RESUMEN
The genome of the Nipah virus (NiV) encodes a variety of structural proteins linked to a diverse array of symptoms, including fevers, headaches, somnolence, and respiratory impairment. In instances of heightened severity, it can also invade the central nervous system (CNS), resulting in more pronounced problems. This work investigates the effects of NiV on the blood-brain barrier (BBB), the vital physiological layer responsible for safeguarding the CNS by regulating the passage of chemicals into the brain selectively. To achieve this, the researchers (MMJAO, AM and MNMD) searched a variety of databases for relevant articles on NiV and BBB disruption, looking for evidence of work on inflammation, immune response (cytokines and chemokines), tight junctions (TJs), and basement membranes related to NiV and BBB. Based on these works, it appears that the affinity of NiV for various receptors, including Ephrin-B2 and Ephrin-B3, has seen many NiV infections begin in the respiratory epithelium, resulting in the development of acute respiratory distress syndrome. The virus then gains entry into the circulatory system, offering it the potential to invade brain endothelial cells (ECs). NiV also has the ability to infect the leukocytes and the olfactory pathway, offering it a "Trojan horse" strategy. When NiV causes encephalitis, the CNS generates a strong inflammatory response, which makes the blood vessels more permeable. Chemokines and cytokines all have a substantial influence on BBB disruption, and NiV also has the ability to affect TJs, leading to disturbances in the structural integrity of the BBB. The pathogen's versatility is also shown by its capacity to impact multiple organ systems, despite particular emphasis on the CNS. It is of the utmost importance to comprehend the mechanisms by which NiV impacts the integrity of the BBB, as such comprehension has the potential to inform treatment approaches for NiV and other developing viral diseases. Nevertheless, the complicated pathophysiology and molecular pathways implicated in this phenomenon have offered several difficult challenges to researchers to date, underscoring the need for sustained scientific investigation and collaboration in the ongoing battle against this powerful virus.
Asunto(s)
Barrera Hematoencefálica , Infecciones por Henipavirus , Virus Nipah , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/virología , Virus Nipah/fisiología , Humanos , Infecciones por Henipavirus/metabolismo , Infecciones por Henipavirus/virología , Infecciones por Henipavirus/fisiopatología , Animales , Tropismo Viral/fisiologíaRESUMEN
SARS-CoV-2 has infected hundreds of millions of people with over four million dead, resulting in one of the worst global pandemics in recent history. Neurological symptoms associated with COVID-19 include anosmia, ageusia, headaches, confusion, delirium, and strokes. These may manifest due to viral entry into the central nervous system (CNS) through the blood-brain barrier (BBB) by means of ill-defined mechanisms. Here, we summarize the abilities of SARS-CoV-2 and other neurotropic RNA viruses, including Zika virus and Nipah virus, to cross the BBB into the CNS, highlighting the role of magnetic resonance imaging (MRI) in assessing presence and severity of brain structural changes in COVID-19 patients. We present new insight into key mutations in SARS-CoV-2 variants B.1.1.7 (P681H) and B.1.617.2 (P681R), which may impact on neuropilin 1 (NRP1) binding and CNS invasion. We postulate that SARS-CoV-2 may infect both peripheral cells capable of crossing the BBB and brain endothelial cells to traverse the BBB and spread into the brain. COVID-19 patients can be followed up with MRI modalities to better understand the long-term effects of COVID-19 on the brain.
Asunto(s)
Barrera Hematoencefálica , Infecciones por Henipavirus , Virus Nipah , SARS-CoV-2 , Infección por el Virus Zika , Virus Zika , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/fisiopatología , Barrera Hematoencefálica/virología , COVID-19/epidemiología , COVID-19/genética , COVID-19/metabolismo , COVID-19/fisiopatología , Infecciones por Henipavirus/epidemiología , Infecciones por Henipavirus/genética , Infecciones por Henipavirus/metabolismo , Infecciones por Henipavirus/fisiopatología , Humanos , Mutación , Virus Nipah/genética , Virus Nipah/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Virus Zika/genética , Virus Zika/metabolismo , Infección por el Virus Zika/epidemiología , Infección por el Virus Zika/genética , Infección por el Virus Zika/metabolismo , Infección por el Virus Zika/fisiopatologíaRESUMEN
Hendra virus and Nipah virus (NiV), members of the Henipavirus (HNV) genus, are zoonotic paramyxoviruses known to cause severe disease across six mammalian orders, including humans. We isolated a panel of human monoclonal antibodies (mAbs) from the B cells of an individual with prior exposure to equine Hendra virus (HeV) vaccine, targeting distinct antigenic sites. The most potent class of cross-reactive antibodies achieves neutralization by blocking viral attachment to the host cell receptors ephrin-B2 and ephrin-B3, with a second class being enhanced by receptor binding. mAbs from both classes display synergistic activity in vitro. In a stringent hamster model of NiV Bangladesh (NiVB) infection, antibodies from both classes reduce morbidity and mortality and achieve synergistic protection in combination. These candidate mAbs might be suitable for use in a cocktail therapeutic approach to achieve synergistic potency and reduce the risk of virus escape.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Anticuerpos Neutralizantes/farmacología , Antivirales/farmacología , Efrina-B2/antagonistas & inhibidores , Efrina-B3/antagonistas & inhibidores , Infecciones por Henipavirus/prevención & control , Henipavirus/patogenicidad , Receptores Virales/antagonistas & inhibidores , Animales , Especificidad de Anticuerpos , Chlorocebus aethiops , Reacciones Cruzadas , Modelos Animales de Enfermedad , Quimioterapia Combinada , Efrina-B2/inmunología , Efrina-B2/metabolismo , Efrina-B3/inmunología , Efrina-B3/metabolismo , Femenino , Infecciones por Henipavirus/inmunología , Infecciones por Henipavirus/metabolismo , Infecciones por Henipavirus/virología , Interacciones Huésped-Patógeno , Humanos , Mesocricetus , Receptores Virales/inmunología , Receptores Virales/metabolismo , Células VeroRESUMEN
