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Suppression of RNA interference by adenovirus virus-associated RNA
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
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2005 (English)In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 79, no 15, 9556-65 p.Article in journal (Refereed) Published
Place, publisher, year, edition, pages
2005. Vol. 79, no 15, 9556-65 p.
Keyword [en]
Adenoviruses; Human/*genetics, Base Sequence, Cell Line, Humans, Molecular Sequence Data, Mutation, RNA Interference, RNA; Small Interfering/genetics, RNA; Viral/chemistry/genetics/*metabolism/*physiology, Research Support; Non-U.S. Gov't, Ribonuclease III/antagonists & inhibitors/metabolism, Virus Integration, eIF-2 Kinase/antagonists & inhibitors/metabolism
Identifiers
URN: urn:nbn:se:uu:diva-80238DOI: 10.1128/JVI.79.15.9556-9565.2005PubMedID: 16014917OAI: oai:DiVA.org:uu-80238DiVA: diva2:108152
Available from: 2006-05-04 Created: 2006-05-04 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Construction of Adenovirus Vectors for Studies of Protein Function and RNA Interference
Open this publication in new window or tab >>Construction of Adenovirus Vectors for Studies of Protein Function and RNA Interference
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

During an adenovirus infection the accumulation of alternatively spliced mRNAs is subjected to a tight temporal regulation. The IIIa protein is a structural protein expressed exclusively late after infection. To study the significance of the restricted IIIa protein expression we used a Tet-ON regulated adenoviral vector to overexpress the IIIa protein during the early phase of infection. The results show that unregulated IIIa protein expression caused a reduction in late viral protein accumulation and a slight block of viral DNA replication. Further, the results indicate that IIIa splicing might be subjected to a regulation via a feed back loop stimulating its own expression.

To improve the efficacy of vectors for regulated transgene expression, we constructed binary adenoviral vectors based on the Tet-ON and Tet-OFF systems. These vectors encode both the transcriptional activator and the tetracycline-regulated expression cassette from the same viral unit, ensuring that each infected cell will express both the activator and the reporter gene. In model experiments this system was shown to result in a tight control of gene expression with no detectable background expression of the transgene and induction levels reaching 500-600 fold.

Introduction of dsRNA into a cell will induce a sequence specific degradation of the homologous mRNA via a mechanism named RNA interference (RNAi). The adenovirus VA RNAs are short highly structured RNAs that are expressed in large amounts late during an adenovirus infection. Here we showed that both VA RNAI and VA RNAII functions as virus-encoded suppressors of RNAi, by interfering with the activity of Dicer, the enzyme that cleaves the initial dsRNA to short-interfering RNAs (siRNAs) that mediate RNAi. Further, the VA RNAs themselves are substrates for Dicer and are cleaved into siRNAs in vivo that are incorporated into active RNA-induced silencing complexes.

There is a great interest in developing novel therapeutic strategies based on RNAi. We constructed adenoviral vectors that express short hairpin RNAs, which in vivo will be cleaved to siRNAs that induce sequence-specific RNAi. We compared the efficiency of RNAi induced by vectors based on the viral VA RNAI and the human U6 promoters. Our results suggest that under conditions where the recombinant virus does not replicate, the VA RNA promoter is more effective in down regulating target gene expression, whereas the U6 promoter was more effective under replicative conditions.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2006. 49 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 104
Keyword
Microbiology, Adenovirus, viral vectors, VA RNAI/II, RNAi, Tet-ON/OFF, RNA splicing, Mikrobiologi
National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-6328 (URN)91-554-6458-0 (ISBN)
Public defence
2006-03-03, Room C8:305, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2006-02-09 Created: 2006-02-09 Last updated: 2009-10-14Bibliographically approved
2. Adenoviral Control of RNAi/miRNA Pathways in Human Cells
Open this publication in new window or tab >>Adenoviral Control of RNAi/miRNA Pathways in Human Cells
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

RNA interference (RNAi) is a diverse, conserved regulatory mechanism in eukaryotic cells, which silences the target gene expression in a homology-dependent manner. Although it has been well documented that RNAi is an antiviral mechanism in plants and insects, it is still unclear whether RNAi naturally limits viral infections in vertebrates. Viruses are masters of adopting strategies to subvert cellular defense mechanisms. Not only can viruses use elaborate strategies to suppress the effects of defensive RNAi, but they can also redirect or interfere with cellular functions orchestrated by endogenous small RNAs.

In our work we have focused on studying the relationship of human adenovirus type 5 (Ad5) infection and the RNAi/miRNA pathways. We show that Ad5 infection inhibits RNAi by blocking the activity of Dicer and the RNA-induced silencing complex (RISC). For Dicer inhibition, the virus-associated RNAs, VA RNAI and VA RNAII bind Dicer through their terminal stems and are cleaved by Dicer into functional small RNAs that are incorporated into active RISC.

Furthermore, by cloning small RNAs, we found that approximately 80% of Ago2-containing RISC immunopurified from late infected cells was associated with VA RNA-derived small RNAs (mivaRNAs). Interestingly, the small RNAs derived from VA RNAII, the minor VA RNA species, appear to be the major mivaRNAs occupying RISC and associate with polyribosomes, which indicates their potential roles as miRNAs regulating translation of cellular mRNAs.

During our previous work, we observed that the strand bias of VA RNAI derived small RNA (mivaRI) incorporating into active RISC varied in the different viable Ad5 mutant viruses infected cells. It has been reported that Ad5 VA RNAI had two transcription initiation sites, which produced two clusters of VA RNAI with 3 nt difference at their 5’ end. Our data show that this heterogeneity resulted in a dramatic difference in mivaRI guide strand selection.

Collectively, our data contributes to understanding the interplay between virus and host. This study would be beneficial in designing optimal adenovirus vectors for therapeutic RNAi application.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2008. 66 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 397
Keyword
Adenovirus, RNAi, miRNA, VA RNA
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:uu:diva-9387 (URN)978-91-554-7339-6 (ISBN)
Public defence
2008-12-10, C8:301, Biomedical Center, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2008-11-18 Created: 2008-11-18 Last updated: 2009-10-14Bibliographically approved

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Xu, NingBerenjian, SaidehAkusjärvi, Göran

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