RESUMEN
While an essential role of HIV-1 integrase (IN) for integration of viral cDNA into human chromosome is established, studies with IN mutants and allosteric IN inhibitors (ALLINIs) have suggested that IN can also influence viral particle maturation. However, it has remained enigmatic as to how IN contributes to virion morphogenesis. Here, we demonstrate that IN directly binds the viral RNA genome in virions. These interactions have specificity, as IN exhibits distinct preference for select viral RNA structural elements. We show that IN substitutions that selectively impair its binding to viral RNA result in eccentric, non-infectious virions without affecting nucleocapsid-RNA interactions. Likewise, ALLINIs impair IN binding to viral RNA in virions of wild-type, but not escape mutant, virus. These results reveal an unexpected biological role of IN binding to the viral RNA genome during virion morphogenesis and elucidate the mode of action of ALLINIs.
Asunto(s)
Genoma Viral , Integrasa de VIH/metabolismo , VIH-1/crecimiento & desarrollo , ARN Viral/metabolismo , Virión/crecimiento & desarrollo , Células HEK293 , Integrasa de VIH/genética , Inhibidores de Integrasa VIH/farmacología , VIH-1/efectos de los fármacos , VIH-1/enzimología , Humanos , Morfogénesis , Nucleocápside/efectos de los fármacos , Unión Proteica , Virión/efectos de los fármacos , Virión/enzimología , Integración Viral/efectos de los fármacosRESUMEN
Although vaccines confer protection against influenza A viruses, antiviral treatment becomes the first line of defense during pandemics because there is insufficient time to produce vaccines. Current antiviral drugs are susceptible to drug resistance, and developing new antivirals is essential. We studied host defense peptides from the skin of the South Indian frog and demonstrated that one of these, which we named "urumin," is virucidal for H1 hemagglutinin-bearing human influenza A viruses. This peptide specifically targeted the conserved stalk region of H1 hemagglutinin and was effective against drug-resistant H1 influenza viruses. Using electron microscopy, we showed that this peptide physically destroyed influenza virions. It also protected naive mice from lethal influenza infection. Urumin represents a unique class of anti-influenza virucide that specifically targets the hemagglutinin stalk region, similar to targeting of antibodies induced by universal influenza vaccines. Urumin therefore has the potential to contribute to first-line anti-viral treatments during influenza outbreaks.
Asunto(s)
Proteínas Anfibias/farmacología , Virus de la Influenza A/efectos de los fármacos , Gripe Humana/prevención & control , Infecciones por Orthomyxoviridae/prevención & control , Péptidos/farmacología , Secuencia de Aminoácidos , Proteínas Anfibias/inmunología , Animales , Antivirales/inmunología , Antivirales/farmacología , Perros , Relación Dosis-Respuesta a Droga , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Interacciones Huésped-Patógeno/efectos de los fármacos , Interacciones Huésped-Patógeno/inmunología , Humanos , Virus de la Influenza A/inmunología , Virus de la Influenza A/metabolismo , Gripe Humana/inmunología , Gripe Humana/virología , Células de Riñón Canino Madin Darby , Ratones Endogámicos BALB C , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/virología , Péptidos/inmunología , Ranidae/metabolismo , Análisis de Supervivencia , Resultado del Tratamiento , Virión/efectos de los fármacos , Virión/inmunología , Virión/metabolismoRESUMEN
In persons living with HIV-1 (PLWH) who start antiretroviral therapy (ART), plasma virus decays in a biphasic fashion to below the detection limit. The first phase reflects the short half-life (<1 d) of cells that produce most of the plasma virus. The second phase represents the slower turnover (t1/2 = 14 d) of another infected cell population, whose identity is unclear. Using the intact proviral DNA assay (IPDA) to distinguish intact and defective proviruses, we analyzed viral decay in 17 PLWH initiating ART. Circulating CD4+ T cells with intact proviruses include few of the rapidly decaying first-phase cells. Instead, this population initially decays more slowly (t1/2 = 12.9 d) in a process that largely represents death or exit from the circulation rather than transition to latency. This more protracted decay potentially allows for immune selection. After â¼3 mo, the decay slope changes, and CD4+ T cells with intact proviruses decay with a half-life of 19 mo, which is still shorter than that of the latently infected cells that persist on long-term ART. Two-long-terminal repeat (2LTR) circles decay with fast and slow phases paralleling intact proviruses, a finding that precludes their use as a simple marker of ongoing viral replication. Proviruses with defects at the 5' or 3' end of the genome show equivalent monophasic decay at rates that vary among individuals. Understanding these complex early decay processes is important for correct use of reservoir assays and may provide insights into properties of surviving cells that can constitute the stable latent reservoir.