Cedar virus (CedV) is a nonpathogenic member of the Henipavirus (HNV) genus of emerging viruses, which includes the deadly Nipah (NiV) and Hendra (HeV) viruses. CedV forms syncytia, a hallmark of henipaviral and paramyxoviral infections and pathogenicity. However, the intrinsic fusogenic capacity of CedV relative to NiV or HeV remains unquantified. HNV entry is mediated by concerted interactions between the attachment (G) and fusion (F) glycoproteins. Upon receptor binding by the HNV G head domain, a fusion-activating G stalk region is exposed and triggers F to undergo a conformational cascade that leads to viral entry or cell-cell fusion. Here, we demonstrate quantitatively that CedV is inherently significantly less fusogenic than NiV at equivalent G and F cell surface expression levels. We then generated and tested six headless CedV G mutants of distinct C-terminal stalk lengths, surprisingly revealing highly hyperfusogenic cell-cell fusion phenotypes 3- to 4-fold greater than wild-type CedV levels. Additionally, similarly to NiV, a headless HeV G mutant yielded a less pronounced hyperfusogenic phenotype compared to wild-type HeV. Further, coimmunoprecipitation and cell-cell fusion assays revealed heterotypic NiV/CedV functional G/F bidentate interactions, as well as evidence of HNV G head domain involvement beyond receptor binding or G stalk exposure. All evidence points to the G head/stalk junction being key to modulating HNV fusogenicity, supporting the notion that head domains play several distinct and central roles in modulating stalk domain fusion promotion. Further, this study exemplifies how CedV may help elucidate important mechanistic underpinnings of HNV entry and pathogenicity. IMPORTANCE The Henipavirus genus in the Paramyxoviridae family includes the zoonotic Nipah (NiV) and Hendra (HeV) viruses. NiV and HeV infections often cause fatal encephalitis and pneumonia, but no vaccines or therapeutics are currently approved for human use. Upon viral entry, Henipavirus infections yield the formation of multinucleated cells (syncytia). Viral entry and cell-cell fusion are mediated by the attachment (G) and fusion (F) glycoproteins. Cedar virus (CedV), a nonpathogenic henipavirus, may be a useful tool to gain knowledge on henipaviral pathogenicity. Here, using homotypic and heterotypic full-length and headless CedV, NiV, and HeV G/F combinations, we discovered that CedV G/F are significantly less fusogenic than NiV or HeV G/F, and that the G head/stalk junction is key to modulating cell-cell fusion, refining the mechanism of henipaviral membrane fusion events. Our study exemplifies how CedV may be a useful tool to elucidate broader mechanistic understanding for the important henipaviruses.
Asunto(s)
Henipavirus/metabolismo , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/metabolismo , Células Gigantes/metabolismo , Glicoproteínas/genética , Células HEK293 , Henipavirus/genética , Infecciones por Henipavirus/metabolismo , Infecciones por Henipavirus/virología , Humanos , Fusión de Membrana/fisiología , Receptores Virales/metabolismo , Proteínas del Envoltorio Viral/genética , Proteínas Virales de Fusión/fisiología , Acoplamiento Viral , Internalización del VirusRESUMEN
The Nipah virus (NiV) phosphoprotein (P) gene encodes four proteins. Three of these-P, V, and W-possess a common N-terminal domain but distinct C termini. These proteins interact with immune modulators. Previous studies demonstrated that P, V, and W bind STAT1 and STAT4 and that V also interacts with STAT2 but not with STAT3. The STAT1 and STAT2 interactions block interferon (IFN)-induced STAT tyrosine phosphorylation. To more fully characterize the interactions of P, V, and W with the STATs, we screened for interaction of each viral protein with STATs 1 to 6 by coimmunoprecipitation. We demonstrate that NiV P, V, and W interact with STAT4 through their common N-terminal domain and block STAT4 activity, based on a STAT4 response element reporter assay. Although none of the NiV proteins interact with STAT3 or STAT6, NiV V, but not P or W, interacts with STAT5 through its unique C terminus. Furthermore, the interaction of NiV V with STAT5 was not disrupted by overexpression of the N-terminal binding STAT1 or the C-terminal binding MDA5. NiV V also inhibits a STAT5 response element reporter assay. Residues 114 to 140 of the common N-terminal domain of the NiV P gene products were found to be sufficient to bind STAT1 and STAT4. Analysis of STAT1-STAT3 chimeras suggests that the P gene products target the STAT1 SH2 domain. When fused to GST, the 114-140 peptide is sufficient to decrease STAT1 phosphorylation in IFN-ß-stimulated cells, suggesting that this peptide could potentially be fused to heterologous proteins to confer inhibition of STAT1- and STAT4-dependent responses.IMPORTANCE How Nipah virus (NiV) antagonizes innate immune responses is incompletely understood. The P gene of NiV encodes the P, V, and W proteins. These proteins have a common N-terminal sequence that is sufficient to bind to STAT1 and STAT2 and block IFN-induced signal transduction. This study sought to more fully understand how P, V, and W engage with the STAT family of transcription factors to influence their functions. The results identify a novel interaction of V with STAT5 and demonstrate V inhibition of STAT5 function. We also demonstrate that the common N-terminal residues 114 to 140 of P, V, and W are critical for inhibition of STAT1 and STAT4 function, map the interaction to the SH2 region of STAT1, and show that a fusion construct with this peptide significantly inhibits cytokine-induced STAT1 phosphorylation. These data clarify how these important virulence factors modulate innate antiviral defenses.
