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1.
J Virol ; 94(13)2020 06 16.
Article in English | MEDLINE | ID: covidwho-1583223

ABSTRACT

Fusion with, and subsequent entry into, the host cell is one of the critical steps in the life cycle of enveloped viruses. For Middle East respiratory syndrome coronavirus (MERS-CoV), the spike (S) protein is the main determinant of viral entry. Proteolytic cleavage of the S protein exposes its fusion peptide (FP), which initiates the process of membrane fusion. Previous studies on the related severe acute respiratory syndrome coronavirus (SARS-CoV) FP have shown that calcium ions (Ca2+) play an important role in fusogenic activity via a Ca2+ binding pocket with conserved glutamic acid (E) and aspartic acid (D) residues. SARS-CoV and MERS-CoV FPs share a high sequence homology, and here, we investigated whether Ca2+ is required for MERS-CoV fusion by screening a mutant array in which E and D residues in the MERS-CoV FP were substituted with neutrally charged alanines (A). Upon verifying mutant cell surface expression and proteolytic cleavage, we tested their ability to mediate pseudoparticle (PP) infection of host cells in modulating Ca2+ environments. Our results demonstrate that intracellular Ca2+ enhances MERS-CoV wild-type (WT) PP infection by approximately 2-fold and that E891 is a crucial residue for Ca2+ interaction. Subsequent electron spin resonance (ESR) experiments revealed that this enhancement could be attributed to Ca2+ increasing MERS-CoV FP fusion-relevant membrane ordering. Intriguingly, isothermal calorimetry showed an approximate 1:1 MERS-CoV FP to Ca2+ ratio, as opposed to an 1:2 SARS-CoV FP to Ca2+ ratio, suggesting significant differences in FP Ca2+ interactions of MERS-CoV and SARS-CoV FP despite their high sequence similarity.IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) is a major emerging infectious disease with zoonotic potential and has reservoirs in dromedary camels and bats. Since its first outbreak in 2012, the virus has repeatedly transmitted from camels to humans, with 2,468 confirmed cases causing 851 deaths. To date, there are no efficacious drugs and vaccines against MERS-CoV, increasing its potential to cause a public health emergency. In order to develop novel drugs and vaccines, it is important to understand the molecular mechanisms that enable the virus to infect host cells. Our data have found that calcium is an important regulator of viral fusion by interacting with negatively charged residues in the MERS-CoV FP region. This information can guide therapeutic solutions to block this calcium interaction and also repurpose already approved drugs for this use for a fast response to MERS-CoV outbreaks.


Subject(s)
Calcium/metabolism , Coronavirus Infections/metabolism , Coronavirus Infections/virology , Host-Pathogen Interactions , Ions/metabolism , Membrane Fusion , Middle East Respiratory Syndrome Coronavirus/physiology , Virus Internalization , Amino Acid Sequence , Amino Acid Substitution , Animals , Cell Line , Chlorocebus aethiops , Humans , Middle East Respiratory Syndrome Coronavirus/pathogenicity , Models, Molecular , Mutation , Protein Binding , Proteolysis , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Structure-Activity Relationship , Vero Cells , Virulence , Virus Assembly
2.
Int J Mol Sci ; 21(19)2020 Oct 01.
Article in English | MEDLINE | ID: covidwho-1389385