Asunto(s)
Antirretrovirales/farmacología , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Provirus/efectos de los fármacos , Virión/efectos de los fármacos , Linfocitos T CD4-Positivos/efectos de los fármacos , Células Cultivadas , ADN Viral/efectos de los fármacos , Humanos , Estudios Longitudinales , Carga Viral/efectos de los fármacos , Latencia del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
Enterovirus 70 (EV70) is a human pathogen belonging to the family Picornaviridae. EV70 is transmitted by eye secretions and causes acute hemorrhagic conjunctivitis, a serious eye disease. Despite the severity of the disease caused by EV70, its structure is unknown. Here, we present the structures of the EV70 virion, altered particle, and empty capsid determined by cryo-electron microscopy. The capsid of EV70 is composed of the subunits VP1, VP2, VP3, and VP4. The partially collapsed hydrophobic pocket located in VP1 of the EV70 virion is not occupied by a pocket factor, which is commonly present in other enteroviruses. Nevertheless, we show that the pocket can be targeted by the antiviral compounds WIN51711 and pleconaril, which block virus infection. The inhibitors prevent genome release by stabilizing EV70 particles. Knowledge of the structures of complexes of EV70 with inhibitors will enable the development of capsid-binding therapeutics against this virus. IMPORTANCE Globally distributed enterovirus 70 (EV70) causes local outbreaks of acute hemorrhagic conjunctivitis. The discharge from infected eyes enables the high-efficiency transmission of EV70 in overcrowded areas with low hygienic standards. Currently, only symptomatic treatments are available. We determined the structures of EV70 in its native form, the genome release intermediate, and the empty capsid resulting from genome release. Furthermore, we elucidated the structures of EV70 in complex with two inhibitors that block virus infection, and we describe the mechanism of their binding to the virus capsid. These results enable the development of therapeutics against EV70.
Asunto(s)
Antivirales , Cápside , Enterovirus Humano D , Antivirales/farmacología , Cápside/ultraestructura , Proteínas de la Cápside , Conjuntivitis Hemorrágica Aguda/virología , Microscopía por Crioelectrón , Enterovirus Humano D/efectos de los fármacos , Enterovirus Humano D/ultraestructura , Humanos , Oxadiazoles/farmacología , Oxazoles/farmacología , Virión/efectos de los fármacos , Virión/ultraestructuraRESUMEN
Coxsackievirus A9 (CVA9), an enterovirus, is a common cause of pediatric aseptic meningitis and neonatal sepsis. During cell entry, enterovirus capsids undergo conformational changes leading to expansion, formation of large pores, externalization of VP1 N termini, and loss of the lipid factor from VP1. Factors such as receptor binding, heat, and acidic pH can trigger capsid expansion in some enteroviruses. Here, we show that fatty acid-free bovine serum albumin or neutral endosomal ionic conditions can independently prime CVA9 for expansion and genome release. Our results showed that CVA9 treatment with albumin or endosomal ions generated a heterogeneous population of virions, which could be physically separated by asymmetric flow field flow fractionation and computationally by cryo-electron microscopy (cryo-EM) and image processing. We report cryo-EM structures of CVA9 A-particles obtained by albumin or endosomal ion treatment and a control nonexpanded virion to 3.5, 3.3, and 2.9 Å resolution, respectively. Whereas albumin promoted stable expanded virions, the endosomal ionic concentrations induced unstable CVA9 virions which easily disintegrated, losing their genome. Loss of most of the VP4 molecules and exposure of negatively charged amino acid residues in the capsid's interior after expansion created a repulsive viral RNA-capsid interface, aiding genome release. IMPORTANCE Coxsackievirus A9 (CVA9) is a common cause of meningitis and neonatal sepsis. The triggers and mode of action of RNA release into the cell unusually do not require receptor interaction. Rather, a slow process in the endosome, independent of low pH, is required. Here, we show by biophysical separation, cryogenic electron microscopy, and image reconstruction that albumin and buffers mimicking the endosomal ion composition can separately and together expand and prime CVA9 for uncoating. Furthermore, we show in these expanded particles that VP4 is present at only ~10% of the occupancy found in the virion, VP1 is externalized, and the genome is repelled by the negatively charged, repulsive inner surface of the capsid that occurs due to the expansion. Thus, we can now link observations from cell biology of infection with the physical processes that occur in the capsid to promote genome uncoating.
Asunto(s)
Cationes , Enterovirus Humano B , Humanos , Albúminas/farmacología , Proteínas de la Cápside/metabolismo , Cationes/farmacología , Microscopía por Crioelectrón , Endosomas/metabolismo , Enterovirus Humano B/efectos de los fármacos , Enterovirus Humano B/genética , Enterovirus Humano B/ultraestructura , Infecciones por Enterovirus/patología , Infecciones por Enterovirus/virología , ARN/metabolismo , Virión/efectos de los fármacos , Virión/metabolismo , Virión/ultraestructura , Genoma ViralRESUMEN
The respiratory disease COVID-19 is caused by the coronavirus SARS-CoV-2. Here we report the discovery of ethacridine as a potent drug against SARS-CoV-2 (EC50 ~ 0.08 µM). Ethacridine was identified via high-throughput screening of an FDA-approved drug library in living cells using a fluorescence assay. Plaque assays, RT-PCR and immunofluorescence imaging at various stages of viral infection demonstrate that the main mode of action of ethacridine is through inactivation of viral particles, preventing their binding to the host cells. Consistently, ethacridine is effective in various cell types, including primary human nasal epithelial cells that are cultured in an air-liquid interface. Taken together, our work identifies a promising, potent, and new use of the old drug via a distinct mode of action for inhibiting SARS-CoV-2.
