Enhanced sensitivity of pancreatic tumours cells to 5-FU-chemotherapy mediated by adeno-associated virus type 2 (AAV-2) infection in vitro and in vivo

2000 ◽  
Vol 118 (4) ◽  
pp. A531
Author(s):  
Sven Christian Eisold ◽  
Ruediger Ridder ◽  
Eduard Ryschisch ◽  
Jan Schmidt ◽  
Geeske C. Meyer ◽  
...  
Keyword(s):  
Virus Type ◽  
Adeno Associated Virus ◽  
Pancreatic Tumours ◽  
Enhanced Sensitivity

scholarly journals Adeno-Associated Virus Type 2 Capsids with Externalized VP1/VP2 Trafficking Domains Are Generated prior to Passage through the Cytoplasm and Are Maintained until Uncoating Occurs in the Nucleus

2006 ◽  
Vol 80 (22) ◽  
pp. 11040-11054 ◽  
Author(s):  
Florian Sonntag ◽  
Svenja Bleker ◽  
Barbara Leuchs ◽  
Roger Fischer ◽  
Jürgen A. Kleinschmidt
Keyword(s):  
Virus Type ◽  
Catalytic Domain ◽  
Nuclear Translocation ◽  
Basic Amino Acid ◽  
Nuclear Entry ◽  
Direct Role ◽  
Adeno Associated Virus

ABSTRACT Common features of parvovirus capsids are open pores at the fivefold symmetry axes that traverse the virion shell. Upon limited heat treatment in vitro, the pores can function as portals to externalize VP1/VP2 protein N-terminal sequences which harbor infection-relevant functional domains, such as a phospholipase A2 catalytic domain. Here we show that adeno-associated virus type 2 (AAV2) also exposes its VP1/VP2 N termini in vivo during infection, presumably in the endosomal compartment. This conformational change is influenced by treatment with lysosomotropic reagents. While incubation of cells with bafilomycin A1 reduced exposure of VP1/VP2 N termini, incubation with chloroquine stimulated externalization transiently. N-terminally located basic amino acid clusters with nuclear localization activity also become exposed in this process and are accessible on the virus capsid when it enters the cytoplasm. This is an obligatory step in AAV2 infection. However, a direct role of these sequences in nuclear translocation of viral capsids could not be determined by microinjection of wild-type or mutant viruses. This suggests that further modifications of the capsid have to take place in a precytoplasmic entry step that prepares the virus for nuclear entry. Microinjection of several capsid-specific antibodies into the cell nucleus blocked AAV2 infection completely, supporting the conclusion that AAV2 capsids bring the infectious genome into the nucleus.

scholarly journals The Cellular TATA Binding Protein Is Required for Rep-Dependent Replication of a Minimal Adeno-Associated Virus Type 2 p5 Element

2005 ◽  
Vol 79 (17) ◽  
pp. 11082-11094 ◽  
Author(s):  
Achille François ◽  
Mickaël Guilbaud ◽  
Rafi Awedikian ◽  
Gilliane Chadeuf ◽  
Philippe Moullier ◽  
...  
Keyword(s):  
Virus Type ◽  
Transcription Initiation ◽  
Promoter Region ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Tata Box ◽  
Adeno Associated Virus

ABSTRACT The p5 promoter region of adeno-associated virus type 2 (AAV-2) is a multifunctional element involved in rep gene expression, Rep-dependent replication, and site-specific integration. We initially characterized a 350-bp p5 region by its ability to behave like a cis-acting replication element in the presence of Rep proteins and adenoviral factors. The objective of this study was to define the minimal elements within the p5 region required for Rep-dependent replication. Assays performed in transfected cells (in vivo) indicated that the minimal p5 element was composed by a 55-bp sequence (nucleotides 250 to 304 of wild-type AAV-2) containing the TATA box, the Rep binding site, the terminal resolution site present at the transcription initiation site (trs+1), and a downstream 17-bp region that could potentially form a hairpin structure localizing the trs+1 at the top of the loop. Interestingly, the TATA box was absolutely required for in vivo but dispensable for in vitro, i.e., cell-free, replication. We also demonstrated that Rep binding and nicking at the trs+1 was enhanced in the presence of the cellular TATA binding protein, and that overexpression of this cellular factor increased in vivo replication of the minimal p5 element. Together, these studies identified the minimal replication origin present within the AAV-2 p5 promoter region and demonstrated for the first time the involvement of the TATA box, in cis, and of the TATA binding protein, in trans, for Rep-dependent replication of this viral element.