ABSTRACT

Tom70 is a versatile adaptor protein of 70 kDa anchored in the outer membrane of mitochondria in metazoa, fungi and amoeba. The tertiary structure was resolved for the Tom70 of yeast, showing 26 α-helices, most of them participating in the formation of 11 tetratricopeptide repeat (TPR) motifs. Tom70 serves as a docking site for cytosolic chaperone proteins and co-chaperones and is thereby involved in the uptake of newly synthesized chaperone-bound proteins in mitochondrial biogenesis. In yeast, Tom70 additionally mediates ER-mitochondria contacts via binding to sterol transporter Lam6/Ltc1. In mammalian cells, TOM70 promotes endoplasmic reticulum (ER) to mitochondria Ca2+ transfer by association with the inositol-1,4,5-triphosphate receptor type 3 (IP3R3). TOM70 is specifically targeted by the Bcl-2-related protein MCL-1 that acts as an anti-apoptotic protein in macrophages infected by intracellular pathogens, but also in many cancer cells. By participating in the recruitment of PINK1 and the E3 ubiquitin ligase Parkin, TOM70 can be implicated in the development of Parkinson's disease. TOM70 acts as receptor of the mitochondrial antiviral-signaling protein (MAVS) and thereby participates in the corresponding system of innate immunity against viral infections. The protein encoded by Orf9b in the genome of SARS-CoV-2 binds to TOM70, probably compromising the synthesis of type I interferons.


Subject(s)
Immunity, Innate , Mitochondrial Membrane Transport Proteins/chemistry , Animals , Betacoronavirus/genetics , Binding Sites , Humans , Mitochondrial Membrane Transport Proteins/metabolism , Open Reading Frames , Protein Binding , Protein Transport , SARS-CoV-2
3.
World J Stem Cells ; 13(4): 260-280, 2021 Apr 26.
Article in English | MEDLINE | ID: covidwho-1218873

ABSTRACT

Stem cells hold indefinite self-renewable capability that can be differentiated into all desired cell types. Based on their plasticity potential, they are divided into totipotent (morula stage cells), pluripotent (embryonic stem cells), multipotent (hematopoietic stem cells, multipotent adult progenitor stem cells, and mesenchymal stem cells [MSCs]), and unipotent (progenitor cells that differentiate into a single lineage) cells. Though bone marrow is the primary source of multipotent stem cells in adults, other tissues such as adipose tissues, placenta, amniotic fluid, umbilical cord blood, periodontal ligament, and dental pulp also harbor stem cells that can be used for regenerative therapy. In addition, induced pluripotent stem cells also exhibit fundamental properties of self-renewal and differentiation into specialized cells, and thus could be another source for regenerative medicine. Several diseases including neurodegenerative diseases, cardiovascular diseases, autoimmune diseases, virus infection (also coronavirus disease 2019) have limited success with conventional medicine, and stem cell transplantation is assumed to be the best therapy to treat these disorders. Importantly, MSCs, are by far the best for regenerative medicine due to their limited immune modulation and adequate tissue repair. Moreover, MSCs have the potential to migrate towards the damaged area, which is regulated by various factors and signaling processes. Recent studies have shown that extracellular calcium (Ca2+) promotes the proliferation of MSCs, and thus can assist in transplantation therapy. Ca2+ signaling is a highly adaptable intracellular signal that contains several components such as cell-surface receptors, Ca2+ channels/pumps/exchangers, Ca2+ buffers, and Ca2+ sensors, which together are essential for the appropriate functioning of stem cells and thus modulate their proliferative and regenerative capacity, which will be discussed in this review.

4.
Sci Signal ; 14(675)2021 03 23.
Article in English | MEDLINE | ID: covidwho-1186203

ABSTRACT

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger that releases Ca2+ from acidic organelles through the activation of two-pore channels (TPCs) to regulate endolysosomal trafficking events. NAADP action is mediated by NAADP-binding protein(s) of unknown identity that confer NAADP sensitivity to TPCs. Here, we used a "clickable" NAADP-based photoprobe to isolate human NAADP-binding proteins and identified Jupiter microtubule-associated homolog 2 (JPT2) as a TPC accessory protein required for endogenous NAADP-evoked Ca2+ signaling. JPT2 was also required for the translocation of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus through the endolysosomal system. Thus, JPT2 is a component of the NAADP receptor complex that is essential for TPC-dependent Ca2+ signaling and control of coronaviral entry.


