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Restructured membrane contacts rewire organelles for human cytomegalovirus infection.

ABSTRACT: Membrane contact sites (MCSs) link organelles to coordinate cellular functions across space and time. Although viruses remodel organelles for their replication cycles, MCSs remain largely unexplored during infections. Here, we design a targeted proteomics platform for measuring MCS proteins at all organelles simultaneously and define functional virus-driven MCS alterations by the ancient beta-herpesvirus human cytomegalovirus (HCMV). Integration with super-resolution microscopy and comparisons to herpes simplex virus (HSV-1), Influenza A, and beta-coronavirus HCoV-OC43 infections reveals time-sensitive contact regulation that allows switching anti- to pro-viral organelle functions. We uncover a stabilized mitochondria-ER encapsulation structure (MENC). As HCMV infection progresses, MENCs become the predominant mitochondria-ER contact phenotype and sequentially recruit the tethering partners VAP-B and PTPIP51, supporting virus production. However, premature ER-mitochondria tethering activates STING and interferon response, priming cells against infection. At peroxisomes, ACBD5-mediated ER contacts balance peroxisome proliferation versus membrane expansion, with ACBD5 impacting the titers of each virus tested.

SUBMITTER: Cook KC

PROVIDER: S-EPMC9366835 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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Restructured membrane contacts rewire organelles for human cytomegalovirus infection.

Cook Katelyn C KC Tsopurashvili Elene E Needham Jason M JM Thompson Sunnie R SR Cristea Ileana M IM

Nature communications 20220811 1

Membrane contact sites (MCSs) link organelles to coordinate cellular functions across space and time. Although viruses remodel organelles for their replication cycles, MCSs remain largely unexplored during infections. Here, we design a targeted proteomics platform for measuring MCS proteins at all organelles simultaneously and define functional virus-driven MCS alterations by the ancient beta-herpesvirus human cytomegalovirus (HCMV). Integration with super-resolution microscopy and comparisons t ...[more]

PMID: 35953480

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Project description:Endoplasmic reticulum-mitochondrial contacts, known as mitochondria-associated membranes, regulate important cellular functions including calcium signaling, bioenergetics, and apoptosis. Human cytomegalovirus is a medically important herpesvirus whose growth increases energy demand and depends upon continued cell survival. To gain insight into how human cytomegalovirus infection affects endoplasmic reticulum-mitochondrial contacts, we undertook quantitative proteomics of mitochondria-associated membranes using differential stable isotope labeling by amino acids in cell culture strategy and liquid chromatography-tandem MS analysis. This is the first reported quantitative proteomic analyses of a suborganelle during permissive human cytomegalovirus infection. Human fibroblasts were uninfected or human cytomegalovirus-infected for 72 h. Heavy mitochondria-associated membranes were isolated from paired unlabeled, uninfected cells and stable isotope labeling by amino acids in cell culture-labeled, infected cells and analyzed by liquid chromatography-tandem MS analysis. The results were verified by a reverse labeling experiment. Human cytomegalovirus infection dramatically altered endoplasmic reticulum-mitochondrial contacts by late times. Notable is the increased abundance of several fundamental networks in the mitochondria-associated membrane fraction of human cytomegalovirus-infected fibroblasts. Chaperones, including HSP60 and BiP, which is required for human cytomegalovirus assembly, were prominently increased at endoplasmic reticulum-mitochondrial contacts after infection. Minimal translational and translocation machineries were also associated with endoplasmic reticulum-mitochondrial contacts and increased after human cytomegalovirus infection as were glucose regulated protein 75 and the voltage dependent anion channel, which can form an endoplasmic reticulum-mitochondrial calcium signaling complex. Surprisingly, mitochondrial metabolic enzymes and cytosolic glycolytic enzymes were confidently detected in the mitochondria-associated membrane fraction and increased therein after infection. Finally, proapoptotic regulatory proteins, including Bax, cytochrome c, and Opa1, were augmented in endoplasmic reticulum-mitochondrial contacts after infection, suggesting attenuation of proapoptotic signaling by their increased presence therein. Together, these results suggest that human cytomegalovirus infection restructures the proteome of endoplasmic reticulum-mitochondrial contacts to bolster protein translation at these junctions, calcium signaling to mitochondria, cell survival, and bioenergetics and, thereby, allow for enhanced progeny production.

