ABSTRACT
In a pivotal trial (EPIC-HR), a 5-day course of oral ritonavir-boosted nirmatrelvir, given early during symptomatic SARS-CoV-2 infection (within three days of symptoms onset), decreased hospitalization and death by 89.1% and nasal viral load by 0.87 log relative to placebo in high-risk individuals. Yet, nirmatrelvir/ritonavir failed as post-exposure prophylaxis in a trial, and frequent viral rebound has been observed in subsequent cohorts. We develop a mathematical model capturing viral-immune dynamics and nirmatrelvir pharmacokinetics that recapitulates viral loads from this and another clinical trial (PLATCOV). Our results suggest that nirmatrelvir's in vivo potency is significantly lower than in vitro assays predict. According to our model, a maximally potent agent would reduce the viral load by approximately 3.5 logs relative to placebo at 5 days. The model identifies that earlier initiation and shorter treatment duration are key predictors of post-treatment rebound. Extension of treatment to 10 days for Omicron variant infection in vaccinated individuals, rather than increasing dose or dosing frequency, is predicted to lower the incidence of viral rebound significantly.
Subject(s)
Antiviral Agents , COVID-19 Drug Treatment , COVID-19 , Ritonavir , SARS-CoV-2 , Viral Load , Humans , SARS-CoV-2/drug effects , Ritonavir/therapeutic use , Ritonavir/administration & dosage , COVID-19/prevention & control , COVID-19/virology , COVID-19/immunology , Viral Load/drug effects , Antiviral Agents/administration & dosage , Antiviral Agents/therapeutic use , Antiviral Agents/pharmacology , Indazoles/pharmacology , Models, Theoretical , Post-Exposure Prophylaxis/methods , Lactams , Leucine , Nitriles , ProlineABSTRACT
Existing pharmacodynamic (PD) mathematical models for drug combinations discriminate antagonistic, additive, multiplicative, and synergistic effects, but fail to consider how concentration-dependent drug interaction effects may vary across an entire dose-response matrix. We developed a two-way pharmacodynamic (TWPD) model to capture the PD of two-drug combinations. TWPD captures interactions between upstream and downstream drugs that act on different stages of viral replication, by quantifying upstream drug efficacy and concentration-dependent effects on downstream drug pharmacodynamic parameters. We applied TWPD to previously published in vitro drug matrixes for repurposed potential anti-Ebola and anti-SARS-CoV-2 drug pairs. Depending on the drug pairing, the model recapitulated combined efficacies as or more accurately than existing models and can be used to infer efficacy at untested drug concentrations. TWPD fits the data slightly better in one direction for all drug pairs, meaning that we can tentatively infer the upstream drug. Based on its high accuracy, TWPD could be used in concert with PK models to estimate the therapeutic effects of drug pairs in vivo.
Subject(s)
COVID-19 , Hemorrhagic Fever, Ebola , Humans , Models, Biological , SARS-CoV-2 , Hemorrhagic Fever, Ebola/drug therapy , Drug CombinationsABSTRACT
In a pivotal trial (EPIC-HR), a 5-day course of oral ritonavir-boosted nirmatrelvir, given early during symptomatic SARS-CoV-2 infection (within three days of symptoms onset), decreased hospitalization and death by 89.1% and nasal viral load by 0.87 log relative to placebo in high-risk individuals. Yet, nirmatrelvir/ritonavir failed as post-exposure prophylaxis in a trial, and frequent viral rebound has been observed in subsequent cohorts. We developed a mathematical model capturing viral-immune dynamics and nirmatrelvir pharmacokinetics that recapitulated viral loads from this and another clinical trial (PLATCOV). Our results suggest that nirmatrelvir's in vivo potency is significantly lower than in vitro assays predict. According to our model, a maximally potent agent would reduce the viral load by approximately 3.5 logs relative to placebo at 5 days. The model identifies that earlier initiation and shorter treatment duration are key predictors of post-treatment rebound. Extension of treatment to 10 days for Omicron variant infection in vaccinated individuals, rather than increasing dose or dosing frequency, is predicted to lower the incidence of viral rebound significantly.