Asunto(s)
Antivirales/farmacología , Etacridina/farmacología , Inhibidores de Proteasas/farmacología , Activación Viral/efectos de los fármacos , Animales , Línea Celular , Chlorocebus aethiops , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Humanos , Células Vero , Virión/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
The glycosphingolipid (GSL) globoside (Gb4) is essential for parvovirus B19 (B19V) infection. Historically considered the cellular receptor of B19V, the role of Gb4 and its interaction with B19V are controversial. In this study, we applied artificial viral particles, genetically modified cells, and specific competitors to address the interplay between the virus and the GSL. Our findings demonstrate that Gb4 is not involved in the binding or internalization process of the virus into permissive erythroid cells, a function that corresponds to the VP1u cognate receptor. However, Gb4 is essential at a post-internalization step before the delivery of the single-stranded viral DNA into the nucleus. In susceptible erythroid Gb4 knockout cells, incoming viruses were arrested in the endosomal compartment, showing no cytoplasmic spreading of capsids as observed in Gb4-expressing cells. Hemagglutination and binding assays revealed that pH acts as a switch to modulate the affinity between the virus and the GSL. Capsids interact with Gb4 exclusively under acidic conditions and dissociate at neutral pH. Inducing a specific Gb4-mediated attachment to permissive erythroid cells by acidification of the extracellular environment led to a non-infectious uptake of the virus, indicating that low pH-mediated binding to the GSL initiates active membrane processes resulting in vesicle formation. In summary, this study provides mechanistic insight into the interaction of B19V with Gb4. The strict pH-dependent binding to the ubiquitously expressed GSL prevents the redirection of the virus to nonpermissive tissues while promoting the interaction in acidic intracellular compartments as an essential step in infectious endocytic trafficking.
Asunto(s)
Cápside/metabolismo , Endocitosis/inmunología , Glicoesfingolípidos/metabolismo , Parvovirus B19 Humano/genética , Proteínas de la Cápside/efectos de los fármacos , Proteínas de la Cápside/metabolismo , Endocitosis/fisiología , Globósidos/metabolismo , Humanos , Parvovirus B19 Humano/patogenicidad , Receptores Virales/efectos de los fármacos , Receptores Virales/metabolismo , Virión/efectos de los fármacos , Virión/metabolismo , Internalización del Virus/efectos de los fármacosRESUMEN
Biocidal agents such as formaldehyde and glutaraldehyde are able to inactivate several coronaviruses including SARS-CoV-2. In this article, an insight into one mechanism for the inactivation of these viruses by those two agents is presented, based on analysis of previous observations during electron microscopic examination of several members of the orthocoronavirinae subfamily, including the new virus SARS-CoV-2. This inactivation is proposed to occur through Schiff base reaction-induced conformational changes in the spike glycoprotein leading to its disruption or breakage, which can prevent binding of the virus to cellular receptors. Also, a new prophylactic and therapeutic measure against SARS-CoV-2 using acetoacetate is proposed, suggesting that it could similarly break the viral spike through Schiff base reaction with lysines of the spike protein. This measure needs to be confirmed experimentally before consideration. In addition, a new line of research is proposed to help find a broad-spectrum antivirus against several members of this subfamily.
Asunto(s)
Desinfectantes/farmacología , Cuerpos Cetónicos/farmacología , SARS-CoV-2/efectos de los fármacos , Glicoproteína de la Espiga del Coronavirus/metabolismo , Animales , Antivirales/química , Antivirales/farmacología , Desinfectantes/química , Formaldehído/química , Formaldehído/farmacología , Glutaral/química , Glutaral/farmacología , Humanos , Cuerpos Cetónicos/química , Cuerpos Cetónicos/metabolismo , Cetosis/etiología , Cetosis/virología , SARS-CoV-2/patogenicidad , Virión/efectos de los fármacos , Virión/patogenicidadRESUMEN
HIV-1 maturation involves conversion of the immature Gag polyprotein lattice, which lines the inner surface of the viral membrane, to the mature capsid protein (CA) lattice, which encloses the viral RNA. Maturation inhibitors such as bevirimat (BVM) bind within six-helix bundles, formed by a segment that spans the junction between the CA and spacer peptide 1 (SP1) subunits of Gag, and interfere with cleavage between CA and SP1 catalyzed by the HIV-1 protease (PR). We report solid-state NMR (ssNMR) measurements on spherical virus-like particles (VLPs), facilitated by segmental isotopic labeling, that provide information about effects of BVM on the structure and dynamics of CA-SP1 junction helices in the immature lattice. Although BVM strongly blocks PR-catalyzed CA-SP1 cleavage in VLPs and blocks conversion of VLPs to tubular CA assemblies, 15N and 13C ssNMR chemical shifts of segmentally labeled VLPs with and without BVM are very similar, indicating that interaction with BVM does not alter the six-helix bundle structure appreciably. Only the 15N chemical shift of A280 (the first residue of SP1) changes significantly, consistent with BVM binding to an internal ring of hydrophobic side chains of L279 residues. Measurements of transverse 15N spin relaxation rates reveal a reduction in the amplitudes and/or timescales of backbone N-H bond motions, corresponding to a rigidification of the six-helix bundles. Overall, our data show that inhibition of HIV-1 maturation by BVM involves changes in structure and dynamics that are surprisingly subtle, but still sufficient to produce a large effect on CA-SP1 cleavage.
