scholarly journals Adeno-Associated Virus Type 2 Rep Protein Inhibits Human Papillomavirus Type 16 E2 Recruitment of the Transcriptional Coactivator p300

2000 ◽  
Vol 74 (19) ◽  
pp. 9090-9098 ◽  
Author(s):  
Alessandro Marcello ◽  
Paola Massimi ◽  
Lawrence Banks ◽  
Mauro Giacca
Keyword(s):  
Virus Type ◽  
Protective Factor ◽  
Human Papillomaviruses ◽  
Transactivation Domain ◽  
Transcriptional Coactivator ◽  
Adeno Associated Virus ◽  
Rep Protein ◽  
Coactivator P300

ABSTRACT Infection by human adeno-associated virus type 2 (AAV2) is a possible protective factor in the development of cervical carcinomas associated with human papillomaviruses (HPV). The replicative proteins of AAV2 (Rep) have been implicated in the inhibition of papillomavirus replication and transforming activities, although the molecular events underlying these effects are poorly understood. We observed that each of the four forms of AAV2 Rep inhibited the E1- and E2-driven replication of oncogenic HPV type 16 (HPV16). Rep40, corresponding to the C-terminal domain of all Rep proteins, inhibited both HPV DNA replication and HPV16 E2-mediated transactivation. Rep40 specifically bound the N-terminal transactivation domain of HPV16 E2 both in vitro and in vivo. This interaction was found to specifically disrupt the binding of E2 to the cellular transcriptional coactivator p300. Accordingly, the inhibitory effect of Rep on HPV16 E2 transactivation was rescued by the overexpression of p300. These data indicate a novel role of Rep in the down-regulation of papillomaviruses through inhibition of complex formation between the HPV16 E2 transcriptional activator and its cellular coactivator, p300.

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.

scholarly journals Presence of atrs-Like Motif Promotes Rep-Mediated Wild-Type Adeno-Associated Virus Type 2 Integration

2015 ◽  
Vol 89 (14) ◽  
pp. 7428-7432 ◽  
Author(s):  
Karl Petri ◽  
Richard Gabriel ◽  
Leticia Agundez ◽  
Raffaele Fronza ◽  
Saira Afzal ◽  
...  
Keyword(s):  
Virus Type ◽  
Integration Site ◽  
Dermal Fibroblasts ◽  
Wild Type ◽  
Mobility Shift ◽  
Adeno Associated Virus ◽  
Rep Protein ◽  
Electrophoretic Mobility Shift Assays ◽  
First Time

High-throughput integration site (IS) analysis of wild-type adeno-associated virus type 2 (wtAAV2) in human dermal fibroblasts (HDFs) and HeLa cells revealed that juxtaposition of a Rep binding site (RBS) and terminal resolution site (trs)-like motif leads to a 4-fold-increased probability of wtAAV integration. Electrophoretic mobility shift assays (EMSAs) confirmed binding of Rep to off-target RBSs. For the first time, we show Rep protein off-target nicking activity, highlighting the importance of the nicking substrate for Rep-mediated integration.

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

scholarly journals Factors Affecting the Terminal Resolution Site Endonuclease, Helicase, and ATPase Activities of Adeno-Associated Virus Type 2 Rep Proteins

1999 ◽  
Vol 73 (10) ◽  
pp. 8235-8244 ◽  
Author(s):  
Jianwen Wu ◽  
Michael D. Davis ◽  
Roland A. Owens
Keyword(s):  
Virus Type ◽  
Helicase Activity ◽  
Endonuclease Activity ◽  
Single Stranded Dna ◽  
Adeno Associated Virus ◽  
Site Specific ◽  
Rabbit Reticulocyte Lysate System ◽  
Wide Range

ABSTRACT The Rep68 and Rep78 proteins (Rep68/78) of adeno-associated virus type 2 (AAV) are critical for AAV replication and site-specific integration. They bind specifically to the AAV inverted terminal repeats (ITRs) and possess ATPase, helicase, and strand-specific/site-specific endonuclease activities. In the present study, we further characterized the AAV Rep68/78 helicase, ATPase, and endonuclease activities by using a maltose binding protein-Rep68 fusion (MBP-Rep68Δ) produced in Escherichia coli cells and Rep78 produced in vitro in a rabbit reticulocyte lysate system. We found that the minimal length of single-stranded DNA capable of stimulating the ATPase activity of MBP-Rep68Δ is 100 to 200 bases. The degree of stimulation correlated positively with the length of single-stranded DNA added to the reaction mixture. We then determined the ATP concentration needed for optimal MBP-Rep68Δ helicase activity and showed that the helicase is active over a wide range of ATP concentrations. We determined the directionality of MBP-Rep68Δ helicase activity and found that it appears to move in a 3′ to 5′ direction, which is consistent with a model in which AAV Rep68/78 participates in AAV DNA replication by unwinding DNA ahead of a cellular DNA polymerase. In this report, we also demonstrate that single-stranded DNA is capable of inhibiting the MBP-Rep68Δ or Rep78 endonuclease activity greater than 10-fold. In addition, we show that removal of the secondary Rep68/78 binding site, which is found only in the hairpin form of the AAV ITR, causes a three- to eightfold reduction in the ability of the ITR to be used as a substrate for the Rep78 or MBP-Rep68Δ endonuclease activity. This suggests that contact between Rep68/78 and this secondary element may play an important role in the Rep-mediated endonuclease activity.