Subject(s)
COVID-19/metabolism , COVID-19/virology , Calcium Signaling/physiology , Microtubule-Associated Proteins/metabolism , NADP/analogs & derivatives , SARS-CoV-2/physiology , Affinity Labels , Animals , Calcium Channels/metabolism , Carrier Proteins/metabolism , Click Chemistry/methods , Gene Knockdown Techniques , HEK293 Cells , Humans , Microtubule-Associated Proteins/antagonists & inhibitors , Microtubule-Associated Proteins/genetics , NADP/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Second Messenger Systems/physiology , Transcriptome , Virus Internalization
5.
Blood ; 137(8): 1061-1071, 2021 02 25.
Article in English | MEDLINE | ID: covidwho-1013145

ABSTRACT

The pathophysiology of COVID-19-associated thrombosis seems to be multifactorial. We hypothesized that COVID-19 is accompanied by procoagulant platelets with subsequent alteration of the coagulation system. We investigated depolarization of mitochondrial inner transmembrane potential (ΔΨm), cytosolic calcium (Ca2+) concentration, and phosphatidylserine (PS) externalization. Platelets from COVID-19 patients in the intensive care unit (ICU; n = 21) showed higher ΔΨm depolarization, cytosolic Ca2+, and PS externalization compared with healthy controls (n = 18) and non-ICU COVID-19 patients (n = 4). Moreover, significant higher cytosolic Ca2+ and PS were observed compared with a septic ICU control group (ICU control; n = 5). In the ICU control group, cytosolic Ca2+ and PS externalization were comparable with healthy controls, with an increase in ΔΨm depolarization. Sera from COVID-19 patients in the ICU induced a significant increase in apoptosis markers (ΔΨm depolarization, cytosolic Ca2+, and PS externalization) compared with healthy volunteers and septic ICU controls. Interestingly, immunoglobulin G fractions from COVID-19 patients induced an Fcγ receptor IIA-dependent platelet apoptosis (ΔΨm depolarization, cytosolic Ca2+, and PS externalization). Enhanced PS externalization in platelets from COVID-19 patients in the ICU was associated with increased sequential organ failure assessment score (r = 0.5635) and D-dimer (r = 0.4473). Most importantly, patients with thrombosis had significantly higher PS externalization compared with those without. The strong correlations between markers for apoptosic and procoagulant platelets and D-dimer levels, as well as the incidence of thrombosis, may indicate that antibody-mediated procoagulant platelets potentially contributes to sustained increased thromboembolic risk in ICU COVID-19 patients.


Subject(s)
Apoptosis , Blood Platelets/pathology , COVID-19/pathology , Immunoglobulin G/metabolism , Adult , Aged , Blood Coagulation , Blood Platelets/metabolism , COVID-19/blood , COVID-19/complications , COVID-19/metabolism , Calcium/metabolism , Cohort Studies , Female , Humans , Male , Membrane Potential, Mitochondrial , Middle Aged , Phosphatidylserines/metabolism , SARS-CoV-2/isolation & purification , SARS-CoV-2/physiology , Thrombosis/blood , Thrombosis/etiology , Thrombosis/metabolism , Thrombosis/pathology
6.
ACS Infect Dis ; 6(12): 3174-3189, 2020 12 11.
Article in English | MEDLINE | ID: covidwho-954597