Asunto(s)
Proteínas de la Cápside/química , VIH-1/efectos de los fármacos , Fragmentos de Péptidos/química , Succinatos/farmacología , Triterpenos/farmacología , Virión/efectos de los fármacos , Replicación Viral , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Fármacos Anti-VIH/farmacología , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Farmacorresistencia Viral , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , VIH-1/genética , VIH-1/metabolismo , Humanos , Modelos Moleculares , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Virión/genética , Virión/metabolismo , Ensamble de Virus , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Epstein-Barr virus (EBV) is associated with epithelial and lymphoid malignancies, establishes latent infection in memory B cells, and intermittently produces infectious virions through lytic replication. Released virions play a key role in latent reservoir maintenance and transmission. Lytic EBV transcription differs from cellular transcription in requiring a virus-encoded preinitiation complex that binds to TATT motifs unique to EBV late lytic promoters. Expression of 15 late lytic genes that are important for virion production and infectivity is particularly dependent on the EBV SM protein, a nuclear protein expressed early during lytic reactivation that binds to viral RNAs and enhances RNA stability. We recently discovered that spironolactone blocks EBV virion production by inhibiting EBV SM function. Since spironolactone causes degradation of xeroderma pigmentosum group B-complementing protein (XPB), a component of human transcription factor TFIIH, in both B lymphocytes and epithelial cells, we hypothesized that SM utilizes XPB to specifically activate transcription of SM target promoters. While EBV SM has been thought to act posttranscriptionally, we provide evidence that SM also facilitates EBV gene transcription. We demonstrate that SM binds and recruits XPB to EBV promoters during lytic replication. Depletion of XPB protein, by spironolactone treatment or by siRNA transfection, inhibits SM-dependent late lytic gene transcription but not transcription of other EBV genes or cellular genes. These data indicate that SM acts as a transcriptional activator that has co-opted XPB to specifically target 15 EBV promoters that have uniquely evolved to require XPB for activity, providing an additional mechanism to differentially regulate EBV gene expression.
Asunto(s)
ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Herpesvirus Humano 4/genética , Interacciones Huésped-Patógeno/genética , Fosfoproteínas/metabolismo , Transactivadores/metabolismo , Línea Celular Tumoral , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Infecciones por Virus de Epstein-Barr/tratamiento farmacológico , Infecciones por Virus de Epstein-Barr/virología , Regulación Viral de la Expresión Génica/efectos de los fármacos , Humanos , Regiones Promotoras Genéticas/genética , Proteolisis/efectos de los fármacos , ARN Interferente Pequeño/metabolismo , Espironolactona/farmacología , Espironolactona/uso terapéutico , Activación Transcripcional/efectos de los fármacos , Virión/efectos de los fármacos , Virión/metabolismoRESUMEN
Transmission electron microscopy has historically been indispensable for virology research, as it offers unique insight into virus function. In the past decade, as cryo-electron microscopy (cryo-EM) has matured and become more accessible, we have been able to peer into the structure of viruses at the atomic level and understand how they interact with the host cell, with drugs or with antibodies. Perhaps, there was no time in recent history where cryo-EM was more needed, as SARS-CoV-2 has spread around the globe, causing millions of deaths and almost unquantifiable economic devastation. In this concise review, we aim to mark the most important contributions of cryo-EM to understanding the structure and function of SARS-CoV-2 proteins, from surface spikes to the virus core and from virus-receptor interactions to antibody binding.
