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1.
J Med Virol ; 93(9): 5487-5504, 2021 09.
Article in English | MEDLINE | ID: covidwho-1733919

ABSTRACT

Along with the control and prevention of coronavirus disease 2019 transmission, infected animals might have potential to carry the virus to spark new outbreaks. However, very few studies explore the susceptibility of animals to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Viral attachment as a crucial step for cross-species infection requires angiotensin-converting enzyme 2 (ACE2) as a receptor and depends on TMPRSS2 protease activity. Here, we searched the genomes of metazoans from different classes using an extensive BLASTP survey and found ACE2 and TMPRSS2 occur in vertebrates, but some vertebrates lack Tmprss2. We identified 6 amino acids among 25 known human ACE2 residues are highly associated with the binding of ACE2 to SARS-CoV-2 (p value < .01) by Fisher exact test, and following this, calculated the probability of viral attachment within each species by the randomForest function from R randomForest library. Furthermore, we observed that Ace2 selected from seven animals based on the above analysis lack the hydrophobic contacts identified on human ACE2, indicating less affinity of SARS-CoV-2 to Ace2 in animals than humans. Finally, the alignment of 3D structure between human ACE2 and other animals by I-TASSER and TM-align displayed a reasonable structure for viral attachment within these species. Taken together, our data may shed light on the human-to-animal transmission of SARS-CoV-2.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/virology , Host-Pathogen Interactions , SARS-CoV-2/physiology , Serine Endopeptidases/metabolism , Vertebrates/metabolism , Angiotensin-Converting Enzyme 2/genetics , Animals , COVID-19/genetics , COVID-19/metabolism , Disease Susceptibility , Genetic Predisposition to Disease , Humans , Receptors, Virus/metabolism , SARS-CoV-2/classification , Serine Endopeptidases/genetics , Spike Glycoprotein, Coronavirus/metabolism , Vertebrates/genetics , Virus Attachment , Virus Internalization , Virus Release
2.
Viruses ; 14(2)2022 01 25.
Article in English | MEDLINE | ID: covidwho-1715763

ABSTRACT

Epithelial cells are apico-basolateral polarized cells that line all tubular organs and are often targets for infectious agents. This review focuses on the release of human RNA virus particles from both sides of polarized human cells grown on transwells. Most viruses that infect the mucosa leave their host cells mainly via the apical side while basolateral release is linked to virus propagation within the host. Viruses do this by hijacking the cellular factors involved in polarization and trafficking. Thus, understanding epithelial polarization is essential for a clear understanding of virus pathophysiology.


Subject(s)
Epithelial Cells/virology , RNA Viruses/physiology , Virus Release , Cell Polarity , Humans , Virion/physiology , Virus Assembly , Virus Replication
3.
Signal Transduct Target Ther ; 7(1): 26, 2022 01 27.
Article in English | MEDLINE | ID: covidwho-1655545

ABSTRACT

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the causative agent of the pandemic disease COVID-19, which is so far without efficacious treatment. The discovery of therapy reagents for treating COVID-19 are urgently needed, and the structures of the potential drug-target proteins in the viral life cycle are particularly important. SARS-CoV-2, a member of the Orthocoronavirinae subfamily containing the largest RNA genome, encodes 29 proteins including nonstructural, structural and accessory proteins which are involved in viral adsorption, entry and uncoating, nucleic acid replication and transcription, assembly and release, etc. These proteins individually act as a partner of the replication machinery or involved in forming the complexes with host cellular factors to participate in the essential physiological activities. This review summarizes the representative structures and typically potential therapy agents that target SARS-CoV-2 or some critical proteins for viral pathogenesis, providing insights into the mechanisms underlying viral infection, prevention of infection, and treatment. Indeed, these studies open the door for COVID therapies, leading to ways to prevent and treat COVID-19, especially, treatment of the disease caused by the viral variants are imperative.


