The goal of this project is to define the interactions between CD34-expressing hematopoietic progenitor cells (HPCs) and human cytomegalovirus (HCMV) that impact viral latency and reactivation. HPCs are a prominent site of HCMV latency and a common target of human viruses. Latency enables HCMV to persist indefinitely in infected individuals and prevents pathogen clearance. Reactivation of HCMV from latency in immunosuppressed individuals, including stem cell and solid organ transplant recipients and AIDS patients, is associated with high rates of morbidity and mortality. Despite its critical importance to HCMV pathobiology, the mechanisms underlying HCMV latency remain obscure. Elucidating the mechanisms of viral latency in HPCs will contribute importantly to our understanding of viral disease in immunocompromised individuals. Latency- competent clinical strains of HCMV contain a unique 13-15 kilobase (kb) region of the genome, termed ULb', that has been lost from strains adapted for replication in cultured fibroblasts. These laboratory-adapted strains cannot support a latent infection. We hypothesize that HCMV encodes viral determinants of latency within the ULb'region of the genome that function in latency through interactions with other viral or cellular proteins. Using a novel experimental model we developed to recapitulate latency in primary human HPCs infected in vitro, we have identified a 5 kb region, and more specifically the UL138 open reading frame (ORF), within the ULb'region of the genome that is required for a latent HCMV infection in HPCs. The UL138 ORF has not been previously characterized with respect to HCMV infection or latency and represents the first functional viral determinant of latency. We propose to determine the function of UL138 in promoting a latent infection.
In Aim 1, we will identify and characterize the gene products derived from the UL138 locus that are required for a latent infection. Our preliminary studies have identified two large transcripts and a protein encoded by the UL138 gene locus.
In Aim 2, we will identify novel viral determinants of latency encoded in the ULb'region and determine which determinants are sufficient to restore latency to a latency-incompetent recombinant strain of HCMV.
In Aim 3, we will elucidate the mechanism by which the UL138 protein and other determinants promote latency by identifying viral and cellular proteins that interact with UL138. HPC-specific interactions will be functionally characterized by analyzing the impact of their expression on HCMV latency and replication. Viral and cellular proteins that contribute to HCMV latency will implicate cellular pathways exploited or negotiated by the virus in HPCs. These studies will define key mechanisms governing HCMV latency. Further, this work will identify candidate molecular targets leading to improved antiviral therapies aimed against latently infected cells to prevent HCMV disease and complications associated with reactivation of latent HCMV.

Public Health Relevance

Human cytomegalovirus (CMV) is a herpesvirus that infects bloods cells and establishes a life long infection in up to 90% of the population world-wide (5). CMV is a significant pathogen associated with high rates or morbidity and mortality in immunocompromised individuals, particularly stem cell transplant recipients (3, 4), and unborn children (1, 2) and is associated with heart disease (6) and age-related loss of immune function (7). Our proposal will identify mechanisms by which the virus persists in infected individuals, thereby identifying targets for improved prevention, treatment, or eradication of CMV.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI079059-05
Application #
8298233
Study Section
Virology - B Study Section (VIRB)
Program Officer
Beisel, Christopher E
Project Start
2008-07-08
Project End
2014-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
5
Fiscal Year
2012
Total Cost
$367,689
Indirect Cost
$122,664
Name
University of Arizona
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
806345617
City
Tucson
State
AZ
Country
United States
Zip Code
85721
Collins-McMillen, Donna; Goodrum, Felicia D (2017) The loss of binary: Pushing the herpesvirus latency paradigm. Curr Clin Microbiol Rep 4:124-131
Cheng, Shu; Caviness, Katie; Buehler, Jason et al. (2017) Transcriptome-wide characterization of human cytomegalovirus in natural infection and experimental latency. Proc Natl Acad Sci U S A 114:E10586-E10595
Kim, Jung Heon; Collins-McMillen, Donna; Buehler, Jason C et al. (2017) Human Cytomegalovirus Requires Epidermal Growth Factor Receptor Signaling To Enter and Initiate the Early Steps in the Establishment of Latency in CD34(+) Human Progenitor Cells. J Virol 91:
Leng, Sean X; Kamil, Jeremy; Purdy, John G et al. (2017) Recent advances in CMV tropism, latency, and diagnosis during aging. Geroscience 39:251-259
Gordon, Claire L; Miron, Michelle; Thome, Joseph J C et al. (2017) Tissue reservoirs of antiviral T cell immunity in persistent human CMV infection. J Exp Med 214:651-667
Caviness, Katie; Bughio, Farah; Crawford, Lindsey B et al. (2016) Complex Interplay of the UL136 Isoforms Balances Cytomegalovirus Replication and Latency. MBio 7:e01986
Goodrum, Felicia (2016) Human Cytomegalovirus Latency: Approaching the Gordian Knot. Annu Rev Virol 3:333-357
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
Buehler, Jason; Zeltzer, Sebastian; Reitsma, Justin et al. (2016) Opposing Regulation of the EGF Receptor: A Molecular Switch Controlling Cytomegalovirus Latency and Replication. PLoS Pathog 12:e1005655
Goodrum, Felicia; Bughio, Farah (2015) Viral infection at the endothelium. Oncotarget 6:26541-2

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