RESUMEN
T-cell immunoglobin and mucin domain protein-1 (TIM-1) mediates entry of chikungunya virus (CHIKV) into some mammalian cells through the interaction with envelope phospholipids. While this interaction enhances entry, TIM-1 has been shown to tether newly formed HIV and Ebola virus particles, limiting their efficient release. In this study, we investigate the ability of surface receptors such as TIM-1 to sequester newly budded virions on the surface of infected cells. We established a luminescence reporter system to produce chikungunya viral particles that integrate nano-luciferase and easily quantify viral particles. We found that TIM-1 on the surface of host cells significantly reduced CHIKV release efficiency in comparison to other entry factors. Removal of cell surface TIM-1 through direct cellular knock-out or altering the cellular lipid distribution enhanced CHIKV release. Over the course of infection, CHIKV was able to counteract the tethering effect by gradually decreasing the surface levels of TIM-1 in a process mediated by the nonstructural protein 2. This study highlights the importance of phosphatidylserine receptors in mediating not only the entry of CHIKV but also its release and could aid in developing cell lines capable of enhanced vaccine production. IMPORTANCE: Chikungunya virus (CHIKV) is an enveloped alphavirus transmitted by the bites of infectious mosquitoes. Infection with CHIKV results in the development of fever, joint pain, and arthralgia that can become chronic and last for months after infection. Prevention of this disease is still highly focused on vector control strategies. In December 2023, a new live attenuated vaccine against CHIKV was approved by the FDA. We aimed to study the cellular factors involved in CHIKV release, to better understand CHIKV's ability to efficiently infect and spread among a wide variety of cell lines. We found that TIM-1 receptors can significantly abrogate CHIKV's ability to efficiently exit infected cells. This information can be beneficial for maximizing viral particle production in laboratory settings and during vaccine manufacturing.
Asunto(s)
Fiebre Chikungunya , Virus Chikungunya , Receptor Celular 1 del Virus de la Hepatitis A , Fosfatidilserinas , Liberación del Virus , Virus Chikungunya/fisiología , Virus Chikungunya/metabolismo , Receptor Celular 1 del Virus de la Hepatitis A/metabolismo , Humanos , Fosfatidilserinas/metabolismo , Fiebre Chikungunya/virología , Fiebre Chikungunya/metabolismo , Células HEK293 , Internalización del Virus , Animales , Envoltura Viral/metabolismo , Línea Celular , Virión/metabolismo , Receptores Virales/metabolismoRESUMEN
Synthetic viral nanostructures are useful as materials for analyzing the biological behavior of natural viruses and as vaccine materials. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped virus embedding a spike (S) protein involved in host cell infection. Although nanomaterials modified with an S protein without an envelope membrane have been developed, they are considered unsuitable for stability and functionality. We previously constructed an enveloped viral replica complexed with a cationic lipid bilayer and an anionic artificial viral capsid self-assembled from ß-annulus peptides. In this study, we report the first example of an enveloped viral replica equipped with an S protein derived from SARS-CoV-2. Interestingly, even the S protein equipped on the enveloped viral replica bound strongly to the free angiotensin-converting enzyme 2 (ACE2) receptor as well as ACE2 localized on the cell membrane.
Asunto(s)
Enzima Convertidora de Angiotensina 2 , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Glicoproteína de la Espiga del Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/química , SARS-CoV-2/metabolismo , Humanos , COVID-19/virología , Membrana Dobles de Lípidos/metabolismo , Membrana Dobles de Lípidos/química , Envoltura Viral/metabolismo , Nanoestructuras/químicaRESUMEN
Porcine reproductive and respiratory syndrome (PRRSV) is an enveloped single-stranded positive-sense RNA virus and one of the main pathogens that causes the most significant economical losses in the swine-producing countries. PRRSV is currently divided into two distinct species, PRRSV-1 and PRRSV-2. The PRRSV virion envelope is composed of four glycosylated membrane proteins and three non-glycosylated envelope proteins. Previous work has suggested that PRRSV-linked glycans are critical structural components for virus assembly. In addition, it has been proposed that PRRSV glycans are implicated in the interaction with host cells and critical for virus infection. In contrast, recent findings showed that removal of N-glycans from PRRSV does not influence virus infection of permissive cells. Thus, there are not sufficient evidences to indicate compellingly that N-glycans present in the PRRSV envelope play a direct function in viral infection. To gain insights into the role of N-glycosylation in PRRSV infection, we analysed the specific contribution of the envelope protein-linked N-glycans to infection of permissive cells. For this purpose, we used a novel strategy to modify envelope protein-linked N-glycans that consists of production of monoglycosylated PRRSV and viral glycoproteins with different glycan states. Our results showed that removal or alteration of N-glycans from PRRSV affected virus infection. Specifically, we found that complex N-glycans are required for an efficient infection in cell cultures. Furthermore, we found that presence of high mannose type glycans on PRRSV surface is the minimal requirement for a productive viral infection. Our findings also show that PRRSV-1 and PRRSV-2 have different requirements of N-glycan structure for an optimal infection. In addition, we demonstrated that removal of N-glycans from PRRSV does not affect viral attachment, suggesting that these carbohydrates played a major role in regulating viral entry. In agreement with these findings, by performing immunoprecipitation assays and colocalization experiments, we found that N-glycans present in the viral envelope glycoproteins are not required to bind to the essential viral receptor CD163. Finally, we found that the presence of N-glycans in CD163 is not required for PRRSV infection.
