RESUMO
Ebolavirus (EBOV) belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans. EBOV replication requires the activity of the viral polymerase complex, which includes the cofactor and Interferon antagonist VP35. We previously showed that the covalent ubiquitination of VP35 promotes virus replication by regulating interactions with the polymerase complex. In addition, VP35 can also interact non-covalently with ubiquitin (Ub); however, the function of this interaction is unknown. Here, we report that VP35 interacts with free (unanchored) K63-linked polyUb chains. Ectopic expression of Isopeptidase T (USP5), which is known to degrade unanchored polyUb chains, reduced VP35 association with Ub and correlated with diminished polymerase activity in a minigenome assay. Using computational methods, we modeled the VP35-Ub non-covalent interacting complex, identified the VP35-Ub interacting surface, and tested mutations to validate the interface. Docking simulations identified chemical compounds that can block VP35-Ub interactions leading to reduced viral polymerase activity. Treatment with the compounds reduced replication of infectious EBOV in cells and in vivo in a mouse model. In conclusion, we identified a novel role of unanchored polyUb in regulating Ebola virus polymerase function and discovered compounds that have promising anti-Ebola virus activity.
Assuntos
Ebolavirus , Doença pelo Vírus Ebola , Proteínas do Nucleocapsídeo , Ubiquitina , Replicação Viral , Animais , Humanos , Camundongos , Ebolavirus/genética , Ubiquitina/metabolismo , Proteínas Virais Reguladoras e Acessórias , Replicação Viral/genéticaRESUMO
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein substitution D614G became dominant during the coronavirus disease 2019 (COVID-19) pandemic1,2. However, the effect of this variant on viral spread and vaccine efficacy remains to be defined. Here we engineered the spike D614G substitution in the USA-WA1/2020 SARS-CoV-2 strain, and found that it enhances viral replication in human lung epithelial cells and primary human airway tissues by increasing the infectivity and stability of virions. Hamsters infected with SARS-CoV-2 expressing spike(D614G) (G614 virus) produced higher infectious titres in nasal washes and the trachea, but not in the lungs, supporting clinical evidence showing that the mutation enhances viral loads in the upper respiratory tract of COVID-19 patients and may increase transmission. Sera from hamsters infected with D614 virus exhibit modestly higher neutralization titres against G614 virus than against D614 virus, suggesting that the mutation is unlikely to reduce the ability of vaccines in clinical trials to protect against COVID-19, and that therapeutic antibodies should be tested against the circulating G614 virus. Together with clinical findings, our work underscores the importance of this variant in viral spread and its implications for vaccine efficacy and antibody therapy.
Assuntos
COVID-19/transmissão , COVID-19/virologia , Aptidão Genética , Mutação , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/genética , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/uso terapêutico , COVID-19/imunologia , Vacinas contra COVID-19/imunologia , Cricetinae , Modelos Animais de Doenças , Humanos , Pulmão/virologia , Masculino , Mesocricetus/virologia , Modelos Biológicos , Mucosa Nasal/virologia , Testes de Neutralização , Estabilidade Proteica , SARS-CoV-2/imunologia , SARS-CoV-2/patogenicidade , Técnicas de Cultura de Tecidos , Traqueia/virologia , Carga Viral , Vírion/química , Vírion/patogenicidade , Vírion/fisiologia , Replicação Viral/genéticaRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-a new coronavirus that has led to a worldwide pandemic1-has a furin cleavage site (PRRAR) in its spike protein that is absent in other group-2B coronaviruses2. To explore whether the furin cleavage site contributes to infection and pathogenesis in this virus, we generated a mutant SARS-CoV-2 that lacks the furin cleavage site (ΔPRRA). Here we report that replicates of ΔPRRA SARS-CoV-2 had faster kinetics, improved fitness in Vero E6 cells and reduced spike protein processing, as compared to parental SARS-CoV-2. However, the ΔPRRA mutant had reduced replication in a human respiratory cell line and was attenuated in both hamster and K18-hACE2 transgenic mouse models of SARS-CoV-2 pathogenesis. Despite reduced disease, the ΔPRRA mutant conferred protection against rechallenge with the parental SARS-CoV-2. Importantly, the neutralization values of sera from patients with coronavirus disease 2019 (COVID-19) and monoclonal antibodies against the receptor-binding domain of SARS-CoV-2 were lower against the ΔPRRA mutant than against parental SARS-CoV-2, probably owing to an increased ratio of particles to plaque-forming units in infections with the former. Together, our results demonstrate a critical role for the furin cleavage site in infection with SARS-CoV-2 and highlight the importance of this site for evaluating the neutralization activities of antibodies.
