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Functional characterization of the human adenovirus pVII protein and non-coding VA RNAI
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.ORCID iD: 0000-0003-1344-3962
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Human adenovirus (HAdV) is a common pathogen causing a broad spectrum of diseases. HAdV encodes the pVII protein, which is involved in nuclear delivery, protection and expression of viral DNA. To suppress the cellular interferon (IFN) and RNA interference (RNAi) systems, HAdVs encode non-coding virus-associated (VA) RNAs. In this thesis we have investigated the functional significance of the pVII protein and VA RNAI in HAdV-5 infected cells.

We report that the propeptide module is the destabilizing element targeting the precursor pVII protein for proteasomal degradation. We also found that the Cul3-based E3 ubiquitin ligase complex alter the precursor pVII protein stability via binding to the propeptide sequence. In addition, we show that inhibition of the Cul3 protein reduces HAdV-5 E1A gene expression. Collectively, our results suggest a novel function for the pVII propeptide module and involvement of Cul3 in viral E1A gene expression.

Our studies show that the cellular E3 ubiquitin ligase MKRN1 is a novel pVII interacting protein in HAdV-5 infected cells. MKRN1 expression reduced the pVII protein accumulation in virus-infected cells and affected infectious virus formation. Surprisingly, the endogenous MKRN1 protein underwent proteasomal degradation during the prolonged HAdV-5 infection. Furthermore, the precursor pVII protein enhanced MKRN1 self-ubiquitination, suggesting the direct involvement of pVII in the initiation of MKRN1 degradation. Hence, we propose that the MKRN1 is a novel antiviral protein and that HAdV-5 infection counteracts its antiviral activity.

In papers III and IV, we tested the ability of various plant and animal virus encoded RNAi/miRNA and IFN suppressor proteins to functionally substitute for the HAdV-5 VA RNAI. Our results revealed that the Vaccinia virus E3L protein was able to partially substitute for the HAdV-5 VA RNAI functions in virus-infected cells. Interestingly, the E3L protein rescued the translational defect but did not stimulate viral capsid mRNA accumulation observed with VA RNA. Additionally, we show that the HAdV-4 and HAdV-37 VA RNAI are more effective in virus replication compared to HAdV-5 and HAdV-12 VA RNAI. In paper IV, we employed a novel triplex-specific probing assay, based on the intercalating and cleaving agent benzoquinoquinaxline 1,10-phenanthroline (BQQ-OP), to unravel triplex structure formation in 
VA RNAI. The BQQ-OP cleavage of HAdV-4 VA RNAI indicates that a potential 
triplex is formed involving the highly conserved stem 4 of the central domain and side 
stem 7. Further, the integrity of HAdV-4 VA RNAI stem 7 contributes to the virus growth in vivo.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. , 58 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1339
Keyword [en]
Adenovirus, VA RNA, protein VII, Ubiquitination, proteasome, anti-viral
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Cell and Molecular Biology
Research subject
Medical Biochemistry; Microbiology; Medical Virology
Identifiers
URN: urn:nbn:se:uu:diva-321641ISBN: 978-91-554-9941-9 (print)OAI: oai:DiVA.org:uu-321641DiVA: diva2:1094348
Public defence
2017-09-01, Room C8:305, Biomedical Centrum (BMC), Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2017-06-09 Created: 2017-05-09 Last updated: 2017-08-09
List of papers
1. Adenovirus Precursor pVII Protein Stability Is Regulated By Its Propeptide Sequence
Open this publication in new window or tab >>Adenovirus Precursor pVII Protein Stability Is Regulated By Its Propeptide Sequence
2013 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 8, no 11, e80617- p.Article in journal (Refereed) Published
Abstract [en]