Asunto(s)
Núcleo Celular/química , Infecciones por Henipavirus/metabolismo , Virus Nipah/fisiología , Factores de Transcripción STAT/metabolismo , Proteínas Virales/metabolismo , Células HEK293 , Infecciones por Henipavirus/inmunología , Infecciones por Henipavirus/virología , Humanos , Inmunidad Innata/inmunología , Fosforilación , Factores de Transcripción STAT/genética , Transducción de Señal , Transactivadores/metabolismo , Proteínas Virales/genéticaRESUMEN
Nipah Virus (NiV) has been designated as a priority disease with an urgent need for therapeutic development by World Health Organization. The monoclonal antibody m102.4 binds to the immunodominant NiV receptor-binding glycoprotein (GP), and potently neutralizes NiV, indicating its potential as a therapeutic agent. Although the co-crystal structure of m102.3, an m102.4 derivative, in complex with the GP of the related Hendra Virus (HeV) has been solved, the structural interaction between m102.4 and NiV is uncharacterized. Herein, we used structure-guided alanine-scanning mutagenesis to map the functional epitope and paratope residues that govern the antigen-antibody interaction. Our results revealed that the binding of m102.4 is mediated predominantly by two residues in the HCDR3 region, which is unusually small for an antibody-antigen interaction. We performed computational docking to generate a structural model of m102.4-NiV interaction. Our model indicates that m102.4 targets the common hydrophobic central cavity and a hydrophilic rim on the GP, as observed for the m102.3-HeV co-crystal, albeit with Fv orientation differences. In summary, our study provides insight into the m102.4-NiV interaction, demonstrating that structure-guided alanine-scanning and computational modeling can serve as the starting point for additional antibody reengineering (e.g. affinity maturation) to generate potential therapeutic candidates.
Asunto(s)
Alanina/genética , Anticuerpos Monoclonales/metabolismo , Simulación por Computador , Glicoproteínas/metabolismo , Infecciones por Henipavirus/virología , Virus Nipah/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Alanina/química , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/metabolismo , Complejo Antígeno-Anticuerpo/química , Complejo Antígeno-Anticuerpo/inmunología , Complejo Antígeno-Anticuerpo/metabolismo , Epítopos/inmunología , Glicoproteínas/química , Glicoproteínas/genética , Infecciones por Henipavirus/inmunología , Infecciones por Henipavirus/metabolismo , Humanos , Mutagénesis Sitio-Dirigida , Virus Nipah/inmunología , Virus Nipah/aislamiento & purificación , Elementos Estructurales de las Proteínas/inmunología , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genéticaRESUMEN
Nipah and Hendra viruses are highly pathogenic, zoonotic henipaviruses that encode proteins that inhibit the host's innate immune response. The W protein is one of four products encoded from the P gene and binds a number of host proteins to regulate signalling pathways. The W protein is intrinsically disordered, a structural attribute that contributes to its diverse host protein interactions. Here, we review the role of W in innate immune suppression through inhibition of both pattern recognition receptor (PRR) pathways and interferon (IFN)-responsive signalling. PRR stimulation leading to activation of IRF-3 and IFN release is blocked by henipavirus W, and unphosphorylated STAT proteins are sequestered within the nucleus of host cells by W, thereby inhibiting the induction of IFN stimulated genes. We examine the critical role of nuclear transport in multiple functions of W and how specific binding of importin-alpha (Impα) isoforms, and the 14-3-3 group of regulatory proteins suggests further modulation of these processes. Overall, the disordered nature and multiple functions of W warrant further investigation to understand henipavirus pathogenesis and may reveal insights aiding the development of novel therapeutics.
