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1.
Biochemistry (Mosc) ; 86(7): 800-817, 2021 Jul.
Article in English | MEDLINE | ID: covidwho-1594970

ABSTRACT

COVID-19, a new human respiratory disease that has killed nearly 3 million people in a year since the start of the pandemic, is a global public health challenge. Its infectious agent, SARS-CoV-2, differs from other coronaviruses in a number of structural features that make this virus more pathogenic and transmissible. In this review, we discuss some important characteristics of the main SARS-CoV-2 surface antigen, the spike (S) protein, such as (i) ability of the receptor-binding domain (RBD) to switch between the "standing-up" position (open pre-fusion conformation) for receptor binding and the "lying-down" position (closed pre-fusion conformation) for immune system evasion; (ii) advantage of a high binding affinity of the RBD open conformation to the human angiotensin-converting enzyme 2 (ACE2) receptor for efficient cell entry; and (iii) S protein preliminary activation by the intracellular furin-like proteases for facilitation of the virus spreading across different cell types. We describe interactions between the S protein and cellular receptors, co-receptors, and antagonists, as well as a hypothetical mechanism of the homotrimeric spike structure destabilization that triggers the fusion of the viral envelope with the cell membrane at physiological pH and mediates the viral nucleocapsid entry into the cytoplasm. The transition of the S protein pre-fusion conformation to the post-fusion one on the surface of virions after their treatment with some reagents, such as ß-propiolactone, is essential, especially in relation to the vaccine production. We also compare the COVID-19 pathogenesis with that of severe outbreaks of "avian" influenza caused by the A/H5 and A/H7 highly pathogenic viruses and discuss the structural similarities between the SARS-CoV-2 S protein and hemagglutinins of those highly pathogenic strains. Finally, we touch on the prospective and currently used COVID-19 antiviral and anti-pathogenetic therapeutics, as well as recently approved conventional and innovative COVID-19 vaccines and their molecular and immunological features.


Subject(s)
Angiotensin-Converting Enzyme 2 , COVID-19 , Pandemics , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/epidemiology , COVID-19/genetics , COVID-19/metabolism , Humans , Influenza A virus/chemistry , Influenza A virus/genetics , Influenza A virus/metabolism , Influenza, Human/epidemiology , Influenza, Human/genetics , Influenza, Human/metabolism , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
2.
PLoS Comput Biol ; 17(9): e1009357, 2021 09.
Article in English | MEDLINE | ID: covidwho-1470651

ABSTRACT

Cell culture-derived defective interfering particles (DIPs) are considered for antiviral therapy due to their ability to inhibit influenza A virus (IAV) production. DIPs contain a large internal deletion in one of their eight viral RNAs (vRNAs) rendering them replication-incompetent. However, they can propagate alongside their homologous standard virus (STV) during infection in a competition for cellular and viral resources. So far, experimental and modeling studies for IAV have focused on either the intracellular or the cell population level when investigating the interaction of STVs and DIPs. To examine these levels simultaneously, we conducted a series of experiments using highly different multiplicities of infections for STVs and DIPs to characterize virus replication in Madin-Darby Canine Kidney suspension cells. At several time points post infection, we quantified virus titers, viable cell concentration, virus-induced apoptosis using imaging flow cytometry, and intracellular levels of vRNA and viral mRNA using real-time reverse transcription qPCR. Based on the obtained data, we developed a mathematical multiscale model of STV and DIP co-infection that describes dynamics closely for all scenarios with a single set of parameters. We show that applying high DIP concentrations can shut down STV propagation completely and prevent virus-induced apoptosis. Interestingly, the three observed viral mRNAs (full-length segment 1 and 5, defective interfering segment 1) accumulated to vastly different levels suggesting the interplay between an internal regulation mechanism and a growth advantage for shorter viral RNAs. Furthermore, model simulations predict that the concentration of DIPs should be at least 10000 times higher than that of STVs to prevent the spread of IAV. Ultimately, the model presented here supports a comprehensive understanding of the interactions between STVs and DIPs during co-infection providing an ideal platform for the prediction and optimization of vaccine manufacturing as well as DIP production for therapeutic use.