Subject(s)
Antiviral Agents/therapeutic use , COVID-19/drug therapy , Drug Design/trends , Drug Repositioning , SARS-CoV-2/drug effects , Adrenal Cortex Hormones/chemistry , Adrenal Cortex Hormones/therapeutic use , Antibodies, Viral/chemistry , Antibodies, Viral/therapeutic use , Antiviral Agents/chemistry , Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/therapeutic use , COVID-19/metabolism , COVID-19/pathology , COVID-19/virology , Drugs, Chinese Herbal/chemistry , Drugs, Chinese Herbal/therapeutic use , Humans , Models, Molecular , Nucleosides/chemistry , Nucleosides/therapeutic use , Protein Conformation , SARS-CoV-2/genetics , SARS-CoV-2/growth & development , SARS-CoV-2/metabolism , Virus Internalization/drug effects , Virus Release/drug effects , Virus Replication/drug effects
4.
Life Sci Alliance ; 5(4)2022 04.
Article in English | MEDLINE | ID: covidwho-1614505

ABSTRACT

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The positive-sense single-stranded RNA virus contains a single linear RNA segment that serves as a template for transcription and replication, leading to the synthesis of positive and negative-stranded viral RNA (vRNA) in infected cells. Tools to visualize vRNA directly in infected cells are critical to analyze the viral replication cycle, screen for therapeutic molecules, or study infections in human tissue. Here, we report the design, validation, and initial application of FISH probes to visualize positive or negative RNA of SARS-CoV-2 (CoronaFISH). We demonstrate sensitive visualization of vRNA in African green monkey and several human cell lines, in patient samples and human tissue. We further demonstrate the adaptation of CoronaFISH probes to electron microscopy. We provide all required oligonucleotide sequences, source code to design the probes, and a detailed protocol. We hope that CoronaFISH will complement existing techniques for research on SARS-CoV-2 biology and COVID-19 pathophysiology, drug screening, and diagnostics.


Subject(s)
COVID-19/diagnosis , In Situ Hybridization, Fluorescence/methods , RNA, Viral/genetics , SARS-CoV-2/genetics , Virus Replication/genetics , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/pharmacology , Animals , Antiviral Agents/pharmacology , COVID-19/drug therapy , COVID-19/virology , Caco-2 Cells , Cell Line, Tumor , Chlorocebus aethiops , Humans , In Situ Hybridization/methods , Microscopy, Electron/methods , RNA, Viral/ultrastructure , Reproducibility of Results , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Sensitivity and Specificity , Vero Cells , Virus Release/drug effects , Virus Release/genetics , Virus Release/physiology , Virus Replication/drug effects , Virus Replication/physiology
5.
Microbiol Spectr ; 10(1): e0150421, 2022 02 23.
Article in English | MEDLINE | ID: covidwho-1604818

ABSTRACT

In December 2019, a new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) started spreading worldwide causing the coronavirus disease 2019 (COVID-19) pandemic. The hyperactivation of the immune system has been proposed to account for disease severity and death in COVID-19 patients. Despite several approaches having been tested, no therapeutic protocol has been approved. Given that Cyclosporine A (CsA) is well-known to exert a strong antiviral activity on several viral strains and an anti-inflammatory role in different organs with relevant benefits in diverse pathological contexts, we tested its effects on SARS-CoV-2 infection of lung cells. We found that treatment with CsA either before or after infection of CaLu3 cells by three SARS-CoV-2 variants: (i) reduces the expression of both viral RNA and proteins in infected cells; (ii) decreases the number of progeny virions released by infected cells; (iii) dampens the virus-triggered synthesis of cytokines (including IL-6, IL-8, IL1α and TNF-α) that are involved in cytokine storm in patients. Altogether, these data provide a rationale for CsA repositioning for the treatment of severe COVID-19 patients. IMPORTANCE SARS-CoV-2 is the most recently identified member of the betacoronavirus genus responsible for the COVID-19 pandemic. Repurposing of available drugs has been a "quick and dirty" approach to try to reduce mortality and severe symptoms in affected patients initially, and can still represent an undeniable and valuable approach to face COVID-19 as the continuous appearance and rapid diffusion of more "aggressive"/transmissible variants, capable of eluding antibody neutralization, challenges the effectiveness of some anti-SARS-CoV-2 vaccines. Here, we tested a known antiviral and anti-inflammatory drug, Cyclosporine A (CsA), and found that it dampens viral infection and cytokine release from lung cells upon exposure to three different SARS-CoV-2 variants. Knock down of the main intracellular target of CsA, Cyclophilin A, does not phenocopy the drug inhibition of viral infection. Altogether, these findings shed new light on the cellular mechanisms of SARS-CoV-2 infection and provide the rationale for CsA repositioning to treat severe COVID-19 patients.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Antiviral Agents/pharmacology , COVID-19/virology , Cyclosporine/pharmacology , Cytokines/immunology , Lung/virology , SARS-CoV-2/drug effects , Virus Release/drug effects , COVID-19/genetics , COVID-19/immunology , Cytokine Release Syndrome , Cytokines/genetics , Humans , SARS-CoV-2/genetics , SARS-CoV-2/physiology
6.
Viruses ; 13(12)2021 11 23.
Article in English | MEDLINE | ID: covidwho-1542792

