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Adenoviral small non-coding RNAs: A Structural and Functional Charaterization
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. (Göran Akusärvi)
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Since their discovery in 1953, adenoviruses have significantly contributed to the understanding of virus-host cell interactions, including mechanistic details of cellular processes such as cell cycle control and alternative RNA splicing. Among the first characterized adenoviral genes were the virus-associated RNAs (VA RNAI/II), which are produced in massive amount during a lytic infection. The VA RNAs perform multiple functions and are required for a successful adenovirus life cycle. More recently, it was shown that the VA RNAs are processed into small viral miRNAs, so-called mivaRNAs, which interfere with the function of the cellular RNAi/miRNA machinery.

In papers I and II, we focused on a structural and functional characterization of the mivaRNAs using two approaches. Firstly, we created a model system where the predicted miRNA-like function of mivaRNAI could be investigated, without interfering with other VA RNA functions. This was accomplished by construction of recombinant adenoviruses, in which the seed sequence of mivaRNAI was altered. The results showed that in cell culture experiments the mivaRNAI seed sequence mutants grew as the wild type virus, suggesting that the mivaRNAs are not required during the lytic phase of an adenovirus infection. Secondly, we showed that the VA RNAs from different human adenoviruses (Ad4, Ad5, Ad11 and Ad37) undergo the same type of Dicer-dependent processing into mivaRNAs, which subsequently are loaded onto the RNA induced silencing complex (RISC), albeit with different efficiencies.

In paper III, we demonstrated that the promoter proximal region of the adenovirus major late promoter (MLP) produces a novel non-canonical class of small RNAs, which we termed the MLP-TSS-sRNAs. Surprisingly the MLP-TSS-sRNA maintains the m7G-cap structure while bound to Ago2 containing RISC. These complexes are functional suppressing expression of target mRNAs with complementary binding site. Most importantly, the MLP-TSS-sRNA limits the efficiency of viral DNA replication probably through a targeting of the E2B mRNAs, which are transcribed in the antisense orientation. In conclusion, the MLP-TSS-sRNA represents the first viral small RNA, which has been shown to have a function as a regulator of an adenovirus infection.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. , 49 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1216
Keyword [en]
non-coding RNA miRNA mivaRNA canonical Dicer Ago2 TSS m7G Cap Adenovirus replication E2B
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-283080ISBN: 978-91-554-9556-5OAI: oai:DiVA.org:uu-283080DiVA: diva2:918162
Public defence
2016-06-02, B/A1:111a, BMC, Uppsala, 13:15 (English)
Available from: 2016-05-11 Created: 2016-04-11 Last updated: 2016-06-01
List of papers
1. The adenovirus VA RNA-derived miRNAs are not essential for lytic virus growth in tissue culture cells
Open this publication in new window or tab >>The adenovirus VA RNA-derived miRNAs are not essential for lytic virus growth in tissue culture cells
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2013 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 41, no 9, 4802-4812 p.Article in journal (Refereed) Published
Abstract [en]

At late times during a lytic infection human adenovirus type 5 produces ∼10(8) copies per cell of virus-associated RNA I (VA RNAI). This short highly structured RNA polymerase III transcript has previously been shown to be essential for lytic virus growth. A fraction of VA RNAI is processed by Dicer into small RNAs, so-called mivaRNAIs, which are efficiently incorporated into the RNA-induced silencing complex. Here, we constructed recombinant adenoviruses with mutations in the seed sequence of both the 5'- and the 3'-strand of the mivaRNAI duplex. The results showed that late viral protein synthesis, as well as new virus progeny formation, was essentially unaffected by the seed sequence mutations under lytic replicative conditions in HeLa or HEK293 cells. Collectively, our results suggest that either strand of the mivaRNAI duplex does not have target mRNA interactions that are critical for the establishment of virus growth under lytic conditions. Further, by depletion of protein kinase R (PKR) in HEK293 cells, we show that the suppressive effect of VA RNAI on the interferon-induced PKR pathway is most critical for late gene expression.

National Category
Natural Sciences
urn:nbn:se:uu:diva-198031 (URN)10.1093/nar/gkt172 (DOI)000318570000013 ()23525465 (PubMedID)
Available from: 2013-04-08 Created: 2013-04-08 Last updated: 2016-06-01Bibliographically approved
2. Small RNA Sequence Analysis of Adenovirus VA RNA-Derived MiRNAs Reveals an Unexpected Serotype-Specific Difference in Structure and Abundance
Open this publication in new window or tab >>Small RNA Sequence Analysis of Adenovirus VA RNA-Derived MiRNAs Reveals an Unexpected Serotype-Specific Difference in Structure and Abundance
2014 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 9, no 8, e105746- p.Article in journal (Refereed) Published
Abstract [en]

Human adenoviruses (HAds) encode for one or two highly abundant virus-associated RNAs, designated VA RNAI and VA RNAII, which fold into stable hairpin structures resembling miRNA precursors. Here we show that the terminal stem of the VA RNAs originating from Ad4, Ad5, Ad11 and Ad37, all undergo Dicer dependent processing into virus-specific miRNAs (so-called mivaRNAs). We further show that the mivaRNA duplex is subjected to a highly asymmetric RISC loading with the 3'-strand from all VA RNAs being the favored strand, except for the Ad37 VA RNAII, where the 5'-mivaRNAII strand was preferentially assembled into RISC. Although the mivaRNA seed sequences are not fully conserved between the HAds a bioinformatics prediction approach suggests that a large fraction of the VA RNAII-, but not the VA RNAI-derived mivaRNAs still are able to target the same cellular genes. Using small RNA deep sequencing we demonstrate that the Dicer processing event in the terminal stem of the VA RNAs is not unique and generates 3'-mivaRNAs with a slight variation of the position of the 5'-terminal nucleotide in the RISC loaded guide strand. Also, we show that all analyzed VA RNAs, except Ad37 VA RNAI and Ad5 VA RNAII, utilize an alternative upstream A start site in addition to the classical +1 G start site. Further, the 5'-mivaRNAs with an A start appears to be preferentially incorporated into RISC. Although the majority of mivaRNA research has been done using Ad5 as the model system our analysis demonstrates that the mivaRNAs expressed in Ad11- and Ad37-infected cells are the most abundant mivaRNAs associated with Ago2-containing RISC. Collectively, our results show an unexpected variability in Dicer processing of the VA RNAs and a serotype-specific loading of mivaRNAs into Ago2-based RISC.

National Category
Evolutionary Biology
urn:nbn:se:uu:diva-233020 (URN)10.1371/journal.pone.0105746 (DOI)000341106100115 ()
Swedish Cancer Society, 13 0469, 12 0504Swedish Research Council, K2012-99X-21959-01-3, 2006-5038-36531-16
Available from: 2014-10-07 Created: 2014-09-29 Last updated: 2016-06-01Bibliographically approved
3. Characterization of a non-canonical 5’ capped adenoviral small RNA that is functionally active
Open this publication in new window or tab >>Characterization of a non-canonical 5’ capped adenoviral small RNA that is functionally active
(English)Manuscript (preprint) (Other academic)
National Category
Medical and Health Sciences
urn:nbn:se:uu:diva-282954 (URN)
Available from: 2016-04-11 Created: 2016-04-08 Last updated: 2016-06-01

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