Asunto(s)
Polisacáridos , Síndrome Respiratorio y de la Reproducción Porcina , Virus del Síndrome Respiratorio y Reproductivo Porcino , Virus del Síndrome Respiratorio y Reproductivo Porcino/fisiología , Virus del Síndrome Respiratorio y Reproductivo Porcino/metabolismo , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Glicosilación , Animales , Porcinos , Polisacáridos/metabolismo , Síndrome Respiratorio y de la Reproducción Porcina/virología , Síndrome Respiratorio y de la Reproducción Porcina/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Proteínas del Envoltorio Viral/genética , Línea Celular , Receptores de Superficie Celular/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Antígenos CD/metabolismo , Envoltura Viral/metabolismoRESUMEN
The Red Queen Hypothesis (RQH), derived from Lewis Carroll's "Through the Looking-Glass", postulates that organisms must continually adapt in response to each other to maintain relative fitness. Within the context of host-pathogen interactions, the RQH implies an evolutionary arms race, wherein viruses evolve to exploit hosts and hosts evolve to resist viral invasion. This study delves into the dynamics of the RQH in the context of virus-cell interactions, specifically focusing on virus receptors and cell receptors. We observed multiple virus-host systems and noted patterns of co-evolution. As viruses evolved receptor-binding proteins to effectively engage with cell receptors, cells countered by altering their receptor genes. This ongoing mutual adaptation cycle has influenced the molecular intricacies of receptor-ligand interactions. Our data supports the RQH as a driving force behind the diversification and specialization of both viral and host cell receptors. Understanding this co-evolutionary dance offers insights into the unpredictability of emerging viral diseases and potential therapeutic interventions. Future research is crucial to dissect the nuanced molecular changes and the broader ecological consequences of this ever-evolving battle. Here, we combine phylogenetic inferences, structural modeling, and molecular dynamics analyses to describe the epidemiological characteristics of major Brazilian DENV strains that circulated from 1990 to 2022 from a combined perspective, thus providing us with a more detailed picture on the dynamics of such interactions over time.
Asunto(s)
Moléculas de Adhesión Celular , Virus del Dengue , Evolución Molecular , Interacciones Huésped-Patógeno , Receptores de Superficie Celular , Proteínas del Envoltorio Viral , Envoltura Viral , Humanos , Brasil , Moléculas de Adhesión Celular/metabolismo , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/química , Dengue/virología , Virus del Dengue/genética , Virus del Dengue/metabolismo , Interacciones Huésped-Patógeno/genética , Lectinas Tipo C/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/química , Simulación de Dinámica Molecular , Filogenia , Unión Proteica , Receptores de Superficie Celular/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/química , Receptores Virales/metabolismo , Receptores Virales/química , Receptores Virales/genética , Envoltura Viral/metabolismo , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Proteínas del Envoltorio Viral/químicaRESUMEN
Labeling the genome and envelope of a virus with multicolor quantum dots (QDs) simultaneously enables real-time monitoring of viral uncoating and genome release, contributing to our understanding of virus infection mechanisms. However, current labeling techniques require genetic modification, which alters the virus's composition and infectivity. To address this, we utilized the CRISPR/Cas13 system and a bioorthogonal metabolic method to label the Japanese encephalitis virus (JEV) genome and envelopes with different-colored QDs in situ. This technique allows one-step two-color labeling of the viral envelope and intraviral genome with QDs harnessing virus infection. In combination with single-virus tracking, we visualized JEV uncoating and genome release in real time near the endoplasmic reticulum of live cells. This labeling strategy allows for real-time visualization of uncoating and genome release at the single-virus level, and it is expected to advance the study of other viral infection mechanisms.