ABSTRACT

The ongoing coronavirus disease (COVID-19) pandemic has required a variety of non-medical interventions to limit the transmission of the causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). One such option is over-the-counter nasal sprays that aim to block virus entry and transmission within the nasal cavity. In this study, we assessed the ability of three hydroxypropyl methylcellulose (HPMC)-based powder nasal sprays, produced by Nasaleze, to inhibit SARS-CoV-2 infection and release in vitro. Upon application, the HPMC powder forms a gel-like matrix within the nasal cavity-a process we recapitulated in cell culture. We found that virus release from cells previously infected with SARS-CoV-2 was inhibited by the gel matrix product in a dose-dependent manner, with virus levels reduced by >99.99% over a 72 h period at a dose of 6.4 mg/3.5 cm2. We also show that the pre-treatment of cells with product inhibited SARS-CoV-2 infection, independent of the virus variant. The primary mechanism of action appears to be via the formation of a physical, passive barrier. However, the addition of wild garlic provided additional direct antiviral properties in some formulations. We conclude that HPMC-based nasal sprays may offer an additional component to strategies to limit the spread of respiratory viruses, including SARS-CoV-2.


Subject(s)
COVID-19/prevention & control , Hypromellose Derivatives/pharmacology , SARS-CoV-2/drug effects , Animals , Chlorocebus aethiops , Dose-Response Relationship, Drug , Nasal Sprays , Vero Cells , Virus Internalization/drug effects , Virus Release/drug effects
7.
Cell Rep Med ; 2(12): 100456, 2021 12 21.
Article in English | MEDLINE | ID: covidwho-1500334

ABSTRACT

The ongoing SARS-CoV-2 pandemic continues to lead to high morbidity and mortality. During pregnancy, severe maternal and neonatal outcomes and placental pathological changes have been described. We evaluate SARS-CoV-2 infection at the maternal-fetal interface using precision-cut slices (PCSs) of human placenta. Remarkably, exposure of placenta PCSs to SARS-CoV-2 leads to a full replication cycle with infectious virus release. Moreover, the susceptibility of placental tissue to SARS-CoV-2 replication relates to the expression levels of ACE2. Viral proteins and/or viral RNA are detected in syncytiotrophoblasts, cytotrophoblasts, villous stroma, and possibly Hofbauer cells. While SARS-CoV-2 infection of placenta PCSs does not cause a detectable cytotoxicity or a pro-inflammatory cytokine response, an upregulation of one order of magnitude of interferon type III transcripts is measured. In conclusion, our data demonstrate the capacity of SARS-CoV-2 to infect and propagate in human placenta and constitute a basis for further investigation of SARS-CoV-2 biology at the maternal-fetal interface.


Subject(s)
Placenta/virology , SARS-CoV-2/physiology , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/transmission , COVID-19/virology , Chorionic Villi/virology , Female , Humans , Infectious Disease Transmission, Vertical , Interferons/metabolism , Placenta/cytology , Placenta/metabolism , Pregnancy , RNA, Viral/metabolism , Trophoblasts/cytology , Trophoblasts/virology , Viral Proteins/metabolism , Virus Release , Virus Replication
8.
Dev Cell ; 56(23): 3250-3263.e5, 2021 12 06.
Article in English | MEDLINE | ID: covidwho-1458566