Asunto(s)
Transporte Activo de Núcleo Celular/inmunología , Virus Hendra/metabolismo , Infecciones por Henipavirus/inmunología , Proteínas Intrínsecamente Desordenadas/metabolismo , Virus Nipah/metabolismo , Membrana Nuclear/metabolismo , Transducción de Señal/inmunología , Proteínas Virales/metabolismo , Infecciones por Henipavirus/metabolismo , Infecciones por Henipavirus/virología , Interacciones Microbiota-Huesped/inmunología , Humanos , Inmunidad Innata , Interferones/metabolismo , Proteínas Intrínsecamente Desordenadas/química , Receptores de Reconocimiento de Patrones/metabolismo , Proteínas Virales/químicaRESUMEN
Nipah virus (NiV) and Hendra virus (HeV), members of the Henipavirus genus in the Paramyxoviridae family, are recently emerged, highly lethal zoonotic pathogens. The NiV and HeV nonsegmented, negative-sense RNA genomes encode nine proteins, including the W protein. Expressed from the P gene through mRNA editing, W shares a common N-terminus with P and V but has a unique C-terminus. Expressed alone, W modulates innate immune responses by several mechanisms, and elimination of W from NiV alters the course of infection in experimentally infected ferrets. However, the specific host interactions that allow W to modulate innate immunity are incompletely understood. This study demonstrates that the NiV and HeV W proteins interact with all seven isoforms of the 14-3-3 family, regulatory molecules that preferentially bind phosphorylated target proteins to regulate a wide range of cellular functions. The interaction is dependent on the penultimate amino acid residue in the W sequence, a conserved, phosphorylated serine. The cocrystal structure of the W C-terminal binding motif with 14-3-3 provides only the second structure of a complex containing a mode III interactor, which is defined as a 14-3-3 interaction with a phosphoserine/phosphothreonine at the C-termini of the target protein. Transcriptomic analysis of inducible cell lines infected with an RNA virus and expressing either wild-type W or W lacking 14-3-3 binding, identifies new functions for W. These include the regulation of cellular metabolic processes, extracellular matrix organization, and apoptosis.IMPORTANCE Nipah virus (NiV) and Hendra virus (HeV), members of the Henipavirus genus, are recently emerged, highly lethal zoonotic pathogens that cause yearly outbreaks. NiV and HeV each encode a W protein that has roles in regulating host signaling pathways, including antagonism of the innate immune response. However, the mechanisms used by W to regulate these host responses are not clear. Here, characterization of the interaction of NiV and HeV W with 14-3-3 identifies modulation of 14-3-3-regulated host signaling pathways not previously associated with W, suggesting new avenues of research. The cocrystal structure of the NiV W:14-3-3 complex, as only the second structure of a 14-3-3 mode III interactor, provides further insight into this less-well-understood 14-3-3 binding motif.
Asunto(s)
Proteínas 14-3-3/metabolismo , Regulación de la Expresión Génica , Virus Hendra/metabolismo , Infecciones por Henipavirus/metabolismo , Virus Nipah/metabolismo , Proteínas Virales/metabolismo , Proteínas 14-3-3/genética , Células HEK293 , Virus Hendra/genética , Infecciones por Henipavirus/genética , Humanos , Virus Nipah/genética , Proteínas Virales/genéticaRESUMEN
The emergent zoonotic henipaviruses, Hendra, and Nipah are responsible for frequent and fatal disease outbreaks in domestic animals and humans. Specificity of henipavirus attachment glycoproteins (G) for highly species-conserved ephrin ligands underpins their broad host range and is associated with systemic and neurological disease pathologies. Here, we demonstrate that Cedar virus (CedV)-a related henipavirus that is ostensibly nonpathogenic-possesses an idiosyncratic entry receptor repertoire that includes the common henipaviral receptor, ephrin-B2, but, distinct from pathogenic henipaviruses, does not include ephrin-B3. Uniquely among known henipaviruses, CedV can use ephrin-B1 for cellular entry. Structural analyses of CedV-G reveal a key region of molecular specificity that directs ephrin-B1 utilization, while preserving a universal mode of ephrin-B2 recognition. The structural and functional insights presented uncover diversity within the known henipavirus receptor repertoire and suggest that only modest structural changes may be required to modulate receptor specificities within this group of lethal human pathogens.
