Double-Membrane Vesicles as Platforms for Viral Replication

Evidence is emerging that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect various organs of the body, including cardiomyocytes and cardiac endothelial cells in the heart. This review focuses on the effects of SARS-CoV-2 in the heart after direct infection that can lead to myocarditis and an outline of potential treatment options. The main points are: (1) Viral entry: SARS-CoV-2 uses specific receptors and proteases for docking and priming in cardiac cells. Thus, different receptors or protease inhibitors might be effective in SARS-CoV-2-infected cardiac cells. (2) Viral replication: SARS-CoV-2 uses RNA-dependent RNA polymerase for replication. Drugs acting against ssRNA(+) viral replication for cardiac cells can be effective. (3) Autophagy and double-membrane vesicles: SARS-CoV-2 manipulates autophagy to inhibit viral clearance and promote SARS-CoV-2 replication by creating double-membrane vesicles as replication sites. (4) Immune response: Host immune response is manipulated to evade host cell attacks against SARS-CoV-2 and increased inflammation by dysregulating immune cells. Efficiency of immunosuppressive therapy must be elucidated. (5) Programmed cell death: SARS-CoV-2 inhibits programmed cell death in early stages and induces apoptosis, necroptosis, and pyroptosis in later stages. (6) Energy metabolism: SARS-CoV-2 infection leads to disturbed energy metabolism that in turn leads to a decrease in ATP production and ROS production. (7) Viroporins: SARS-CoV-2 creates viroporins that lead to an imbalance of ion homeostasis. This causes apoptosis, altered action potential, and arrhythmia.

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Identification of Anti-Severe Acute Respiratory Syndrome-Related Coronavirus 2 (SARS-CoV-2) Oxysterol Derivatives In Vitro

International Journal of Molecular Sciences ◽

10.3390/ijms22063163 ◽

2021 ◽

Vol 22 (6) ◽

pp. 3163

Author(s):

Hirofumi Ohashi ◽

Feng Wang ◽

Frank Stappenbeck ◽

Kana Tsuchimoto ◽

Chisa Kobayashi ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Viral Replication ◽

Cultured Cells ◽

Peak Plasma ◽

Plasma Concentrations ◽

Membrane Vesicles ◽

Drug Candidates ◽

Increased Risk ◽

Derivatives Of

The development of effective antiviral drugs targeting the severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is urgently needed to combat the coronavirus disease 2019 (COVID-19). We have previously studied the use of semi-synthetic derivatives of oxysterols, oxidized derivatives of cholesterol as drug candidates for the inhibition of cancer, fibrosis, and bone regeneration. In this study, we screened a panel of naturally occurring and semi-synthetic oxysterols for anti-SARS-CoV-2 activity using a cell culture infection assay. We show that the natural oxysterols, 7-ketocholesterol, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 27-hydroxycholesterol, substantially inhibited SARS-CoV-2 propagation in cultured cells. Among semi-synthetic oxysterols, Oxy210 and Oxy232 displayed more robust anti-SARS-CoV-2 activities, reducing viral replication more than 90% at 10 μM and 99% at 15 μM, respectively. When orally administered in mice, peak plasma concentrations of Oxy210 fell into a therapeutically relevant range (19 μM), based on the dose-dependent curve for antiviral activity in our cell-based assay. Mechanistic studies suggest that Oxy210 reduced replication of SARS-CoV-2 by disrupting the formation of double-membrane vesicles (DMVs); intracellular membrane compartments associated with viral replication. Our study warrants further evaluation of Oxy210 and Oxy232 as a safe and reliable oral medication, which could help protect vulnerable populations with increased risk of developing COVID-19.