Asunto(s)
Puntos Cuánticos , Virosis , Virus , Humanos , Envoltura Viral/metabolismo , Proteínas del Envoltorio ViralRESUMEN
Membrane-associated RING-CH (MARCH) family proteins were recently reported to inhibit viral replication through multiple modes. Previous work showed that human MARCH8 blocked Ebola virus (EBOV) glycoprotein (GP) maturation. Our study here demonstrates that human MARCH1 and MARCH2 share a similar pattern to MARCH8 in restricting EBOV GP-pseudotyped viral infection. Human MARCH1 and MARCH2 retain EBOV GP at the trans-Golgi network, reduce its cell surface display, and impair EBOV GP-pseudotyped virions infectivity. Furthermore, we uncover that the host proprotein convertase furin could interact with human MARCH1/2 and EBOV GP intracellularly. Importantly, the furin P domain is verified to be recognized by MARCH1/2/8, which is critical for their blocking activities. Besides, bovine MARCH2 and murine MARCH1 also impair EBOV GP proteolytic processing. Altogether, our findings confirm that MARCH1/2 proteins of different mammalian origins showed a relatively conserved feature in blocking EBOV GP cleavage, which could provide clues for subsequent MARCHs antiviral studies and may facilitate the development of novel strategies to antagonize enveloped virus infection.
Asunto(s)
Ebolavirus , Fiebre Hemorrágica Ebola , Animales , Bovinos , Humanos , Ratones , Línea Celular , Furina/metabolismo , Glicoproteínas , Mamíferos/metabolismo , Proteínas de la Membrana/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Envoltura Viral/metabolismo , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Intracellular pathogens rely on host metabolic networks for multiplication. Enveloped viruses need lipids for formation of the viral envelope and positive sense RNA viruses that replicate in membranous inclusions require lipids for formation of the replication compartments. In addition, all intracellular pathogens need energy for their replicative cycle. As triglycerides in lipid droplets are the main energy storage unit of cells and major source of membrane lipids, it is not surprising that viruses have evolved various strategies to exploit different aspects of lipid droplet biology.
Asunto(s)
Gotas Lipídicas , Replicación Viral , Gotas Lipídicas/metabolismo , Gotas Lipídicas/virología , Humanos , Animales , Envoltura Viral/metabolismo , Virus ARN/fisiología , Virus ARN/metabolismo , Virus ARN/genética , Metabolismo de los Lípidos , Triglicéridos/metabolismoRESUMEN
Enterovirus D68 (EV-D68) is a re-emerging enterovirus that causes acute respiratory illness in infants and has recently been linked to Acute Flaccid Myelitis. Here, we show that the histone deacetylase, SIRT-1, is essential for autophagy and EV-D68 infection. Knockdown of SIRT-1 inhibits autophagy and reduces EV-D68 extracellular titers. The proviral activity of SIRT-1 does not require its deacetylase activity or functional autophagy. SIRT-1's proviral activity is, we demonstrate, mediated through the repression of endoplasmic reticulum stress (ER stress). Inducing ER stress through thapsigargin treatment or SERCA2A knockdown in SIRT-1 knockdown cells had no additional effect on EV-D68 extracellular titers. Knockdown of SIRT-1 also decreases poliovirus and SARS-CoV-2 titers but not coxsackievirus B3. In non-lytic conditions, EV-D68 is primarily released in an enveloped form, and SIRT-1 is required for this process. Our data show that SIRT-1, through its translocation to the cytosol, is critical to promote the release of enveloped EV-D68 viral particles.