Asunto(s)
Efrina-B1/metabolismo , Infecciones por Henipavirus/metabolismo , Henipavirus/fisiología , Proteínas Virales de Fusión/metabolismo , Internalización del Virus , Animales , Quirópteros/virología , Chlorocebus aethiops , Efrina-B1/genética , Efrina-B2/genética , Efrina-B2/metabolismo , Células HEK293 , Henipavirus/aislamiento & purificación , Infecciones por Henipavirus/virología , Humanos , Ligandos , Unión Proteica , Estructura Secundaria de Proteína , Receptores Virales/metabolismo , Transfección , Células VeroRESUMEN
Nipah virus (NiV) is a pathogenic paramyxovirus and zoononis with very high human fatality rates. Previous protein over-expression studies have shown that various mutations to the common N-terminal STAT1-binding motif of the NiV P, V, and W proteins affected the STAT1-binding ability of these proteins thus interfering with he JAK/STAT pathway and reducing their ability to inhibit type-I IFN signaling, but due to differing techniques it was unclear which amino acids were most important in this interaction or what impact this had on pathogenesis in vivo. We compared all previously described mutations in parallel and found the amino acid mutation Y116E demonstrated the greatest reduction in binding to STAT1 and the greatest reduction in interferon antagonism. A similar reduction in binding and activity was seen for a deletion of twenty amino acids constituting the described STAT1-binding domain. To investigate the contribution of this STAT1-binding motif in NiV-mediated disease, we produced rNiVs with complete deletion of the STAT1-binding motif or the Y116E mutation for ferret challenge studies (rNiVM-STAT1blind). Despite the reduced IFN inhibitory function, ferrets challenged with these rNiVM-STAT1blind mutants had a lethal, albeit altered, NiV-mediated disease course. These data, together with our previously published data, suggest that the major role of NiV P, V, and W in NiV-mediated disease in the ferret model are likely to be in the inhibition of viral recognition/innate immune signaling induction with a minor role for inhibition of IFN signaling.
Asunto(s)
Infecciones por Henipavirus/patología , Infecciones por Henipavirus/virología , Virus Nipah/fisiología , Fosfoproteínas/metabolismo , Factor de Transcripción STAT1/antagonistas & inhibidores , Proteínas Virales/metabolismo , Proteínas Estructurales Virales/metabolismo , Animales , Anticuerpos Neutralizantes/inmunología , Sitios de Unión , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Hurones , Infecciones por Henipavirus/metabolismo , Fosfoproteínas/genética , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/inmunología , Factor de Transcripción STAT1/metabolismo , Proteínas Virales/genética , Proteínas Estructurales Virales/genéticaRESUMEN
Nipah virus (NiV) and Hendra virus (HeV) are zoonotic henipaviruses (HNVs) responsible for outbreaks of encephalitis and respiratory illness with fatality rates of 50-100%. No vaccines or licensed therapeutics currently exist to protect humans against NiV or HeV. HNVs enter host cells by fusing the viral and cellular membranes via the concerted action of the attachment (G) and fusion (F) glycoproteins, the main targets of the humoral immune response. Here, we describe the isolation and humanization of a potent monoclonal antibody cross-neutralizing NiV and HeV. Cryo-electron microscopy, triggering and fusion studies show the antibody binds to a prefusion-specific quaternary epitope, conserved in NiV F and HeV F glycoproteins, and prevents membrane fusion and viral entry. This work supports the importance of the HNV prefusion F conformation for eliciting a robust immune response and paves the way for using this antibody for prophylaxis and post-exposure therapy with NiV- and HeV-infected individuals.
Asunto(s)
Anticuerpos Neutralizantes/farmacología , Antivirales/farmacología , Virus Hendra/efectos de los fármacos , Infecciones por Henipavirus/tratamiento farmacológico , Virus Nipah/efectos de los fármacos , Proteínas Virales de Fusión/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales Humanizados/farmacología , Células HEK293 , Virus Hendra/metabolismo , Infecciones por Henipavirus/metabolismo , Infecciones por Henipavirus/virología , Humanos , Modelos Moleculares , Virus Nipah/metabolismo , Proteínas Virales de Fusión/metabolismo , Internalización del Virus/efectos de los fármacosRESUMEN
Cedar virus (CedV) is a bat-borne henipavirus related to Nipah virus (NiV) and Hendra virus (HeV), zoonotic agents of fatal human disease. CedV receptor-binding protein (G) shares only â¼30% sequence identity with those of NiV and HeV, although they can all use ephrin-B2 as an entry receptor. We demonstrate that CedV also enters cells through additional B- and A-class ephrins (ephrin-B1, ephrin-A2, and ephrin-A5) and report the crystal structure of the CedV G ectodomain alone and in complex with ephrin-B1 or ephrin-B2. The CedV G receptor-binding site is structurally distinct from other henipaviruses, underlying its capability to accommodate additional ephrin receptors. We also show that CedV can enter cells through mouse ephrin-A1 but not human ephrin-A1, which differ by 1 residue in the key contact region. This is evidence of species specific ephrin receptor usage by a henipavirus, and implicates additional ephrin receptors in potential zoonotic transmission.
