Human cytomegalovirus is a significant human pathogen that establishes a life- long latent reservoir in undifferentiated cells of the myeloid lineage in part by suppressing viral immediate early (IE) lytic phase gene expression when it enters these cells. While it was known for some time that the viral tegument protein pp71 was the master regulator of IE gene expression, how this critical transcription factor actually worked remained enigmatic. In the last funding period of this grant, we determined the mechanism through which pp71 activates IE gene expression. The protein delivered from the virion tegument translocates to the nucleus of differentiated cells such as fibroblasts and degrades Daxx, a cellular transcriptional repressor and intrinsic defense protein that, prior to or in the absence of pp71 function, transcriptionally silences infecting viral genomes. By degrading Daxx, pp71 activates IE gene expression and lytic replication ensues. We further showed during the last funding period that the Daxx intrinsic defense also represses IE gene expression during the establishment of latency, and in this context is not inactivated by pp71. Daxx represses cellular gene expression in at least three ways: blocking the activity of cellular transcription factors, recruiting modifying proteins to targeted promoters, and depositing histone variant H3.3 onto non- replicating DNA. We propose in Aim 1 to decipher how each individual activity of Daxx contributes to its ability to silence viral IE gene expression at the start of both lytic and laten infections. In lytically infected fibroblasts, the Daxx intrinsic defense is quickly inactivated by pp71, but remains active in latently infected undifferentiated cells. In the last funding period we showed that Daxx is not inactivated when latency is established because tegument-delivered pp71 remains in the cytoplasm. In broad terms, this means that the entry processes into differentiated and undifferentiated cells must have at least some differences. Specifically, the subcellular localization of tegument-delivered proteins is different. In the last funding period, w used heterologous fusions between differentiated and undifferentiated cells to show that differentiated cells express a factor that permits the nuclear trafficking of tegument delivered pp71 in undifferentiated cells. We propose in Aim 2 to define the entry process HCMV uses to infect undifferentiated cells and establish latency, and to identify the factor found in differentiated cells that permits tegument-delivered pp71 access to the nucleus.

Public Health Relevance

Human cytomegalovirus infects almost everyone. It causes birth defects and disease in people sick with cancer, AIDS, and those undergoing organ transplants. It also seems to exacerbate cardiovascular diseases, to adversely affect the ability of the elderly to fight off other infectious diseases, and may be directly related to human glioblastoma multiforme brain tumors. HCMV can hide in the body in a latent state and reactivate at any time to cause disease and to spread from person to person. This work will study how HCMV establishes this latent state, with the hopes of identifying ways to fight the virus with antiviral drugs.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI074984-10
Application #
9388941
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Beisel, Christopher E
Project Start
2007-07-01
Project End
2018-11-30
Budget Start
2017-12-01
Budget End
2018-11-30
Support Year
10
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Microbiology/Immun/Virology
Type
Graduate Schools
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Lee, Song Hee; Caviness, Katie; Albright, Emily R et al. (2016) Long and Short Isoforms of the Human Cytomegalovirus UL138 Protein Silence IE Transcription and Promote Latency. J Virol 90:9483-94
Albright, Emily R; Kalejta, Robert F (2016) Canonical and variant forms of histone H3 are deposited onto the human cytomegalovirus genome during lytic and latent infections. J Virol :
Lee, Song Hee; Albright, Emily R; Lee, Jeong-Hee et al. (2015) Cellular defense against latent colonization foiled by human cytomegalovirus UL138 protein. Sci Adv 1:e1501164
Winkler, Laura L; Kalejta, Robert F (2014) The 19S proteasome activator promotes human cytomegalovirus immediate early gene expression through proteolytic and nonproteolytic mechanisms. J Virol 88:11782-90
Qin, Qingsong; Lee, Song Hee; Liang, Ruibin et al. (2014) Insertion of myeloid-active elements into the human cytomegalovirus major immediate early promoter is not sufficient to drive its activation upon infection of undifferentiated myeloid cells. Virology 448:125-32
Penkert, Rhiannon R; Kalejta, Robert F (2013) Human embryonic stem cell lines model experimental human cytomegalovirus latency. MBio 4:e00298-13
Qin, Qingsong; Penkert, Rhiannon R; Kalejta, Robert F (2013) Heterologous viral promoters incorporated into the human cytomegalovirus genome are silenced during experimental latency. J Virol 87:9886-94
Albright, Emily R; Kalejta, Robert F (2013) Myeloblastic cell lines mimic some but not all aspects of human cytomegalovirus experimental latency defined in primary CD34+ cell populations. J Virol 87:9802-12
Winkler, Laura L; Hwang, Jiwon; Kalejta, Robert F (2013) Ubiquitin-independent proteasomal degradation of tumor suppressors by human cytomegalovirus pp71 requires the 19S regulatory particle. J Virol 87:4665-71
Penkert, Rhiannon R; Kalejta, Robert F (2012) Tale of a tegument transactivator: the past, present and future of human CMV pp71. Future Virol 7:855-869

Showing the most recent 10 out of 19 publications