Asunto(s)
Enterovirus Humano D , Infecciones por Enterovirus , Sirtuina 1 , Activación Viral , Humanos , COVID-19 , Enterovirus/genética , Enterovirus/fisiología , Enterovirus Humano D/genética , Enterovirus Humano D/fisiología , Infecciones por Enterovirus/genética , Infecciones por Enterovirus/fisiopatología , Enfermedades Neuromusculares , Provirus , SARS-CoV-2 , Envoltura Viral/metabolismo , Envoltura Viral/fisiología , Activación Viral/genética , Activación Viral/fisiología , Sirtuina 1/genética , Sirtuina 1/fisiologíaRESUMEN
Autophagy serves as a defense mechanism against intracellular pathogens, but several microorganisms exploit it for their own benefit. Accordingly, certain herpesviruses include autophagic membranes into their infectious virus particles. In this study, we analyzed the composition of purified virions of the Epstein-Barr virus (EBV), a common oncogenic γ-herpesvirus. In these, we found several components of the autophagy machinery, including membrane-associated LC3B-II, and numerous viral proteins, such as the capsid assembly proteins BVRF2 and BdRF1. Additionally, we showed that BVRF2 and BdRF1 interact with LC3B-II via their common protein domain. Using an EBV mutant, we identified BVRF2 as essential to assemble mature capsids and produce infectious EBV. However, BdRF1 was sufficient for the release of noninfectious viral envelopes as long as autophagy was not compromised. These data suggest that BVRF2 and BdRF1 are not only important for capsid assembly but together with the LC3B conjugation complex of ATG5-ATG12-ATG15L1 are also critical for EBV envelope release.
Asunto(s)
Cápside , Infecciones por Virus de Epstein-Barr , Humanos , Cápside/metabolismo , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Envoltura Viral/metabolismo , Infecciones por Virus de Epstein-Barr/metabolismo , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismoRESUMEN
Flaviviruses encompass many important human pathogens, including Dengue, Zika, West Nile, Yellow fever, Japanese encephalitis, and Tick-borne encephalitis viruses as well as several emerging viruses that affect millions of people worldwide. They enter cells by endocytosis, fusing their membrane with the late endosomal one in a pH-dependent manner, so membrane fusion is one of the main targets for obtaining new antiviral inhibitors. The envelope E protein, a class II membrane fusion protein, is responsible for fusion and contains different domains involved in the fusion mechanism, including the fusion peptide. However, other segments, apart from the fusion peptide, have been implicated in the mechanism of membrane fusion, in particular a segment containing a His residue supposed to act as a specific pH sensor. We have used atomistic molecular dynamics to study the binding of the envelope E protein segment containing the conserved His residue in its three different tautomer forms with a complex membrane mimicking the late-endosomal one. We show that this His-containing segment is capable of spontaneous membrane binding, preferentially binds electronegatively charged phospholipids and does not bind cholesterol. Since Flaviviruses have caused epidemics in the past, continue to do so and will undoubtedly continue to do so, this specific segment could characterise a new target that would allow finding effective antiviral molecules against DENV virus in particular and Flaviviruses in general.
Asunto(s)
Dengue , Flavivirus , Infección por el Virus Zika , Virus Zika , Humanos , Envoltura Viral/metabolismo , Proteínas del Envoltorio Viral/química , Flavivirus/química , Flavivirus/metabolismo , Virus Zika/metabolismo , Péptidos , Antivirales , FosfolípidosRESUMEN
African swine fever (ASF) is an acute and hemorrhagic infectious disease caused by African swine fever virus (ASFV), which is listed as an animal epidemic disease that must be reported by The World Organization for Animal Health and that causes serious economic losses to China and even the whole world. Currently, the entry mechanism of ASFV is not fully understood. Especially in the early stages of virus entry, the host factors required for ASFV entry have not yet been identified and characterized. In this study, we demonstrated that ASFV externalized phosphatidylserine (PS) on the envelope functioned as viral apoptotic mimicry, which interacts with AXL, a tyrosine kinase receptor, to mediate ASFV entry into porcine alveolar macrophages (PAMs). We found that AXL was the most pronounced phosphatidylserine receptor (PSR) affecting ASFV entry in PAMs by RNA interference screening. Knockout AXL gene expression remarkably decreased ASFV internalization and replication in MA104 cells. Furthermore, the antibody against AXL extracellular domains effectively inhibited the ASFV entry. Consistent with these results, the deletion of the intracellular kinase domain of AXL and the treatment of the AXL inhibitor, R428, significantly inhibited the internalization of ASFV. Mechanistically, AXL facilitated the internalization of ASFV virions via macropinocytosis. Collectively, we provide evidence that AXL is a coreceptor for ASFV entry into PAMs, which expands our knowledge of ASFV entry and provides a theoretical basis for identifying new antiviral targets. IMPORTANCE African swine fever (ASF) is a highly contagious infectious disease caused by the ASF virus (ASFV), with a mortality rate of up to 100%. ASFV has caused huge economic losses to pig farming worldwide. Specific cellular surface receptors are considered crucial determinants of ASFV tropism. However, the host factors required for ASFV entry have not yet been identified, and the molecular mechanism of its entry remains unclear. Here, we found that ASFV utilized phosphatidylserine (PS) on the surface of virions to masquerade as apoptotic mimicry and facilitated virus entry by interacting with host factor AXL. We found that knockout of AXL remarkably decreased ASFV internalization and replication. The antibody against AXL extracellular domains and AXL inhibitor R428 significantly inhibited the internalization of ASFV via macropinocytosis. The current work deepens our understanding of ASFV entry and provides clues for the development of antiviral drugs to control ASFV infection.