Asunto(s)
Efrina-B1/metabolismo , Efrina-B2/metabolismo , Efrina-B3/metabolismo , Infecciones por Henipavirus/virología , Henipavirus/fisiología , Receptores Virales/metabolismo , Proteínas del Envoltorio Viral/química , Animales , Fusión Celular , Efrina-B1/genética , Efrina-B2/genética , Efrina-B3/genética , Infecciones por Henipavirus/genética , Infecciones por Henipavirus/metabolismo , Humanos , Ratones , Mutación , Unión Proteica , Conformación Proteica , Receptores Virales/genética , Especificidad de la Especie , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Internalización del VirusRESUMEN
Hendra virus (HeV) is a zoonotic paramyxovirus that utilizes a trimeric fusion (F) protein within its lipid bilayer to mediate membrane merger with a cell membrane for entry. Previous HeV F studies showed that transmembrane domain (TMD) interactions are important for stabilizing the prefusion conformation of the protein prior to triggering. Thus, the current model for HeV F fusion suggests that modulation of TMD interactions is critical for initiation and completion of conformational changes that drive membrane fusion. HeV F constructs (T483C/V484C, V484C/N485C, and N485C/P486C) were generated with double cysteine substitutions near the N-terminal region of the TMD to study the effect of altered flexibility in this region. Oligomeric analysis showed that the double cysteine substitutions successfully promoted intersubunit disulfide bond formation in HeV F. Subsequent fusion assays indicated that the introduction of disulfide bonds in the mutants prohibited fusion events. Further testing confirmed that T483C/V484C and V484C/N485C were expressed at the cell surface at levels that would allow for fusion. Attempts to restore fusion with a reducing agent were unsuccessful, suggesting that the introduced disulfide bonds were likely buried in the membrane. Conformational analysis showed that T483C/V484C and V484C/N485C were able to bind a prefusion conformation-specific antibody prior to cell disruption, indicating that the introduced disulfide bonds did not significantly affect protein folding. This study is the first to report that TMD dissociation is required for HeV F fusogenic activity and strengthens our model for HeV fusion.IMPORTANCE The paramyxovirus Hendra virus (HeV) causes severe respiratory illness and encephalitis in humans. To develop therapeutics for HeV and related viral infections, further studies are needed to understand the mechanisms underlying paramyxovirus fusion events. Knowledge gained in studies of the HeV fusion (F) protein may be applicable to a broad span of enveloped viruses. In this study, we demonstrate that disulfide bonds introduced between the HeV F transmembrane domains (TMDs) block fusion. Depending on the location of these disulfide bonds, HeV F can still fold properly and bind a prefusion conformation-specific antibody prior to cell disruption. These findings support our current model for HeV membrane fusion and expand our knowledge of the TMD and its role in HeV F stability and fusion promotion.
Asunto(s)
Virus Hendra/metabolismo , Infecciones por Henipavirus/metabolismo , Proteínas Virales de Fusión/metabolismo , Secuencia de Aminoácidos/genética , Animales , Línea Celular , Chlorocebus aethiops , Virus Hendra/genética , Humanos , Fusión de Membrana/fisiología , Paramyxovirinae/metabolismo , Dominios Proteicos/genética , Pliegue de Proteína , Células Vero , Proteínas Virales de Fusión/genética , Internalización del VirusRESUMEN
Formation of cytoplasmic inclusion bodies (IBs) is a hallmark of infections with non-segmented negative-strand RNA viruses (order Mononegavirales). We show here that Nipah virus (NiV), a bat-derived highly pathogenic member of the Paramyxoviridae family, differs from mononegaviruses of the Rhabdo-, Filo- and Pneumoviridae families by forming two types of IBs with distinct localizations, formation kinetics, and protein compositions. IBs in the perinuclear region form rapidly upon expression of the nucleocapsid proteins. These IBperi are highly mobile and associate with the aggresome marker y-tubulin. IBperi can recruit unrelated overexpressed cytosolic proteins but do not contain the viral matrix (M) protein. Additionally, NiV forms an as yet undescribed IB population at the plasma membrane (IBPM) that is y-tubulin-negative but contains the M protein. Infection studies with recombinant NiV revealed that IBPM require the M protein for their formation, and most likely represent sites of NiV assembly and budding. The identification of this novel type of plasma membrane-associated IBs not only provides new insights into NiV biology and may open new avenues to develop novel antiviral approaches to treat these highly pathogenic viruses, it also provides a basis for a more detailed characterization of IBs and their role in virus assembly and replication in infections with other Mononegavirales.
Asunto(s)
Membrana Celular/virología , Infecciones por Henipavirus/virología , Cuerpos de Inclusión Viral/virología , Virus Nipah/patogenicidad , Proteínas de la Matriz Viral/metabolismo , Animales , Chlorocebus aethiops , Glicoproteínas/metabolismo , Infecciones por Henipavirus/metabolismo , Infecciones por Henipavirus/patología , Humanos , Cuerpos de Inclusión Viral/metabolismo , Cuerpos de Inclusión Viral/patología , Células Vero , Ensamble de Virus , Internalización del VirusRESUMEN
In 1998 an outbreak of fatal encephalitis among pig farm workers in Malaysia and Singapore led to the discovery of Nipah henipavirus (NiV), a novel paramyxovirus closely related to Hendra henipavirus with case fatality rates of nearly 40%. Following its initial emergence nearly annual outbreaks of NiV have occurred in Bangladesh with a different, NiV Bangladesh, genotype, where the role of pigs in its transmission remains unknown. The present study provides the first report on susceptibility of domestic pigs to NiV Bangladesh following experimental infection, characterizing acute and long-term phases of disease and pathogenesis. All pigs were successfully infected with NiV Bangladesh following oronasal inoculation, with viral shedding confirmed by a novel genotype-specific qRT-PCR in oral, nasal and rectal excretions and dissemination from the upper respiratory tract to the brain, lungs, and associated lymphatic tissues. Unlike previous NiV Malaysia findings in pigs, clinical signs were absent, viremia was undetectable throughout the study, and only low level neutralizing antibody titers were measured by 28/29 days post-NiV-B infection. Results obtained highlight the need for continued and enhanced NiV surveillance in pigs in endemic and at-risk regions, and raise questions regarding applicability of current serological assays to detect animals with previous NiV-B exposure.