Adenovirus encodes for the pVII protein, which interacts and modulates virus DNA structure in the infected cells. The pVII protein is synthesized as the precursor protein and undergoes proteolytic processing by viral proteinase Avp, leading to release of a propeptide sequence and accumulation of the mature VII protein. Here we elucidate the molecular functions of the propeptide sequence present in the precursor pVII protein. The results show that the propeptide is the destabilizing element targeting the precursor pVII protein for proteasomal degradation. Our data further indicate that the propeptide sequence and the lysine residues K26 and K27 regulate the precursor pVII protein stability in a co-dependent manner. We also provide evidence that the Cullin-3 E3 ubiquitin ligase complex alters the precursor pVII protein stability by association with the propeptide sequence. In addition, we show that inactivation of the Cullin-3 protein activity reduces adenovirus E1A gene expression during early phase of virus infection. Collectively, our results indicate a novel function of the adenovirus propeptide sequence and involvement of Cullin-3 in adenovirus gene expression.

Keyword
Adenovirus, pro-peptide, pVII, cullin3, protein stability
National Category
Microbiology
Research subject
Microbiology; Biochemistry
Identifiers
urn:nbn:se:uu:diva-212439 (URN)10.1371/journal.pone.0080617 (DOI)000327258600074 ()
Funder
Swedish Research Council, K2012-99X-21959-01-3, 2006-5038-36531-16Swedish Cancer Society, 12 0504
Available from: 2013-12-10 Created: 2013-12-10 Last updated: 2017-05-09Bibliographically approved
2. Human adenovirus infection counteracts the anti-viral activity of the cellular MKRN1 E3 ubiquitin ligase
Open this publication in new window or tab >>Human adenovirus infection counteracts the anti-viral activity of the cellular MKRN1 E3 ubiquitin ligase
Show others...
(English)Article in journal (Refereed) Submitted
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-321638 (URN)
Available from: 2017-05-09 Created: 2017-05-09 Last updated: 2017-05-09
3. Complementation of the human adenovirus type 5 VA RNAI defect by the Vaccinia virus E3L protein and serotype-specific VA RNAIs
Open this publication in new window or tab >>Complementation of the human adenovirus type 5 VA RNAI defect by the Vaccinia virus E3L protein and serotype-specific VA RNAIs
2015 (English)In: Virology, ISSN 0042-6822, E-ISSN 1096-0341, Vol. 485, 25-35 p.Article in journal (Refereed) Published
Abstract [en]

Human adenoviruses (HAdVs) encode for multifunctional non-coding virus-associated (VA) RNAs, which function as powerful suppressors of the cellular interferon (IFN) and RNA interference (RNAi) systems. In this study we tested the ability of various plant and animal virus encoded RNAi and IFN suppressor proteins to functionally substitute for the HAdV-5 VA RNAI. Our results revealed that only the Vaccinia virus (VACV) E3L protein was able to substitute for the HAdV-5 VA RNAI functions in virus-infected cells. Interestingly, the E3L protein rescues the translational defect but does not stimulate viral capsid mRNA accumulation observed with VA RNA. We further show that the E3L C-terminal region containing the dsRNA-binding domain is needed to enhance VA RNAI mutant virus replication. Additionally, we show that the HAdV-4 and HAdV-37 VA RNAI are more effective than the HAdV-5 VA RNAI in rescuing virus replication.

Keyword
Adenovirus, Vaccinia virus, interferon, PKR, RNAi
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Medical Virology
Identifiers
urn:nbn:se:uu:diva-258879 (URN)10.1016/j.virol.2015.07.002 (DOI)000363993100003 ()
Funder
Swedish Cancer Society, 12 0504, 13 0469Swedish Research Council, K2012–999X-21959-01-301–3Åke Wiberg Foundation, M14-01555
Available from: 2015-07-21 Created: 2015-07-21 Last updated: 2017-05-09Bibliographically approved
4. RNA triplex formation in human adenovirus type 4 VA RNAI and its implication on virus growth
Open this publication in new window or tab >>RNA triplex formation in human adenovirus type 4 VA RNAI and its implication on virus growth
(English)Article in journal (Refereed) Submitted
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-321639 (URN)
Available from: 2017-05-09 Created: 2017-05-09 Last updated: 2017-05-09

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