Asunto(s)
Fiebre Porcina Africana , Tirosina Quinasa del Receptor Axl , Interacciones Microbiota-Huesped , Internalización del Virus , Animales , Fiebre Porcina Africana/virología , Virus de la Fiebre Porcina Africana/genética , Porcinos , Tirosina Quinasa del Receptor Axl/genética , Tirosina Quinasa del Receptor Axl/metabolismo , Macrófagos Alveolares/virología , Técnicas de Inactivación de Genes , Línea Celular , Envoltura Viral/metabolismo , Acoplamiento Viral , Dominios ProteicosRESUMEN
SARS-CoV-2 viremia is associated with increased acute lung injury (ALI) and mortality in children and adults. The mechanisms by which viral components in the circulation mediate ALI in COVID-19 remain unclear. We tested the hypothesis that the SARS-CoV-2 envelope (E) protein induces Toll-like receptor (TLR)-mediated ALI and lung remodeling in a model of neonatal COVID-19. Neonatal C57BL6 mice given intraperitoneal E protein injections revealed a dose-dependent increase in lung cytokines [interleukin 6 (Il6), tumor necrosis factor (Tnfα), and interleukin 1 beta (Il1ß)] and canonical proinflammatory TLR signaling. Systemic E protein induced endothelial immune activation, immune cell influx, and TGFß signaling and lung matrix remodeling inhibited alveolarization in the developing lung. E protein-mediated ALI and transforming growth factor beta (TGFß) signaling was repressed in Tlr2-/-, but not Tlr4-/- mice. A single dose of intraperitoneal E protein injection induced chronic alveolar remodeling as evidenced by a decrease in radial alveolar counts and increase in mean linear intercepts. Ciclesonide, a synthetic glucocorticoid, inhibited E protein-induced proinflammatory TLR signaling and ALI. In vitro, E protein-mediated inflammation and cell death were TLR2-dependent in human primary neonatal lung endothelial cells and were rescued by ciclesonide. This study provides insight into the pathogenesis of ALI and alveolar remodeling with SARS-CoV-2 viremia in children, whereas revealing the efficacy of steroids.NEW & NOTEWORTHY We reveal that the envelope protein of SARS-CoV-2 mediates acute lung injury (ALI) and alveolar remodeling through Toll-like receptor activation, which is rescued by the glucocorticoid, ciclesonide.
Asunto(s)
Lesión Pulmonar Aguda , COVID-19 , Animales , Niño , Humanos , Ratones , Lesión Pulmonar Aguda/inducido químicamente , COVID-19/complicaciones , Células Endoteliales/metabolismo , Glucocorticoides , Lipopolisacáridos/efectos adversos , Ratones Endogámicos C57BL , SARS-CoV-2/metabolismo , Receptor Toll-Like 2 , Receptor Toll-Like 4/metabolismo , Receptores Toll-Like , Factor de Crecimiento Transformador beta , Viremia/complicaciones , Envoltura Viral/metabolismoRESUMEN
Pathogenic enveloped viruses are covered with a glycan shield that provides a dual function: the glycan structures contribute to virus protection as well as host cell recognition. The three classical types of N-glycans, in particular complex glycans, high-mannose glycans, and hybrid glycans, together with some O-glycans, participate in the glycan shield of the Ebola virus, influenza virus, human cytomegalovirus, herpes virus, human immunodeficiency virus, Lassa virus, and MERS-CoV, SARS-CoV, and SARS-CoV-2, which are responsible for respiratory syndromes. The glycans are linked to glycoproteins that occur as metastable prefusion glycoproteins on the surface of infectious virions such as gp120 of HIV, hemagglutinin of influenza, or spike proteins of beta-coronaviruses. Plant lectins with different carbohydrate-binding specificities and, especially, mannose-specific lectins from the Vicieae tribe, such as pea lectin and lentil lectin, can be used as glycan probes for targeting the glycan shield because of their specific interaction with the α1,6-fucosylated core Man3GlcNAc2, which predominantly occurs in complex and hybrid glycans. Other plant lectins with Neu5Ac specificity or GalNAc/T/Tn specificity can also serve as potential glycan probes for the often sialylated complex glycans and truncated O-glycans, respectively, which are abundantly distributed in the glycan shield of enveloped viruses. The biomedical and therapeutical potential of plant lectins as antiviral drugs is discussed.
