  
Microtiter Plate Format

Abstract Background: The phospholipase A2 (PLA2) family comprises multiple isoenzymes that vary in their physicochemical properties, cellular localizations, calcium sensitivities, and substrate specificities. Despite these differences, PLA2s share the ability to catalyze the synthesis of the precursors of the proinflammatory mediators. To investigate the potential of PLA2 as a biomarker in screening neuroinflammatory disorders in both clinical and research settings, we developed a PLA2 assay and determined the predominant types of PLA2 activity in cerebrospinal fluid (CSF). Methods: We used liposomes composed of a fluorescent probe (bis-Bodipy® FL C11-PC [1,2-bis-(4,4- difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-undecanoyl)-sn-glycero-3-phosphocholine]) and 1,2-dioleoyl-l-α-phosphatidylcholine as a substrate to measure CSF PLA2 activity in a 96-well microtiter plate format. We established the type of CSF PLA2 activity using type-specific inhibitors of PLA2. Results: Using 5 μL CSF per assay, our PLA2 activity assay was reproducible with CVs <15% in 2 CSF samples and for recombinant secretory Ca2+-dependent PLA2 (sPLA2) in concentrations ranging from 0.25 to 1 μmol/L. This PLA2 assay allowed identification of sPLA2 activity in lumbar CSF from healthy individuals 20–77 years old that did not depend on either sex or age. Additionally, CSF sPLA2 activity was found to be increased (P = 0.0008) in patients with Alzheimer disease. Conclusions: Adult human CSF has sPLA2 activity that can be measured reliably with the assay described. This enzyme activity in the CSF is independent of both sex and age and might serve as a valuable biomarker of neuroinflammation, as we demonstrated in Alzheimer disease.

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