Assuntos
COVID-19/virologia , Furina/metabolismo , Mutação , SARS-CoV-2/genética , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/imunologia , COVID-19/patologia , COVID-19/fisiopatologia , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Feminino , Humanos , Pneumopatias/patologia , Pneumopatias/fisiopatologia , Pneumopatias/virologia , Masculino , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteólise , SARS-CoV-2/química , SARS-CoV-2/metabolismo , Serina Endopeptidases/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero , Replicação Viral/genéticaRESUMO
Nipah virus (NiV) is a highly lethal, zoonotic Henipavirus (HNV) that causes respiratory and neurological signs and symptoms in humans. Similar to other paramyxoviruses, HNVs mediate entry into host cells through the concerted actions of two surface glycoproteins: a receptor-binding protein (RBP) that mediates attachment and a fusion glycoprotein (F) that triggers fusion in an RBP-dependent manner. NiV uses ephrin-B2 (EFNB2) and ephrin-B3 (EFNB3) as entry receptors. Ghana virus (GhV), a novel HNV identified in a Ghanaian bat, uses EFNB2 but not EFNB3. In this study, we employ a structure-informed approach to identify receptor-interfacing residues and systematically introduce GhV-RBP residues into a NiV-RBP backbone to uncover the molecular determinants of EFNB3 usage. We reveal two regions that severely impair EFNB3 binding by NiV-RBP and EFNB3-mediated entry by NiV pseudotyped viral particles. Further analyses uncovered two-point mutations (NiVN557SGhV and NiVY581TGhV) pivotal for this phenotype. Moreover, we identify NiV interaction with Y120 of EFNB3 as important for the usage of this receptor. Beyond these EFNB3-related findings, we reveal two domains that restrict GhV binding of EFNB2, confirm the HNV-head as an immunodominant target for polyclonal and monoclonal antibodies, and describe putative epitopes for GhV- and NiV-specific monoclonal antibodies. Cumulatively, the work presented here generates useful reagents and tools that shed insight to residues important for NiV usage of EFNB3, reveal regions critical for GhV binding of EFNB2, and describe putative HNV antibody-binding epitopes. IMPORTANCE: Hendra virus and Nipah virus (NiV) are lethal, zoonotic Henipaviruses (HNVs) that cause respiratory and neurological clinical features in humans. Since their initial outbreaks in the 1990s, several novel HNVs have been discovered worldwide, including Ghana virus. Additionally, there is serological evidence of zoonotic transmission, lending way to concerns about future outbreaks. HNV infection of cells is mediated by the receptor-binding protein (RBP) and the Fusion protein (F). The work presented here identifies NiV RBP amino acids important for the usage of ephrin-B3 (EFNB3), a receptor highly expressed in neurons and predicted to be important for neurological clinical features caused by NiV. This study also characterizes epitopes recognized by antibodies against divergent HNV RBPs. Together, this sheds insight to amino acids critical for HNV receptor usage and antibody binding, which is valuable for future studies investigating determinants of viral pathogenesis and developing antibody therapies.