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 Nonstructural Protein Rep78 Suppresses Translation in Vitro

Virology ◽  
2000 ◽  
Vol 266 (1) ◽  
pp. 196-202 ◽  
Author(s):  
Takamasa Takeuchi ◽  
Takuyou Kozuka ◽  
Keiichi Nakagawa ◽  
Yukimasa Aoki ◽  
Kuni Ohtomo ◽  
...  
Keyword(s):  
Virus Type ◽  
Nonstructural Protein ◽  
Adeno Associated Virus

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 Methylation Status of the Adeno-Associated Virus Type 2 (AAV2)

Viruses ◽  
2019 ◽  
Vol 11 (1) ◽  
pp. 38 ◽  
Author(s):  
Renáta Tóth ◽  
István Mészáros ◽  
Daniela Hüser ◽  
Barbara Forró ◽  
Szilvia Marton ◽  
...  
Keyword(s):  
Virus Type ◽  
Viral Genome ◽  
Methylation Status ◽  
Replication Initiation ◽  
Wild Type ◽  
Adeno Associated Virus ◽  
Cpg Site ◽  
Cg Dinucleotides

To analyze the methylation status of wild-type adeno-associated virus type 2 (AAV2), bisulfite PCR sequencing (BPS) of the packaged viral genome and its integrated form was performed and 262 of the total 266 CG dinucleotides (CpG) were mapped. In virion-packaged DNA, the ratio of the methylated cytosines ranged between 0–1.7%. In contrast, the chromosomally integrated AAV2 genome was hypermethylated with an average of 76% methylation per CpG site. The methylation level showed local minimums around the four known AAV2 promoters. To study the effect of methylation on viral rescue and replication, the replication initiation capability of CpG methylated and non-CpG methylated AAV DNA was compared. The in vitro hypermethylation of the viral genome does not inhibit its rescue and replication from a plasmid transfected into cells. This insensitivity of the viral replicative machinery to methylation may permit the rescue of the integrated heavily methylated AAV genome from the host’s chromosomes.

scholarly journals The Adeno-Associated Virus Type 2 Rep Protein Regulates RNA Processing via Interaction with the Transcription Template

2002 ◽  
Vol 22 (11) ◽  
pp. 3639-3652 ◽  
Author(s):  
Jianming Qiu ◽  
David J. Pintel
Keyword(s):  
Rna Polymerase Ii ◽  
Virus Type ◽  
Rna Processing ◽  
Transcription Initiation ◽  
Transcription Unit ◽  
Rna Pol Ii ◽  
Adeno Associated Virus ◽  
Rep Protein ◽  
Rep Proteins

ABSTRACT The adeno-associated virus type 2 (AAV) large Rep proteins can act to increase the ratio of spliced to unspliced AAV RNA when they are targeted to the transcription template via a Rep binding element. The required Rep binding site is both location and orientation independent; however, Rep enhancement decreases as the distance between the promoter and the intron of the affected transcription unit increases. Only the AAV intron and an extended polyadenylation site must remain for the AAV transcription unit to manifest responsiveness to Rep. A number of promoters, when driving the AAV capsid gene transcription unit, were responsive to targeted Rep, though to various degrees. Transactivation of transcription initiation is not sufficient for the enhancement of RNA processing, because activation of the P40 transcription unit by other activators targeted to this transcription template did not result in enhancement of the ratio of spliced to unspliced AAV RNA. These results suggest that Rep may act as a trans regulator of RNA processing by modulating such functions coupled to RNA polymerase II (RNA pol II) transcription, perhaps by affecting the composition of the transcription complex either prior to or during elongation. These results reveal another way in which gene expression can be regulated by trans-acting proteins and help explain an important feature of the parvovirus life cycle.

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