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The Origin, Dynamic Morphology, and PI4P-Independent Formation of Encephalomyocarditis Virus Replication Organelles

mBio ◽

10.1128/mbio.00420-18 ◽

2018 ◽

Vol 9 (2) ◽

pp. e00420-18 ◽

Cited By ~ 11

Author(s):

C. E. Melia ◽

H. M. van der Schaar ◽

A. W. M. de Jong ◽

H. R. Lyoo ◽

E. J. Snijder ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Rna Synthesis ◽

Electron Tomography ◽

Membrane Vesicles ◽

Encephalomyocarditis Virus ◽

Escape Mutant ◽

Membrane Structures ◽

Double Membrane ◽

Single Membrane ◽

Phosphatidylinositol 4

ABSTRACTPicornaviruses induce dramatic rearrangements of endomembranes in the cells that they infect to produce dedicated platforms for viral replication. These structures, termed replication organelles (ROs), have been well characterized for theEnterovirusgenus of thePicornaviridae. However, it is unknown whether the diverse RO morphologies associated with enterovirus infection are conserved among other picornaviruses. Here, we use serial electron tomography at different stages of infection to assess the three-dimensional architecture of ROs induced by encephalomyocarditis virus (EMCV), a member of theCardiovirusgenus of the family of picornaviruses that is distantly related. Ultrastructural analyses revealed connections between early single-membrane EMCV ROs and the endoplasmic reticulum (ER), establishing the ER as a likely donor organelle for their formation. These early single-membrane ROs appear to transform into double-membrane vesicles (DMVs) as infection progresses. Both single- and double-membrane structures were found to support viral RNA synthesis, and progeny viruses accumulated in close proximity, suggesting a spatial association between RNA synthesis and virus assembly. Further, we explored the role of phosphatidylinositol 4-phosphate (PI4P), a critical host factor for both enterovirus and cardiovirus replication that has been recently found to expedite enterovirus RO formation rather than being strictly required. By exploiting an EMCV escape mutant, we found that low-PI4P conditions could also be overcome for the formation of cardiovirus ROs. Collectively, our data show that despite differences in the membrane source, there are striking similarities in the biogenesis, morphology, and transformation of cardiovirus and enterovirus ROs, which may well extend to other picornaviruses.IMPORTANCELike all positive-sense RNA viruses, picornaviruses induce the rearrangement of host cell membranes to form unique structures, or replication organelles (ROs), that support viral RNA synthesis. Here, we investigate the architecture and biogenesis of cardiovirus ROs and compare them with those induced by enteroviruses, members of the well-characterized picornavirus genusEnterovirus. The origins and dynamic morphologies of cardiovirus ROs are revealed using electron tomography, which points to the endoplasmic reticulum as the donor organelle usurped to produce single-membrane tubules and vesicles that transform into double-membrane vesicles. We show that PI4P, a critical lipid for cardiovirus and enterovirus replication, is not strictly required for the formation of cardiovirus ROs, as functional ROs with typical morphologies are formed under phosphatidylinositol 4-kinase type III alpha (PI4KA) inhibition in cells infected with an escape mutant. Our data show that the transformation from single-membrane structures to double-membrane vesicles is a conserved feature of cardiovirus and enterovirus infections that likely extends to other picornavirus genera.

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Ultrastructural Evidence for Direct Renal Infection with SARS-CoV-2

Journal of the American Society of Nephrology ◽

10.1681/asn.2020040432 ◽

2020 ◽

Vol 31 (8) ◽

pp. 1683-1687 ◽

Cited By ~ 84

Author(s):

Evan A. Farkash ◽

Allecia M. Wilson ◽

Jeffrey M. Jentzen

Keyword(s):

