The Papillomavirus E2 proteins are multifunctional regulatory factors known to play pivotal roles in various stages of the papillomavirus life cycle. These proteins consist of three structural domains: Carboxy-Terminal DNA-Binding Domain: This domain forms homodimers and binds to 12 bp palindromic target sequences within the viral genome. Amino-Terminal Interaction Domain: This domain serves as a platform for interacting with host proteins and the viral helicase E1, facilitating the formation of the di-hexameric E1 complex that regulates viral replication in conjunction with host proteins. Unstructured Hinge Region: This region links the amino and carboxyl domains, contributing to the overall structural flexibility and functionality of E2.
The E2 protein has three crucial roles during the papillomavirus life cycle: E2 facilitates the recruitment of the viral helicase E1 to the origin of replication, promoting the formation of the di-hexameric E1 complex, which is essential for regulating viral replication in coordination with host proteins. E2 can regulate transcription from both the viral and host genomes, ensuring the promotion of the viral life cycle. E2 acts as a plasmid segregation factor, ensuring that viral genomes are accurately distributed into daughter nuclei following cell division. Despite our understanding of these roles, the precise host proteins that mediate E2 functions remain incompletely characterized. This study primarily focuses on enhancing our comprehension of the plasmid segregation function of HPV16 E2.
During mitosis, a complex series of events takes place, including the breakdown and reformation of the nuclear envelope, which results in the formation of nuclei in daughter cells. The HPV E2 protein plays a pivotal role in this process, acting as a “bridge” during mitosis. Its carboxy-terminal domain binds to the viral genome, while the amino-terminal interaction domain binds to mitotic chromatin. This interaction with mitotic chromatin is mediated by the E2 amino-terminal domain and involves host mitotic proteins.
One notable interaction is between bovine papillomavirus 1 E2 (BPV1 E2) and BRD4, which promotes the interaction between BPV1 E2 and mitotic chromatin. This interaction is significant as it also regulates the transcriptional and replication properties of E2. However, the specific role of BRD4 in mediating the mitotic interaction and plasmid segregation function of HPV16 E2 has been a subject of debate in the scientific community. Some studies have suggested that BRD4 is not the mitotic chromatin receptor for E2, while others have proposed a direct role for BRD4 in mediating HPV16 E2’s association with mitotic chromatin.
In a recent study published in the peer-reviewed Journal Virology, scientists from Virginia Commonwealth University, led by Professor Iain M. Morgan, have shed new light on the intricate mechanisms underlying the functions of Papillomavirus E2 proteins during mitosis and plasmid segregation. This research, carried out by Apurva T Prabhakar, Claire James, Christian Fontan, Raymonde Otoa, Xu Wang, Molly L Bristol, Calvin Yeager, Ronald Hill, Aanchal Dubey, Shwu-Yuan Wu, and Cheng-Ming Chiang, is a significant contribution to our understanding of the molecular intricacies of Papillomavirus, particularly focusing on the human papillomavirus 16 (HPV16) E2 protein.
The researchers have recently uncovered that the interaction with TopBP1 is critical for E2’s association with mitotic chromatin and plasmid segregation. This discovery was made through a yeast two-hybrid screen, which revealed that the phosphorylation of E2 on serine 23 by CK2 promotes its interaction with TopBP1, thereby facilitating its association with mitotic chromatin. Additionally, this interaction with TopBP1 can also regulate the DNA replication properties of E2, similar to the role of BRD4. TopBP1 is a highly active protein during mitosis, participating in various nucleic acid processes, including DNA replication and repair. The study also showed that there is a complex interplay between TopBP1 and BRD4, as they coexist in the same cellular complex. This coexistence suggests that BRD4 plays a critical role in papillomavirus life cycles, partially due to its direct interaction with E2 proteins.
The research unveiled a complex interplay between E2, TopBP1, and BRD4. To investigate the roles of these proteins in mitotic chromatin association and plasmid segregation function, the scientists generated stable cell lines expressing various E2 mutants. These mutants included E2-WT (wild-type E2), E2-S23A (a mutant with serine 23 mutated to alanine, compromising TopBP1 binding), E2-R37A (a mutant with arginine 37 mutated to alanine, preventing BRD4 binding), and E2-S23A/R37A (a double mutant).
The authors’ results demonstrated that E2-S23A showed a significant reduction in its association with mitotic chromatin, indicating the importance of serine 23 phosphorylation for this interaction. In contrast, E2-R37A exhibited increased chromatin association during mitosis, reinforcing the notion that BRD4 negatively regulates E2’s association with mitotic chromatin. The E2-S23A/R37A double mutant had compromised interaction with both TopBP1 and BRD4, leading to significantly reduced chromatin association. Interestingly, in the presence of the E2-R37A mutant, the interaction between E2 and TopBP1 was enhanced, suggesting a competitive relationship between BRD4 and TopBP1 for E2 binding.
This new study highlights the intricate interplay of Papillomavirus E2 proteins, particularly HPV16 E2, with host proteins during mitosis and plasmid segregation. By elucidating the role of TopBP1 in mediating E2’s association with mitotic chromatin and the competitive relationship with BRD4, this research advances our understanding of the molecular mechanisms governing E2 function in these critical processes.
These findings not only expand our knowledge of HPV biology but also have potential implications for the development of targeted therapies against HPV-associated diseases, such as cervical cancer. Understanding the precise molecular interactions between viral proteins and host factors during mitosis could open new avenues for therapeutic interventions aimed at disrupting these interactions and ultimately combating HPV-related malignancies. Moreover, this research underscores the complexity of host-virus interactions, emphasizing the need for further investigations to unravel the intricacies of viral replication and segregation processes.
Prabhakar AT, James CD, Fontan CT, Otoa R, Wang X, Bristol ML, Yeager C, Hill RD, Dubey A, Wu SY, Chiang CM, Morgan IM. Direct interaction with the BRD4 carboxyl-terminal motif (CTM) and TopBP1 is required for human papillomavirus 16 E2 association with mitotic chromatin and plasmid segregation function. J Virol. 2023 Sep 15:e0078223. doi: 10.1128/jvi.00782-23.