ABSTRACT

Viral entry and egress are important determinants of virus infectivity and pathogenicity. ß-coronaviruses, including the COVID-19 virus SARS-CoV-2 and mouse hepatitis virus (MHV), exploit the lysosomal exocytosis pathway for egress. Here, we show that SARS-CoV-2 ORF3a, but not SARS-CoV ORF3a, promotes lysosomal exocytosis. SARS-CoV-2 ORF3a facilitates lysosomal targeting of the BORC-ARL8b complex, which mediates trafficking of lysosomes to the vicinity of the plasma membrane, and exocytosis-related SNARE proteins. The Ca2+ channel TRPML3 is required for SARS-CoV-2 ORF3a-mediated lysosomal exocytosis. Expression of SARS-CoV-2 ORF3a greatly elevates extracellular viral release in cells infected with the coronavirus MHV-A59, which itself lacks ORF3a. In SARS-CoV-2 ORF3a, Ser171 and Trp193 are critical for promoting lysosomal exocytosis and blocking autophagy. When these residues are introduced into SARS-CoV ORF3a, it acquires the ability to promote lysosomal exocytosis and inhibit autophagy. Our results reveal a mechanism by which SARS-CoV-2 interacts with host factors to promote its extracellular egress.


Subject(s)
ADP-Ribosylation Factors/metabolism , Autophagy , Exocytosis , Lysosomes/physiology , Transient Receptor Potential Channels/metabolism , Viroporin Proteins/metabolism , Virus Release , ADP-Ribosylation Factors/genetics , Animals , COVID-19/virology , HeLa Cells , Humans , Mice , SARS-CoV-2/isolation & purification , Transient Receptor Potential Channels/genetics , Viroporin Proteins/genetics
9.
Cell Mol Life Sci ; 78(7): 3565-3576, 2021 Apr.
Article in English | MEDLINE | ID: covidwho-1384325

ABSTRACT

Many studies on SARS-CoV-2 have been performed over short-time scale, but few have focused on the ultrastructural characteristics of infected cells. We used TEM to perform kinetic analysis of the ultrastructure of SARS-CoV-2-infected cells. Early infection events were characterized by the presence of clusters of single-membrane vesicles and stacks of membrane containing nuclear pores called annulate lamellae (AL). A large network of host cell-derived organelles transformed into virus factories was subsequently observed in the cells. As previously described for other RNA viruses, these replication factories consisted of double-membrane vesicles (DMVs) located close to the nucleus. Viruses released at the cell surface by exocytosis harbored the typical crown of spike proteins, but viral particles without spikes were also observed in intracellular compartments, possibly reflecting incorrect assembly or a cell degradation process.


Subject(s)
SARS-CoV-2/growth & development , Viral Replication Compartments/ultrastructure , Virus Release/physiology , Virus Replication/physiology , Animals , COVID-19/pathology , Cell Line , Chlorocebus aethiops , Microscopy, Electron, Transmission , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells , Viral Replication Compartments/physiology
10.
Int J Mol Sci ; 22(10)2021 May 17.
Article in English | MEDLINE | ID: covidwho-1383880

ABSTRACT

Numerous viruses hijack cellular protein trafficking pathways to mediate cell entry or to rearrange membrane structures thereby promoting viral replication and antagonizing the immune response. Adaptor protein complexes (AP), which mediate protein sorting in endocytic and secretory transport pathways, are one of the conserved viral targets with many viruses possessing AP-interacting motifs. We present here different mechanisms of viral interference with AP complexes and the functional consequences that allow for efficient viral propagation and evasion of host immune defense. The ubiquity of this phenomenon is evidenced by the fact that there are representatives for AP interference in all major viral families, covered in this review. The best described examples are interactions of human immunodeficiency virus and human herpesviruses with AP complexes. Several other viruses, like Ebola, Nipah, and SARS-CoV-2, are pointed out as high priority disease-causative agents supporting the need for deeper understanding of virus-AP interplay which can be exploited in the design of novel antiviral therapies.


