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Principal Investigator: JOSHUA ZIMMERBERG
Organization: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
Fiscal Year: 2024
Award: $1,356,672
Funding agency: Eunice Kennedy Shriver National Institute of Child Health and Human Development
The first project in this report focuses on PIP2, a minor component of the PM in mammalian cells (~1% of total lipid) yet as the most abundant polyphosphoinositide it plays an outsized role in cell function. PIP2 mediates many intracellular processes, such as endo- and exocytosis, actin cytoskeleton regulation, cytoskeleton PM adhesion, and many others. PIP2 clusters at the PM and these clusters are known to interact with various cytoskeletal proteins such as actin and actin-binding proteins. Proteins that have been purified from IAV are also known to interact with PIP2, and IAV exploits several PIP2-dependent pathways. Because other viruses, such as HIV and Ebola, use PIP2 to mediate the membrane binding of their capsid proteins, and because IAV HA and NP have known interactions with PIP2, it is reasonable to investigate whether PIP2 and M1 might also interact.
We recently showed that the quaternary ammonium compound cetylpyridinium chloride (CPC) at non-cytotoxic concentrations modulates the membrane association and clustering of PIP2-binding proteins such as myristoylated alanine-rich C-kinase substrate (MARCKS) and phospholipase C (PLC)- through its pleckstrin homology (PH)-domain. CPC contains a positively charged head group and a hydrophobic tail, allowing it to efficiently associate with membrane bilayers and micelles. The detergent action of CPC is observed when the concentration of CPC is above its critical micelle concentration (CMC) in water, which has been quantified by various methods in different buffers and temperatures to be in the range ~600–900 M.
Although CPC has been previously shown to possess antibacterial and antiviral properties through micelle formation at millimolar concentrations, there is much less information on how low micromolar concentrations of CPC could affect interactions between IAV components. Our recent CPC study showed that HA and PH (PIP2) coclustering were modulated by CPC and suggested a mechanism for antiviral properties of CPC at non-cytotoxic concentrations. In this study, we build on these findings and test the hypothesis that CPC modulates the assembly of HA and M1. Using the localization based super-resolution microscopy technique of fluorescence photoactivation localization microscopy (FPALM) with total internal reflection fluorescence (TIRF) excitation, we also shed light on the effect of M1 on the PM distribution of PIP2 and the effect of HA in the clustering of M1 adjacent to the PM.
The interaction of M1 with lipid bilayers is considered to be primarily electrostatic and M1 can bind lipid bilayers through multiple residues. M1 is known to interact with PS in cells and cell models, but M1 interaction with other lipids has not been extensively studied. If interactions between M1 and lipid bilayers are primarily electrostatic, it is plausible that M1 could interact with lipids that are more negatively charged than PS. We tested this hypothesis and found M1 colocalized with the PLC -PH domain, an established marker of PIP2. The finding that PIP2 clustering was significantly enhanced in the presence of M1 is consistent with the response of PIP2 clustering to other positively charged membrane-associated molecules and ions and the positively charged face of M1, supporting the notion that M1 interacts with PIP2 electrostatically at the PM.
The quaternary ammonium compound CPC, found previously to disrupt PIP2 clustering and reduce the PM association of PIP2-binding proteins at low micromolar concentrations, significantly disrupted the colocalization, clustering, and co-clustering of HA and M1, as well as the colocalization, clustering, and co-clustering of M1 and PIP2. CPC also improved the outcomes of IAV infection in vivo. The implicated role of PIP2 in HA-M1 interaction and M1 association with the PM is novel, suggesting that it may be fruitful to explore strategies which modulate host cell PIP2 or other phosphoinositides to prevent or inhibit IAV infection.
A second project studies large numbers of viral spike protein sequences from several viral families. We analyzed the degree to which viral spike proteins contain a particular motif within their vSID: one or more basic amino acids in close proximity to a potentially acylated cysteine, serine, glycine, or hydrophobic amino acid. We abbreviate this generalized feature as (C+/CX+), with the reverse of the features also being included (i.e., +XC or + C). We chose to analyze a subset of viruses with particular relevance to human health: influenza viruses A and B, with particular attention to avian influenza virus strains, SARS-CoV-2 and other β-coronaviruses, Ebolavirus, HIV1, RSV, and measles. While gag is not technically a spike protein, it does have known interactions with anionic lipid membranes, and is thus of interest. HIV1 envelope protein was also included as a comparison.
