The Human Cytomegalovirus vGPCR UL33 is Essential for Efficient Lytic Replication in Epithelial Cells.

Human cytomegalovirus (HCMV) is a ß-herpesvirus which is ubiquitous in the human population. HCMV has the largest genome of all known human herpesviruses, and thus encodes a large array of proteins that affect pathogenesis in different cell types. Given the large genome and the ability of HCMV to replicate in a range of cells, investigators have begun to identify viral proteins required for cell type-specific replication. There are four proteins encoded in the HCMV genome that are homologous to human G protein-coupled receptors (GPCRs); these viral-encoded GPCRs (vGPCRs) are UL33, UL78, US27, and US28. In the current study, we find that deletion of all four vGPCR genes from a clinical isolate of HCMV severely attenuates lytic replication in both primary human salivary gland epithelial cells, as well as ARPE-19 retinal epithelial cells as evidenced by significant decreases in immediate early gene expression and virus production. Deletion of UL33 from the HCMV genome also results in a failure to efficiently replicate in epithelial cells, and this defect is manifested by decreased levels of immediate early, early, and late gene expression, as well as reduced viral production. We find that similar to US28, UL33 constitutively activates Gαq-dependent PLC-ß signaling to high levels in these epithelial cells. We also find that UL33 transcription is more complicated than originally believed, and there is the potential for the virus to utilize various 5' UTRs to create novel UL33 proteins that are all capable of constitutive Gαq signaling. Taken together, these studies suggest that UL33 driven signaling is important for lytic HCMV replication in cells of epithelial origin.

Human cytomegalovirus infection coopts chromatin organization to diminish TEAD1 transcription factor activity.

Human cytomegalovirus (HCMV) infects up to 80% of the world's population. Here, we show that HCMV infection leads to widespread changes in human chromatin accessibility and chromatin looping, with hundreds of thousands of genomic regions affected 48 hours after infection. Integrative analyses reveal HCMV-induced perturbation of Hippo signaling through drastic reduction of TEAD1 transcription factor activity. We confirm extensive concordant loss of TEAD1 binding, active H3K27ac histone marks, and chromatin looping interactions upon infection. Our data position TEAD1 at the top of a hierarchy involving multiple altered important developmental pathways. HCMV infection reduces TEAD1 activity through four distinct mechanisms: closing of TEAD1-bound chromatin, reduction of YAP1 and phosphorylated YAP1 levels, reduction of TEAD1 transcript and protein levels, and alteration of TEAD1 exon-6 usage. Altered TEAD1-based mechanisms are highly enriched at genetic risk loci associated with eye and ear development, providing mechanistic insight into HCMV's established roles in these processes.

Rescue of Pentamer-Null Strains of Human Cytomegalovirus in Epithelial Cells by Use of Histone Deacetylase Inhibitors Reveals an Additional Postentry Function for the Pentamer Complex.

Human cytomegalovirus (HCMV) tropism for epithelial cells is determined by the pentameric glycoprotein complex found on the viral envelope. Laboratory-adapted strains, such as AD169, typically develop loss-of-function mutations for the pentamer, thus losing the ability to efficiently initiate lytic replication in epithelial cells. Using our human salivary gland-derived epithelial (hSGE) cell model, we observed that 3 chemically distinct histone deacetylase (HDAC) inhibitors can rescue infection in hSGE cells using pentamer-null strains of HCMV. Additionally, infection in ARPE-19 epithelial cells was rescued in a similar manner. We isolated nuclei from AD169-infected cells, quantified viral genomes by quantitative PCR (qPCR), and discovered that while HDAC inhibitors increased immediate early (IE) gene expression, they did not increase the amount of viral DNA in the nucleus. Using immunofluorescence microscopy, we observed that pentamer-null strains showed punctate patterning of pp71 in proximity to the nucleus of infected cells, while pp71 was localized to the nucleus after infection with pentamer-containing strains. Upon treatment with HDAC inhibitors, these punctae remained perinuclear, while more cells displayed entry into the lytic cycle, noted by increased IE-positive nuclei. Taken together, our data indicate that HCMV pentamer-null viruses are able to infect epithelial cells (albeit less efficiently than pentamer-positive viruses) and traffic to the nucleus but fail to initiate lytic gene expression once there. These studies reveal a novel postentry function of the pentamer in addition to the recognized role of pentamer in mediating entry. IMPORTANCE Human cytomegalovirus has a wide cellular tropism, which is driven by one of its glycoprotein complexes, the pentamer. Laboratory-adapted strains continuously passaged on fibroblasts readily lose pentamer function and thus lose their ability to infect diverse cell types such as epithelial cells. Pentamer has been attributed an entry function during infection, but mechanistic details as to how this is achieved have not been definitely demonstrated. In this study, we investigate how pharmacological rescue of pentamer-null strains during epithelial infection by histone deacetylase inhibitors implicates a novel role for the pentamer downstream of entry. This work expands on potential functions of the pentamer, will drive future studies to understand mechanistically how it affects tropism, and provides a new target for future therapeutics.