Subject(s)
Adaptor Proteins, Vesicular Transport/metabolism , HIV-1/metabolism , Herpesviridae/metabolism , SARS-CoV-2/metabolism , Ebolavirus/metabolism , Endocytosis , Humans , Nipah Virus/metabolism , Protein Transport , Virus Release , Virus Replication
12.
Nat Commun ; 12(1): 4629, 2021 07 30.
Article in English | MEDLINE | ID: covidwho-1333939

ABSTRACT

Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified viral components and inactivated viruses. However, structural and ultrastructural evidence on how the SARS-CoV-2 infection progresses in the native cellular context is scarce, and there is a lack of comprehensive knowledge on the SARS-CoV-2 replicative cycle. To correlate cytopathic events induced by SARS-CoV-2 with virus replication processes in frozen-hydrated cells, we established a unique multi-modal, multi-scale cryo-correlative platform to image SARS-CoV-2 infection in Vero cells. This platform combines serial cryoFIB/SEM volume imaging and soft X-ray cryo-tomography with cell lamellae-based cryo-electron tomography (cryoET) and subtomogram averaging. Here we report critical SARS-CoV-2 structural events - e.g. viral RNA transport portals, virus assembly intermediates, virus egress pathway, and native virus spike structures, in the context of whole-cell volumes revealing drastic cytppathic changes. This integrated approach allows a holistic view of SARS-CoV-2 infection, from the whole cell to individual molecules.


Subject(s)
COVID-19/immunology , SARS-CoV-2/immunology , Virus Assembly/immunology , Virus Release/immunology , Virus Replication/immunology , Animals , COVID-19/epidemiology , COVID-19/virology , Chlorocebus aethiops , Cryoelectron Microscopy , Electron Microscope Tomography , Humans , Pandemics/prevention & control , SARS-CoV-2/physiology , SARS-CoV-2/ultrastructure , Vero Cells , Virus Assembly/physiology , Virus Release/physiology , Virus Replication/physiology
13.
Microbiol Spectr ; 9(1): e0043921, 2021 09 03.
Article in English | MEDLINE | ID: covidwho-1329042

ABSTRACT

Hepatitis C virus (HCV) can cause acute and chronic infection that is associated with considerable liver-related morbidity and mortality. In recent years, there has been a shift in the treatment paradigm with the discovery and approval of agents that target specific proteins vital for viral replication. We employed a cell culture-adapted strain of HCV and human hepatoma-derived cells lines to test the effects of our novel small-molecule compound (AO13) on HCV. Virus inhibition was tested by analyzing RNA replication, protein expression, and virus production in virus-infected cells treated with AO13. Treatment with AO13 inhibited virus spread in cell culture and showed a 100-fold reduction in the levels of infectious virus production. AO13 significantly reduced the level of viral RNA contained within cell culture fluids and reduced the cellular levels of HCV core protein, suggesting that the compound might act on a late step in the viral life cycle. Finally, we observed that AO13 did not affect the release of infectious virus from infected cells. Docking studies and molecular dynamics analyses suggested that AO13 might target the NS5B RNA polymerase, however, real-time RT-PCR analyses of cellular levels of HCV RNA showed only an ∼2-fold reduction in viral RNA levels in the presence of AO13. Taken together, this study revealed that AO13 showed consistent, but low-level antiviral effect against HCV, although the mechanism of action remains unclear. IMPORTANCE The discovery of curative antiviral drugs for a chronic disease such as HCV infection has encouraged drug discovery in the context of other viruses for which no curative drugs currently exist. Since we currently face a novel virus that has caused a pandemic, the need for new antiviral agents is more apparent than ever. We describe here a novel compound that shows a modest antiviral effect against HCV that could serve as a lead compound for future drug development against other important viruses such as SARS-CoV-2.


Subject(s)
Antiviral Agents/pharmacology , Cell Culture Techniques , Hepacivirus/drug effects , Virus Replication/drug effects , Antiviral Agents/therapeutic use , Carcinoma, Hepatocellular , Cell Line , Hepacivirus/genetics , Hepacivirus/physiology , Hepatitis C/drug therapy , Hepatitis C/virology , Humans , Life Cycle Stages , Liver , Liver Neoplasms , Molecular Docking Simulation , RNA, Viral , SARS-CoV-2 , Viral Nonstructural Proteins , Virus Release/drug effects
14.
J Med Virol ; 93(9): 5487-5504, 2021 09.
Article in English | MEDLINE | ID: covidwho-1222642

