Biology and Biomedical Sciences: Molecular Microbiology and Microbial Pathogenesis
Date of Award
Doctor of Philosophy (PhD)
Chair and Committee
Deborah J Lenschow
Human cytomegalovirus (HCMV) is a ubiquitous pathogen that infects the majority of the world's population. Even though HCMV infection is usually asymptomatic, it acts as an opportunistic pathogen and is the major cause of morbidity and mortality in immunocompromised individuals, including transplant recipients and AIDS/HIV patients. Understanding HCMV biology is critical to the development of HCMV therapeutics. However, our current knowledge of HCMV biology is limited by the fact that only less than half of the HCMV genes have been characterized, especially viral essential and augmenting genes which play critical roles in viral replication.
To study the function of HCMV essential and augmenting genes, we devised a conditional approach to facilitate the analysis. In this approach, we constructed recombinant virus where the viral open reading frame (ORF) of interest is tagged with the destabilization domain FKBP (ddFKBP) which targets the fusion protein for rapid degradation. However, the fusion protein can be stabilized by the synthetic ligand Shield-1 (Shld-1). This system allows us to monitor the effects of the viral protein on the HCMV life cycle simply by infecting human fibroblasts in the presence or absence of Shld-1.
We adopted this conditional protein genetic approach to characterize the role of the human cytomegalovirus (HCMV) gene UL79 during virus infection. We constructed ADddUL79, a recombinant HCMV in which the annotated UL79 open reading frame (ORF) was tagged with ddFKBP. ADddUL79 failed to replicate without Shield-1, but it grew at wild-type levels with Shield-1 or in human foreskin fibroblasts overexpressing hemagglutinin (HA)-tagged UL79 (HF-UL79HA cells), indicating an essential role of UL79 and the effectiveness of this approach. Without Shield-1, representative immediate-early and early viral proteins as well as viral DNA accumulated normally, but late transcripts and proteins were markedly reduced. UL79 was transcribed with early- late kinetics, and was also regulated via a positive-feedback loop. We also found that the UL79 protein localized to viral replication compartments during HCMV infection. Moreover, we created a second UL79 mutant virus (ADinUL79stop) in which the UL79 ORF was disrupted by a stop codon mutation and found that ADinUL79stop phenocopied ADddUL79 under the destabilizing condition. Taking together, we showed that UL79 acts after viral DNA replication to promote the accumulation of late viral transcripts.
To test how pUL79 regulates viral late gene transcription, we analyzed the proteome associated with pUL79 during virus infection by mass spectrometry. We identified both cellular transcriptional factors, including multiple RNA polymerase II (RNAP II) subunits, and novel viral transactivators, including pUL87, pUL92, and pUL95, as protein binding partners of pUL79. Co-immunoprecipitation (co-IP) followed by immunoblot analysis confirmed the pUL79-RNAP II interaction, and this interaction was independent of any other viral proteins. We showed that this interaction did not alter the total levels of RNAP II or its recruitment to viral late promoters. Furthermore, pUL79 did not alter the phosphorylation profiles of the RNAP II C-terminal domain, which is critical for transcriptional regulation. Rather, nuclear run-on assay indicated that, in the absence of pUL79, RNAP II failed to elongate and stalled on viral DNA. Surprisingly, pUL79-dependent RNAP II elongation was required for transcription from all three kinetic classes of viral genes (i.e. immediate-early, early, and late) at late times during virus infection. In contrast, host gene transcription during HCMV infection was independent of pUL79. In summary, we have identified a novel viral mechanism by which pUL79 regulates the rate of RNAP II transcription machinery on viral transcription during late stages of HCMV infection.
Together these data provide important insight into how HCMV uses pUL79 to promote viral transcription specifically during late stages of viral infection. Understanding the mechanisms by which pUL79 regulates RNAP II elongation as well as its association with other viral factors will aid in the development of future therapeutics against HCMV infection.
Perng, Yi-Chieh, "The Role of Novel Gene UL79 Encoded by Human Cytomegalovirus in Viral Replication" (2014). All Theses and Dissertations (ETDs). 1333.