Asunto(s)
Enzima Convertidora de Angiotensina 2/química , Anticuerpos Antivirales/química , Vacunas contra la COVID-19/química , COVID-19/prevención & control , Receptores Virales/química , Glicoproteína de la Espiga del Coronavirus/química , Enzima Convertidora de Angiotensina 2/inmunología , Enzima Convertidora de Angiotensina 2/metabolismo , Anticuerpos Antivirales/biosíntesis , COVID-19/inmunología , COVID-19/virología , Vacunas contra la COVID-19/administración & dosificación , Vacunas contra la COVID-19/biosíntesis , Microscopía por Crioelectrón , Epítopos/química , Epítopos/inmunología , Epítopos/metabolismo , Humanos , Modelos Moleculares , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Receptores Virales/inmunología , Receptores Virales/metabolismo , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/patogenicidad , SARS-CoV-2/ultraestructura , Serina Endopeptidasas/química , Serina Endopeptidasas/inmunología , Serina Endopeptidasas/metabolismo , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Virión/efectos de los fármacos , Virión/patogenicidad , Virión/ultraestructuraRESUMEN
Several next-generation (universal) influenza vaccines and broadly neutralizing antibodies (bNAbs) are in clinical development. Some of these mediate inhibitions of virus replication at the postentry stage or use Fc-dependent mechanisms. Nonneutralizing antibodies have the potential to mediate enhancement of viral infection or disease. In the current study, two monoclonal antibodies (MAbs) 72/8 and 69/1, enhanced respiratory disease (ERD) in mice following H3N2 virus challenge by demonstrating increased lung pathology and changes in lung cytokine/chemokine levels. MAb 78/2 caused changes in the lung viral loads in a dose-dependent manner. Both MAbs increased HA sensitivity to trypsin cleavage at a higher pH range, suggesting MAb-induced conformational changes. pHrodo-labeled virus particles' entry and residence time in the endocytic compartment were tracked during infection of Madin-Darby canine kidney (MDCK) cells. Both MAbs reduced H3N2 virus residence time in the endocytic pathway, suggesting faster virus fusion kinetics. Structurally, 78/2 and 69/1 Fabs bound the globular head or base of the head domain of influenza hemagglutinin (HA), respectively, and induced destabilization of the HA stem domain. Together, this study describes Mab-induced destabilization of the influenza HA stem domain, faster kinetics of influenza virus fusion, and ERD in vivo. The in vivo animal model and in vitro assays described could augment preclinical safety evaluation of antibodies and next-generation influenza vaccines that generate antibodies which do not block influenza virus-receptor interaction.
Asunto(s)
Anticuerpos Monoclonales/efectos adversos , Anticuerpos Antivirales/efectos adversos , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Pulmón/virología , Infecciones por Orthomyxoviridae/virología , Internalización del Virus/efectos de los fármacos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Anticuerpos Antivirales/química , Anticuerpos Antivirales/metabolismo , Sitios de Unión , Perros , Endocitosis/efectos de los fármacos , Endocitosis/inmunología , Femenino , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Subtipo H3N2 del Virus de la Influenza A/efectos de los fármacos , Subtipo H3N2 del Virus de la Influenza A/inmunología , Subtipo H3N2 del Virus de la Influenza A/patogenicidad , Pulmón/inmunología , Pulmón/patología , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos BALB C , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/patología , Unión Proteica , Proteolisis , Carga Viral/efectos de los fármacos , Virión/efectos de los fármacos , Virión/inmunología , Virión/patogenicidad , Replicación Viral/efectos de los fármacosRESUMEN
Ebola virus (EBOV) entry into cells is mediated by its spike glycoprotein (GP). Following attachment and internalization, virions traffic to late endosomes where GP is cleaved by host cysteine proteases. Cleaved GP then binds its cellular receptor, Niemann-Pick C1. In response to an unknown cellular trigger, GP undergoes conformational rearrangements that drive fusion of viral and endosomal membranes. The temperature-dependent stability (thermostability) of the prefusion conformers of class I viral fusion glycoproteins, including those of filovirus GPs, has provided insights into their propensity to undergo fusion-related rearrangements. However, previously described assays have relied on soluble glycoprotein ectodomains. Here, we developed a simple enzyme-linked immunosorbent assay (ELISA)-based assay that uses the temperature-dependent loss of conformational epitopes to measure thermostability of GP embedded in viral membranes. The base and glycan cap subdomains of all filovirus GPs tested suffered a concerted loss of prefusion conformation at elevated temperatures but did so at different temperature ranges, indicating virus-specific differences in thermostability. Despite these differences, all of these GPs displayed reduced thermostability upon cleavage to GP conformers (GPCL). Surprisingly, acid pH enhanced, rather than decreased, GP thermostability, suggesting it could enhance viral survival in hostile endo/lysosomal compartments. Finally, we confirmed and extended previous findings that some small-molecule inhibitors of filovirus entry destabilize EBOV GP and uncovered evidence that the most potent inhibitors act through multiple mechanisms. We establish the epitope-loss ELISA as a useful tool for studies of filovirus entry, engineering of GP variants with enhanced stability for use in vaccine development, and discovery of new stability-modulating antivirals.IMPORTANCE The development of Ebola virus countermeasures is challenged by our limited understanding of cell entry, especially at the step of membrane fusion. The surface-exposed viral protein, GP, mediates membrane fusion and undergoes major structural rearrangements during this process. The stability of GP at elevated temperatures (thermostability) can provide insights into its capacity to undergo these rearrangements. Here, we describe a new assay that uses GP-specific antibodies to measure GP thermostability under a variety of conditions relevant to viral entry. We show that proteolytic cleavage and acid pH have significant effects on GP thermostability that shed light on their respective roles in viral entry. We also show that the assay can be used to study how small-molecule entry inhibitors affect GP stability. This work provides a simple and readily accessible assay to engineer stabilized GP variants for antiviral vaccines and to discover and improve drugs that act by modulating GP stability.
