scholarly journals Adeno-Associated Virus Small Rep Proteins Are Modified with at Least Two Types of Polyubiquitination

2009 ◽  
Vol 84 (2) ◽  
pp. 1206-1211 ◽  
Author(s):  
K. David Farris ◽  
Olufemi Fasina ◽  
Loretta Sukhu ◽  
Long Li ◽  
David J. Pintel
Keyword(s):  
Small Interfering Rna ◽  
Adenovirus Type ◽  
Proteasomal Degradation ◽  
Transient Transfection ◽  
Adeno Associated Virus ◽  
Rep Proteins ◽  
Cullin 5 ◽  
Interfering Rna ◽  
Adenovirus Type 5

ABSTRACT Adeno-associated virus (AAV) type 2 and 5 proteins Rep52 and Rep40 were polyubiquitinated during AAV-adenovirus type 5 (Ad5) coinfection and during transient transfection in either the presence or absence of Ad5 E4orf6 and E1b-55k. Polyubiquitination of small Rep proteins via lysine 48 (K48) linkages, normally associated with targeting of proteins for proteasomal degradation, was detected only in the presence of E4orf6. The small Rep proteins were ubiquitinated via lysine 63 (K63) following transfection in either the presence or absence of E4orf6 or following coinfection with Ad5. E4orf6/E1b-55k-dependent K48-specific polyubiquitination of small Rep proteins could be inhibited using small interfering RNA (siRNA) to cullin 5.

scholarly journals Adeno-Associated Viruses Can Induce Phosphorylation of eIF2α via PKR Activation, Which Can Be Overcome by Helper Adenovirus Type 5 Virus-Associated RNA

2007 ◽  
Vol 81 (21) ◽  
pp. 11908-11916 ◽  
Author(s):  
Ramnath Nayak ◽  
David J. Pintel
Keyword(s):  
Cellular Response ◽  
Adenovirus Type ◽  
Capsid Gene ◽  
Protein Accumulation ◽  
Protein Kinase R ◽  
Adeno Associated Virus ◽  
Simplex Virus ◽  
Rna I ◽  
Interfering Rna ◽  
Adenovirus Type 5

ABSTRACT Mutants of adenovirus type 5 (Ad5) virus-associated RNA I deficient in inhibiting the activation and subsequent phosphorylation of protein kinase R (PKR) could neither function as helpers for adeno-associated virus type 5 (AAV5) replication nor enhance AAV5 protein accumulation in either the presence or absence of Ad5 E4Orf6 and E2a. Furthermore, a short region of the AAV5 capsid gene RNA leader sequence surrounding the AUG of VP1 could induce the phosphorylation of eIF2α. Both short interfering RNA directed against PKR and the addition of the herpes simplex virus ICP34.5 protein enhanced the accumulation of AAV5 capsid protein in the presence of the AAV5 capsid gene PKR-inducing element, suggesting that VA RNA acted to overcome direct AAV5-induced activation of PKR that led to the phosphorylation of eIF2α. The expression of both the closely related goat-derived AAV and the prototype AAV2 capsid gene transcription units also induced the phosphorylation of eIF2α, suggesting that the induction of the PKR/eIF2α cellular response may be a previously unrecognized general feature of at least the Dependovirus genus of the Parvovirinae.

scholarly journals E4Orf6-E1B-55k-Dependent Degradation of De Novo-Generated Adeno-Associated Virus Type 5 Rep52 and Capsid Proteins Employs a Cullin 5-Containing E3 Ligase Complex

2008 ◽  
Vol 82 (7) ◽  
pp. 3803-3808 ◽  
Author(s):  
Ramnath Nayak ◽  
K. David Farris ◽  
David J. Pintel
Keyword(s):  
Virus Type ◽  
De Novo ◽  
E3 Ligase ◽  
Adenovirus Type ◽  
Dominant Negative ◽  
Capsid Proteins ◽  
Chain Elongation ◽  
Adeno Associated Virus ◽  
Cullin 5 ◽  
Interfering Rna

ABSTRACT Degradation of de novo-generated adeno-associated virus type 5 (AAV5) Rep52 and capsid proteins is part of the limited target specificity displayed by adenovirus type 5 E4Orf6-E1B-55k as part of a cullin 5-containing E3 ligase complex. Both Rep and capsid proteins can be found in the ligase complex, and their presence is dependent on interaction between E4Orf6 and elongins B and C. Degradation of AAV5 proteins can be inhibited by a dominant-negative ubiquitin that prevents chain elongation or by small interfering RNA directed against cullin 5.

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 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 Ubiquitin-Proteasomal Degradation of COX-2 in TGF-β Stimulated Human Endometrial Cells Is Mediated Through Endoplasmic Reticulum Mannosidase I

Endocrinology ◽  
2012 ◽  
Vol 153 (1) ◽  
pp. 426-437 ◽  
Author(s):  
Mohan Singh ◽  
Parvesh Chaudhry ◽  
Sophie Parent ◽  
Eric Asselin
Keyword(s):  
Endoplasmic Reticulum ◽  
Stromal Cells ◽  
Small Interfering Rna ◽  
Proteasomal Degradation ◽  
26S Proteasome ◽  
Dependent Manner ◽  
Endometrial Stromal Cells ◽  
Endometrial Cells ◽  
Cox 2 ◽  
Interfering Rna