ABSTRACT
There are at least 21 families of enveloped viruses that infect mammals, and many contain members of high concern for global human health. All enveloped viruses have a dedicated fusion protein or fusion complex that enacts the critical genome-releasing membrane fusion event that is essential before viral replication within the host cell interior can begin. Because all enveloped viruses enter cells by fusion, it behooves us to know how viral fusion proteins function. Viral fusion proteins are also major targets of neutralizing antibodies, and hence they serve as key vaccine immunogens. Here we review current concepts about viral membrane fusion proteins focusing on how they are triggered, structural intermediates between pre- and postfusion forms, and their interplay with the lipid bilayers they engage. We also discuss cellular and therapeutic interventions that thwart virus-cell membrane fusion.
Subject(s)
Virus Internalization , Viruses , Animals , Humans , Viral Fusion Proteins/chemistry , Membrane Fusion , Viruses/genetics , Lipids , Mammals/metabolismABSTRACT
Ebola virus (EBOV) causes a hemorrhagic fever with fatality rates up to 90%. The EBOV entry process is complex and incompletely understood. Following attachment to host cells, EBOV is trafficked to late endosomes/lysosomes where its glycoprotein (GP) is processed to a 19-kDa form, which binds to the EBOV intracellular receptor Niemann-Pick type C1. We previously showed that the cathepsin protease inhibitor, E-64d, blocks infection by pseudovirus particles bearing 19-kDa GP, suggesting that further cathepsin action is needed to trigger fusion. This, however, has not been demonstrated directly. Since 19-kDa Ebola GP fusion occurs in late endosomes, we devised a system in which enriched late endosomes are used to prepare supported planar endosomal membranes (SPEMs), and fusion of fluorescent (pseudo)virus particles is monitored by total internal reflection fluorescence microscopy. We validated the system by demonstrating the pH dependencies of influenza virus hemagglutinin (HA)-mediated and Lassa virus (LASV) GP-mediated fusion. Using SPEMs, we showed that fusion mediated by 19-kDa Ebola GP is dependent on low pH, enhanced by Ca2+, and augmented by the addition of cathepsins. Subsequently, we found that E-64d inhibits full fusion, but not lipid mixing, mediated by 19-kDa GP, which we corroborated with the reversible cathepsin inhibitor VBY-825. Hence, we provide both gain- and loss-of-function evidence that further cathepsin action enhances the fusion activity of 19-kDa Ebola GP. In addition to providing new insights into how Ebola GP mediates fusion, the approach we developed employing SPEMs can now be broadly used for studies of virus and toxin entry through endosomes. IMPORTANCE Ebola virus is the causative agent of Ebola virus disease, which is severe and frequently lethal. EBOV gains entry into cells via late endosomes/lysosomes. The events immediately preceding fusion of the viral and endosomal membranes are incompletely understood. In this study, we report a novel in vitro system for studying virus fusion with endosomal membranes. We validated the system by demonstrating the low pH dependencies of influenza and Lassa virus fusion. Moreover, we show that further cathepsin B action enhances the fusion activity of the primed Ebola virus glycoprotein. Finally, this model endosomal membrane system should be useful in studying the mechanisms of bilayer breaching by other enveloped viruses, by non-enveloped viruses, and by acid-activated bacterial toxins.
ABSTRACT
The host restriction factor, Serinc5, incorporates into budding HIV particles and inhibits their infection by an incompletely understood mechanism. We have previously reported that Serinc5 but not its paralogue, Serinc2, blocks HIV cell entry by membrane fusion, specifically by inhibiting fusion pore formation and dilation. A body of work suggests that Serinc5 may alter the conformation and clustering of the HIV fusion protein, Env. To contribute an additional perspective to the developing model of Serinc5 restriction, we assessed Serinc2 and Serinc5's effects on HIV pseudoviral membranes. By measuring pseudoviral membrane thickness via cryo-electron microscopy and order via the fluorescent dye, FLIPPER-TR, Serinc5 was found to increase membrane heterogeneity, skewing the distribution toward a larger fraction of the viral membrane in an ordered phase. We also directly observed for the first time the coexistence of membrane domains within individual viral membrane envelopes. Using a total internal reflection fluorescence-based single particle fusion assay, we found that treatment of HIV pseudoviral particles with phosphatidylethanolamine (PE) rescued HIV pseudovirus fusion from restriction by Serinc5, which was accompanied by decreased membrane heterogeneity and order. This effect was specific for PE and did not depend on acyl chain length or saturation. Together, these data suggest that Serinc5 alters multiple interrelated properties of the viral membraneâlipid chain order, rigidity, line tension, and lateral pressureâwhich decrease the accessibility of fusion intermediates and disfavor completion of fusion. These biophysical insights into Serinc5 restriction of HIV infectivity could contribute to the development of novel antivirals that exploit the same weaknesses.
