The relationship between the adenovirus early region 1B55K (E1B55K) protein and the cellular DNA damage response

Abualfaraj, Tareq ORCID: 0000-0003-2747-679X (2023). The relationship between the adenovirus early region 1B55K (E1B55K) protein and the cellular DNA damage response. University of Birmingham. Ph.D.

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Abstract

Adenoviruses are members of the small DNA tumour virus family; there are more than 100 identified Human Adenovirus (HAdV) types which are assigned into 7 groups designated A to G. The adenoviral early region 1B55K (E1B55K) protein is essential for complete HAdV- mediated transformation of cells in culture and for efficient adenovirus replication. During infection, E1B55K forms a complex with early region 4 open reading frame 6 (E4orf6) protein; together they bind and target several cellular proteins, many of which are involved in the DNA damage response (DDR), for proteasomal-mediated degradation. This is the case for HAdV5 and HAdV12 although other HAdV types such as HAdV16 (group B), HAdV9 (group D), HAdV4 (group E), and HAdV40 (group F) do not cause degradation of many of the same proteins. Most previous research has focused on the activities of the HAdV5-E1B55K protein during viral infection while in collaboration with E4orf6 and functioning as part of the E3 ubiquitin ligase. Here, we examined effects of E1B55K both as part of the E3 ligase and in isolation. Furthermore, we have concentrated on the properties of the group A, B, D, E, and F E1B55K proteins rather than HAdV5. Initially, we confirmed the interactions between HAdV5-E1B55K, and proteins previously identified as part of mass spectroscopy screens and showed that some, but not all, are degraded during viral infection. We have further shown binding of the same proteins to HAdV12-E1B55K, although degradation during infection was more limited. Novel association of HAdV12-E1B55K with replisome components has also been demonstrated. In the second part of the study, we have confirmed that HAdV12-E1B55K can induce genome instability in human fibroblasts in the absence of any other viral proteins. We investigated the effects of HAdV12-E1B55K protein on the DNA damage response (DDR) and on genomic stability, using human skin fibroblasts (HSF) expressing only HAdV12-E1B55K (55K+HSF). Our results show that HAdV12-E1B55K interferes with DNA replication dynamics, seen as a significant increase in stalled forks and R-loops, both of which are indicative of increased replication stress. We have also demonstrated that HAdV12-E1B55K expression sensitises cells to genotoxic agents, as shown by an increase in DNA damage foci formation and ATM activation. Furthermore, our findings are in line with previous observations which indicated that HAdV12 induces genomic instability in human cells, represented by an increase in chromosomal breaks. We hypothesise that interactions of HAdV12-E1B55K with different DDR and replication complex proteins is responsible for majority of these effects. Similarly, the expression of E1B55K protein from groups B, D, E and F HAdVs increased genomic instability in human tumour cells, seen as an increase in micronuclei formation. The viral proteins from these HAdV types associate with several homologous recombination DNA damage repair components, although with a limited effect, compared to HAdV12. Additionally, we have shown that HAdV-E1B55K from group B, D, E and F inhibits DNA double strand break repair as seen by the increased DNA damage repair foci after induced DNA damage. Using proteomic analysis, we have also identified novel E1B55K-interacting cellular substrates that bind these viral proteins, although the significance of these interactions is yet to be determined.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

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