Asunto(s)
COVID-19/metabolismo , Fabaceae/metabolismo , Lectinas de Plantas/metabolismo , Polisacáridos/metabolismo , SARS-CoV-2/metabolismo , Envoltura Viral/metabolismo , COVID-19/epidemiología , COVID-19/virología , Humanos , Manosa/metabolismo , Unión Proteica , SARS-CoV-2/fisiología , Virión/metabolismo , Internalización del VirusRESUMEN
About 8% of our genome is composed of sequences from Human Endogenous Retroviruses (HERVs). The HERV-K (HML.2) family, here abbreviated HML.2, is able to produce virus particles that were detected in cell lines, malignant tumors and in autoimmune diseases. Parameters and properties of HML.2 released from teratocarcinoma cell lines GH and Tera-1 were investigated in detail. In most experiments, analyzed viruses were purified by density gradient centrifugation. HML.2 structural proteins, reverse transcriptase (RT) activity, viral RNA (vRNA) and particle morphology were analyzed. The HML.2 markers were predominantly detected in fractions with a buoyant density of 1.16 g/cm3. Deglycosylation of TM revealed truncated forms of transmembrane (TM) protein. Free virions and extracellular vesicles (presumably microvesicles-MVs) with HML.2 elements, including budding intermediates, were detected by electron microscopy. Viral elements and assembled virions captured and exported by MVs can boost specific immune responses and trigger immunomodulation in recipient cells. Sequencing of cDNA clones demonstrated exclusive presence of HERV-K108 env in HML.2 from Tera-1 cells. Not counting two recombinant variants, four known env sequences were found in HML.2 from GH cells. Obtained results shed light on parameters and morphology of HML.2. A possible mechanism of HML.2-induced diseases is discussed.
Asunto(s)
Cápside/metabolismo , Retrovirus Endógenos/genética , Retrovirus Endógenos/metabolismo , Vesículas Extracelulares/virología , Productos del Gen env/metabolismo , Genes env , ARN Viral/genética , Teratocarcinoma/metabolismo , Teratocarcinoma/virología , Envoltura Viral/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/virología , Centrifugación por Gradiente de Densidad/métodos , Retrovirus Endógenos/aislamiento & purificación , Productos del Gen env/genética , Células HEK293 , Humanos , Proteínas de la Membrana/metabolismo , ARN Viral/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Teratocarcinoma/patología , Transfección , Ensamble de Virus/genéticaRESUMEN
Gag, the primary structural protein of HIV-1, is recruited to the plasma membrane for virus assembly by its matrix (MA) domain. Gag is subsequently cleaved into its component domains, causing structural maturation to repurpose the virion for cell entry. We determined the structure and arrangement of MA within immature and mature HIV-1 through cryo-electron tomography. We found that MA rearranges between two different hexameric lattices upon maturation. In mature HIV-1, a lipid extends out of the membrane to bind with a pocket in MA. Our data suggest that proteolytic maturation of HIV-1 not only assembles the viral capsid surrounding the genome but also repurposes the membrane-bound MA lattice for an entry or postentry function and results in the partial removal of up to 2500 lipids from the viral membrane.