Subject(s)
HIV Infections , Membrane Proteins , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Cryoelectron Microscopy , Membrane Fusion , LipidsABSTRACT
Niemann-Pick C1 protein (NPC1) is a membrane protein that primarily resides in late endosomes and lysosomes, and plays an important role in cholesterol homeostasis in the cell. The second luminal domain of NPC1 (NPC1-C) serves as the intracellular receptor for Ebola and Marburg viruses. Here, the recombinant production of nonglycosylated and glycosylated NPC1-C and a new crystal form of the nonglycosylated protein are reported. The crystals belonged to space group P21 and diffracted to 2.3â Å resolution. The structure is similar to other reported structures of NPC1-C, with differences observed in the protruding loops when compared with NPC1-C in complex with Ebola virus glycoprotein or NPC2.
Subject(s)
Membrane Glycoproteins , Niemann-Pick C1 Protein , Humans , Membrane Glycoproteins/genetics , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/metabolism , Niemann-Pick C1 Protein/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Crystallography, X-Ray , Glycoproteins/chemistry , Lysosomes/metabolismABSTRACT
Three directly acting antivirals (DAAs) demonstrated substantial reduction in COVID-19 hospitalizations and deaths in clinical trials. However, these agents did not completely prevent severe illness and are associated with cases of rebound illness and viral shedding. Combination regimens can enhance antiviral potency, reduce the emergence of drug-resistant variants, and lower the dose of each component in the combination. Concurrently targeting virus entry and virus replication offers opportunities to discover synergistic drug combinations. While combination antiviral drug treatments are standard for chronic RNA virus infections, no antiviral combination therapy has been approved for SARS-CoV-2. Here, we demonstrate that combining host-targeting antivirals (HTAs) that target TMPRSS2 and hence SARS-CoV-2 entry, with the DAA molnupiravir, which targets SARS-CoV-2 replication, synergistically suppresses SARS-CoV-2 infection in Calu-3 lung epithelial cells. Strong synergy was observed when molnupiravir, an oral drug, was combined with three TMPRSS2 (HTA) oral or inhaled inhibitors: camostat, avoralstat, or nafamostat. The combination of camostat plus molnupiravir was also effective against the beta and delta variants of concern. The pyrimidine biosynthesis inhibitor brequinar combined with molnupiravir also conferred robust synergistic inhibition. These HTA+DAA combinations had similar potency to the synergistic all-DAA combination of molnupiravir plus nirmatrelvir, the protease inhibitor found in paxlovid. Pharmacodynamic modeling allowed estimates of antiviral potency at all possible concentrations of each agent within plausible therapeutic ranges, suggesting possible in vivo efficacy. The triple combination of camostat, brequinar, and molnupiravir further increased antiviral potency. These findings support the development of HTA+DAA combinations for pandemic response and preparedness. IMPORTANCE Imagine a future viral pandemic where if you test positive for the new virus, you can quickly take some medicines at home for a few days so that you do not get too sick. To date, only single drugs have been approved for outpatient use against SARS-CoV-2, and we are learning that these have some limitations and may succumb to drug resistance. Here, we show that combinations of two oral drugs are better than the single ones in blocking SARS-CoV-2, and we use mathematical modeling to show that these drug combinations are likely to work in people. We also show that a combination of three oral drugs works even better at eradicating the virus. Our findings therefore bode well for the development of oral drug cocktails for at home use at the first sign of an infection by a coronavirus or other emerging viral pathogens.