Assuntos
Infecções por Henipavirus , Henipavirus , Receptores Virais , Humanos , Aminoácidos/genética , Anticorpos Monoclonais/metabolismo , Proteínas de Transporte/metabolismo , Efrina-B3/genética , Efrina-B3/química , Efrina-B3/metabolismo , Epitopos/genética , Epitopos/metabolismo , Gana , Vírus Hendra/metabolismo , Henipavirus/classificação , Henipavirus/genética , Henipavirus/metabolismo , Mutagênese , Vírus Nipah/metabolismo , Proteínas do Envelope Viral/genética , Internalização do Vírus , Receptores Virais/metabolismoRESUMO
Ebola virus (EBOV) VP35 is a polyfunctional protein involved in viral genome packaging, viral polymerase function, and host immune antagonism. The mechanisms regulating VP35's engagement in different functions are not well-understood. We previously showed that the host E3 ubiquitin ligase TRIM6 ubiquitinates VP35 at lysine 309 (K309) to facilitate virus replication. However, how K309 ubiquitination regulates the function of VP35 as the viral polymerase co-factor and the precise stage(s) of the EBOV replication cycle that require VP35 ubiquitination are not known. Here, we generated recombinant EBOVs encoding glycine (G) or arginine (R) mutations at VP35/K309 (rEBOV-VP35/K309G/-R) and show that both mutations prohibit VP35/K309 ubiquitination. The K309R mutant retains dsRNA binding and efficient type-I Interferon (IFN-I) antagonism due to the basic residue conservation. The rEBOV-VP35/K309G mutant loses the ability to efficiently antagonize the IFN-I response, while the rEBOV-VP35/K309R mutant's suppression is enhanced. The replication of both mutants was significantly attenuated in both IFN-competent and -deficient cells due to impaired interactions with the viral polymerase. The lack of ubiquitination on VP35/K309 or TRIM6 deficiency disrupts viral transcription with increasing severity along the transcriptional gradient. This disruption of the transcriptional gradient results in unbalanced viral protein production, including reduced synthesis of the viral transcription factor VP30. In addition, lack of ubiquitination on K309 results in enhanced interactions with the viral nucleoprotein and premature nucleocapsid packaging, leading to dysregulation of virus assembly. Overall, we identified a novel role of VP35 ubiquitination in coordinating viral transcription and assembly.
Assuntos
Ebolavirus , Doença pelo Vírus Ebola , Ebolavirus/metabolismo , Humanos , Lisina/genética , Lisina/metabolismo , Proteínas do Nucleocapsídeo/metabolismo , Ubiquitinação , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/metabolismo , Transcrição ViralRESUMO
Rift Valley fever virus (RVFV), an emerging arboviral and zoonotic bunyavirus, causes severe disease in livestock and humans. Here, we report the isolation of a panel of monoclonal antibodies (mAbs) from the B cells of immune individuals following natural infection in Kenya or immunization with MP-12 vaccine. The B cell responses of individuals who were vaccinated or naturally infected recognized similar epitopes on both Gc and Gn proteins. The Gn-specific mAbs and two mAbs that do not recognize either monomeric Gc or Gn alone but recognized the hetero-oligomer glycoprotein complex (Gc+Gn) when Gc and Gn were coexpressed exhibited potent neutralizing activities in vitro, while Gc-specific mAbs exhibited relatively lower neutralizing capacity. The two Gc+Gn-specific mAbs and the Gn domain A-specific mAbs inhibited RVFV fusion to cells, suggesting that mAbs can inhibit the exposure of the fusion loop in Gc, a class II fusion protein, and thus prevent fusion by an indirect mechanism without direct fusion loop contact. Competition-binding analysis with coexpressed Gc/Gn and mutagenesis library screening indicated that these mAbs recognize four major antigenic sites, with two sites of vulnerability for neutralization on Gn. In experimental models of infection in mice, representative mAbs recognizing three of the antigenic sites reduced morbidity and mortality when used at a low dose in both prophylactic and therapeutic settings. This study identifies multiple candidate mAbs that may be suitable for use in humans against RVFV infection and highlights fusion inhibition against bunyaviruses as a potential contributor to potent antibody-mediated neutralization.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vírus da Febre do Vale do Rift/imunologia , Proteínas Virais de Fusão/imunologia , Animais , Anticorpos Monoclonais/imunologia , Linhagem Celular Tumoral , Células Cultivadas , Chlorocebus aethiops , Epitopos/química , Epitopos/imunologia , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Células Vero , Proteínas Virais de Fusão/químicaRESUMO
The common marmoset (Callithrix jacchus) is increasingly recognized as an ideal nonhuman primate (NHP) at high biocontainment due to its smaller size and relative ease of handling. Here, we evaluated the susceptibility and pathogenesis of Nipah virus Bangladesh strain (NiVB) infection in marmosets at biosafety level 4. Infection via the intranasal and intratracheal route resulted in fatal disease in all 4 infected marmosets. Three developed pulmonary edema and hemorrhage as well as multifocal hemorrhagic lymphadenopathy, while 1 recapitulated neurologic clinical manifestations and cardiomyopathy on gross pathology. Organ-specific innate and inflammatory responses were characterized by RNA sequencing in 6 different tissues from infected and control marmosets. Notably, a unique transcriptome was revealed in the brainstem of the marmoset exhibiting neurological signs. Our results provide a more comprehensive understanding of NiV pathogenesis in an accessible and novel NHP model, closely reflecting clinical disease as observed in NiV patients.