Asunto(s)
Antígenos VIH/química , Antígenos VIH/metabolismo , VIH-1/química , VIH-1/fisiología , Envoltura Viral/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/química , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Cápside/química , Cápside/fisiología , Tomografía con Microscopio Electrónico , VIH-1/ultraestructura , Membrana Dobles de Lípidos , Lípidos de la Membrana/metabolismo , Modelos Moleculares , Conformación Proteica , Dominios Proteicos , Estructura Secundaria de Proteína , Envoltura Viral/química , Envoltura Viral/ultraestructura , Virión/química , Virión/fisiología , Virión/ultraestructura , Ensamble de Virus , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Because most of animal viruses are enveloped, cytoplasmic entry of these viruses via fusion with cellular membrane initiates their invasion. However, the strategies in which host cells counteract cytoplasmic entry of such viruses are incompletely understood. Pore-forming toxin aerolysin-like proteins (ALPs) exist throughout the animal kingdom, but their functions are mostly unknown. In this study, we report that ßγ-crystallin fused aerolysin-like protein and trefoil factor complex (ßγ-CAT), an ALP and trefoil factor complex from the frog Bombina maxima, directly blocks enveloped virus invasion by interfering with cytoplasmic entry. ßγ-CAT targeted acidic glycosphingolipids on the HSV type 1 (HSV-1) envelope to induce pore formation, as indicated by the oligomer formation of protein and potassium and calcium ion efflux. Meanwhile, ßγ-CAT formed ring-like oligomers of â¼10 nm in diameter on the liposomes and induced dye release from liposomes that mimic viral envelope. Unexpectedly, transmission electron microscopy analysis showed that the ßγ-CAT-treated HSV-1 was visibly as intact as the vehicle-treated HSV-1, indicating that ßγ-CAT did not lyse the viral envelope. However, the cytoplasmic entry of the ßγ-CAT-treated HSV-1 into HeLa cells was totally hindered. In vivo, topical application of ßγ-CAT attenuated the HSV-1 corneal infection in mice. Collectively, these results uncovered that ßγ-CAT possesses the capacity to counteract enveloped virus invasion with its featured antiviral-acting manner. Our findings will also largely help to illustrate the putative antiviral activity of animal ALPs.
Asunto(s)
Proteínas Anfibias/metabolismo , Antivirales/metabolismo , Córnea/patología , Herpes Simple/inmunología , Herpesvirus Humano 1/fisiología , Complejos Multiproteicos/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Factores Trefoil/metabolismo , Proteínas Anfibias/genética , Animales , Anuros , Toxinas Bacterianas/genética , Córnea/virología , Femenino , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Ratones , Microscopía Electrónica de Transmisión , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/genética , Envoltura Viral/metabolismo , Envoltura Viral/ultraestructura , Internalización del Virus , gamma-Cristalinas/químicaRESUMEN
To date, there are no broad-spectrum antivirals available to treat infections with flaviviruses such as dengue (DENV) and Zika virus (ZIKV). In this study, we determine the broad antiviral activity of the lantibiotic Labyrinthopeptin A1. We show that Laby A1 inhibits all DENV serotypes and various ZIKV strains with IC50 around 1 µM. The structurally related Laby A2 also displayed a consistent, but about tenfold lower, antiviral activity. Furthermore, Laby A1 inhibits many viruses from divergent families such as HIV, YFV, RSV and Punta Torovirus. Of interest, Laby A1 does not show activity against non-enveloped viruses. Its antiviral activity is independent of the cell line or the used evaluation method, and can also be observed in MDDC, a physiologically relevant primary cell type. Furthermore, Laby A1 demonstrates low cellular toxicity and has a more favorable SI compared to duramycin, a well-described lantibiotic with broad-spectrum antiviral activity. Time-of-drug addition experiments demonstrate that Laby A1 inhibits infection and entry processes of ZIKV and DENV. We reveal that Laby A1 performs its broad antiviral activity by interacting with a viral factor rather than a cellular factor, and that it has virucidal properties. Finally, using SPR interaction studies we demonstrate that Laby A1 interacts with several phospholipids (i.e. PE and PS) present in the viral envelope. Together with other recent Labyrinthopeptin antiviral publications, this work validates the activity of Laby A1 as broad antiviral entry inhibitor with a unique mechanism of action and demonstrates its potential value as antiviral agent against emerging flaviviruses.