ABSTRACT

Along with the control and prevention of coronavirus disease 2019 transmission, infected animals might have potential to carry the virus to spark new outbreaks. However, very few studies explore the susceptibility of animals to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Viral attachment as a crucial step for cross-species infection requires angiotensin-converting enzyme 2 (ACE2) as a receptor and depends on TMPRSS2 protease activity. Here, we searched the genomes of metazoans from different classes using an extensive BLASTP survey and found ACE2 and TMPRSS2 occur in vertebrates, but some vertebrates lack Tmprss2. We identified 6 amino acids among 25 known human ACE2 residues are highly associated with the binding of ACE2 to SARS-CoV-2 (p value < .01) by Fisher exact test, and following this, calculated the probability of viral attachment within each species by the randomForest function from R randomForest library. Furthermore, we observed that Ace2 selected from seven animals based on the above analysis lack the hydrophobic contacts identified on human ACE2, indicating less affinity of SARS-CoV-2 to Ace2 in animals than humans. Finally, the alignment of 3D structure between human ACE2 and other animals by I-TASSER and TM-align displayed a reasonable structure for viral attachment within these species. Taken together, our data may shed light on the human-to-animal transmission of SARS-CoV-2.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/virology , Host-Pathogen Interactions , SARS-CoV-2/physiology , Serine Endopeptidases/metabolism , Vertebrates/metabolism , Angiotensin-Converting Enzyme 2/genetics , Animals , COVID-19/genetics , COVID-19/metabolism , Disease Susceptibility , Genetic Predisposition to Disease , Humans , Receptors, Virus/metabolism , SARS-CoV-2/classification , Serine Endopeptidases/genetics , Spike Glycoprotein, Coronavirus/metabolism , Vertebrates/genetics , Virus Attachment , Virus Internalization , Virus Release
15.
Viruses ; 13(5)2021 04 30.
Article in English | MEDLINE | ID: covidwho-1217120

ABSTRACT

Repurposing clinically available drugs to treat the new coronavirus disease 2019 (COVID-19) is an urgent need in the course of the Severe Acute Respiratory Syndrome coronavirus (SARS-CoV-2) pandemic, as very few treatment options are available. The iminosugar Miglustat is a well-characterized drug for the treatment of rare genetic lysosome storage diseases, such as Gaucher and Niemann-Pick type C, and has also been described to be active against a variety of enveloped viruses. The activity of Miglustat is here demonstrated in the micromolar range for SARS-CoV-2 in vitro. The drug acts at the post-entry level and leads to a marked decrease of viral proteins and release of infectious viruses. The mechanism resides in the inhibitory activity toward α-glucosidases that are involved in the early stages of glycoprotein N-linked oligosaccharide processing in the endoplasmic reticulum, leading to a marked decrease of the viral Spike protein. Indeed, the antiviral potential of protein glycosylation inhibitors against SARS-CoV-2 is further highlighted by the low-micromolar activity of the investigational drug Celgosivir. These data point to a relevant role of this approach for the treatment of COVID-19.


Subject(s)
1-Deoxynojirimycin/analogs & derivatives , Antiviral Agents/pharmacology , Drug Repositioning , Glycoside Hydrolase Inhibitors/pharmacology , Indolizines/pharmacology , SARS-CoV-2/drug effects , 1-Deoxynojirimycin/pharmacology , A549 Cells , Animals , COVID-19/drug therapy , Chlorocebus aethiops , Glycosylation/drug effects , HEK293 Cells , Humans , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells , Virus Release/drug effects
16.
Mol Cell ; 81(12): 2656-2668.e8, 2021 06 17.
Article in English | MEDLINE | ID: covidwho-1179919

ABSTRACT

A deficient interferon (IFN) response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been implicated as a determinant of severe coronavirus disease 2019 (COVID-19). To identify the molecular effectors that govern IFN control of SARS-CoV-2 infection, we conducted a large-scale gain-of-function analysis that evaluated the impact of human IFN-stimulated genes (ISGs) on viral replication. A limited subset of ISGs were found to control viral infection, including endosomal factors inhibiting viral entry, RNA binding proteins suppressing viral RNA synthesis, and a highly enriched cluster of endoplasmic reticulum (ER)/Golgi-resident ISGs inhibiting viral assembly/egress. These included broad-acting antiviral ISGs and eight ISGs that specifically inhibited SARS-CoV-2 and SARS-CoV-1 replication. Among the broad-acting ISGs was BST2/tetherin, which impeded viral release and is antagonized by SARS-CoV-2 Orf7a protein. Overall, these data illuminate a set of ISGs that underlie innate immune control of SARS-CoV-2/SARS-CoV-1 infection, which will facilitate the understanding of host determinants that impact disease severity and offer potential therapeutic strategies for COVID-19.