ABSTRACT

Human coronaviruses (hCoVs) have become a threat to global health and society, as evident from the SARS outbreak in 2002 caused by SARS-CoV-1 and the most recent COVID-19 pandemic caused by SARS-CoV-2. Despite a high sequence similarity between SARS-CoV-1 and -2, each strain has a distinctive virulence. A better understanding of the basic molecular mechanisms mediating changes in virulence is needed. Here, we profile the virus-host protein-protein interactions of two hCoV nonstructural proteins (nsps) that are critical for virus replication. We use tandem mass tag-multiplexed quantitative proteomics to sensitively compare and contrast the interactomes of nsp2 and nsp4 from three betacoronavirus strains: SARS-CoV-1, SARS-CoV-2, and hCoV-OC43-an endemic strain associated with the common cold. This approach enables the identification of both unique and shared host cell protein binding partners and the ability to further compare the enrichment of common interactions across homologues from related strains. We identify common nsp2 interactors involved in endoplasmic reticulum (ER) Ca2+ signaling and mitochondria biogenesis. We also identify nsp4 interactors unique to each strain, such as E3 ubiquitin ligase complexes for SARS-CoV-1 and ER homeostasis factors for SARS-CoV-2. Common nsp4 interactors include N-linked glycosylation machinery, unfolded protein response associated proteins, and antiviral innate immune signaling factors. Both nsp2 and nsp4 interactors are strongly enriched in proteins localized at mitochondria-associated ER membranes suggesting a new functional role for modulating host processes, such as calcium homeostasis, at these organelle contact sites. Our results shed light on the role these hCoV proteins play in the infection cycle, as well as host factors that may mediate the divergent pathogenesis of OC43 from SARS strains. Our mass spectrometry workflow enables rapid and robust comparisons of multiple bait proteins, which can be applied to additional viral proteins. Furthermore, the identified common interactions may present new targets for exploration by host-directed antiviral therapeutics.


Subject(s)
COVID-19/metabolism , Host-Pathogen Interactions/genetics , SARS-CoV-2/pathogenicity , Viral Nonstructural Proteins/metabolism , COVID-19/virology , Coronavirus OC43, Human/pathogenicity , Endoplasmic Reticulum/metabolism , HEK293 Cells , Humans , Membrane Proteins/metabolism , Mitochondria/metabolism , Protein Binding , Protein Interaction Maps/genetics , SARS Virus/pathogenicity , Severe Acute Respiratory Syndrome/metabolism , Severe Acute Respiratory Syndrome/virology , Transfection , Viral Nonstructural Proteins/genetics , Virulence/genetics , Virus Replication/genetics
7.
ACS Chem Neurosci ; 11(15): 2145-2148, 2020 08 05.
Article in English | MEDLINE | ID: covidwho-646274

ABSTRACT

Studies have shown that the calcium ion (Ca2+) plays important roles both in Alzheimer's dementia and SARS-CoV S-mediated fusion to host cell entry. An elevated level of intracellular calcium causes neuronal dysfunction, cell death, and apoptosis. Dysregulation of calcium has also been shown to increase the production of amyloid beta (Aß) protein, the hallmark of Alzheimer's dementia. Reversely, deposition of Aß is also responsible for calcium dysregulation. On the other hand, it has been well investigated that viruses can disturb host cell Ca2+ homeostasis as well as modulate signal transduction mechanisms. Viruses can also hijack the host cell calcium channels and pumps to release more intracellular Ca2+ to utilize for their life cycle. Even though evidence has not been reported on SARS-CoV-2 concerning Ca2+ regulation, however, it has been well established that Ca2+ is essential for viral entry, viral gene replication, and virion maturation and release. Recent reports suggest that SARS-CoV needs two Ca2+ ions to fuse with the host cell at the entry step. Furthermore, some calcium channel blockers (CCBs), such as nimodipine, memantine, etc., have been reported to be effective in the treatment of dementia in Alzheimer's disease (AD) as well as have shown inhibition in various virus infections.


Subject(s)
Alzheimer Disease/drug therapy , Betacoronavirus , Calcium Channel Blockers/therapeutic use , Coronavirus Infections/drug therapy , Pneumonia, Viral/drug therapy , Alzheimer Disease/metabolism , Alzheimer Disease/psychology , COVID-19 , Calcium/metabolism , Calcium Channel Blockers/chemistry , Calcium Channel Blockers/pharmacology , Calcium Channels/metabolism , Coronavirus Infections/metabolism , Coronavirus Infections/psychology , Humans , Pandemics , Pneumonia, Viral/metabolism , Pneumonia, Viral/psychology , SARS-CoV-2 , Treatment Outcome
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