Biology subjects

Miller, W. E.

Publications and source records attributed to Miller, W. E..

5 recordsLinked to original sources

bioRxiv2024

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

Human cytomegalovirus (HCMV) is a {beta}-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 Gq-dependent PLC-{beta} 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 Gq signaling. Taken together, these studies suggest that UL33 driven signaling is important for lytic HCMV replication in cells of epithelial origin.

microbiology↗

bioRxiv2024

Human cytomegalovirus extensively re-organizes the human genome, diminishing TEAD1 transcription factor activity

Human cytomegalovirus (HCMV) infects up to 80% of the worlds 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 HCMVs established roles in these processes.

genomics↗

bioRxiv2021

Activation of GPR116/ADGRF5 by its tethered agonist requires key amino acids in extracellular loop 2 of the transmembrane domain

The mechanistic details of the tethered agonist mode of activation for adhesion GPCRs has not been completely deciphered. We set out to investigate the physiologic importance of autocatalytic cleavage upstream of the agonistic peptide sequence, an event necessary for NTF displacement and subsequent receptor activation. To examine this hypothesis, we characterized tethered agonist-mediated activation of GPR116 in vitro and in vivo. A knock-in mouse expressing a non-cleavable GPR116 mutant phenocopies the pulmonary phenotype of GPR116 knock-out mice, demonstrating that tethered agonist-mediated receptor activation is indispensable for function in vivo. Using site-directed mutagenesis and species swapping approaches we identified key conserved amino acids for GPR116 activation in the tethered agonist sequence and in extracellular loops 2/3 (ECL2/3). We further highlight residues in transmembrane7 (TM7) that mediate stronger signaling in mouse versus human GPR116 and recapitulate these findings in a model supporting tethered agonist:ECL2 interactions for GPR116 activation. Grant supportThis work was supported in part by HL131634 (JPB) from the National Heart, Lung and Blood Institute of the National Institutes of Health.

cell biology↗

bioRxiv2019

Fetal stage melanopsin (OPN4) and GNAQ (Gαq) signaling regulates vascular development of the eye

Maturation of sensory systems in mammals is regulated by appropriate sensory stimulation. Developmental refinement of the eye and visual system is regulated by light and visual stimulation. One compelling example is that fetal mouse pups deprived of light exhibit altered vascular development in their eyes. Previous work demonstrated that light activation of the photopigment melanopsin (Opn4), an atypical opsin expressed in intrinsically photosensitive retinal ganglion cells (ipRGCs), is crucial to normal vascular development. This suggested the unusual hypothesis that vascular development of the eye was regulated by ipRGC responses in the fetal eye by light that traveled through the body wall of the mother. Here, we test the requirement of OPN4 during fetal stages using genetic approaches. The G-protein GNAQ (Gq) is a candidate mediator of melanopsin signaling. We show that ipRGC-specific deletion of Gnaq phenocopies both hyaloid and retinal vascular development of the Opn4 null mouse. Furthermore, GNAQ gain-of-function in Opn4-expressing cells only during late gestation was sufficient to reverse the consequences for vascular development of either dark rearing or Opn4 loss-of-function. We conclude that melanopsin-dependent signaling in the fetal mouse eye is necessary and sufficient for vascular maturation.

developmental biology↗

bioRxiv2018

The human cytomegalovirus G-protein coupled receptor US28 promotes latency by attenuating c-fos

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that undergoes latency in cells of the hematopoietic compartment, though the mechanisms underlying establishment and maintenance of latency remain elusive. We previously reported that the HCMV-encoded G-protein coupled receptor (GPCR) homolog, US28 is required for successful latent infection. We now show that US28 protein (pUS28) provided in trans complements the US28{Delta} lytic phenotype in myeloid cells, suggesting that sustained US28 expression is necessary for long-term latency. Furthermore, expression of pUS28 at the time of infection represses transcription from the major immediate early promoter (MIEP) within 24 hours. However, this repression is only maintained in the presence of continual pUS28 expression provided in trans. Our data also reveal that pUS28-mediated signaling attenuates both expression and phosphorylation of cellular fos (c-fos), an AP-1 transcription factor subunit, to repress MIEP-driven transcription. AP-1 binds to the MIEP and promotes lytic replication, and in line with this, we find that US28{Delta} infection results in an increase in AP-1 binding to the MIEP, compared to wild type latent infection. Pharmacological inhibition of c-fos represses the MIEP during US28{Delta} infection to similar levels we observe during wild type latent infection. Together, our data reveal that US28 is required for both establishment and long-term maintenance of HCMV latency, which is modulated, at least in part, by repressing functional AP-1 binding to the MIEP.\n\nSignificance StatementHuman cytomegalovirus (HCMV) is a wise-spread pathogen that remains with an individual for life in a quiescent/latent state, posing little threat to an otherwise healthy person. However, when an individuals immune system is severely compromised, HCMV can reactivate to its active/lytic state, resulting in viral spread and disease that is often fatal. The biological mechanisms underlying HCMV latency and reactivation remain poorly understood. Herein we show that the viral-encoded G-protein coupled receptor (GPCR) US28 aids in the establishment and the maintenance of viral latency. Furthermore, we find that US28 modulates host cell proteins to suppress viral processes associated with active/lytic replication, thereby promoting latent infection. This work provides mechanism by which HCMV modulates the host cell environment to its advantage.

microbiology↗