The long-term goal of this project is to develop an understanding of the cellular and molecular mechanisms of human cytomegalovirus (HCMV) persistence in the host. HCMV is a significant pathogen in immune compromised patients and the leading viral cause of congenital defects. Our group and others have shown that HCMV and other herpesviruses encode multiple microRNAs (miRNAs) that are small 21-24 base pair (bp) single-stranded RNA species that regulate gene expression through post-transcriptional mechanisms. Herpesvirus miRNAs have been shown to target many different cellular and viral processes involved in viral immune recognition, apoptosis, cell cycle regulation, as well as viral latency and lytic replication. During the previous funding period our group has made significant advances elucidating functional roles for HCMV miRNAs. We have demonstrated that one of the HCMV miRNAs efficiently targets and reduces expression the IE72 protein that significantly reduces viral replication suggesting a role for maintenance of latent virus. Secondly, we have also found that the HCMV miRNAs coordinately work together to efficiently down-regulate individual genes that may explain why individual miRNA knockouts in the viral genome do not show phenotypic effects. Additionally, the HCMV miRNAs appear to also target multiple individual cellular genes in the same cellular pathways including two potential antiviral cytokines IL-1 and TNF-? as well as the NF?B inhibitor IkBa . Lastly, we have observed that a double HCMV miRNA mutation results in an increase in viral reactivation while two other viral miRNA double mutants fail to reactivate in a human progenitor cell (HPC) culture system in vitro. Interestingly, one of the HCMV miRNAs in the double-mutants that fail to reactivate targets two NF?B activators suggesting that NFkB activation is deleterious for reactivation of virus. Therefore, in the current proposal we will fully characterize the IL-1 and TNF-? signaling pathways targeted by the HCMV miRNAs and their functional relevance for viral latency and replication in a CD34+ HPC. We will also examine the function of HCMV miRNA targeting of the IL-1 and TNF-? signaling pathways during latency and reactivation in a newly developed human CD34+-engrafted NOD-scidIL2Rgc null mouse model that is able to support latent HCMV infection as well as reactivation from latency. We hypothesize that HCMV miRNA repression of viral replication is related to NFkB activation through the IL-1 and TNF-? pathways.

Public Health 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) inhibit viral replication and hypothesize that this repression is related to NFkB activation through the IL-1 and TNF-alpha pathways. This project will use HCMV miRNA mutants as well as miRNA inhibitory molecules in an in vitro CD34+ human progenitor cell system and a humanized mouse model to examine the role of the viral miRNAs in latency and reactivation. !

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI021640-29
Application #
8606384
Study Section
Virology - B Study Section (VIRB)
Program Officer
Beisel, Christopher E
Project Start
1984-12-01
Project End
2018-01-31
Budget Start
2014-02-01
Budget End
2015-01-31
Support Year
29
Fiscal Year
2014
Total Cost
$449,647
Indirect Cost
$179,850
Name
Oregon Health and Science University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
096997515
City
Portland
State
OR
Country
United States
Zip Code
97239
Hakki, Morgan; Goldman, Devorah C; Streblow, Daniel N et al. (2014) HCMV infection of humanized mice after transplantation of G-CSF-mobilized peripheral blood stem cells from HCMV-seropositive donors. Biol Blood Marrow Transplant 20:132-5
Hook, Lauren M; Grey, Finn; Grabski, Robert et al. (2014) Cytomegalovirus miRNAs target secretory pathway genes to facilitate formation of the virion assembly compartment and reduce cytokine secretion. Cell Host Microbe 15:363-73
Hook, Lauren; Hancock, Meaghan; Landais, Igor et al. (2014) Cytomegalovirus microRNAs. Curr Opin Virol 7:40-6
MacManiman, Jason D; Meuser, Andrew; Botto, Sara et al. (2014) Human cytomegalovirus-encoded pUL7 is a novel CEACAM1-like molecule responsible for promotion of angiogenesis. MBio 5:e02035
Landais, Igor; Nelson, Jay A (2013) Functional genomics approaches to understand cytomegalovirus replication, latency and pathogenesis. Curr Opin Virol 3:408-15
Hancock, Meaghan H; Tirabassi, Rebecca S; Nelson, Jay A (2012) Rhesus cytomegalovirus encodes seventeen microRNAs that are differentially expressed in vitro and in vivo. Virology 425:133-42
Viswanathan, Kasinath; Smith, M Shane; Malouli, Daniel et al. (2011) BST2/Tetherin enhances entry of human cytomegalovirus. PLoS Pathog 7:e1002332
Umashankar, Mahadevaiah; Petrucelli, Alex; Cicchini, Louis et al. (2011) A novel human cytomegalovirus locus modulates cell type-specific outcomes of infection. PLoS Pathog 7:e1002444
Grey, Finn; Tirabassi, Rebecca; Meyers, Heather et al. (2010) A viral microRNA down-regulates multiple cell cycle genes through mRNA 5'UTRs. PLoS Pathog 6:e1000967
Wille, Paul T; Knoche, Amber J; Nelson, Jay A et al. (2010) A human cytomegalovirus gO-null mutant fails to incorporate gH/gL into the virion envelope and is unable to enter fibroblasts and epithelial and endothelial cells. J Virol 84:2585-96

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