Asunto(s)
Ebolavirus/efectos de los fármacos , Proteína Niemann-Pick C1/antagonistas & inhibidores , Receptores Virales/antagonistas & inhibidores , Proteínas del Envoltorio Viral/antagonistas & inhibidores , Proteínas Virales de Fusión/antagonistas & inhibidores , Virión/efectos de los fármacos , Animales , Sitios de Unión , Bioensayo , Chlorocebus aethiops , Clomifeno/química , Clomifeno/farmacología , Ebolavirus/química , Ebolavirus/genética , Ebolavirus/metabolismo , Epítopos/química , Epítopos/genética , Epítopos/metabolismo , Calor , Concentración de Iones de Hidrógeno , Simulación del Acoplamiento Molecular , Proteína Niemann-Pick C1/química , Proteína Niemann-Pick C1/genética , Proteína Niemann-Pick C1/metabolismo , Unión Proteica/efectos de los fármacos , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Estructura Terciaria de Proteína , Receptores Virales/química , Receptores Virales/genética , Receptores Virales/metabolismo , Tamoxifeno/análogos & derivados , Tamoxifeno/química , Tamoxifeno/farmacología , Toremifeno/química , Toremifeno/farmacología , Células Vero , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Proteínas Virales de Fusión/química , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/metabolismo , Virión/química , Virión/genética , Virión/metabolismoRESUMEN
HIV-1 Nef and the unrelated mouse leukaemia virus glycosylated Gag (glycoGag) strongly enhance the infectivity of HIV-1 virions produced in certain cell types in a clathrin-dependent manner. Here we show that Nef and glycoGag prevent the incorporation of the multipass transmembrane proteins serine incorporator 3 (SERINC3) and SERINC5 into HIV-1 virions to an extent that correlates with infectivity enhancement. Silencing of both SERINC3 and SERINC5 precisely phenocopied the effects of Nef and glycoGag on HIV-1 infectivity. The infectivity of nef-deficient virions increased more than 100-fold when produced in double-knockout human CD4(+) T cells that lack both SERINC3 and SERINC5, and re-expression experiments confirmed that the absence of SERINC3 and SERINC5 accounted for the infectivity enhancement. Furthermore, SERINC3 and SERINC5 together restricted HIV-1 replication, and this restriction was evaded by Nef. SERINC3 and SERINC5 are highly expressed in primary human HIV-1 target cells, and inhibiting their downregulation by Nef is a potential strategy to combat HIV/AIDS.
Asunto(s)
VIH-1/química , VIH-1/fisiología , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Línea Celular , Regulación hacia Abajo , Eliminación de Gen , Productos del Gen gag/metabolismo , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , VIH-1/crecimiento & desarrollo , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Virus de la Leucemia Murina/química , Glicoproteínas de Membrana , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/farmacología , Proteínas de Neoplasias/deficiencia , Proteínas de Neoplasias/farmacología , Transporte de Proteínas , Receptores de Superficie Celular/deficiencia , Virión/química , Virión/efectos de los fármacos , Virión/crecimiento & desarrollo , Virión/fisiología , Replicación Viral/efectos de los fármacos , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/deficienciaRESUMEN
Enterovirus D68 (EV-D68) belongs to a group of enteroviruses that contain a single positive-sense RNA genome surrounded by an icosahedral capsid. Like common cold viruses, EV-D68 mainly causes respiratory infections and is acid-labile. The molecular mechanism by which the acid-sensitive EV-D68 virions uncoat and deliver their genome into a host cell is unknown. Using cryoelectron microscopy (cryo-EM), we have determined the structures of the full native virion and an uncoating intermediate [the A (altered) particle] of EV-D68 at 2.2- and 2.7-Å resolution, respectively. These structures showed that acid treatment of EV-D68 leads to particle expansion, externalization of the viral protein VP1 N termini from the capsid interior, and formation of pores around the icosahedral twofold axes through which the viral RNA can exit. Moreover, because of the low stability of EV-D68, cryo-EM analyses of a mixed population of particles at neutral pH and following acid treatment demonstrated the involvement of multiple structural intermediates during virus uncoating. Among these, a previously undescribed state, the expanded 1 ("E1") particle, shows a majority of internal regions (e.g., the VP1 N termini) to be ordered as in the full native virion. Thus, the E1 particle acts as an intermediate in the transition from full native virions to A particles. Together, the present work delineates the pathway of EV-D68 uncoating and provides the molecular basis for the acid lability of EV-D68 and of the related common cold viruses.
Asunto(s)
Ácidos/farmacología , Enterovirus Humano D/fisiología , Enterovirus Humano D/ultraestructura , Desencapsidación Viral/efectos de los fármacos , Cápside/efectos de los fármacos , Cápside/metabolismo , Cápside/ultraestructura , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Microscopía por Crioelectrón , Enterovirus Humano D/efectos de los fármacos , Enterovirus Humano D/genética , Infecciones por Enterovirus/virología , Humanos , Concentración de Iones de Hidrógeno , Virión/efectos de los fármacos , Virión/genética , Virión/fisiología , Virión/ultraestructuraRESUMEN
Viral infections cause a host of fatal diseases and seriously affect every form of life from bacteria to humans. Although most viral infections can receive appropriate treatment thereby limiting damage to life and livelihood with modern medicine and early diagnosis, new types of viral infections are continuously emerging that need to be properly and timely treated. As time is the most important factor in the progress of many deadly viral diseases, early detection becomes of paramount importance for effective treatment. Aptamers are small oligonucleotide molecules made by the systematic evolution of ligands by exponential enrichment (SELEX). Aptamers are characterized by being able to specifically bind to a target, much like antibodies. However, unlike antibodies, aptamers are easily synthesized, modified, and are able to target a wider range of substances, including proteins and carbohydrates. With these advantages in mind, many studies on aptamer-based viral diagnosis and treatments are currently in progress. The use of aptamers for viral diagnosis requires a system that recognizes the binding of viral molecules to aptamers in samples of blood, serum, plasma, or in virus-infected cells. From a therapeutic perspective, aptamers target viral particles or host cell receptors to prevent the interaction between the virus and host cells or target intracellular viral proteins to interrupt the life cycle of the virus within infected cells. In this paper, we review recent attempts to use aptamers for the diagnosis and treatment of various viral infections.
