Advisory Committee Chair
Sunnie R Thompson
Advisory Committee Members
Igor Chesnovkov
Louise T Chow
Elliot J Lefkowitz
Bradley K Yoder
Document Type
Dissertation
Date of Award
2023
Degree Name by School
Doctor of Philosophy (PhD) Heersink School of Medicine
Abstract
Cell cycle is a universal necessity of cellular life and impacts nearly every process within the cell. However, despite over one hundred years of research, there still remain questions concerning the mechanisms and regulation of eukaryotic cell cycle. From the beginning, viruses have informed our knowledge of cell cycle. Many key regulators such as the E2F transcription factors, Src kinase, and p53 were originally discovered while studying the interactions between viruses and the cell. Herein, we continue the investigation of viral interactions with cell cycle machinery by researching an RNA virus and a DNA virus. These viruses, human coronavirus OC43 and BK polyomavirus, generate vastly different cell cycle phenotypes, but ultimately demonstrate both the manipulation and dependence of viruses on host cell cycle. Human coronaviruses are large, positive-sense RNA viruses with great pandemic potential. Despite replicating in the cytoplasm, human and nonhuman coronaviruses have been reported to cause cell cycle arrest. However, many of these studies report conflicting phenotypes and mechanisms. While investigating the human coronavirus OC43 in primary human cells, we found that a variety of human coronaviruses generate the same, iii multifaceted cell cycle arrest. Not only does this include a G1/S and G2/M arrest, we also observed a rapid S phase collapse. These findings both unite the previous reports under a single phenotype and suggest that cell cycle may directly impact coronavirus replication. By inhibiting cell cycle related kinases, we found that OC43 required Cdk1 for replication and phosphorylation of its structural protein nucleocapsid. Furthermore, we developed a single-cell technique for quantifying the timing and production of viral progeny. Using this technique, we discovered that S phase was antiviral and increased the likelihood of a nonproductive infection. From this work, we have not only characterized the necessity of cell cycle for coronaviruses, but also build on the growing consensus that RNA viruses are affected by host cell cycle. In contrast, human polyomaviruses are well known to manipulate cell cycle. As a small double-stranded DNA virus, BK polyomavirus (BKPyV) requires S phase proteins for its own replication. To gain access to these proteins, BKPyV encodes the viral oncogene large tumor-antigen (TAg), which induces S phase in the host cell. Replication of the viral genome is then thought to activate the DNA damage response (DDR) and prevent mitosis. Although typically activated in the response to genotoxic stress, DNA damage has not been observed during a normal BKPyV infection. Through the expression of TAg and the activation of the DDR, BKPyV causes the cell to re-replicate cellular DNA beyond 4N. Paradoxically, DDR activation is known to prevent eukaryotic DNA replication. The first clue to this dilemma came from investigating the role of a nonessential ribosomal protein, eS25, in a BKPyV infection. Although known to regulate iv noncanonical translation initiation, we found no evidence that BKPyV requires noncanonical translation. Instead, we found that eS25 was required to maintain cellular replication and that BKPyV infection was delayed in the absence of eS25. If BKPyV replication only relied on S phase proteins, viral replication should be insensitive to eS25 loss, as TAg alone would be capable of upregulating S phase proteins. In attempting to resolve this, we found that the viral expression of TAg required an initial host S phase. Furthermore, markers of viral replication such as DDR activation and late gene expression were found only in the re-replicating cells. Finally, we found that variation in the host cell type and mitogenic signaling was predictive of viral production; culminating in the discovery of a link between initial host mitogenic signaling and viral production later during infection. These findings not only fundamentally change the paradigm of a BKPyV infection, but also further demonstrate that even DNA tumor viruses are sensitive to the intrinsic cell cycle.
Recommended Citation
Needham, Jason M., "Manipulation and Dependence on Host Cell Cycle by Human RNA and DNA Viruses: Human Coronavirus OC43 and BK Polyomavirus" (2023). All ETDs from UAB. 386.
https://digitalcommons.library.uab.edu/etd-collection/386