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Comparison of protein expression during wild-type, and E1B-55k-deletion, adenovirus infection using quantitative time-course proteomics.

ABSTRACT: Adenovirus has evolved strategies to usurp host-cell factors and machinery to facilitate its life cycle, including cell entry, replication, assembly and egress. Adenovirus continues, therefore, to be an important model system for investigating fundamental cellular processes. The role of adenovirus E1B-55k in targeting host-cell proteins that possess antiviral activity for proteasomal degradation is now well established. To expand our understanding of E1B-55k in regulating the levels of host-cell proteins, we performed comparative proteome analysis of wild-type, and E1B-55k-deletion, adenovirus-infected cancer cells. As such we performed quantitative MS/MS analysis to monitor protein expression changes affected by viral E1B-55k. We identified 5937 proteins, and of these, 69 and 58 proteins were down-regulated during wild-type and E1B-55k (dl1520) adenovirus infection, respectively. This analysis revealed that there are many, previously unidentified, cellular proteins subjected to degradation by adenovirus utilizing pathways independent of E1B-55k expression. Moreover, we found that ALCAM, EPHA2 and PTPRF, three cellular proteins that function in the regulation of cell-cell contacts, appeared to be degraded by E1B-55k/E4orf3 and/or E1B-55k/E4orf6 complexes. These molecules, like integrin ?3 (a known substrate of E1B-55k/E4orf6), are critical regulators of cell signalling, cell adhesion and cell surface modulation, and their degradation during infection is, potentially, pertinent to adenovirus propagation. The data presented in this study illustrate the broad nature of protein down-regulation mediated by adenovirus.

SUBMITTER: Fu YR

PROVIDER: S-EPMC5656791 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Comparison of protein expression during wild-type, and E1B-55k-deletion, adenovirus infection using quantitative time-course proteomics.

Fu Yen Rong YR Turnell Andrew S AS Davis Simon S Heesom Kate J KJ Evans Vanessa C VC Matthews David A DA

The Journal of general virology 20170620 6

Adenovirus has evolved strategies to usurp host-cell factors and machinery to facilitate its life cycle, including cell entry, replication, assembly and egress. Adenovirus continues, therefore, to be an important model system for investigating fundamental cellular processes. The role of adenovirus E1B-55k in targeting host-cell proteins that possess antiviral activity for proteasomal degradation is now well established. To expand our understanding of E1B-55k in regulating the levels of host-cell ...[more]

PMID: 28631589

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Project description:Human adenovirus (HAdV) E1B-55K is a multifunctional regulator of productive viral replication and oncogenic transformation in nonpermissive mammalian cells. These functions depend on E1B-55K's posttranslational modification with the SUMO protein and its binding to HAdV E4orf6. Both early viral proteins recruit specific host factors to form an E3 ubiquitin ligase complex that targets antiviral host substrates for proteasomal degradation. Recently, we reported that the PML-NB-associated factor Daxx represses efficient HAdV productive infection and is proteasomally degraded via a SUMO-E1B-55K-dependent, E4orf6-independent pathway, the details of which remained to be established. RNF4, a cellular SUMO-targeted ubiquitin ligase (STUbL), induces ubiquitinylation of specific SUMOylated proteins and plays an essential role during DNA repair. Here, we show that E1B-55K recruits RNF4 to the insoluble nuclear matrix fraction of the infected cell to support RNF4/Daxx association, promoting Daxx PTM and thus inhibiting this antiviral factor. Removing RNF4 from infected cells using RNA interference resulted in blocking the proper establishment of viral replication centers and significantly diminished viral gene expression. These results provide a model for how HAdV antagonize the antiviral host responses by exploiting the functional capacity of cellular STUbLs. Thus, RNF4 and its STUbL function represent a positive factor during lytic infection and a novel candidate for future therapeutic antiviral intervention strategies.IMPORTANCE Daxx is a PML-NB-associated transcription factor that was recently shown to repress efficient HAdV productive infection. To counteract this antiviral measurement during infection, Daxx is degraded via a novel pathway including viral E1B-55K and host proteasomes. This virus-mediated degradation is independent of the classical HAdV E3 ubiquitin ligase complex, which is essential during viral infection to target other host antiviral substrates. To maintain a productive viral life cycle, HAdV E1B-55K early viral protein inhibits the chromatin-remodeling factor Daxx in a SUMO-dependent manner. In addition, viral E1B-55K protein recruits the STUbL RNF4 and sequesters it into the insoluble fraction of the infected cell. E1B-55K promotes complex formation between RNF4- and E1B-55K-targeted Daxx protein, supporting Daxx posttranslational modification prior to functional inhibition. Hence, RNF4 represents a novel host factor that is beneficial for HAdV gene expression by supporting Daxx counteraction. In this regard, RNF4 and other STUbL proteins might represent novel targets for therapeutic intervention.

Project description:Potato late blight is one the most important crop diseases worldwide. Even though potato has been studied for many years, the potato disease late blight still has a vast negative effect on the potato production [1], [2], [3]. Late blight is caused by the pathogen Phytophthora infestans (P. infestans), which initiates infection through leaves. However, the biological activities during different stages of infection are poorly described, and could enable novel or improved ways of defeating late blight infection [4]. Therefore, we investigated the interactions between P. infestans (mixed strain culture) and potato (Solanum tuberosum). Three commercially available field potato cultivars of different resistance to late blight infection; Kuras (moderate), Sarpo Mira (highly resistant) and Bintje (very susceptable) were grown under controlled green house conditions and inoculated with a diversity of P. infestans populations. We used label-free quantitative proteomics to investigate the infection with P. infestans in a time-course study over 258 h. Several key issues limits proteome analysis of potato leaf tissue [5], [6], [7]. Firstly, the immense complexity of the plant proteome, which is further complicated by the presence of highly abundant proteins, such as ribulose bisphosphate carboxylase/oxygenase (RuBisCO). Secondly, plant leaf and potato, in particular, contain abundant levels amounts of phenols and polyphenols, which hinder or completely prevent a successful protein extraction. Hitherto, protein profiling of potato leaf tissues have been limited to few proteome studies and only 1484 proteins have been extracted and comprehensively described [5], [8], [9]. We here present the detailed methods and raw data by optimized gel-enhanced label free quantitative approach. The methodology enabled us to detect and quantify between 3248 and 3529 unique proteins from each cultivar, and up to 758 P. infestans derived proteins. The complete dataset is available via ProteomeXchange, with the identifier PXD002767.

Dataset Information

Comparison of protein expression during wild-type, and E1B-55k-deletion, adenovirus infection using quantitative time-course proteomics.

Publications

Comparison of protein expression during wild-type, and E1B-55k-deletion, adenovirus infection using quantitative time-course proteomics.

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