Assuntos
Infecções por Henipavirus , Vírus Nipah , Edema Pulmonar , Animais , Callithrix , BangladeshRESUMO
Interferons (IFN) are essential antiviral cytokines that establish the cellular antiviral state through upregulation of hundreds of interferon-stimulated genes (ISGs), most of which have uncharacterized functions and mechanisms. We identified cholesterol-25-hydroxylase (CH25H) as a broadly antiviral ISG. CH25H converts cholesterol to a soluble antiviral factor, 25-hydroxycholesterol (25HC). 25HC treatment in cultured cells broadly inhibited growth of enveloped viruses including VSV, HSV, HIV, and MHV68 and acutely pathogenic EBOV, RVFV, RSSEV, and Nipah viruses under BSL4 conditions. It suppressed viral growth by blocking membrane fusion between virus and cell. In animal models, Ch25h-deficient mice were more susceptible to MHV68 lytic infection. Moreover, administration of 25HC in humanized mice suppressed HIV replication and reversed T cell depletion. Thus, our studies demonstrate a unique mechanism by which IFN achieves its antiviral state through the production of a natural oxysterol to inhibit viral entry and implicate membrane-modifying oxysterols as potential antiviral therapeutics.
Assuntos
Antivirais/farmacologia , Hidroxicolesteróis/metabolismo , Interferons/farmacologia , Esteroide Hidroxilases/metabolismo , Internalização do Vírus/efeitos dos fármacos , Animais , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/virologia , Vírus de DNA/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hidroxicolesteróis/farmacologia , Fusão de Membrana/efeitos dos fármacos , Camundongos , Camundongos Knockout , Vírus de RNA/efeitos dos fármacos , Esteroide Hidroxilases/genética , Proteínas Virais/metabolismoRESUMO
Several members of the tripartite motif (TRIM) family of E3 ubiquitin ligases regulate immune pathways, including the antiviral type I interferon (IFN-I) system. Previously, we demonstrated that TRIM6 is involved in IFN-I induction and signaling. In the absence of TRIM6, optimal IFN-I signaling is reduced, allowing increased replication of interferon-sensitive viruses. Despite having evolved numerous mechanisms to restrict the vertebrate host's IFN-I response, West Nile virus (WNV) replication is sensitive to pretreatment with IFN-I. However, the regulators and products of the IFN-I pathway that are important in regulating WNV replication are incompletely defined. Consistent with WNV's sensitivity to IFN-I, we found that in TRIM6 knockout (TRIM6-KO) A549 cells, WNV replication is significantly increased and IFN-I induction and signaling are impaired compared to wild-type (wt) cells. IFN-ß pretreatment was more effective in protecting against subsequent WNV infection in wt cells than TRIM6-KO, indicating that TRIM6 contributes to the establishment of an IFN-induced antiviral response against WNV. Using next-generation sequencing, we identified VAMP8 as a potential factor involved in this TRIM6-mediated antiviral response. VAMP8 knockdown resulted in reduced JAK1 and STAT1 phosphorylation and impaired induction of several interferon-stimulated genes (ISGs) following WNV infection or IFN-ß treatment. Furthermore, VAMP8-mediated STAT1 phosphorylation required the presence of TRIM6. Therefore, the VAMP8 protein is a novel regulator of IFN-I signaling, and its expression and function are dependent on TRIM6 activity. Overall, these results provide evidence that TRIM6 contributes to the antiviral response against WNV and identify VAMP8 as a novel regulator of the IFN-I system.IMPORTANCE WNV is a mosquito-borne flavivirus that poses a threat to human health across large discontinuous areas throughout the world. Infection with WNV results in febrile illness, which can progress to severe neurological disease. Currently, there are no approved treatment options to control WNV infection. Understanding the cellular immune responses that regulate viral replication is important in diversifying the resources available to control WNV. Here, we show that the elimination of TRIM6 in human cells results in an increase in WNV replication and alters the expression and function of other components of the IFN-I pathway through VAMP8. Dissecting the interactions between WNV and host defenses both informs basic molecular virology and promotes the development of host- and virus-targeted antiviral strategies.