Subject(s)
COVID-19 Drug Treatment , SARS-CoV-2 , Humans , Antiviral Agents/pharmacology , Protease Inhibitors/pharmacology , Drug Combinations , PyrimidinesABSTRACT
Broadly effective antiviral therapies must be developed to be ready for clinical trials, which should begin soon after the emergence of new life-threatening viruses. Here, we pave the way towards this goal by reviewing conserved druggable virus-host interactions, mechanisms of action, immunomodulatory properties of available broad-spectrum antivirals (BSAs), routes of BSA delivery, and interactions of BSAs with other antivirals. Based on the review, we concluded that the range of indications of BSAs can be expanded, and new pan- and cross-viral mono- and combinational therapies can be developed. We have also developed a new scoring algorithm that can help identify the most promising few of the thousands of potential BSAs and BSA-containing drug cocktails (BCCs) to prioritize their development during the critical period between the identification of a new virus and the development of virus-specific vaccines, drugs, and therapeutic antibodies.
ABSTRACT
Most enveloped viruses infect cells by binding receptors at the cell surface and undergo trafficking through the endocytic pathway to a compartment with the requisite conditions to trigger fusion with a host endosomal membrane. Broad categories of compartments in the endocytic pathway include early and late endosomes, which can be further categorized into subpopulations with differing rates of maturation and motility characteristics. Endocytic compartments have varying protein and lipid components, luminal ionic conditions and pH that provide uniquely hospitable environments for specific viruses to fuse. In order to characterize compartments that permit fusion, we studied the trafficking and fusion of viral particles pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G) on their surface and equipped with a novel pH sensor and a fluorescent content marker to measure pH, motion and fusion at the single particle level in live cells. We found that the VSV-G particles fuse predominantly from more acidic and more motile endosomes, and that a significant fraction of particles is trafficked to more static and less acidic endosomes that do not support their fusion. Moreover, the fusion-supporting endosomes undergo directed motion.
Subject(s)
Vesicular Stomatitis , Virus Internalization , Animals , Endocytosis , Endosomes/metabolism , Glycoproteins/metabolism , Hydrogen-Ion Concentration , Vesicular Stomatitis/metabolismABSTRACT
The world was unprepared for coronavirus disease 2019 (COVID-19) and remains ill-equipped for future pandemics. While unprecedented strides have been made developing vaccines and treatments for COVID-19, there remains a need for highly effective and widely available regimens for ambulatory use for novel coronaviruses and other viral pathogens. We posit that a priority is to develop pan-family drug cocktails to enhance potency, limit toxicity, and avoid drug resistance. We urge cocktail development for all viruses with pandemic potential both in the short term (<1 to 2 years) and longer term with pairs of drugs in advanced clinical testing or repurposed agents approved for other indications. While significant efforts were launched against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in vitro and in the clinic, many studies employed solo drugs and had disappointing results. Here, we review drug combination studies against SARS-CoV-2 and other viruses and introduce a model-driven approach to assess drug pairs with the highest likelihood of clinical efficacy. Where component agents lack sufficient potency, we advocate for synergistic combinations to achieve therapeutic levels. We also discuss issues that stymied therapeutic progress against COVID-19, including testing of agents with low likelihood of efficacy late in clinical disease and lack of focus on developing virologic surrogate endpoints. There is a need to expedite efficient clinical trials testing drug combinations that could be taken at home by recently infected individuals and exposed contacts as early as possible during the next pandemic, whether caused by a coronavirus or another viral pathogen. The approach herein represents a proactive plan for global viral pandemic preparedness.