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Antivirales/uso terapéutico , Aptámeros de Nucleótidos/uso terapéutico , Virosis/diagnóstico , Virosis/tratamiento farmacológico , Animales , Virus ADN/efectos de los fármacos , Humanos , Virus ARN/efectos de los fármacos , Proteínas Virales/efectos de los fármacos , Virión/efectos de los fármacosRESUMEN
Acquired immune deficiency syndrome (AIDS) has prevailed over the last 30 years. Although highly active antiretroviral therapy (HAART) has decreased mortality and efficiently controlled the progression of disease, no vaccine or curative drugs have been approved until now. A viral inactivator is expected to inactivate cell-free virions in the absence of target cells. Previously, we identified a gp120-binding protein, mD1.22, which can inactivate laboratory-adapted HIV-1. In this study, we have found that the gp41 N-terminal heptad repeat (NHR)-binding antibody D5 single-chain variable fragment (scFv) alone cannot inactivate HIV-1 at the high concentration tested. However, D5 scFv in the combination could enhance inactivation activity of mD1.22 against divergent HIV-1 strains, including HIV-1 laboratory-adapted strains, primary HIV-1 isolates, T20- and AZT-resistant strains, and LRA-reactivated virions. Combining mD1.22 and D5 scFv exhibited synergistic effect on inhibition of infection by divergent HIV-1 strains. These results suggest good potential to develop the strategy of combining a gp120-binding protein and a gp41-binding antibody for the treatment of HIV-1 infection.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/virología , Proteínas Portadoras/farmacología , Proteína gp120 de Envoltorio del VIH/antagonistas & inhibidores , Proteína gp41 de Envoltorio del VIH/antagonistas & inhibidores , Inhibidores de Fusión de VIH/farmacología , Proteínas Recombinantes/farmacología , Virión/efectos de los fármacos , Anticuerpos Antivirales/inmunología , Sitios de Unión , Línea Celular , VIH-1/inmunología , Humanos , Anticuerpos de Cadena Única/inmunologíaRESUMEN
Ebola virus (EBOV), pathogen of Ebola hemorrhagic fever (EHF), is an enveloped filamental RNA virus. Recently, the EHF crisis occurred in the Democratic Republic of the Congo again highlights the urgency for its clinical treatments. However, no Food and Drug Administration (FDA)-approved therapeutics are currently available. Drug repurposing screening is a time- and cost-effective approach for identifying anti-EBOV therapeutics. Here, by combinatorial screening using pseudovirion and minigenome replicon systems we have identified several FDA-approved drugs with significant anti-EBOV activities. These potential candidates include azithromycin, clomiphene, chloroquine, digitoxin, epigallocatechin-gallate, fluvastatin, tetrandrine and tamoxifen. Mechanistic studies revealed that fluvastatin inhibited EBOV pseudovirion entry by blocking the pathway of mevalonate biosynthesis, while the inhibitory effect of azithromycin on EBOV maybe due to its intrinsic cationic amphiphilic structure altering the homeostasis of later endosomal vesicle similar as tamoxifen. Moreover, based on structure and pathway analyses, the anti-EBOV activity has been extended to other family members of statins, such as simvastatin, and multiple other cardiac glycoside drugs, some of which exhibited even stronger activities. More importantly, in searching for drug interaction, we found various synergy between several anti-EBOV drug combinations, showing substantial and powerful synergistic against EBOV infection. In conclusion, our work illustrates a successful and productive approach to identify new mechanisms and targets for treating EBOV infection by combinatorial screening of FDA-approved drugs.