Asunto(s)
Infecciones por Henipavirus , Virus Nipah/patogenicidad , Enfermedades de los Porcinos , Porcinos , Animales , Bangladesh/epidemiología , Infecciones por Henipavirus/epidemiología , Infecciones por Henipavirus/metabolismo , Infecciones por Henipavirus/virología , Porcinos/metabolismo , Porcinos/virología , Enfermedades de los Porcinos/epidemiología , Enfermedades de los Porcinos/metabolismo , Enfermedades de los Porcinos/virologíaRESUMEN
Ebola virus (EBOV) and Nipah virus (NiV) infection of humans can cause fatal disease and constitutes a public health threat. In contrast, EBOV and NiV infection of fruit bats, the putative (EBOV) or proven (NiV) natural reservoir, is not associated with disease, and it is currently unknown how these animals control the virus. The human interferon (IFN)-stimulated antiviral effector protein tetherin (CD317, BST-2) blocks release of EBOV- and NiV-like particles from cells and is counteracted by the EBOV glycoprotein (GP). In contrast, it is unknown whether fruit bat tetherin restricts virus infection and is susceptible to GP-driven antagonism. Here, we report the sequence of fruit bat tetherin and show that its expression is IFN stimulated and associated with strong antiviral activity. Moreover, we demonstrate that EBOV-GP antagonizes tetherin orthologues of diverse species but fails to efficiently counteract fruit bat tetherin in virus-like particle (VLP) release assays. However, unexpectedly, tetherin was dispensable for robust IFN-mediated inhibition of EBOV spread in fruit bat cells. Thus, the VLP-based model systems mimicking tetherin-mediated inhibition of EBOV release and its counteraction by GP seem not to adequately reflect all aspects of EBOV release from IFN-stimulated fruit bat cells, potentially due to differences in tetherin expression levels that could not be resolved by the present study. In contrast, tetherin expression was essential for IFN-dependent inhibition of NiV infection, demonstrating that IFN-induced fruit bat tetherin exerts antiviral activity and may critically contribute to control of NiV and potentially other highly virulent viruses in infected animals.IMPORTANCE Ebola virus and Nipah virus (EBOV and NiV) can cause fatal disease in humans. In contrast, infected fruit bats do not develop symptoms but can transmit the virus to humans. Why fruit bats but not humans control infection is largely unknown. Tetherin is an antiviral host cell protein and is counteracted by the EBOV glycoprotein in human cells. Here, employing model systems, we show that tetherin of fruit bats displays higher antiviral activity than human tetherin and is largely resistant against counteraction by the Ebola virus glycoprotein. Moreover, we demonstrate that induction of tetherin expression is critical for interferon-mediated inhibition of NiV but, for at present unknown reasons, not EBOV spread in fruit bat cells. Collectively, our findings identify tetherin as an antiviral effector of innate immune responses in fruit bats, which might allow these animals to control infection with NiV and potentially other viruses that cause severe disease in humans.
Asunto(s)
Antivirales/farmacología , Antígeno 2 del Estroma de la Médula Ósea/farmacología , Ebolavirus/efectos de los fármacos , Fiebre Hemorrágica Ebola/virología , Infecciones por Henipavirus/prevención & control , Virus Nipah/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Animales , Quirópteros , Fiebre Hemorrágica Ebola/metabolismo , Infecciones por Henipavirus/metabolismo , Infecciones por Henipavirus/virología , Humanos , Inmunidad Innata/efectos de los fármacos , Interferones/farmacología , Primates , Roedores , Liberación del VirusRESUMEN
Nipah virus (NiV) is a highly pathogenic paramyxovirus that causes lethal encephalitis in humans. We previously reported that the V protein, one of the three accessory proteins encoded by the P gene, is one of the key determinants of the pathogenesis of NiV in a hamster infection model. Satterfield B.A. et al. have also revealed that V protein is required for the pathogenicity of henipavirus in a ferret infection model. However, the complete functions of NiV V have not been clarified. In this study, we identified UBX domain-containing protein 1 (UBXN1), a negative regulator of RIG-I-like receptor signaling, as a host protein that interacts with NiV V. NiV V interacted with the UBX domain of UBXN1 via its proximal zinc-finger motif in the C-terminal domain. NiV V increased the level of UBXN1 protein by suppressing its proteolysis. Furthermore, NiV V suppressed RIG-I and MDA5-dependent interferon signaling by stabilizing UBXN1 and increasing the interaction between MAVS and UBXN1 in addition to directly interrupting the activation of MDA5. Our results suggest a novel molecular mechanism by which the induction of interferon is potentially suppressed by NiV V protein via UBXN1.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Infecciones por Henipavirus/metabolismo , Interferón beta/metabolismo , Virus Nipah/fisiología , Fosfoproteínas/metabolismo , Proteínas Estructurales Virales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Células HeLa , Infecciones por Henipavirus/virología , Humanos , Conformación Proteica , Estabilidad ProteicaRESUMEN