Subject(s)
Antiviral Agents/pharmacology , Coronavirus/drug effects , Drug Combinations , Animals , Coronavirus/classification , Coronavirus/pathogenicity , Coronavirus Infections/drug therapy , Humans , Mice , Pandemics/prevention & control , SARS-CoV-2/drug effects , COVID-19 Drug TreatmentABSTRACT
Host plasma membrane protein SERINC5 is incorporated into budding retrovirus particles where it blocks subsequent entry into susceptible target cells. Three structurally unrelated proteins encoded by diverse retroviruses, human immunodeficiency virus type 1 (HIV-1) Nef, equine infectious anemia virus (EIAV) S2, and ecotropic murine leukemia virus (MLV) GlycoGag, disrupt SERINC5 antiviral activity by redirecting SERINC5 from the site of virion assembly on the plasma membrane to an internal RAB7+ endosomal compartment. Pseudotyping retroviruses with particular glycoproteins, e.g., vesicular stomatitis virus glycoprotein (VSV G), renders the infectivity of particles resistant to inhibition by virion-associated SERINC5. To better understand viral determinants for SERINC5-sensitivity, the effect of SERINC5 was assessed using HIV-1, MLV, and Mason-Pfizer monkey virus (M-PMV) virion cores, pseudotyped with glycoproteins from Arenavirus, Coronavirus, Filovirus, Rhabdovirus, Paramyxovirus, and Orthomyxovirus genera. SERINC5 restricted virions pseudotyped with glycoproteins from several retroviruses, an orthomyxovirus, a rhabdovirus, a paramyxovirus, and an arenavirus. Infectivity of particles pseudotyped with HIV-1, amphotropic-MLV (A-MLV), or influenza A virus (IAV) glycoproteins, was decreased by SERINC5, whether the core was provided by HIV-1, MLV, or M-PMV. In contrast, particles pseudotyped with glycoproteins from M-PMV, parainfluenza virus 5 (PIV5), or rabies virus (RABV) were sensitive to SERINC5, but only with particular retroviral cores. Resistance to SERINC5 did not correlate with reduced SERINC5 incorporation into particles, route of viral entry, or absolute infectivity of the pseudotyped virions. These findings indicate that some non-retroviruses may be sensitive to SERINC5 and that, in addition to the viral glycoprotein, the retroviral core influences sensitivity to SERINC5.
Subject(s)
Host-Pathogen Interactions , Membrane Proteins/genetics , Viral Envelope Proteins , Virion/metabolism , Viruses/metabolism , HEK293 Cells , HIV-1/metabolism , Humans , Leukemia Virus, Murine/metabolism , Membrane Proteins/immunology , Retroviridae/classification , Retroviridae/metabolism , Viral Envelope Proteins/genetics , Viral Envelope Proteins/immunology , Virion/genetics , Virus Internalization , Viruses/chemistry , Viruses/classification , Viruses/geneticsABSTRACT
Outbreaks of Ebola ebolavirus (EBOV) have been associated with high morbidity and mortality. Milestones have been reached recently in the management of EBOV disease (EVD) with licensure of an EBOV vaccine and two monoclonal antibody therapies. However, neither vaccines nor therapies are available for other disease-causing filoviruses. In preparation for such outbreaks, and for more facile and cost-effective management of EVD, we seek a cocktail containing orally available and room temperature stable drugs with strong activity against multiple filoviruses. We previously showed that (bepridil + sertraline) and (sertraline + toremifene) synergistically suppress EBOV in cell cultures. Here, we describe steps towards testing these combinations in a mouse model of EVD. We identified a vehicle suitable for oral delivery of the component drugs and determined that, thus formulated the drugs are equally active against EBOV as preparations in DMSO, and they maintain activity upon storage in solution for up to seven days. Pharmacokinetic (PK) studies indicated that the drugs in the oral delivery vehicle are well tolerated in mice at the highest doses tested. Collectively the data support advancement of these combinations to tests for synergy in a mouse model of EVD. Moreover, mathematical modeling based on human oral PK projects that the combinations would be more active in humans than their component single drugs.
ABSTRACT
Neglected diseases caused by arenaviruses such as Lassa virus (LASV) and filoviruses like Ebola virus (EBOV) primarily afflict resource-limited countries, where antiviral drug development is often minimal. Previous studies have shown that many approved drugs developed for other clinical indications inhibit EBOV and LASV and that combinations of these drugs provide synergistic suppression of EBOV, often by blocking discrete steps in virus entry. We hypothesize that repurposing of combinations of orally administered approved drugs provides effective suppression of arenaviruses. In this report, we demonstrate that arbidol, an approved influenza antiviral previously shown to inhibit EBOV, LASV, and many other viruses, inhibits murine leukemia virus (MLV) reporter viruses pseudotyped with the fusion glycoproteins (GPs) of other arenaviruses (Junin virus [JUNV], lymphocytic choriomeningitis virus [LCMV], and Pichinde virus [PICV]). Arbidol and other approved drugs, including aripiprazole, amodiaquine, sertraline, and niclosamide, also inhibit infection of cells by infectious PICV, and arbidol, sertraline, and niclosamide inhibit infectious LASV. Combining arbidol with aripiprazole or sertraline results in the synergistic suppression of LASV and JUNV GP-bearing pseudoviruses. This proof-of-concept study shows that arenavirus infection in vitro can be synergistically inhibited by combinations of approved drugs. This approach may lead to a proactive strategy with which to prepare for and control known and new arenavirus outbreaks.