Renal Failure ◽

Light Microscopy ◽

Virus Assembly ◽

Light And Electron Microscopy ◽

Membrane Vesicles ◽

Renal Tissue ◽

Double Membrane ◽

Renal Tubular Epithelium ◽

Renal Tubular ◽

Renal Infection

BackgroundA significant fraction of patients with coronavirus disease 2019 (COVID-19) display abnormalities in renal function. Retrospective studies of patients hospitalized with COVID-19 in Wuhan, China, report an incidence of 3%–7% progressing to ARF, a marker of poor prognosis. The cause of the renal failure in COVID-19 is unknown, but one hypothesized mechanism is direct renal infection by the causative virus, SARS-CoV-2.MethodsWe performed an autopsy on a single patient who died of COVID-19 after open repair of an aortic dissection, complicated by hypoxic respiratory failure and oliguric renal failure. We used light and electron microscopy to examine renal tissue for evidence of SARS-CoV-2 within renal cells.ResultsLight microscopy of proximal tubules showed geographic isometric vacuolization, corresponding to a focus of tubules with abundant intracellular viral arrays. Individual viruses averaged 76 µm in diameter and had an envelope studded with crown-like, electron-dense spikes. Vacuoles contained double-membrane vesicles suggestive of partially assembled virus.ConclusionsThe presence of viral particles in the renal tubular epithelium that were morphologically identical to SARS-CoV-2, and with viral arrays and other features of virus assembly, provide evidence of a productive direct infection of the kidney by SARS-CoV-2. This finding offers confirmatory evidence that direct renal infection occurs in the setting of AKI in COVID-19. However, the frequency and clinical significance of direct infection in COVID-19 is unclear. Tubular isometric vacuolization observed with light microscopy, which correlates with double-membrane vesicles containing vacuoles observed with electronic microscopy, may be a useful histologic marker for active SARS-CoV-2 infection in kidney biopsy or autopsy specimens.

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A molecular pore spans the double membrane of the coronavirus replication organelle

Science ◽

10.1126/science.abd3629 ◽

2020 ◽

Vol 369 (6509) ◽

pp. 1395-1398 ◽

Cited By ~ 26

Author(s):

Georg Wolff ◽

Ronald W. A. L. Limpens ◽

Jessika C. Zevenhoven-Dobbe ◽

Ulrike Laugks ◽

Shawn Zheng ◽

...

Keyword(s):

Drug Target ◽

Rna Synthesis ◽

Transmembrane Protein ◽

Membrane Vesicle ◽

Membrane Vesicles ◽

Genome Replication ◽

Messenger Rnas ◽

Double Membrane ◽

The Core ◽

Cryo Electron Microscopy

Coronavirus genome replication is associated with virus-induced cytosolic double-membrane vesicles, which may provide a tailored microenvironment for viral RNA synthesis in the infected cell. However, it is unclear how newly synthesized genomes and messenger RNAs can travel from these sealed replication compartments to the cytosol to ensure their translation and the assembly of progeny virions. In this study, we used cellular cryo–electron microscopy to visualize a molecular pore complex that spans both membranes of the double-membrane vesicle and would allow export of RNA to the cytosol. A hexameric assembly of a large viral transmembrane protein was found to form the core of the crown-shaped complex. This coronavirus-specific structure likely plays a key role in coronavirus replication and thus constitutes a potential drug target.

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Virus-induced double-membrane vesicles

Cellular Microbiology ◽

10.1111/cmi.12372 ◽

2014 ◽

Vol 17 (1) ◽

pp. 45-50 ◽

Cited By ~ 30

Author(s):

Emmanuelle Blanchard ◽

Philippe Roingeard

Keyword(s):

Membrane Vesicles ◽

Double Membrane

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Autophagy in Skin Diseases

Dermatology ◽

10.1159/000500470 ◽

2019 ◽

Vol 235 (5) ◽

pp. 380-389 ◽

Cited By ~ 5

Author(s):

Yeye Guo ◽

Xu Zhang ◽

Tianhao Wu ◽

Xing Hu ◽

Juan Su ◽

...

Keyword(s):

Skin Diseases ◽

Membrane Vesicles ◽

Cellular Homeostasis ◽

Double Membrane ◽

Adaptive Process ◽

Evolutionarily Conserved

Autophagy, or self-eating, is an evolutionarily conserved process in which cytosol and organelles are sequestered within double-membrane vesicles that deliver the contents to the lysosome/vacuole for the degradation and recycling of cytoplasmic components in eukaryotes. It is well recognized that autophagy plays an important role in maintaining cellular homeostasis under physiological and pathophysiological conditions and the upregulation of autophagy may serve as an adaptive process to provide nutrients and energy when under stresses. Recently, studies have illustrated that autophagy is intricately related to skin diseases. This review provides a brief synopsis of the process of autophagy and aims to elucidate the roles of autophagy in different skin diseases and to highlight the need for increased research in the field.