Subject(s)
Antigens, CD/genetics , Host-Pathogen Interactions/genetics , Interferon Regulatory Factors/genetics , Interferon Type I/genetics , SARS-CoV-2/genetics , Viral Proteins/genetics , Animals , Antigens, CD/chemistry , Antigens, CD/immunology , Binding Sites , Cell Line, Tumor , Chlorocebus aethiops , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum/immunology , Endoplasmic Reticulum/virology , GPI-Linked Proteins/chemistry , GPI-Linked Proteins/genetics , GPI-Linked Proteins/immunology , Gene Expression Regulation , Golgi Apparatus/genetics , Golgi Apparatus/immunology , Golgi Apparatus/virology , HEK293 Cells , Host-Pathogen Interactions/immunology , Humans , Immunity, Innate , Interferon Regulatory Factors/classification , Interferon Regulatory Factors/immunology , Interferon Type I/immunology , Molecular Docking Simulation , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , SARS-CoV-2/immunology , Signal Transduction , Vero Cells , Viral Proteins/chemistry , Viral Proteins/immunology , Virus Internalization , Virus Release/genetics , Virus Release/immunology , Virus Replication/genetics , Virus Replication/immunology
17.
Viruses ; 13(3)2021 02 26.
Article in English | MEDLINE | ID: covidwho-1115433

ABSTRACT

Ubiquitination of proteins is a post-translational modification process with many different cellular functions, including protein stability, immune signaling, antiviral functions and virus replication. While ubiquitination of viral proteins can be used by the host as a defense mechanism by destroying the incoming pathogen, viruses have adapted to take advantage of this cellular process. The ubiquitin system can be hijacked by viruses to enhance various steps of the replication cycle and increase pathogenesis. Emerging viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), flaviviruses like Zika and dengue, as well as highly pathogenic viruses like Ebola and Nipah, have the ability to directly use the ubiquitination process to enhance their viral-replication cycle, and evade immune responses. Some of these mechanisms are conserved among different virus families, especially early during virus entry, providing an opportunity to develop broad-spectrum antivirals. Here, we discuss the mechanisms used by emergent viruses to exploit the host ubiquitin system, with the main focus on the role of ubiquitin in enhancing virus replication.


Subject(s)
Ubiquitin/metabolism , Virus Diseases/metabolism , Virus Replication , Viruses/metabolism , Immune Evasion , Ubiquitination , Viral Proteins/metabolism , Virus Assembly , Virus Diseases/immunology , Virus Diseases/virology , Virus Internalization , Virus Release , Viruses/classification , Viruses/immunology , Viruses/pathogenicity
18.
Cells ; 10(3)2021 02 26.
Article in English | MEDLINE | ID: covidwho-1107394

ABSTRACT

Coronaviruses (CoVs) assemble by budding into the lumen of the intermediate compartment (IC) at the endoplasmic reticulum (ER)-Golgi interface. However, why CoVs have chosen the IC as their intracellular site of assembly and how progeny viruses are delivered from this compartment to the extracellular space has remained unclear. Here we address these enigmatic late events of the CoV life cycle in light of recently described properties of the IC. Of particular interest are the emerging spatial and functional connections between IC elements and recycling endosomes (REs), defined by the GTPases Rab1 and Rab11, respectively. The establishment of IC-RE links at the cell periphery, around the centrosome and evidently also at the noncompact zones of the Golgi ribbon indicates that-besides traditional ER-Golgi communication-the IC also promotes a secretory process that bypasses the Golgi stacks, but involves its direct connection with the endocytic recycling system. The initial confinement of CoVs to the lumen of IC-derived large transport carriers and their preferential absence from Golgi stacks is consistent with the idea that they exit cells following such an unconventional route. In fact, CoVs may share this pathway with other intracellularly budding viruses, lipoproteins, procollagen, and/or protein aggregates experimentally introduced into the IC lumen.