scholarly journals Selective Cleavage of AAVS1 Substrates by the Adeno-Associated Virus Type 2 Rep68 Protein Is Dependent on Topological and Sequence Constraints

2000 ◽  
Vol 74 (19) ◽  
pp. 8831-8842 ◽  
Author(s):  
Stefania Lamartina ◽  
Gennaro Ciliberto ◽  
Carlo Toniatti
Keyword(s):  
Virus Type ◽  
Adeno Associated Virus ◽  
Site Specific ◽  
Site Specific Integration ◽  
A Site ◽  
First Time ◽  
Sequence Constraints

ABSTRACT The adeno-associated virus type 2 (AAV-2) Rep78 and Rep68 proteins are required for replication of the virus as well as its site-specific integration into a unique site, called AAVS1, of human chromosome 19. Rep78 and Rep68 initiate replication by binding to a Rep binding site (RBS) contained in the AAV-2 inverted terminal repeats (ITRs) and then specifically nicking at a nearby site called the terminal resolution site (trs). Similarly, Rep78 and Rep68 are postulated to trigger the integration process by binding and nicking RBS andtrs homologues present in AAVS1. However, Rep78 and Rep68 cleave in vitro AAVS1 duplex-linear substrates much less efficiently than hairpinned ITRs. In this study, we show that the AAV-2 Rep68 endonuclease activity is affected by the topology of the substrates in that it efficiently cleaves in vitro in a site- and strand-specific manner the AAVS1 trs only if this sequence is in a supercoiled (SC) conformation. DNA sequence mutagenesis in the context of SC templates allowed us to elucidate for the first time the AAVS1trs sequence and position requirements for Rep68-mediated cleavage. Interestingly, Rep68 did not cleave SC templates containing RBS from other sites of the human genome. These findings have intriguing implications for AAV-2 site-specific integration in vivo.

scholarly journals Charge-to-Alanine Mutagenesis of the Adeno-Associated Virus Type 2 Rep78/68 Proteins Yields Temperature-Sensitive and Magnesium-Dependent Variants

1999 ◽  
Vol 73 (11) ◽  
pp. 9433-9445 ◽  
Author(s):  
Denise K. Gavin ◽  
Samuel M. Young ◽  
Weidong Xiao ◽  
Brenda Temple ◽  
Corinne R. Abernathy ◽  
...  
Keyword(s):  
Virus Type ◽  
Specific Binding ◽  
Temperature Sensitive ◽  
Helicase Activity ◽  
Endonuclease Activity ◽  
Adeno Associated Virus ◽  
Rep Protein

ABSTRACT The adeno-associated virus type 2 (AAV) replication (Rep) proteins Rep78 and 68 (Rep78/68) exhibit a number of biochemical activities required for AAV replication, including specific binding to a 22-bp region of the terminal repeat, site-specific endonuclease activity, and helicase activity. Individual and clusters of charged amino acids were converted to alanines in an effort to generate a collection of conditionally defective Rep78/68 proteins. Rep78 variants were expressed in human 293 cells and analyzed for their ability to mediate replication of recombinant AAV vectors at various temperatures. The biochemical activities of Rep variants were further characterized in vitro by using Rep68 His-tagged proteins purified from bacteria. The results of these analyses identified a temperature-sensitive (ts) Rep protein (D40,42,44A-78) that exhibited a delayed replication phenotype at 32°C, which exceeded wild-type activity by 48 h. Replication activity was reduced by more than threefold at 37°C and was undetectable at 39°C. Stability of the Rep78 protein paralleled replication levels at each temperature, further supporting ats phenotype. Replication differences resulted in a 3-log-unit difference in virus yields between the permissive and nonpermissive temperatures (2.2 × 106 and 3 × 103, respectively), demonstrating that this is a relatively tight mutant. In addition to the ts Rep mutant, we identified a nonconditional mutant with a reduced ability to support viral replication in vivo. Additional characterization of this mutant demonstrated an Mg2+-dependent phenotype that was specific to Rep endonuclease activity and did not affect helicase activity. The two mutants described here are unique, in that Rep tsmutants have not previously been described and the D412A Rep mutant represents the first mutant in which the helicase and endonuclease functions can be distinguished biochemically. Further understanding of these mutants should facilitate our understanding of AAV replication and integration, as well as provide novel strategies for production of viral vectors.