Asunto(s)
Antivirales/farmacología , Bacteriocinas/farmacología , Virus del Dengue/efectos de los fármacos , Fosfolípidos/metabolismo , Envoltura Viral/efectos de los fármacos , Virus Zika/efectos de los fármacos , Animales , Antivirales/metabolismo , Bacteriocinas/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Péptidos/farmacología , Envoltura Viral/metabolismo , Internalización del Virus/efectos de los fármacos , Virus/clasificación , Virus/efectos de los fármacosRESUMEN
Inhibition of the binding of enveloped viruses surface glycoproteins to host cell receptor(s) is a major target of vaccines and constitutes an efficient strategy to block viral entry and infection of various host cells and tissues. Cellular entry usually requires the fusion of the viral envelope with host plasma membranes. Such entry mechanism is often preceded by "priming" and/or "activation" steps requiring limited proteolysis of the viral surface glycoprotein to expose a fusogenic domain for efficient membrane juxtapositions. The 9-membered family of Proprotein Convertases related to Subtilisin/Kexin (PCSK) serine proteases (PC1, PC2, Furin, PC4, PC5, PACE4, PC7, SKI-1/S1P, and PCSK9) participate in post-translational cleavages and/or regulation of multiple secretory proteins. The type-I membrane-bound Furin and SKI-1/S1P are the major convertases responsible for the processing of surface glycoproteins of enveloped viruses. Stefan Kunz has considerably contributed to define the role of SKI-1/S1P in the activation of arenaviruses causing hemorrhagic fever. Furin was recently implicated in the activation of the spike S-protein of SARS-CoV-2 and Furin-inhibitors are being tested as antivirals in COVID-19. Other members of the PCSK-family are also implicated in some viral infections, such as PCSK9 in Dengue. Herein, we summarize the various functions of the PCSKs and present arguments whereby their inhibition could represent a powerful arsenal to limit viral infections causing the present and future pandemics.
Asunto(s)
Regulación Viral de la Expresión Génica , Proproteína Convertasas/metabolismo , Virosis/virología , Internalización del Virus , Virus/genética , Transporte Biológico , Furina/metabolismo , Humanos , Proproteína Convertasa 9/metabolismo , Proproteína Convertasas/genética , Proteolisis , SARS-CoV-2/enzimología , SARS-CoV-2/metabolismo , Serina Endopeptidasas/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Envoltura Viral/metabolismo , Virus/metabolismoRESUMEN
Antiviral action of various photosensitizers is already summarized in several comprehensive reviews, and various mechanisms have been proposed for it. However, a critical consideration of the matter of the area is complicated, since the exact mechanisms are very difficult to explore and clarify, and most publications are of an empirical and "phenomenological" nature, reporting a dependence of the antiviral action on illumination, or a correlation of activity with the photophysical properties of the substances. Of particular interest is substance-assisted photogeneration of highly reactive singlet oxygen (1O2). The damaging action of 1O2 on the lipids of the viral envelope can probably lead to a loss of the ability of the lipid bilayer of enveloped viruses to fuse with the lipid membrane of the host cell. Thus, lipid bilayer-affine 1O2 photosensitizers have prospects as broad-spectrum antivirals against enveloped viruses. In this short review, we want to point out the main types of antiviral photosensitizers with potential affinity to the lipid bilayer and summarize the data on new compounds over the past three years. Further understanding of the data in the field will spur a targeted search for substances with antiviral activity against enveloped viruses among photosensitizers able to bind to the lipid membranes.
Asunto(s)
Antivirales , Lípidos de la Membrana/metabolismo , Fármacos Fotosensibilizantes , Envoltura Viral/metabolismo , Virosis , Virus/metabolismo , Animales , Antivirales/química , Antivirales/farmacocinética , Antivirales/uso terapéutico , Humanos , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/farmacocinética , Fármacos Fotosensibilizantes/uso terapéutico , Oxígeno Singlete , Virosis/tratamiento farmacológico , Virosis/metabolismoRESUMEN
Envelope phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtr) have been shown to mediate binding of enveloped viruses. However, commonly used PtdSer binding molecules such as Annexin V cannot block PtdSer-mediated viral infection. Lack of reagents that can conceal envelope PtdSer and PtdEtr and subsequently inhibit infection hinders elucidation of the roles of the envelope phospholipids in viral infection. Here, we developed sTIM1dMLDR801, a reagent capable of blocking PtdSer- and PtdEtr-dependent infection of enveloped viruses. Using sTIM1dMLDR801, we found that envelope PtdSer and/or PtdEtr can support ZIKV infection of not only human but also mosquito cells. In a mouse model for ZIKV infection, sTIM1dMLDR801 reduced ZIKV load in serum and the spleen, indicating envelope PtdSer and/or PtdEtr support in viral infection in vivo. sTIM1dMLDR801 will enable elucidation of the roles of envelope PtdSer and PtdEtr in infection of various virus species, thereby facilitating identification of their receptors and transmission mechanisms.