Subject(s)
Antiviral Agents/therapeutic use , Arenaviridae Infections/drug therapy , Arenavirus/drug effects , Administration, Oral , Animals , Arenaviridae Infections/virology , Cell Line , Chlorocebus aethiops , Drug Synergism , Drug Therapy, Combination/methods , HEK293 Cells , Humans , Mice , Proof of Concept Study , Vero CellsABSTRACT
Cholesterol serves critical roles in enveloped virus fusion by modulating membrane properties. The glycoprotein (GP) of Ebola virus (EBOV) promotes fusion in the endosome, a process that requires the endosomal cholesterol transporter NPC1. However, the role of cholesterol in EBOV fusion is unclear. Here we show that cholesterol in GP-containing membranes enhances fusion and the membrane-proximal external region and transmembrane (MPER/TM) domain of GP interacts with cholesterol via several glycine residues in the GP2 TM domain, notably G660. Compared to wild-type (WT) counterparts, a G660L mutation caused a more open angle between MPER and TM domains in an MPER/TM construct, higher probability of stalling at hemifusion for GP2 proteoliposomes and lower cell entry of virus-like particles (VLPs). VLPs with depleted cholesterol show reduced cell entry, and VLPs produced under cholesterol-lowering statin conditions show less frequent entry than respective controls. We propose that cholesterol-TM interactions affect structural features of GP2, thereby facilitating fusion and cell entry.
Subject(s)
Cholesterol/metabolism , Ebolavirus/physiology , Hemorrhagic Fever, Ebola/virology , Membrane Fusion , Viral Envelope Proteins/metabolism , Virus Internalization , HEK293 Cells , Humans , Protein Binding , Protein DomainsABSTRACT
Many acute viral infections target tissue MÏs, yet the mechanisms of MÏ-mediated control of viruses are poorly understood. Here, we report that CD40 expressed by peritoneal MÏs restricts early infection of a broad range of RNA viruses. Loss of CD40 expression enhanced virus replication as early as 12-24 h of infection and, conversely, stimulation of CD40 signaling with an agonistic Ab blocked infection. With peritoneal cell populations infected with the filovirus, wild-type (WT) Ebola virus (EBOV), or a BSL2 model virus, recombinant vesicular stomatitis virus encoding Ebola virus glycoprotein (rVSV/EBOV GP), we examined the mechanism conferring protection. Here, we demonstrate that restricted virus replication in MÏs required CD154/CD40 interactions that stimulated IL-12 production through TRAF6-dependent signaling. In turn, IL-12 production resulted in IFN-γ production, which induced proinflammatory polarization of MÏs, protecting the cells from infection. These CD40-dependent events protected mice against virus challenge. CD40-/- mice were exquisitely sensitive to intraperitoneal challenge with a dose of rVSV/EBOV GP that was sublethal to CD40+/+ mice, exhibiting viremia within 12 h of infection and rapidly succumbing to infection. This study identifies a previously unappreciated role for MÏ-intrinsic CD40 signaling in controlling acute virus infection.
Subject(s)
CD40 Antigens/metabolism , Immunity, Innate , Macrophages/immunology , Macrophages/virology , RNA Viruses/physiology , Signal Transduction , Virus Diseases/immunology , Virus Replication/physiology , Acute Disease , Animals , CD40 Ligand/metabolism , Ebolavirus/physiology , Glycoproteins/immunology , Humans , Interferon-gamma/metabolism , Interleukin-12/biosynthesis , Mice, Inbred C57BL , Models, Biological , Peritoneum/pathology , Peritoneum/virology , TNF Receptor-Associated Factor 6/metabolism , Virus Diseases/virologyABSTRACT
To enter a cell and establish infection, HIV must first fuse its lipid envelope with the host cell plasma membrane. Whereas the process of HIV membrane fusion can be tracked by fluorescence microscopy, the 3D configuration of proteins and lipids at intermediate steps can only be resolved with cryo-electron tomography (cryoET). However, cryoET of whole cells is technically difficult. To overcome this problem, we have adapted giant plasma membrane vesicles (or blebs) from native cell membranes expressing appropriate receptors as targets for fusion with HIV envelope glycoprotein-expressing pseudovirus particles with and without Serinc host restriction factors. The fusion behavior of these particles was probed by TIRF microscopy on bleb-derived supported membranes. Timed snapshots of fusion of the same particles with blebs were examined by cryo-ET. The combination of these methods allowed us to characterize the structures of various intermediates on the fusion pathway and showed that when Serinc3 or Serinc5 (but not Serinc2) were present, later fusion products were more prevalent, suggesting that Serinc3/5 act at multiple steps to prevent progression to full fusion. In addition, the antifungal amphotericin B reversed Serinc restriction, presumably by intercalation into the fusing membranes. Our results provide a highly detailed view of Serinc restriction of HIV-cell membrane fusion and thus extend current structural and functional information on Serinc as a lipid-binding protein.