Subject(s)
Endoplasmic Reticulum/virology , Extracellular Space/virology , Golgi Apparatus/virology , Intracellular Membranes/virology , SARS-CoV-2/physiology , Secretory Pathway , Virus Release , Animals , COVID-19/therapy , COVID-19/virology , Centrosome/metabolism , Extracellular Space/metabolism , Golgi Apparatus/metabolism , Humans , Protein Transport
19.
J Biol Chem ; 296: 100111, 2021.
Article in English | MEDLINE | ID: covidwho-1066049

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a ß-coronavirus, is the causative agent of the COVID-19 pandemic. Like for other coronaviruses, its particles are composed of four structural proteins: spike (S), envelope (E), membrane (M), and nucleoprotein (N) proteins. The involvement of each of these proteins and their interactions are critical for assembly and production of ß-coronavirus particles. Here, we sought to characterize the interplay of SARS-CoV-2 structural proteins during the viral assembly process. By combining biochemical and imaging assays in infected versus transfected cells, we show that E and M regulate intracellular trafficking of S as well as its intracellular processing. Indeed, the imaging data reveal that S is relocalized at endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) or Golgi compartments upon coexpression of E or M, as observed in SARS-CoV-2-infected cells, which prevents syncytia formation. We show that a C-terminal retrieval motif in the cytoplasmic tail of S is required for its M-mediated retention in the ERGIC, whereas E induces S retention by modulating the cell secretory pathway. We also highlight that E and M induce a specific maturation of N-glycosylation of S, independently of the regulation of its localization, with a profile that is observed both in infected cells and in purified viral particles. Finally, we show that E, M, and N are required for optimal production of virus-like-particles. Altogether, these results highlight how E and M proteins may influence the properties of S proteins and promote the assembly of SARS-CoV-2 viral particles.


Subject(s)
Coronavirus Envelope Proteins/genetics , Nucleocapsid Proteins/genetics , SARS-CoV-2/growth & development , Spike Glycoprotein, Coronavirus/genetics , Viral Matrix Proteins/genetics , Virion/growth & development , Virus Assembly/physiology , Animals , Biomimetic Materials/chemistry , Biomimetic Materials/metabolism , Cell Line, Tumor , Chlorocebus aethiops , Coronavirus Envelope Proteins/metabolism , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/ultrastructure , Endoplasmic Reticulum/virology , Gene Expression , Golgi Apparatus/metabolism , Golgi Apparatus/ultrastructure , Golgi Apparatus/virology , HEK293 Cells , Hepatocytes/metabolism , Hepatocytes/ultrastructure , Hepatocytes/virology , Host-Pathogen Interactions/genetics , Humans , Nucleocapsid Proteins/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells , Viral Matrix Proteins/metabolism , Virion/genetics , Virion/metabolism , Virus Internalization , Virus Release/physiology
20.
Cell Mol Life Sci ; 78(7): 3565-3576, 2021 Apr.
Article in English | MEDLINE | ID: covidwho-1033048

ABSTRACT

Many studies on SARS-CoV-2 have been performed over short-time scale, but few have focused on the ultrastructural characteristics of infected cells. We used TEM to perform kinetic analysis of the ultrastructure of SARS-CoV-2-infected cells. Early infection events were characterized by the presence of clusters of single-membrane vesicles and stacks of membrane containing nuclear pores called annulate lamellae (AL). A large network of host cell-derived organelles transformed into virus factories was subsequently observed in the cells. As previously described for other RNA viruses, these replication factories consisted of double-membrane vesicles (DMVs) located close to the nucleus. Viruses released at the cell surface by exocytosis harbored the typical crown of spike proteins, but viral particles without spikes were also observed in intracellular compartments, possibly reflecting incorrect assembly or a cell degradation process.


Subject(s)
SARS-CoV-2/growth & development , Viral Replication Compartments/ultrastructure , Virus Release/physiology , Virus Replication/physiology , Animals , COVID-19/pathology , Cell Line , Chlorocebus aethiops , Microscopy, Electron, Transmission , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells , Viral Replication Compartments/physiology
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