Investigating the regulation of B cell growth and survival genes by Epstein-Barr virus

Khasnis, Sarika Jayant (2019) Investigating the regulation of B cell growth and survival genes by Epstein-Barr virus. Doctoral thesis (PhD), University of Sussex.

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Abstract

The cancer-associated Epstein Barr virus (EBV) infects and immortalises Bcells and alters the expression of numerous cellular genes involved in the regulation of cell cycle and multiple growth and survival pathways. We investigated cell cycle regulation by EBV focusing on RGC-32, a cell cycle regulator that is translationally upregulated in EBV-infected cells. RGC-32 interacts with two mitotic kinases, cyclin-dependent kinase 1 (CDK1) and polo-like kinase 1 (PLK1) and overexpression of RGC-32 in B cells disrupts cell cycle arrest. Using Surface Plasmon Resonance and Microscale Thermophoresis (MST) and recombinant proteins, we demonstrated that RGC-32 directly interacts with CDK1. The affinity of this interaction is increased in the presence of Cyclin B1. Pull-down studies also identified the kinetochore subunit Spc24-25 as a new binding partner of RGC-32 and we confirmed this interaction using recombinant proteins and MST. The role of RGC-32 in regulating the G2/M transition was investigated using live-cell imaging. We generated a stable transgenic ‘knock-in’ RGC-32 inducible cell line in an ATP analogue-sensitive CDK1 background. Using this cell line arrested in G2 through CDK1 inhibition by the ATP analogue we demonstrated the ability of RGC-32to promote mitotic entry and activate CDK1 in vivo. CDK1 activation was associated with decreased ‘inactivating’ phosphorylation ontyrosine 15 of CDK1. We also identified the B-cell receptor (BCR) signalling pathway to be potentially targeted by the EBV transcription factors Epstein-Barr virus nuclear antigens (EBNA) 2, 3A, 3B and 3C. To confirm regulation of BCR genes bound by these EBNAs, we performed gene expression profiling. We showed that CD19, SYK, LYN, INPP5D, PIK3R1, PIK3R3, PIK3R5 are regulated by EBNA2 and EBNA3s. Our data demonstrated transcriptional repression of the PI3K-AKT pathway genes and decreased ‘activating’ phosphorylation of Akt by EBNA2, EBNA3A and EBNA3C. Our data provide new insights into B cell reprogramming by EBV.

Item Type: Thesis (Doctoral)
Schools and Departments: School of Life Sciences > Biochemistry
Subjects: Q Science > QR Microbiology > QR0355 Virology
R Medicine > RC Internal medicine > RC0109 Infectious and parasitic diseases > RC0141.5 Epstein-Barr virus disease
Depositing User: Library Cataloguing
Date Deposited: 08 Apr 2019 12:20
Last Modified: 11 May 2021 09:43
URI: http://sro.sussex.ac.uk/id/eprint/83089

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