Subject(s)
Cell Membrane/metabolism , Cryoelectron Microscopy , HIV-1/metabolism , Membrane Glycoproteins/metabolism , Membrane Proteins/metabolism , Cell Line , HEK293 Cells , HeLa Cells , Host-Pathogen Interactions , Humans , Membrane Fusion , Microscopy, Fluorescence , env Gene Products, Human Immunodeficiency Virus/metabolismABSTRACT
Influenza virus is an enveloped virus wrapped in a lipid bilayer derived from the host cell plasma membrane. Infection by influenza virus is dependent on these host cell lipids, which include sphingolipids. Here we examined the role of the sphingolipid, glucosylceramide, in influenza virus infection by knocking out the enzyme responsible for its synthesis, glucosylceramide synthase (UGCG). We observed diminished influenza virus infection in HEK 293 and A549 UGCG knockout cells and demonstrated that this is attributed to impaired viral entry. We also observed that entry mediated by the glycoproteins of other enveloped viruses that enter cells by endocytosis is also impaired in UGCG knockout cells, suggesting a broader role for UGCG in viral entry by endocytosis.
Subject(s)
Glucosyltransferases/genetics , Influenza A virus/physiology , A549 Cells , CRISPR-Cas Systems/genetics , Gene Editing , Glucosyltransferases/deficiency , HEK293 Cells , Humans , Macrolides/pharmacology , Virus Internalization/drug effectsABSTRACT
Ebolaviruses cause an often rapidly fatal syndrome known as Ebola virus disease (EVD), with average case fatality rates of ~50%. There is no licensed vaccine or treatment for EVD, underscoring the urgent need to develop new anti-ebolavirus agents, especially in the face of an ongoing outbreak in the Democratic Republic of the Congo and the largest ever outbreak in Western Africa in 2013-2016. Lectins have been investigated as potential antiviral agents as they bind glycans present on viral surface glycoproteins, but clinical use of them has been slowed by concerns regarding their mitogenicity, i.e. ability to cause immune cell proliferation. We previously engineered a banana lectin (BanLec), a carbohydrate-binding protein, such that it retained antiviral activity but lost mitogenicity by mutating a single amino acid, yielding H84T BanLec (H84T). H84T shows activity against viruses containing high-mannose N-glycans, including influenza A and B, HIV-1 and -2, and hepatitis C virus. Since ebolavirus surface glycoproteins also contain many high-mannose N-glycans, we assessed whether H84T could inhibit ebolavirus replication. H84T inhibited Ebola virus (EBOV) replication in cell cultures. In cells, H84T inhibited both virus-like particle (VLP) entry and transcription/replication of the EBOV mini-genome at high micromolar concentrations, while inhibiting infection by transcription- and replication-competent VLPs, which measures the full viral life cycle, in the low micromolar range. H84T did not inhibit assembly, budding, or release of VLPs. These findings suggest that H84T may exert its anti-ebolavirus effect(s) by blocking both entry and transcription/replication. In a mouse model, H84T partially (maximally, ~50-80%) protected mice from an otherwise lethal mouse-adapted EBOV infection. Interestingly, a single dose of H84T pre-exposure to EBOV protected ~80% of mice. Thus, H84T shows promise as a new anti-ebolavirus agent with potential to be used in combination with vaccination or other agents in a prophylactic or therapeutic regimen.