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 Randomization and In Vivo Selection Reveal a GGRG Motif Essential for Packaging Human Immunodeficiency Virus Type 2 RNA

2008 ◽  
Vol 83 (2) ◽  
pp. 802-810 ◽  
Author(s):  
Tayyba T. Baig ◽  
Jean-Marc Lanchy ◽  
J. Stephen Lodmell
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Consensus Sequence ◽  
Rna Packaging ◽  
Stem Loop ◽  
Immunodeficiency Virus

ABSTRACT The packaging signal (ψ) of human immunodeficiency virus type 2 (HIV-2) is present in the 5′ noncoding region of RNA and contains a 10-nucleotide palindrome (pal; 5′-392-GGAGUGCUCC) located upstream of the dimerization signal stem-loop 1 (SL1). pal has been shown to be functionally important in vitro and in vivo. We previously showed that the 3′ side of pal (GCUCC-3′) is involved in base-pairing interactions with a sequence downstream of SL1 to make an extended SL1, which is important for replication in vivo and the regulation of dimerization in vitro. However, the role of the 5′ side of pal (5′-GGAGU) was less clear. Here, we characterized this role using an in vivo SELEX approach. We produced a population of HIV-2 DNA genomes with random sequences within the 5′ side of pal and transfected these into COS-7 cells. Viruses from COS-7 cells were used to infect C8166 permissive cells. After several weeks of serial passage in C8166 cells, surviving viruses were sequenced. On the 5′ side of pal there was a striking convergence toward a GGRGN consensus sequence. Individual clones with consensus and nonconsensus sequences were tested in infectivity and packaging assays. Analysis of individuals that diverged from the consensus sequence showed normal viral RNA and protein synthesis but had replication defects and impaired RNA packaging. These findings clearly indicate that the GGRG motif is essential for viral replication and genomic RNA packaging.

scholarly journals Incorporation of Adeno-Associated Virus in a Calcium Phosphate Coprecipitate Improves Gene Transfer to Airway Epithelia In Vitro and In Vivo

2000 ◽  
Vol 74 (1) ◽  
pp. 535-540 ◽  
Author(s):  
Robert W. Walters ◽  
Dongsheng Duan ◽  
John F. Engelhardt ◽  
Michael J. Welsh
Keyword(s):  
Calcium Phosphate ◽  
Gene Transfer ◽  
Mouse Lung ◽  
Apical Surface ◽  
Airway Epithelia ◽  
Adeno Associated Virus ◽  
Human Airway

ABSTRACT Adeno-associated virus (AAV) is inefficient at infecting differentiated airway epithelia because of a lack of receptors at the apical surface. We hypothesized that incorporation of AAV in a calcium phosphate coprecipitate would circumvent this barrier. Interestingly, coprecipitation of AAV type 2 improved gene transfer to differentiated human airway epithelia in vitro and to the mouse lung in vivo. These results suggest that delivery of AAV as a CaPicoprecipitate may significantly enhance its utility for gene transfer to the airway epithelia in vivo.

scholarly journals Assembly of adeno-associated virus type 2 capsids in vitro.

1997 ◽  
Vol 78 (6) ◽  
pp. 1453-1462 ◽  
Author(s):  
S Steinbach ◽  
J A Kleinschmidt ◽  
A Wistuba ◽  
T Bock
Keyword(s):  
Virus Type ◽  
Adeno Associated Virus
Sign in / Sign up

Export Citation Format

Share Document