Human cytomegalovirus (HCMV) establishes lifelong latent infections in a host and reactivates with devastating consequences within immunosuppressed patients, underscoring the need to identify the conditions that dictate HCMV latency. To this end, we have developed and reported a novel HCMV latency model system utilizing Kasumi 3 cells (K3s) that recapitulates ALL the key stages of HCMV reactivation allowing us to experimentally test conditions critical for HCMV latency that were previously not possible. This proposal aims to test the hypothesis that the antagonistic relationship between miRNA-induced suppression of HCMV IE genes and cytokine-mediated induction of IE genes is a major determinant of the switch from latent to lytic infection. We base this concept on our exciting observations that cellular encoded hsa-miR-200 family members target the essential HCMV Immediate Early (IE) 2 protein. Additionally, we show that IE2 can induce viral Early (E) promoters within the context of the genome and independently of additional viral proteins, providing the first confirmation that IE2 is a key regulator of HCMV viral lytic transcription. In addition, we show that inflammatory cytokines potently initiate lytic reactivation of HCMV in our K3 cels that can be blocked by treatment with an NF-kB inhibitor. Based on these observations we favor a model (derived from published observations with Herpes Simplex Virus) wherein latency is established by chromatization of the viral genome resulting in suppression of viral transcription. However, an added level of suppression mediated by miRNA targeting of IE transcripts would suppress viral translation assuring latency maintenance. We offer the novel concept that HCMV has co-opted cellular miRNAs to additionally restrict viral reactivation. We seek to extend these studies on the mechanisms underlying HCMV latency and reactivation.
In Aim 1, we will determine the biological role of cellular encoded miRNAs on restricting HCMV IE translation as well as evaluate the requirement of viral encoded miRNAs on HCMV latency.
Aim 2 will address the mechanisms by which inflammatory cytokines function to reactivate HCMV as well as define the requirements of cellular transcription factors on promoting the latent to lytic switch. Completion of the aims above will contribute significantly to our knowledge on the mechanisms of viral latency and lytic reactivation. This work will lay the foundation for identifyig novel therapeutic targets for a virus that is a significant global pathogen.

Public Health Relevance

Human Cytomegalovirus (HCMV) is a major cause of morbidity and mortality upon viral reactivation within individuals with suppressed immune systems. With the recent development of our viral latency model we can now interrogate the conditions that regulate viral reactivation and subsequent disease. Understanding the role of these factors will provide insight on viral pathogenesis and identify potential targets for novel therapeutics.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI101080-02
Application #
8707958
Study Section
Virology - B Study Section (VIRB)
Program Officer
Beisel, Christopher E
Project Start
2013-08-01
Project End
2017-07-31
Budget Start
2014-08-01
Budget End
2015-07-31
Support Year
2
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Cleveland Clinic Lerner
Department
Other Basic Sciences
Type
Schools of Medicine
DUNS #
City
Cleveland
State
OH
Country
United States
Zip Code
44195
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Nukui, Masatoshi; Mori, Yasuko; Murphy, Eain A (2015) A human herpesvirus 6A-encoded microRNA: role in viral lytic replication. J Virol 89:2615-27
O'Connor, Christine M; Vanicek, Jiri; Murphy, Eain A (2014) Host microRNA regulation of human cytomegalovirus immediate early protein translation promotes viral latency. J Virol 88:5524-32
Moorman, Nathaniel J; Murphy, Eain A (2014) Roseomics: a blank slate. Curr Opin Virol 9:188-93
O'Connor, Christine M; Murphy, Eain A (2012) A myeloid progenitor cell line capable of supporting human cytomegalovirus latency and reactivation, resulting in infectious progeny. J Virol 86:9854-65
Lee, Song Hee; Kalejta, Robert F; Kerry, Julie et al. (2012) BclAF1 restriction factor is neutralized by proteasomal degradation and microRNA repression during human cytomegalovirus infection. Proc Natl Acad Sci U S A 109:9575-80