Human Cytomegalovirus UL99-Encoded pp28 Is Required for the Cytoplasmic Envelopment of Tegument-Associated Capsids

4EBP1 is phosphorylated by the mTORC1 kinase. When mTORC1 activity is inhibited, hypophosphorylated 4EBP1 binds and sequesters eIF4E, a component of the mRNA cap-binding complex, and blocks translation. As a consequence, mTORC1 activity is needed to maintain active translation. The human cytomegalovirus pUL38 protein preserves mTORC1 activity, keeping most of the E4BP1 in the infected cell in a hyperphosphorylated, inactive state. Here we report that a second viral protein, pUL69, also antagonizes the activity of 4EBP1, but by a separate mechanism. pUL69 interacts directly with eIF4A1, an element of the cap-binding complex, and the poly(A)-binding protein, which binds to the complex. When pUL69 accumulates during infection with wild-type virus, 4EBP1 is excluded from the complex. However, 4EBP1 is present in the cap-binding complex after infection with a pUL69-deficient virus, coincident with reduced accumulation of several late virus-coded proteins. We propose that pUL69 supports translation in human cytomegalovirus-infected cells by excluding hypophosphorylated 4EBP1 from the cap-binding complex.translational control | two-hybrid screen | eukaryotic initiation factor 4A

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Human cytomegalovirus UL69 protein facilitates translation by associating with the mRNA cap-binding complex and excluding 4EBP1

Aoyagi

Gaspar

Shenk

2010

“…Nuclear-cytoplasmic extraction was performed as described previously (69), except 350,000 cells were harvested. Primary antibodies to the following HCMV proteins were used: IE1 (clone 1B12) (70), IE2 (clone 3A9) (71), pUL26 (clone 7H1-5) (68), pUL38 (clone 8D6) (72), pUL44 (clone 10D8; Virusys), pUL83 (clone 8F5) (73), and pUL99 (clone 10B4-29) (74). Cellular proteins were assayed using antibodies to: ERRα (Novus), PGC-1α (Sigma-Aldrich), PGC-1β (Active Motif), PARP-1 (Cell Signaling), β-actin (Abcam), and tubulin (Sigma-Aldrich).…”

Section: Methodsmentioning

confidence: 99%

Estrogen-related receptor α is required for efficient human cytomegalovirus replication

Hwang¹,

Purdy

Wu³

et al. 2014

Self Cite

An shRNA-mediated screen of the 48 human nuclear receptor genes identified multiple candidates likely to influence the production of human cytomegalovirus in cultured human fibroblasts, including the estrogen-related receptor α (ERRα), an orphan nuclear receptor. The 50-kDa receptor and a 76-kDa variant were induced posttranscriptionally following infection. Genetic and pharmacological suppression of the receptor reduced viral RNA, protein, and DNA accumulation, as well as the yield of infectious progeny. In addition, RNAs encoding multiple metabolic enzymes, including enzymes sponsoring glycolysis (enolase 1, triosephosphate isomerase 1, and hexokinase 2), were reduced when the function of ERRα was inhibited in infected cells. Consistent with the effect on RNAs, a substantial number of metabolites, which are normally induced by infection, were either not increased or were increased to a reduced extent in the absence of normal ERRα activity. We conclude that ERRα is needed for the efficient production of cytomegalovirus progeny, and we propose that the nuclear receptor contributes importantly to the induction of a metabolic environment that supports optimal cytomegalovirus replication.herpesvirus | metabolism | nuclear receptor | mass spectrometry H uman cytomegalovirus (HCMV) belongs to the β-herpesvirus subfamily, and although most healthy individuals remain asymptomatic subsequent to infection, the pathogen is a major contributor to birth defects and to life-threatening disease in immunocompromised patients (1-3). As with all viruses, HCMV depends on the host cell to provide macromolecular building blocks for virion production, and throughout the course of its evolution, HCMV has adapted to manipulate numerous fundamental cellular processes, including RNA accumulation (4), translation (5), metabolism (6, 7), secretory pathways (8), and the cell cycle (9).The nuclear receptor gene family, of which there are 48 members in the human genome, encodes transcription factors (10). Some bind to specific hydrophobic ligands such as steroids, vitamin D, fatty acids, and lipids, providing a means to sense changes in the extracellular or intracellular environment; others are "orphans" with no known ligand, and are often constitutively active (11). As transcription factors that modulate broad gene regulatory networks, nuclear receptors control numerous physiological processes, including aspects of metabolism (cell autonomously and at the whole-organism level), development, and cellular differentiation, cancer, circadian rhythm, and immunity (11).The first indication that nuclear receptor signaling modulates HCMV replication surfaced after the identification of a retinoic acid response element within the major immediate-early promoter (MIEP) that caused a dose-dependent response to retinoic acid in reporter assays (12). Retinoic acid was further shown to enhance viral DNA replication and progeny production in human embryonal carcinoma cells and foreskin fibroblasts (13,14). The ligand also exacerbated symptoms of infection,...

“…ARPE-19 cells were exposed to virus at 4°C for 1 h, unbound virus was removed by two washes with cold PBS, growth medium (37°C) was added for 15 min, cells were rinsed with PBS (PBS), fixed and processed for electron microscopy (51,52), and examined with an FEI Tecnai-T12 microscope at 80 kv.…”

Section: Methodsmentioning

confidence: 99%

Human cytomegalovirus uses two distinct pathways to enter retinal pigmented epithelial cells

Wang

Schröer

et al. 2007

Self Cite

Human cytomegalovirus infects multiple cell types, including fibroblasts and epithelial cells. It penetrates fibroblasts by fusion at the cell surface but is endocytosed into epithelial cells. In this report, we demonstrate by electron microscopy that the virus uses two different routes to enter retinal pigmented epithelial cells, depending on the cell type in which the infecting virus was produced. Virus produced in epithelial cells preferentially fuses with the plasma membrane, whereas fibroblast-derived virus mostly enters by receptor-mediated endocytosis. Treatment of epithelial cells with agents that block endosome acidification inhibited infection by virus produced in fibroblasts but had only a modest effect on infection by virus from epithelial cells. Epithelial cell-generated virions had higher intrinsic ''fusion-from-without'' activity than fibroblast-generated particles, and the two virus preparations triggered different cellular signaling responses, as evidenced by markedly different transcriptional profiles. We propose that the cell type in which a human cytomegalovirus particle is produced likely influences its subsequent spread and its contribution to pathogenesis.host cell transcriptome ͉ pathogenesis ͉ virus spread