Subject(s)
Defective Viruses , Influenza A virus , Models, Biological , Orthomyxoviridae Infections/virology , Virus Replication/physiology , Animals , Antiviral Agents , Cell Culture Techniques , Defective Viruses/chemistry , Defective Viruses/genetics , Defective Viruses/pathogenicity , Dogs , Influenza A virus/chemistry , Influenza A virus/genetics , Influenza A virus/pathogenicity , Madin Darby Canine Kidney Cells , RNA, Viral/genetics
3.
ACS Chem Biol ; 15(9): 2331-2337, 2020 09 18.
Article in English | MEDLINE | ID: covidwho-1387140

ABSTRACT

We report on using the synthetic aminoadamantane-CH2-aryl derivatives 1-6 as sensitive probes for blocking M2 S31N and influenza A virus (IAV) M2 wild-type (WT) channels as well as virus replication in cell culture. The binding kinetics measured using electrophysiology (EP) for M2 S31N channel are very dependent on the length between the adamantane moiety and the first ring of the aryl headgroup realized in 2 and 3 and the girth and length of the adamantane adduct realized in 4 and 5. Study of 1-6 shows that, according to molecular dynamics (MD) simulations and molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) calculations, all bind in the M2 S31N channel with the adamantyl group positioned between V27 and G34 and the aryl group projecting out of the channel with the phenyl (or isoxazole in 6) embedded in the V27 cluster. In this outward binding configuration, an elongation of the ligand by only one methylene in rimantadine 2 or using diamantane or triamantane instead of adamantane in 4 and 5, respectively, causes incomplete entry and facilitates exit, abolishing effective block compared to the amantadine derivatives 1 and 6. In the active M2 S31N blockers 1 and 6, the phenyl and isoxazolyl head groups achieve a deeper binding position and high kon/low koff and high kon/high koff rate constants, compared to inactive 2-5, which have much lower kon and higher koff. Compounds 1-5 block the M2 WT channel by binding in the longer area from V27-H37, in the inward orientation, with high kon and low koff rate constants. Infection of cell cultures by influenza virus containing M2 WT or M2 S31N is inhibited by 1-5 or 1-4 and 6, respectively. While 1 and 6 block infection through the M2 block mechanism in the S31N variant, 2-4 may block M2 S31N virus replication in cell culture through the lysosomotropic effect, just as chloroquine is thought to inhibit SARS-CoV-2 infection.


Subject(s)
Adamantane/pharmacology , Influenza A virus/drug effects , Influenza, Human/prevention & control , Ion Channels/antagonists & inhibitors , Molecular Probes/chemistry , Viral Matrix Proteins/antagonists & inhibitors , Adamantane/analogs & derivatives , Adamantane/chemistry , Adamantane/metabolism , Betacoronavirus/drug effects , Binding Sites , COVID-19 , Cells, Cultured , Chloroquine/pharmacology , Coronavirus Infections/drug therapy , Coronavirus Infections/prevention & control , Genetic Variation , Humans , Influenza A virus/chemistry , Influenza A virus/genetics , Influenza, Human/drug therapy , Kinetics , Molecular Probes/metabolism , Pandemics/prevention & control , Pneumonia, Viral/drug therapy , Pneumonia, Viral/prevention & control , Protein Binding , SARS-CoV-2 , Structure-Activity Relationship , Virus Replication/drug effects
4.
ACS Appl Mater Interfaces ; 13(26): 30295-30305, 2021 Jul 07.
Article in English | MEDLINE | ID: covidwho-1337092

ABSTRACT

As viruses have been threatening global public health, fast diagnosis has been critical to effective disease management and control. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) is now widely used as the gold standard for detecting viruses. Although a multiplex assay is essential for identifying virus types and subtypes, the poor multiplicity of RT-qPCR makes it laborious and time-consuming. In this paper, we describe the development of a multiplex RT-qPCR platform with hydrogel microparticles acting as independent reactors in a single reaction. To build target-specific particles, target-specific primers and probes are integrated into the particles in the form of noncovalent composites with boron nitride nanotubes (BNNTs) and carbon nanotubes (CNTs). The thermal release characteristics of DNA, primer, and probe from the composites of primer-BNNT and probe-CNT allow primer and probe to be stored in particles during particle production and to be delivered into the reaction. In addition, BNNT did not absorb but preserved the fluorescent signal, while CNT protected the fluorophore of the probe from the free radicals present during particle production. Bicompartmental primer-incorporated network (bcPIN) particles were designed to harness the distinctive properties of two nanomaterials. The bcPIN particles showed a high RT-qPCR efficiency of over 90% and effective suppression of non-specific reactions. 16-plex RT-qPCR has been achieved simply by recruiting differently coded bcPIN particles for each target. As a proof of concept, multiplex one-step RT-qPCR was successfully demonstrated with a simple reaction protocol.