BACKGROUND: Hendra virus and Nipah virus are zoonotic viruses that have caused severe to fatal disease in livestock and human populations. The isolation of Cedar virus, a non-pathogenic virus species in the genus Henipavirus, closely-related to the highly pathogenic Hendra virus and Nipah virus offers an opportunity to investigate differences in pathogenesis and receptor tropism among these viruses. METHODS: We constructed full-length cDNA clones of Cedar virus from synthetic oligonucleotides and rescued two replication-competent, recombinant Cedar virus variants: a recombinant wild-type Cedar virus and a recombinant Cedar virus that expresses a green fluorescent protein from an open reading frame inserted between the phosphoprotein and matrix genes. Replication kinetics of both viruses and stimulation of the interferon pathway were characterized in vitro. Cellular tropism for ephrin-B type ligands was qualitatively investigated by microscopy and quantitatively by a split-luciferase fusion assay. RESULTS: Successful rescue of recombinant Cedar virus expressing a green fluorescent protein did not significantly affect virus replication compared to the recombinant wild-type Cedar virus. We demonstrated that recombinant Cedar virus stimulated the interferon pathway and utilized the established Hendra virus and Nipah virus receptor, ephrin-B2, but not ephrin-B3 to mediate virus entry. We further characterized virus-mediated membrane fusion kinetics of Cedar virus with the known henipavirus receptors ephrin-B2 and ephrin-B3. CONCLUSIONS: The recombinant Cedar virus platform may be utilized to characterize the determinants of pathogenesis across the henipaviruses, investigate their receptor tropisms, and identify novel pan-henipavirus antivirals. Moreover, these experiments can be conducted safely under BSL-2 conditions.
Asunto(s)
Efrina-B2/metabolismo , Infecciones por Henipavirus/virología , Henipavirus/fisiología , Receptores Virales/metabolismo , Fusión Celular , Línea Celular , Efecto Citopatogénico Viral , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Henipavirus/genética , Henipavirus/metabolismo , Henipavirus/patogenicidad , Infecciones por Henipavirus/metabolismo , Interferón Tipo I/genética , Pruebas de Neutralización , Unión Proteica , Recombinación Genética , Genética Inversa , Proteínas del Envoltorio Viral/metabolismo , Tropismo Viral , Internalización del Virus , Replicación ViralRESUMEN
Hendra virus (HeV) is a paramyxovirus that causes lethal disease in humans, for which no vaccine or antiviral agent is available. HeV V protein is central to pathogenesis through its ability to interact with cytoplasmic host proteins, playing key antiviral roles. Here we use immunoprecipitation, siRNA knockdown and confocal laser scanning microscopy to show that HeV V shuttles to and from the nucleus through specific host nuclear transporters. Spectroscopic and small angle X-ray scattering studies reveal HeV V undergoes a disorder-to-order transition upon binding to either importin α/ß1 or exportin-1/Ran-GTP, dependent on the V N-terminus. Importantly, we show that specific inhibitors of nuclear transport prevent interaction with host transporters, and reduce HeV infection. These findings emphasize the critical role of host-virus interactions in HeV infection, and potential use of compounds targeting nuclear transport, such as the FDA-approved agent ivermectin, as anti-HeV agents.
Asunto(s)
Virus Hendra/fisiología , Infecciones por Henipavirus/metabolismo , Infecciones por Henipavirus/virología , Interacciones Huésped-Patógeno , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Antivirales/química , Antivirales/farmacología , Núcleo Celular/metabolismo , Descubrimiento de Drogas , Técnicas de Silenciamiento del Gen , Virus Hendra/efectos de los fármacos , Infecciones por Henipavirus/genética , Humanos , Carioferinas/química , Carioferinas/genética , Carioferinas/metabolismo , Modelos Moleculares , Conformación Molecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Relación Estructura-Actividad , Proteínas Virales/química , Proteínas Virales/metabolismo , Proteína Exportina 1RESUMEN
Hendra and Nipah viruses (family Paramyxoviridae, genus Henipavirus) are zoonotic RNA viruses that cause lethal disease in humans and are designated as Biosafety Level 4 (BSL4) agents. Moreover, henipaviruses belong to the same group of viruses that cause disease more commonly in humans such as measles, mumps and respiratory syncytial virus. Due to the relatively recent emergence of the henipaviruses and the practical constraints of performing functional genomics studies at high levels of containment, our understanding of the henipavirus infection cycle is incomplete. In this chapter we describe recent loss-of-function (i.e. RNAi) functional genomics screens that shed light on the henipavirus-host interface at a genome-wide level. Further to this, we cross-reference RNAi results with studies probing host proteins targeted by henipavirus proteins, such as nuclear proteins and immune modulators. These functional genomics studies join a growing body of evidence demonstrating that nuclear and nucleolar host proteins play a crucial role in henipavirus infection. Furthermore these studies will underpin future efforts to define the role of nucleolar host-virus interactions in infection and disease.