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The Porcine Deltacoronavirus Replication Organelle Comprises Double-Membrane Vesicles and Zippered Endoplasmic Reticulum with Double-Membrane Spherules

Viruses ◽

10.3390/v11111030 ◽

2019 ◽

Vol 11 (11) ◽

pp. 1030 ◽

Cited By ~ 10

Author(s):

Nicole Doyle ◽

Philippa C. Hawes ◽

Jennifer Simpson ◽

Lorin H. Adams ◽

Helena J. Maier

Keyword(s):

Endoplasmic Reticulum ◽

Rna Synthesis ◽

Membrane Vesicles ◽

Viral Rna ◽

Double Membrane ◽

Severe Diarrhea ◽

The Usa ◽

History Of ◽

Host Cell Interactions ◽

Positive Strand Rna

Porcine deltacoronavirus (PDCoV) was first identified in Hong Kong in 2012 from samples taken from pigs in 2009. PDCoV was subsequently identified in the USA in 2014 in pigs with a history of severe diarrhea. The virus has now been detected in pigs in several countries around the world. Following the development of tissue culture adapted strains of PDCoV, it is now possible to address questions regarding virus–host cell interactions for this genera of coronavirus. Here, we presented a detailed study of PDCoV-induced replication organelles. All positive-strand RNA viruses induce the rearrangement of cellular membranes during virus replication to support viral RNA synthesis, forming the replication organelle. Replication organelles for the Alpha-, Beta-, and Gammacoronavirus genera have been characterized. All coronavirus genera induced the formation of double-membrane vesicles (DMVs). In addition, Alpha- and Betacoronaviruses induce the formation of convoluted membranes, while Gammacoronaviruses induce the formation of zippered endoplasmic reticulum (ER) with tethered double-membrane spherules. However, the structures induced by Deltacoronaviruses, particularly the presence of convoluted membranes or double-membrane spherules, are unknown. Initially, the dynamics of PDCoV strain OH-FD22 replication were assessed with the onset of viral RNA synthesis, protein synthesis, and progeny particle release determined. Subsequently, virus-induced membrane rearrangements were identified in infected cells by electron microscopy. As has been observed for all other coronaviruses studied to date, PDCoV replication was found to induce the formation of double-membrane vesicles. Significantly, however, PDCoV replication was also found to induce the formation of regions of zippered endoplasmic reticulum, small associated tethered vesicles, and double-membrane spherules. These structures strongly resemble the replication organelle induced by avian Gammacoronavirus infectious bronchitis virus.

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Severe Acute Respiratory Syndrome Coronavirus Nonstructural Proteins 3, 4, and 6 Induce Double-Membrane Vesicles

mBio ◽

10.1128/mbio.00524-13 ◽

2013 ◽

Vol 4 (4) ◽

Cited By ~ 146

Author(s):