Project description:BackgroundHuman cytomegalovirus (HCMV) is the most prevalent congenital viral infection in the United States and Europe causing significant morbidity and mortality to both mother and child. HCMV is also an opportunistic pathogen in immunocompromised individuals, including human immunodeficiency virus (HIV)- infected patients with AIDS, and solid organ and allogeneic stem cell transplantation recipients. Current treatments for HCMV-associated diseases are insufficient due to the emergence of drug-induced resistance and cytotoxicity, necessitating novel approaches to limit HCMV infection. The aim of this study was to develop therapeutic peptides targeting glycoprotein B (gB), a major glycoprotein of HCMV that is highly conserved across the Herpesviridae family, that specifically inhibit fusion of the viral envelope with the host cell membrane preventing HCMV entry and infection.ResultsUsing the Wimley-White Interfacial Hydrophobicity Scale (WWIHS), several regions within gB were identified that display a high potential to interact with lipid bilayers of cell membranes and hydrophobic surfaces within proteins. The ability of synthetic peptides analogous to WWIHS-positive sequences of HCMV gB to inhibit viral infectivity was evaluated. Human foreskin fibroblasts (HFF) were infected with the Towne-GFP strain of HCMV (0.5 MOI), preincubated with peptides at a range of concentrations (78 nm to 100 μM), and GFP-positive cells were visualized 48 hours post-infection by fluorescence microscopy and analyzed quantitatively by flow cytometry. Peptides that inhibited HCMV infection demonstrated different inhibitory concentration curves indicating that each peptide possesses distinct biophysical properties. Peptide 174-200 showed 80% inhibition of viral infection at a concentration of 100 μM, and 51% and 62% inhibition at concentrations of 5 μM and 2.5 μM, respectively. Peptide 233-263 inhibited infection by 97% and 92% at concentrations of 100 μM and 50 μM, respectively, and 60% at a concentration of 2.5 μM. While peptides 264-291 and 297-315, individually failed to inhibit viral infection, when combined, they showed 67% inhibition of HCMV infection at a concentration of 0.125 μM each.ConclusionsPeptides designed to target putative fusogenic domains of gB provide a basis for the development of novel therapeutics that prevent HCMV infection.

Project description:Zika virus (ZIKV) is an enveloped positive stranded RNA virus belonging to the genus Flavivirus in the family Flaviviridae that emerged in recent decades causing pandemic outbreaks of human infections occasionally associated with severe neurological disorders in adults and newborns. The intracellular steps of flavivirus multiplication are associated to cellular membranes and their bound organelles leading to an extensive host cell reorganization. Importantly, the association of organelle dysfunction with diseases caused by several human viruses has been widely reported in recent studies. With the aim to increase the knowledge about the impact of ZIKV infection on the host cell functions, the present study was focused on the evaluation of the reorganization of three cell components, promyelocytic leukemia nuclear bodies (PML-NBs), mitochondria, and lipid droplets (LDs). Relevant human cell lines including neural progenitor cells (NPCs), hepatic Huh-7, and retinal pigment epithelial (RPE) cells were infected with the Argentina INEVH116141 ZIKV strain and the organelle alterations were studied by using fluorescent cell imaging analysis. Our results have shown that these three organelles are targeted and structurally modified during ZIKV infection. Considering the nuclear reorganization, the analysis by confocal microscopy of infected cells showed a significantly reduced number of PML-NBs in comparison to uninfected cells. Moreover, a mitochondrial morphodynamic perturbation with an increased fragmentation and the loss of mitochondrial membrane potential was observed in ZIKV infected RPE cells. Regarding lipid structures, a decrease in the number and volume of LDs was observed in ZIKV infected cells. Given the involvement of these organelles in host defense processes, the reported perturbations may be related to enhanced virus replication through protection from innate immunity. The understanding of the cellular remodeling will enable the design of new host-targeted antiviral strategies.

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Restructured membrane contacts rewire organelles for human cytomegalovirus infection.

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Restructured membrane contacts rewire organelles for human cytomegalovirus infection.

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