Isolation of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers.

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to restore function of glands damaged by radiation therapies or due to pathologies associated with Sjögren's syndrome. A second goal of researchers is to define the mechanisms by which viruses replicate within glandular tissue where they can then gain access to salivary fluids important for horizontal transmission. These goals highlight the need for a robust and accessible in vitro salivary gland model that can be utilized by researchers interested in the above mentioned as well as related research areas. Here we discuss a simple protocol to isolate epithelial cells from human salivary glands and propagate them in vitro. Our protocol can be further optimized to meet the needs of individual studies. Briefly, salivary tissue is mechanically and enzymatically separated to isolate single cells or small clusters of cells. Selection for epithelial cells occurs by plating onto a basement membrane matrix in the presence of media optimized to promote epithelial cell growth. These resulting cultures can be maintained as three-dimensional clusters, termed "salispheres", or grown as a monolayer on treated plastic tissue culture dishes. This protocol results in the outgrowth of a heterogenous population of mainly epithelial cells that can be propagated for 5-8 passages (15-20 population doublings) before undergoing cellular senescence.

Development of a Primary Human Cell Model for the Study of Human Cytomegalovirus Replication and Spread within Salivary Epithelium.

Various aspects of human cytomegalovirus (HCMV) pathogenesis, including its ability to replicate in specific cells and tissues and the mechanism(s) of horizontal transmission, are not well understood, predominantly because of the strict species specificity exhibited by HCMV. Murine CMV (MCMV), which contains numerous gene segments highly similar to those of HCMV, has been useful for modeling some aspects of CMV pathogenesis; however, it remains essential to build relevant human cell-based systems to investigate how the HCMV counterparts function. The salivary gland epithelium is a site of persistence for both human and murine cytomegaloviruses, and salivary secretions appear to play an important role in horizontal transmission. Therefore, it is important to understand how HCMV is replicating within the glandular epithelial cells so that it might be possible to therapeutically prevent transmission. In the present study, we describe the development of a salivary epithelial model derived from primary human "salispheres." Initial infection of these primary salivary cells with HCMV occurs in a manner similar to that reported for established epithelial lines, in that gH/gL/UL128/UL130/UL131A (pentamer)-positive strains can infect and replicate, while laboratory-adapted pentamer-null strains do not. However, while HCMV enters the lytic phase and produces virus in salivary epithelial cells, it fails to exhibit robust spread throughout the culture and persists in a low percentage of salivary cells. The present study demonstrates the utility of these primary tissue-derived cells for studying HCMV replication in salivary epithelial cells in vitroIMPORTANCE Human cytomegalovirus (HCMV) infects the majority of the world's population, and although it typically establishes a quiescent infection with little to no disease in most individuals, the virus is responsible for a variety of devastating sequelae in immunocompromised adults and in developing fetuses. Therefore, identifying the viral properties essential for replication, spread, and horizontal transmission is an important area of medical science. Our studies use novel human salivary gland-derived cellular models to investigate the molecular details by which HCMV replicates in salivary epithelial cells and provide insight into the mechanisms by which the virus persists in the salivary epithelium, where it gains access to fluids centrally important for horizontal transmission.

Macrophage activation by IFN-γ triggers restriction of phagosomal copper from intracellular pathogens.

Copper toxicity and copper limitation can both be effective host defense mechanisms against pathogens. Tolerance of high copper by fungi makes toxicity as a defense mechanism largely ineffective against fungal pathogens. A forward genetic screen for Histoplasma capsulatum mutant yeasts unable to replicate within macrophages showed the Ctr3 copper transporter is required for intramacrophage proliferation. Ctr3 mediates copper uptake and is required for growth in low copper. Transcription of the CTR3 gene is induced by differentiation of H. capsulatum into pathogenic yeasts and by low available copper, but not decreased iron. Low expression of a CTR3 transcriptional reporter by intracellular yeasts implies that phagosomes of non-activated macrophages have moderate copper levels. This is further supported by the replication of Ctr3-deficient yeasts within the phagosome of non-activated macrophages. However, IFN-γ activation of phagocytes causes restriction of phagosomal copper as shown by upregulation of the CTR3 transcriptional reporter and by the failure of Ctr3-deficient yeasts, but not Ctr3 expressing yeasts, to proliferate within these macrophages. Accordingly, in a respiratory model of histoplasmosis, Ctr3-deficient yeasts are fully virulent during phases of the innate immune response but are attenuated after the onset of adaptive immunity. Thus, while technical limitations prevent direct measurement of phagosomal copper concentrations and copper-independent factors can influence gene expression, both the CTR3 promoter induction and the attenuation of Ctr3-deficient yeasts indicate activation of macrophages switches the phagosome from a copper-replete to a copper-depleted environment, forcing H. capsulatum reliance on Ctr3 for copper acquisition.