Subject(s)
Hydrogels/chemistry , Multiplex Polymerase Chain Reaction/methods , Nanotubes, Carbon/chemistry , RNA, Viral/analysis , Reverse Transcriptase Polymerase Chain Reaction/methods , Boron Compounds/chemistry , Coronavirus/chemistry , DNA Primers/chemistry , DNA, Single-Stranded/chemistry , Fluorescent Dyes/chemistry , Graphite/chemistry , Influenza A virus/chemistry , Newcastle disease virus/chemistry , Proof of Concept Study , RNA, Viral/chemistry , Virus Diseases/diagnosis
5.
Antioxid Redox Signal ; 35(13): 1081-1092, 2021 11 01.
Article in English | MEDLINE | ID: covidwho-1306508

ABSTRACT

Aims: Influenza A virus hemagglutinin (HA) binding to sialic acid on lung epithelial cells triggers membrane fusion and infection. Host thiol isomerases have been shown to play a role in influenza A virus infection, and we hypothesized that this role involved manipulation of disulfide bonds in HA. Results: Analysis of HA crystal structures revealed that three of the six HA disulfides occur in high-energy conformations and four of the six bonds can exist in unformed states, suggesting that the disulfide landscape of HA is generally strained and the bonds may be labile. We measured the redox state of influenza A virus HA disulfide bonds and their susceptibility to cleavage by vascular thiol isomerases. Using differential cysteine alkylation and mass spectrometry, we show that all six HA disulfide bonds exist in unformed states in ∼1 in 10 recombinant and viral surface HA molecules. Four of the six H1 and H3 HA bonds are cleaved by the vascular thiol isomerases, thioredoxin and protein disulphide isomerase, in recombinant proteins, which correlated with surface exposure of the disulfides in crystal structures. In contrast, viral surface HA disulfide bonds are impervious to five different vascular thiol isomerases. Innovation: It has been assumed that the disulfide bonds in mature HA protein are intact and inert. We show that all six HA disulfide bonds can exist in unformed states. Conclusion: These findings indicate that influenza A virus HA disulfides are naturally labile but not substrates for thiol isomerases when expressed on the viral surface.


Subject(s)
Disulfides/metabolism , Hemagglutinins/metabolism , Influenza A virus/chemistry , Disulfides/chemistry , Hemagglutinins/chemistry , Influenza A virus/metabolism , Models, Molecular
6.
J Biol Chem ; 296: 100017, 2021.
Article in English | MEDLINE | ID: covidwho-910220

ABSTRACT

Through annual epidemics and global pandemics, influenza A viruses (IAVs) remain a significant threat to human health as the leading cause of severe respiratory disease. Within the last century, four global pandemics have resulted from the introduction of novel IAVs into humans, with components of each originating from avian viruses. IAVs infect many avian species wherein they maintain a diverse natural reservoir, posing a risk to humans through the occasional emergence of novel strains with enhanced zoonotic potential. One natural barrier for transmission of avian IAVs into humans is the specificity of the receptor-binding protein, hemagglutinin (HA), which recognizes sialic-acid-containing glycans on host cells. HAs from human IAVs exhibit "human-type" receptor specificity, binding exclusively to glycans on cells lining the human airway where terminal sialic acids are attached in the α2-6 configuration (NeuAcα2-6Gal). In contrast, HAs from avian viruses exhibit specificity for "avian-type" α2-3-linked (NeuAcα2-3Gal) receptors and thus require adaptive mutations to bind human-type receptors. Since all human IAV pandemics can be traced to avian origins, there remains ever-present concern over emerging IAVs with human-adaptive potential that might lead to the next pandemic. This concern has been brought into focus through emergence of SARS-CoV-2, aligning both scientific and public attention to the threat of novel respiratory viruses from animal sources. In this review, we summarize receptor-binding adaptations underlying the emergence of all prior IAV pandemics in humans, maintenance and evolution of human-type receptor specificity in subsequent seasonal IAVs, and potential for future human-type receptor adaptation in novel avian HAs.


Subject(s)
Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Influenza A virus/metabolism , Influenza in Birds/epidemiology , Influenza, Human/epidemiology , Pandemics , Polysaccharides/chemistry , Receptors, Virus/metabolism , Adaptation, Physiological , Animals , Binding Sites , Biological Coevolution , Birds/virology , Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Humans , Influenza A virus/chemistry , Influenza A virus/genetics , Influenza in Birds/transmission , Influenza in Birds/virology , Influenza, Human/transmission , Influenza, Human/virology , Models, Molecular , Polysaccharides/metabolism , Protein Binding , Receptors, Virus/chemistry , Receptors, Virus/genetics , Respiratory System/virology , Sialic Acids/chemistry , Sialic Acids/metabolism , Species Specificity
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