Cyclooxygenase (COX)-2 is a key regulatory enzyme in the production of prostaglandins (PG) during various physiological processes. Mechanisms of COX-2 regulation in human endometrial stromal cells (human endometrial stromal cells) are not fully understood. In this study, we investigate the role of TGF-β in the regulation of COX-2 in human uterine stromal cells. Each TGF-β isoform decreases COX-2 protein level in human uterine stromal cells in Smad2/3-dependent manner. The decrease in COX-2 is accompanied by a decrease in PG synthesis. Knockdown of Smad4 using specific small interfering RNA prevents the decrease in COX-2 protein, confirming that Smad pathway is implicated in the regulation of COX-2 expression in human endometrial stromal cells. Pretreatment with 26S proteasome inhibitor, MG132, significantly restores COX-2 protein and PG synthesis, indicating that COX-2 undergoes proteasomal degradation in the presence of TGF-β. In addition, each TGF-β isoform up-regulates endoplasmic reticulum (ER)-mannosidase I (ERManI) implying that COX-2 degradation is mediated through ER-associated degradation pathway in these cells. Furthermore, inhibition of ERManI activity using the mannosidase inhibitor (kifunensine), or small interfering RNA-mediated knockdown of ERManI, prevents TGF-β-induced COX-2 degradation. Taken together, these studies suggest that TGF-β promotes COX-2 degradation in a Smad-dependent manner by up-regulating the expression of ERManI and thereby enhancing ER-associated degradation and proteasomal degradation pathways.

scholarly journals Rescue and Autonomous Replication of Adeno-Associated Virus Type 2 Genomes Containing Rep-Binding Site Mutations in the Viral p5 Promoter

1998 ◽  
Vol 72 (6) ◽  
pp. 4811-4818 ◽  
Author(s):  
Xu-Shan Wang ◽  
Arun Srivastava
Keyword(s):  
Binding Site ◽  
Virus Type ◽  
Hela Cells ◽  
Biologically Active ◽  
Viral Gene ◽  
Adeno Associated Virus ◽  
Autonomous Replication ◽  
293 Cells ◽  
Rep Proteins

ABSTRACT The Rep proteins encoded by the adeno-associated virus type 2 (AAV) play a crucial role in the rescue, replication, and integration of the viral genome. In the absence of a helper virus, little expression of the AAV Rep proteins occurs, and the AAV genome fails to undergo DNA replication. Since previous studies have established that expression of the Rep78 and Rep68 proteins from the viral p5 promoter is controlled by the Rep-binding site (RBS) and the YY1 factor-binding site (YBS), we constructed a number of recombinant AAV plasmids containing mutations and/or deletions of the RBS and the YBS in the p5 promoter. These plasmids were transfected in HeLa or 293 cells and analyzed for the potential to undergo AAV DNA rescue and replication. Our studies revealed that (i) a low-level rescue and autonomous replication of the wild-type AAV genome occurred in 293 but not in HeLa cells; (ii) mutations in the RBS resulted in augmented expression from the p5 promoter, leading to more efficient rescue and/or replication of the AAV genome in 293 but not in HeLa cells; (iii) little rescue and/or replication occurred from plasmids containing mutations in the YBS alone in the absence of coinfection with adenovirus; (iv) expression of the adenovirus E1A gene products was insufficient to mediate rescue and/or replication of the AAV genome in HeLa cells; (v) autonomously replicated AAV genomes in 293 cells were successfully encapsidated in mature progeny virions that were biologically active in secondary infection of HeLa cells in the presence of adenovirus; and (vi) stable transfection of recombinant AAV plasmids containing a gene for resistance to neomycin significantly affected stable integration only in 293 cells, presumably because rescue and autonomous replication of the AAV genome from these plasmids occurred in 293 cells but not in HeLa or KB cells. These data suggest that in the absence of adenovirus, the AAV Rep protein-RBS interaction plays a dominant role in down-regulating viral gene expression from the p5 promoter and that perturbation in this interaction is sufficient to confer autonomous replication competence to AAV in 293 cells.

scholarly journals Adeno-associated virus Type 2 Rep proteins mediate integration of lentiviral vectors

2014 ◽  
Author(s):  
Victor J McAlister ◽  
Anthony T Craig ◽  
Roland A Owens
Keyword(s):  
Virus Type ◽  
Lentiviral Vector ◽  
Lentiviral Vectors ◽  
Adeno Associated Virus ◽  
Gene Therapy Vector ◽  
Specific Pcr ◽  
Rep Proteins ◽  
Dividing Cells ◽  
Lentivirus Vectors

Aims: Adeno-associated virus type 2 (AAV2) is a naturally defective human parvovirus that is being developed as a gene therapy vector. In dividing cells, AAV2 DNA persists by integration into the host chromosomes. AAV2 is unique among mammalian viruses in its ability to integrate preferentially into a particular locus within human chromosome 19, designated AAVS1(also known as Mbs 85). The AAV2 Rep68 and Rep78 proteins mediate this integration. Recent data suggest that Rep68 and Rep78 can mediate integration of non-AAV2 DNA with free ends. To test this hypothesis, we targeted insertion of different lentiviral vectors to AAVS1. Methods: Cells were co-infected with wild-type AAV2, and integrase-proficient or integrase-deficient lentivirus vectors. A highly specific PCR-based assay was used to detect lentivirus integration at AAVS1. Similar experiments were performed using lentiviral vectors containing the AAV2 rep gene. Results: All lentiviral vectors tested integrated at AAVS1, if the rep gene was present either within the lentiviral vector or supplied in trans. All that was required for integration at AAVS1 was the amino acid sequence shared between Rep68 and Rep78. The results were similar with integrase-proficient or integrase-deficient lentiviral vectors. Conclusions. The inclusion of the rep gene with lentiviral vectors may produce more predictable integration patterns.

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