Investigating how adenovirus E1A oncoproteins regulate host cell signalling pathways

Omran, Asma ORCID: 0000-0001-7378-1248 (2024). Investigating how adenovirus E1A oncoproteins regulate host cell signalling pathways. University of Birmingham. M.Sc.

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Abstract

The adenovirus E1A 13S and 12S transcripts give rise to protein products of 289 and 243 amino acids, respectively, in Ad2/5, and differ by conserved region 3 which is present only in the 289-residue protein. These proteins are, functionally, well characterised: the CR3 region of the 13S gene product is essential for transactivation of other viral early promoters through interaction with the cellular basal transcriptional machinery, transcription factors and transcriptional regulators; whilst the 12S gene product also regulates transcription through interaction with an overlapping set of transcriptional regulators, which can bind to other regions of the E1A protein, as well as CR3, to regulate cellular transcription programmes.
To study cellular functions of the Ad5 and Ad12 13S gene product, in the absence of 12S and other E1A gene products, we have made clonal, tetracycline (doxycycline)-inducible Ad5 and Ad12 13S E1A U2OS cell lines. In this regard, we have also made tetracycline-inducible Ad5 13S E1A L1920A and Ad12 13S E1A RG2 U2OS cell lines where the function of the N-terminal region has been inactivated through mutation. Co-immunoprecipitation/Western blot studies with these cell lines revealed that E1A expressed in response to doxycycline was functional as it was able to bind known E1A-binding proteins, CBP/p300, the pRB family of proteins as well as CtBP, although we were unable to confirm the ability of potential E1A-interacting proteins, identified by mass spectrometry, to interact with the 13S E1A gene product. These cell lines do have the potential, however, to study 13S E1A function in greater detail.
In order to study the function of the 9S E1A gene product we similarly attempted to make tetracycline-inducible Ad5 and Ad12 9S E1A U2OS cell lines, though such attempts were unsuccessful. We therefore utilised GFP-tagged Ad2 and Ad12 9S E1A constructs that could be expressed transiently to study their function. GFP pulldowns from U2OS cells expressing either GFP alone or GFP-tagged Ad12 9S E1A, coupled to mass spectrometry identified a number of cellular proteins that isolated specifically with the Ad12 9S gene product. Conventional IP-Western blot studies will need to be performed however, to identify bona fide 9S-interacting proteins before functional assays can be employed to determine the consequences of 9S interactions with these proteins.
Very little is known about the structure of E1A proteins other than that E1A function is dictated by molecular recognition features that interact with partner proteins. The 13S and 12S E1A gene products, are thought to be largely intrinsically-disordered proteins that assume structure on binding to partner proteins. We therefore utilised Alphafold 2 to determine putative structures for Ad2, Ad5 and Ad12 13S gene products. Consistent with the idea that E1A is mostly unstructured, large regions of the E1A were deemed not to have structural integrity. Interestingly, however, the proposed Zn-finger component of CR3 from these Ad species are predicted to form finger like projections, with an α-helical region separated by a short unstructured region that runs into a smaller α-helix, with the 4 Cys residues, responsible for coordinating Zn2+ binding, forming the base of the finger. These data suggest that CR3 from different adenovirus types are very well conserved at the structural level.
We also determined putative structures for the Ad2, Ad5 and Ad12 9S gene products, which have not been considered previously. Interestingly, 9S gene products were proposed to be highly structured, although structures generated were different for the 3 adenovirus types. Ad2 9S E1A was proposed to form a loop composed of 2 α-helices that ran parallel to each other, whilst Ad5 9S was also composed of 2 α-helices, though were proposed to be arranged perpendicular to one another. Ad12 9S was also composed of 2 α-helices that ran in series. Although these species might adopt similar structure when in association with partner proteins these data suggest that 9S from different Ad types might possess different functions.
Taken together, these studies have been useful in generating cell lines that can be used to probe E1A function in more detail and identifying putative 9S E1A-interacting proteins. It will be useful in the future to consider E1A function and interaction with cellular partner proteins in relation to specific amino acids within its predicted structure.

Type of Work:

Thesis (Masters by Research > M.Sc.)

Award Type:

Masters by Research > M.Sc.

Supervisor(s):