We used several approaches: sequence analysis to identify patterns with putative phosphoinositide interaction, all-atom molecular dynamics to test for interactions between phosphoinositides and these residues in viral proteins, and super-resolution microscopy to test for nanoscale interactions between these proteins and phosphoinositides in fixed cells. We found highly (>99%) conserved patterns within their intracellular domains. The patterns generally consist of one or more basic amino acids (arginine or lysine) adjacent to a cysteine, many of which are known to undergo acylation. These patterns were not enriched in cellular proteins in general.
Molecular dynamics simulations show direct electrostatic and hydrophobic interactions between these conserved residues in hemagglutinin (HA) from influenza A and B and the phosphoinositide PIP2. Super-resolution microscopy shows nanoscale colocalization of PIP2 and several of the same viral proteins. We propose the hypothesis that these conserved viral spike protein features can interact with phosphoinositides such as PIP2.
In conclusion, we analyzed viral spike proteins from a number of viral families and found that they contain highly conserved sequence features in their cytoplasmic tails, typically consisting of one or more basic amino acids in proximity to one or more cysteines. We hypothesized that these basic amino acids, together with the cysteines, when acylated, could interact with phosphoinositides as many cellular proteins are known to do. Using molecular dynamics simulations, we show that in HA from influenza A and B, these features do interact directly with PIP2 through a combination of electrostatic interactions and association of the acylated cysteines with the PIP2 tails. Experimental evidence from super-resolution microscopy verifies that several of these same spike proteins colocalize with PIP2 at the nanoscale. Taken together, these results are suggestive of a mechanism of interaction between viral spike proteins and host cell phosphoinositides. Further work is needed to test the generality of this proposed mechanism.
Terms: <2019 novel corona virus><2019 novel coronavirus><2019-nCoV><47KDa platelet protein><ACAMP-81><AIDS Virus><Abbreviations><Abscission><Acquired Immune Deficiency Syndrome Virus><Acquired Immunodeficiency Syndrome Virus><Actin-Binding Protein><Actins><Acylation><Adhesions><Affect><Amino Acid Sequence><Amino Acids><Amino Acylation><Aminoacetic Acid><Aminoacylation><Anti-Bacterial Agents><Anti-viral Agents><Anti-viral Therapy><Area><Arginine><Attention><Avian Influenza A Virus><Avian Influenza Virus><Avian Orthomyxovirus Type A><Basic Amino Acids><Binding><Binding Proteins><Buffers><COVID-19 virus><COVID19 virus><Capsid Proteins><Cell Body><Cell Components><Cell Function><Cell Membrane Lipids><Cell Physiology><Cell Process><Cell Structure><Cell membrane><Cell model><Cells><Cellular Function><Cellular Matrix><Cellular Physiology><Cellular Process><Cellular Structures><Cellular model><Cetylpyridinium Chloride><Charge><Cholesterol><CoV-2><CoV2><Coat Proteins><Conserved Sequence><Cysteine><Cytoplasmic Domain><Cytoplasmic Membrane><Cytoplasmic Tail><Cytoskeletal Gene><Cytoskeletal Proteins><Cytoskeletal System><Cytoskeleton><Detergents><Development><Drug Design><Drug Targeting><Drug resistance><EBOV><Ebola><Ebola virus><Ebola-like Viruses><Electrostatics><Endoplasmic Reticulum><Envelope Protein><Ergastoplasm><Excision><Exocytosis><Extirpation><Face><Family><Fluorescence><Fowl Plague Virus><Genetic Alteration><Genetic Change><Genetic defect><Glycine><Glycoproteins><HIV><HIV-1><HIV-I><HIV1><Half-Cystine><Head><Health><Hemagglutinin><Human><Human Immunodeficiency Virus Type 1><Human Immunodeficiency Viruses><Human immunodeficiency virus 1><Hydrogen Oxide><Hydrophobic Interactions><Hydrophobicity><Impairment><Influenza A><Influenza A virus><Influenza B><Influenza B Virus><Influenza HA><Influenza Hemagglutinin><Influenza Virus><Influenza Viruses Type A><Influenza Viruses Type B><Influenzavirus A><Inositide Phospholipids><Inositol Phosphoglycerides><Inositol Phospholipids><Interruption><Ions><L-Arginine><L-Cysteine><L-Lysine><L-Serine><LAV-HTLV-III><Lecithinase C><Life Cycle><Life Cycle Stages><Ligand Binding Protein><Ligand Binding Protein Gene><Lipid Bilayers><Lipid Binding><Lipids><Location><Lymphadenopathy-Associated Virus><Lysine><MARCKS gene><MARCKS protein><Mammalian