Megan M. Angelini ◽

Marzieh Akhlaghpour ◽

Benjamin W. Neuman ◽

Michael J. Buchmeier

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Host Cell ◽

Rna Viruses ◽

Membrane Vesicles ◽

Full Length ◽

Nonstructural Proteins ◽

Human Coronavirus ◽

Double Membrane ◽

Infected Cells ◽

Proliferation Ability

ABSTRACTCoronaviruses (CoV), like other positive-stranded RNA viruses, redirect and rearrange host cell membranes for use as part of the viral genome replication and transcription machinery. Specifically, coronaviruses induce the formation of double-membrane vesicles in infected cells. Although these double-membrane vesicles have been well characterized, the mechanism behind their formation remains unclear, including which viral proteins are responsible. Here, we use transfection of plasmid constructs encoding full-length versions of the three transmembrane-containing nonstructural proteins (nsps) of the severe acute respiratory syndrome (SARS) coronavirus to examine the ability of each to induce double-membrane vesicles in tissue culture. nsp3 has membrane disordering and proliferation ability, both in its full-length form and in a C-terminal-truncated form. nsp3 and nsp4 working together have the ability to pair membranes. nsp6 has membrane proliferation ability as well, inducing perinuclear vesicles localized around the microtubule organizing center. Together, nsp3, nsp4, and nsp6 have the ability to induce double-membrane vesicles that are similar to those observed in SARS coronavirus-infected cells. This activity appears to require the full-length form of nsp3 for action, as double-membrane vesicles were not seen in cells coexpressing the C-terminal truncation nsp3 with nsp4 and nsp6.IMPORTANCEAlthough the majority of infections caused by coronaviruses in humans are relatively mild, the SARS outbreak of 2002 to 2003 and the emergence of the human coronavirus Middle Eastern respiratory syndrome (MERS-CoV) in 2012 highlight the ability of these viruses to cause severe pathology and fatality. Insight into the molecular biology of how coronaviruses take over the host cell is critical for a full understanding of any known and possible future outbreaks caused by these viruses. Additionally, since membrane rearrangement is a tactic used by all known positive-sense single-stranded RNA viruses, this work adds to that body of knowledge and may prove beneficial in the development of future therapies not only for human coronavirus infections but for other pathogens as well.

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NS5A Domain 1 and Polyprotein Cleavage Kinetics Are Critical for Induction of Double-Membrane Vesicles Associated with Hepatitis C Virus Replication

mBio ◽

10.1128/mbio.00759-15 ◽

2015 ◽

Vol 6 (4) ◽

Cited By ~ 47

Author(s):

Inés Romero-Brey ◽

Carola Berger ◽

Stephanie Kallis ◽

Androniki Kolovou ◽

David Paul ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Viruses ◽

Nonstructural Protein ◽

Membrane Vesicles ◽

Rna Replication ◽

Concerted Action ◽

Double Membrane ◽

Positive Strand ◽

Positive Strand Rna

ABSTRACTInduction of membrane rearrangements in the cytoplasm of infected cells is a hallmark of positive-strand RNA viruses. These altered membranes serve as scaffolds for the assembly of viral replication factories (RFs). We have recently shown that hepatitis C virus (HCV) infection induces endoplasmic reticulum-derived double-membrane vesicles (DMVs) representing the major constituent of the RF within the infected cell. RF formation requires the concerted action of nonstructural action of nonstructural protein (NS)3, -4A, protein (NS)3 -4A, -4B, -5A, and -5B. Although the sole expression of NS5A is sufficient to induce DMV formation, its efficiency is very low. In this study, we dissected the determinants within NS5A responsible for DMV formation and found that RNA-binding domain 1 (D1) and the amino-terminal membrane anchor are indispensable for this process. In contrast, deletion of NS5A D2 or D3 did not affect DMV formation but disrupted RNA replication and virus assembly, respectively. To identifycis- andtrans-acting factors of DMV formation, we established atranscleavage assay. We found that induction of DMVs requires full-length NS3, whereas a helicase-lacking mutant was unable to trigger DMV formation in spite of efficient polyprotein cleavage. Importantly, a mutation accelerating cleavage kinetics at the NS4B-5A site diminished DMV formation, while the insertion of an internal ribosome entry site mimicking constitutive cleavage at this boundary completely abolished this process. These results identify key determinants governing the biogenesis of the HCV RF with possible implications for our understanding of how RFs are formed in other positive-strand RNA viruses.IMPORTANCELike all positive-strand RNA viruses, hepatitis C virus (HCV) extensively reorganizes intracellular membranes to allow efficient RNA replication. Double-membrane vesicles (DMVs) that putatively represent sites of HCV RNA amplification are induced by the concerted action of viral and cellular factors. However, the contribution of individual proteins to this process remains poorly understood. Here we identify determinants in the HCV replicase that are required for DMV biogenesis. Major contributors to this process are domain 1 of nonstructural protein 5A and the helicase domain of nonstructural protein 3. In addition, efficient DMV induction depends onciscleavage of the viral polyprotein, as well as tightly regulated cleavage kinetics. These results identify key determinants governing the biogenesis of the HCV replication factory with possible implications for our understanding of how this central compartment is formed in other positive-strand RNA viruses.

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