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Antivirales/análisis , Antivirales/farmacología , Técnicas Químicas Combinatorias , Aprobación de Drogas , Evaluación Preclínica de Medicamentos , Ebolavirus/efectos de los fármacos , Azitromicina/farmacología , Glicósidos Cardíacos/farmacología , Línea Celular , Colesterol/biosíntesis , Sinergismo Farmacológico , Ebolavirus/fisiología , Fluvastatina/farmacología , Humanos , Ácido Mevalónico/metabolismo , Modelos Biológicos , Tensoactivos/química , Virión/efectos de los fármacos , Virión/fisiología , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
Tetherin is an interferon-inducible, antiviral host factor that broadly restricts enveloped virus release by tethering budded viral particles to the plasma membrane. In response, many viruses have evolved tetherin antagonists. The human tetherin gene can express two isoforms, long and short, due to alternative translation initiation sites in the N-terminal cytoplasmic tail. The long isoform (L-tetherin) contains 12 extra amino acids in its N terminus, including a dual tyrosine motif (YDYCRV) that is an internalization signal for clathrin-mediated endocytosis and a determinant of NF-κB activation. Tetherin restricts alphaviruses, which are highly organized enveloped RNA viruses that bud from the plasma membrane. L-tetherin is more efficient than S-tetherin in inhibiting alphavirus release in 293 cells. Here, we demonstrated that alphaviruses do not encode an antagonist for either of the tetherin isoforms. Instead, the isoform specificity reflected a requirement for tetherin endocytosis. The YXY motif in L-tetherin was necessary for alphavirus restriction in 293 cells but was not required for rhabdovirus restriction. L-tetherin's inhibition of alphavirus release correlated with its internalization but did not involve NF-κB activation. In contrast, in U-2 OS cells, the YXY motif and the L-tetherin N-terminal domain were not required for either robust tetherin internalization or alphavirus inhibition. Tetherin forms that were negative for restriction accumulated at the surface of infected cells, while the levels of tetherin forms that restrict were decreased. Together, our results suggest that tetherin-mediated virus internalization plays an important role in the restriction of alphavirus release and that cell-type-specific cofactors may promote tetherin endocytosis.IMPORTANCE The mechanisms of tetherin's antiviral activities and viral tetherin antagonism have been studied in detail for a number of different viruses. Although viral countermeasures against tetherin can differ significantly, overall, tetherin's antiviral activity correlates with physical tethering of virus particles to prevent their release. While tetherin can mediate virus endocytic uptake and clearance, this has not been observed to be required for restriction. Here we show that efficient tetherin inhibition of alphavirus release requires efficient tetherin endocytosis. Our data suggest that this endocytic uptake can be mediated by tetherin itself or by a tetherin cofactor that promotes uptake of an endocytosis-deficient variant of tetherin.
Asunto(s)
Alphavirus/efectos de los fármacos , Antígeno 2 del Estroma de la Médula Ósea/farmacología , Liberación del Virus/efectos de los fármacos , Infecciones por Alphavirus/tratamiento farmacológico , Infecciones por Alphavirus/metabolismo , Infecciones por Alphavirus/virología , Animales , Línea Celular , Cricetinae , Endocitosis/efectos de los fármacos , Células HEK293 , Humanos , FN-kappa B/metabolismo , Isoformas de Proteínas/metabolismo , Virión/efectos de los fármacosRESUMEN
A betulinic acid-based compound, bevirimat (BVM), inhibits HIV-1 maturation by blocking a late step in protease-mediated Gag processing: the cleavage of the capsid-spacer peptide 1 (CA-SP1) intermediate to mature CA. Previous studies showed that mutations conferring resistance to BVM cluster around the CA-SP1 cleavage site. Single amino acid polymorphisms in the SP1 region of Gag and the C terminus of CA reduced HIV-1 susceptibility to BVM, leading to the discontinuation of BVM's clinical development. We recently reported a series of "second-generation" BVM analogs that display markedly improved potency and breadth of activity relative to the parent molecule. Here, we demonstrate that viral clones bearing BVM resistance mutations near the C terminus of CA are potently inhibited by second-generation BVM analogs. We performed de novo selection experiments to identify mutations that confer resistance to these novel compounds. Selection experiments with subtype B HIV-1 identified an Ala-to-Val mutation at SP1 residue 1 and a Pro-to-Ala mutation at CA residue 157 within the major homology region (MHR). In selection experiments with subtype C HIV-1, we identified mutations at CA residue 230 (CA-V230M) and SP1 residue 1 (SP1-A1V), residue 5 (SP1-S5N), and residue 10 (SP1-G10R). The positions at which resistance mutations arose are highly conserved across multiple subtypes of HIV-1. We demonstrate that the mutations confer modest to high-level maturation inhibitor resistance. In most cases, resistance was not associated with a detectable increase in the kinetics of CA-SP1 processing. These results identify mutations that confer resistance to second-generation maturation inhibitors and provide novel insights into the mechanism of resistance.IMPORTANCE HIV-1 maturation inhibitors are a class of small-molecule compounds that block a late step in the viral protease-mediated processing of the Gag polyprotein precursor, the viral protein responsible for the formation of virus particles. The first-in-class HIV-1 maturation inhibitor bevirimat was highly effective in blocking HIV-1 replication, but its activity was compromised by naturally occurring sequence polymorphisms within Gag. Recently developed bevirimat analogs, referred to as "second-generation" maturation inhibitors, overcome this issue. To understand more about how these second-generation compounds block HIV-1 maturation, here we selected for HIV-1 mutants that are resistant to these compounds. Selections were performed in the context of two different subtypes of HIV-1. We identified a small set of mutations at highly conserved positions within the capsid and spacer peptide 1 domains of Gag that confer resistance. Identification and analysis of these maturation inhibitor-resistant mutants provide insights into the mechanisms of resistance to these compounds.