Related Experiment Videos
Nuclear matrix localization and SUMO-1 modification of adenovirus type 5 E1b 55K protein are controlled by E4 Orf6
Katherine J Lethbridge1, Gillian E Scott1, Keith N Leppard1
1Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK.
Abstract:
Human adenovirus serotype 5 encodes three proteins, E1b 55K, E4 Orf3 and E4 Orf6, which interact with each other and with components of the nucleus to regulate mRNA processing and export, viral DNA replication and p53-dependent apoptosis. Previous studies have shown that, during wild-type infection, 55K associates initially with structures termed ND10, which are sites of localization of the promyelocytic leukaemia protein, and then moves, dependent upon its interaction with Orf6, to the establishing virus replication centres. Absence of either Orf3 or Orf6 affects the localization of 55K and so may affect its function. In this study, the influence of Orf3 and Orf6 expression on the association of 55K with the insoluble matrix fraction of the nucleus and with ND10 particularly was examined. Overexpression of Orf6 was sufficient to block the association of 55K with this fraction, irrespective of the presence of Orf3. This effect depended upon the two proteins being able to interact. However, the association of 55K with ND10, which persists throughout infection in the absence of Orf6, required Orf3 to be present, thus distinguishing two subsets of matrix-associated 55K. A modified form of 55K, formation of which was blocked by mutating the known site of SUMO-1 attachment, was more abundant in the absence of Orf6 but unaffected by the absence of Orf3. Thus, this modification is favoured when 55K remains associated with the matrix but does not correlate with its stable association with ND10, many components of which are modified by SUMO-1.
Insights
Human adenovirus type 5 proteins E4 Orf3 and E4 Orf6 differentially regulate the nuclear localization of E1b 55K. Orf6 disrupts E1b 55K association with nuclear matrix, while Orf3 is required for ND10 localization.
Area of Science:
- Molecular Virology
- Cellular Biology
- Nuclear Organization
Background:
- Human adenovirus serotype 5 utilizes proteins E1b 55K, E4 Orf3, and E4 Orf6 to modulate host cell processes.
- E1b 55K interacts with Orf3 and Orf6, influencing mRNA processing, viral replication, and apoptosis.
- During infection, E1b 55K initially localizes to ND10 nuclear structures before moving to replication centers, a process dependent on Orf6.
Purpose of the Study:
- To investigate how Orf3 and Orf6 expression impacts the association of E1b 55K with the nuclear matrix and ND10.
- To differentiate the roles of Orf3 and Orf6 in E1b 55K nuclear localization and modification.
Main Methods:
- Overexpression of Orf6 and Orf3 in relevant cellular contexts.
- Analysis of E1b 55K association with insoluble nuclear matrix fractions.
- Examination of E1b 55K localization at ND10 structures.
- Assessment of E1b 55K modification, including SUMO-1 conjugation, using mutated proteins.
Main Results:
- Orf6 overexpression alone was sufficient to prevent E1b 55K association with the nuclear matrix, provided Orf6 and E1b 55K could interact.
- E1b 55K association with ND10 persisted in the absence of Orf6 but required the presence of Orf3.
- A modified form of E1b 55K, SUMO-1 conjugated, was more abundant when Orf6 was absent, indicating Orf6 inhibits this modification.
- SUMO-1 modification of E1b 55K correlated with matrix association but not stable ND10 localization.
Conclusions:
- Orf3 and Orf6 play distinct roles in regulating E1b 55K nuclear localization and association with nuclear substructures.
- Orf6 mediates the dissociation of E1b 55K from the nuclear matrix, while Orf3 is crucial for ND10 targeting.
- SUMO-1 modification of E1b 55K is favored by matrix association and is inhibited by Orf6, suggesting a regulatory mechanism linked to nuclear compartmentalization.