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Adenovirus Precursor pVII Protein Stability Is Regulated By Its Propeptide Sequence
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.
2013 (English)In: PLoS ONE, ISSN 1932-6203, E-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.

Place, publisher, year, edition, pages
2013. Vol. 8, no 11, e80617- p.
Keyword [en]
Adenovirus, pro-peptide, pVII, cullin3, protein stability
National Category
Microbiology
Research subject
Microbiology; Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-212439DOI: 10.1371/journal.pone.0080617ISI: 000327258600074OAI: oai:DiVA.org:uu-212439DiVA: diva2:677772
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: 2017-12-06Bibliographically approved
In thesis
1. Functional characterization of the human adenovirus pVII protein and non-coding VA RNAI
Open this publication in new window or tab >>Functional characterization of the human adenovirus pVII protein and non-coding VA RNAI
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
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:nbn:se:uu:diva-321641 (URN)978-91-554-9941-9 (ISBN)
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

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