Cell><Measles><Mediating><Membrane><Membrane Lipids><Membrane Protein Gene><Membrane Proteins><Membrane-Associated Proteins><Methods><Micelles><Microscopy><Minor><Modern Man><Molecular Dynamics Simulation><Molecular Interaction><Morbidity><Morbidity - disease rate><Morphology><Mutate><Mutation><Myristoylated alanine-rich protein kinase C substrate><Myxovirus pestis galli><Orthomyxovirus Type A><Orthomyxoviruses Type B><PH Domain><PIP2><PRKCSL><Pathway interactions><Pattern><Phosphatidyl Inositol><Phosphatidyl Inositol Phosphates><Phosphatidylinositol 4,5-Biphosphate><Phosphatidylinositol 4,5-Diphosphate><Phosphatidylinositol Phosphates><Phosphatidylinositol-4,5-Bisphosphate><Phosphatidylinositols><Phosphoinositides><Phospholipase C><Plasma Membrane><Play><Pleckstrin-Homology Domain><Polyphosphoinositides><Population><Primary Protein Structure><Process><Property><Protein Binding><Proteins><PtIns 4,5-P2><PtdIns><PtdInsP2><Regulation><Removal><Reporting><Role><Rubeola><SARS corona virus 2><SARS-CO-V2><SARS-COVID-2><SARS-CoV-2><SARS-CoV2><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><SEQ-AN><Sequence Analyses><Sequence Analysis><Serine><Severe Acute Respiratory Coronavirus 2><Severe Acute Respiratory Distress Syndrome CoV 2><Severe Acute Respiratory Distress Syndrome Corona Virus 2><Severe Acute Respiratory Distress Syndrome Coronavirus 2><Severe Acute Respiratory Syndrome CoV 2><Severe Acute Respiratory Syndrome-associated coronavirus 2><Severe Acute Respiratory Syndrome-related coronavirus 2><Severe acute respiratory syndrome associated corona virus 2><Severe acute respiratory syndrome coronavirus 2><Severe acute respiratory syndrome related corona virus 2><Site><Site-Directed Mutagenesis><Site-Specific Mutagenesis><Subcellular Process><Surface><Surface Proteins><Surgical Removal><TM Domain><Tail><Targeted DNA Modification><Targeted Modification><Techniques><Temperature><Testing><Transmembrane Domain><Transmembrane Region><Type A Influenza><Viral><Viral Coat Proteins><Viral Diseases><Viral Gene Products><Viral Gene Proteins><Viral Outer Coat Protein><Viral Proteins><Virus><Virus Assembly><Virus Diseases><Virus-HIV><Virus-like particle><Water><Work><Wuhan coronavirus><aminoacid><anti-bacterial><anti-viral compound><anti-viral development><anti-viral drug development><anti-viral drugs><anti-viral medication><anti-viral therapeutic><anti-viral therapeutic development><anti-viral therapy development><anti-virals><antiviral development><antiviral drug development><antiviral therapeutic development><antiviral therapy development><avian flu virus><beta CoV><beta coronavirus><betaCoV><betacoronavirus><bird flu virus><bound protein><cell fixing><coronavirus disease 2019 virus><coronavirus disease-19 virus><density><developing anti-viral agent><developing anti-viral drug><developing anti-viral therapeutic><developing anti-viral therapy><developing antiviral agent><developing antiviral drug><developing antiviral therapeutic><developing antiviral therapy><developmental><drug resistant><env Antigens><env Gene Products><env Polyproteins><env Protein><faces><facial><flu HA><flu hemagglutinin><flu serotype><flu strain><flu subtype><flu viral strain><flu virus strain><genome mutation><hCoV19><improved outcome><in vivo><influenza serotype><influenza strain><influenza subtype><influenza viral HA><influenza viral hemagglutinin><influenza viral strain><influenza virus HA><influenza virus hemagglutinin><influenza virus strain><influenzavirus><interest><intracellular skeleton><life course><lipid bilayer membrane><lipid bound><lipophosphodiesterase I><membrane structure><molecular dynamics><morbilli><mortality><myristoylated alanine-rich C kinase substrate><nCoV2><nano meter scale><nano meter sized><nanometer scale><nanometer sized><nanoscale><novel><palmitoylation><pathway><phosphatidylcholine cholinephosphohydrolase><photoactivation><plasmalemma><platelet 47k protein><platelet protein P47><pleckstrin><prevent><preventing><protein kinase C substrate, 80-KD, light chain><protein kinase substrate 80KD protein, light chain><protein sequence><quaternary ammonium compound><resection><resistance to Drug><resistant to Drug><response><social role><superresolution microscopy><trafficking><vaccine efficacy><viral assembly><viral infection><viral infectious disease treatment><virus infection><virus protein><virus-induced disease><virus-like nanoparticles><viruslike particle><β CoV><β coronavirus><βCoV>