The long-term goal of this project is to develop an understanding of the cellular and molecular mechanisms of human cytomegalovirus (HCMV) latency in CD34+ hematopoietic progenitor cells (HPCs) and the effect of latent and reactivating virus on hematopoiesis. The ability of HCMV to reactivate in CD34+ HPCs is exquisitely linked to HPC differentiation and hematopoiesis. Understanding how HCMV regulates hematopoiesis is crucial to identifying mechanisms of latency and reactivation as well as disease. HCMV is a species-specific virus that establishes a persistent/latent infection in the host after primary infection. Our group and others have shown that HCMV encodes several microRNAs (miRNAs), small 21-24 base pair single-stranded RNA species that regulate gene expression and multiple cellular processes involved in the virus life-cycle. In preliminary work we have observed that two HCMV latently expressed miRNAs up-regulate and one expressed during lytic replication down-regulates Early Growth Response Gene-1 (Egr-1) that is a transcription factor induced by MEK/ERK signaling that regulates the maintenance human progenitor cells (HPC) or ?Stemness? in the bone marrow through the induction and suppression of key cytokines and co-stimulatory molecules. Deletion of these two HCMV miRNAs that up-regulate Egr-1 results in a virus that fails to reactivate in vitro or in a humanized mouse model. Additionally, we have shown that the miRNAs that regulate Egr-1 signaling pathway alter HPC myelopoiesis suggesting link between HPC latency and reactivation and HPC myelopoiesis. We also observe that Egr-1 up-regulates UL138 expression a gene shown to maintain HCMV latency. Lastly, we show that HCMV latent infection of HPC up-regulates expression of TGF-? that is regulated by Egr-1. Latent HCMV infection of HPC inhibits myelopoiesis in vitro that is restored by neutralization of TGF-?. We hypothesize that HCMV miRNAs that up-regulate Egr-1 maintain CD34+ HPC stemness and latency through expression of UL138 and miRNAs that down-regulate Egr-1 induce reactivation thus providing a regulatory switch for viral latency or reactivation. In the first aim we will identify and characterize additional HCMV miRNA signaling targets that regulate Egr-1 and their role in viral latency and reactivation. In the second aim we will characterize HCMV miRNA regulation of Egr-1 mediated UL136 and UL138 expression in latency. In the last aim we will characterize HCMV miRNA regulation of Egr-1 and TGF-? on HPC stemness and myelopoiesis using both in vitro systems and a unique humanized mouse model. These studies have significant implications on HCMV induced myelosupression observed in bone marrow transplant patients.

Public Health Relevance

/Relevance: HCMV is a significant pathogen in immune compromised patients and the leading viral cause of congenital defects. We have observed that HCMV microRNAs (miRNAs) affect latency, reactivation and hematopoiesis and hypothesize that this is due to targeting components within the Egr-1 signaling pathway. This project will use HCMV miRNA mutants as well as miRNA mutants expressing shRNAs against their known targets in an in vitro CD34+ human progenitor cell system and humanized mouse models to examine the role of the viral miRNAs and their targets in latency, reactivation and hematopoiesis.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Method to Extend Research in Time (MERIT) Award (R37)
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Special Emphasis Panel (ZRG1)
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Beisel, Christopher E
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Oregon Health and Science University
Overall Medical
United States
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Diggins, Nicole L; Hancock, Meaghan H (2018) HCMV miRNA Targets Reveal Important Cellular Pathways for Viral Replication, Latency, and Reactivation. Noncoding RNA 4:
Hancock, Meaghan H; Skalsky, Rebecca L (2018) Roles of Non-coding RNAs During Herpesvirus Infection. Curr Top Microbiol Immunol 419:243-280