Assuntos
Imunidade Inata , Interferon Tipo I/imunologia , Proteínas R-SNARE/imunologia , Proteínas com Motivo Tripartido/imunologia , Ubiquitina-Proteína Ligases/imunologia , Replicação Viral/imunologia , Febre do Nilo Ocidental/imunologia , Vírus do Nilo Ocidental/fisiologia , Células A549 , Deleção de Genes , Células HEK293 , Humanos , Janus Quinase 1/genética , Janus Quinase 1/imunologia , Fosforilação/genética , Fosforilação/imunologia , Proteínas R-SNARE/genética , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/imunologia , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/genética , Replicação Viral/genética , Febre do Nilo Ocidental/genética , Febre do Nilo Ocidental/patologiaRESUMO
We identified and explored the structure-activity relationship (SAR) of a novel heterocyclic chemical series of arenavirus cell entry inhibitors. Optimized lead compounds, including diphenyl-substituted imidazo[1,2-a]pyridines, benzimidazoles, and benzotriazoles exhibited low to sub-nanomolar potency against both pseudotyped and infectious Old and New World arenaviruses, attractive metabolic stability in human and most nonhuman liver microsomes as well as a lack of hERG K + channel or CYP enzyme inhibition. Moreover, the straightforward synthesis of several lead compounds (e.g., the simple high yield 3-step synthesis of imidazo[1,2-a]pyridine 37) could provide a cost-effective broad-spectrum arenavirus therapeutic that may help to minimize the cost-prohibitive burdens associated with treatments for emerging viruses in economically challenged geographical settings.
Assuntos
Antivirais/farmacologia , Arenavirus/efeitos dos fármacos , Descoberta de Drogas , Compostos Heterocíclicos/farmacologia , Proteínas do Envelope Viral/antagonistas & inibidores , Antivirais/síntese química , Antivirais/química , Arenavirus/metabolismo , Relação Dose-Resposta a Droga , Compostos Heterocíclicos/síntese química , Compostos Heterocíclicos/química , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-Atividade , Proteínas do Envelope Viral/metabolismoRESUMO
Enteric viruses exploit bacterial components, including lipopolysaccharides (LPS) and peptidoglycan (PG), to facilitate infection in humans. Because of their origin in the bat enteric system, we wondered if severe acute respiratory syndrome coronavirus (SARS-CoV) or Middle East respiratory syndrome CoV (MERS-CoV) also use bacterial components to modulate infectivity. To test this question, we incubated CoVs with LPS and PG and evaluated infectivity, finding no change following LPS treatment. However, PG from Bacillus subtilis reduced infection >10,000-fold, while PG from other bacterial species failed to recapitulate this. Treatment with an alcohol solvent transferred inhibitory activity to the wash, and mass spectrometry revealed surfactin, a cyclic lipopeptide antibiotic, as the inhibitory compound. This antibiotic had robust dose- and temperature-dependent inhibition of CoV infectivity. Mechanistic studies indicated that surfactin disrupts CoV virion integrity, and surfactin treatment of the virus inoculum ablated infection in vivo Finally, similar cyclic lipopeptides had no effect on CoV infectivity, and the inhibitory effect of surfactin extended broadly to enveloped viruses, including influenza, Ebola, Zika, Nipah, chikungunya, Una, Mayaro, Dugbe, and Crimean-Congo hemorrhagic fever viruses. Overall, our results indicate that peptidoglycan-associated surfactin has broad viricidal activity and suggest that bacteria by-products may negatively modulate virus infection.IMPORTANCE In this article, we consider a role for bacteria in shaping coronavirus infection. Taking cues from studies of enteric viruses, we initially investigated how bacterial surface components might improve CoV infection. Instead, we found that peptidoglycan-associated surfactin is a potent viricidal compound that disrupts virion integrity with broad activity against enveloped viruses. Our results indicate that interactions with commensal bacterial may improve or disrupt viral infections, highlighting the importance of understanding these microbial interactions and their implications for viral pathogenesis and treatment.
Assuntos
Lipopeptídeos/farmacologia , Peptídeos Cíclicos/farmacologia , Peptidoglicano/metabolismo , Vírus de RNA/efeitos dos fármacos , Animais , Linhagem Celular , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Flaviviridae/efeitos dos fármacos , Lipopeptídeos/imunologia , Lipopeptídeos/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Peptídeos Cíclicos/imunologia , Peptídeos Cíclicos/metabolismo , Peptidoglicano/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/metabolismo , Síndrome Respiratória Aguda Grave/virologia , Células Vero , Viroses/metabolismoRESUMO
Quinacrine hydrochloride is a small-molecule, orally bioavailable drug that has been used clinically as an antimalarial and for many other applications. A machine learning model trained on Ebola virus (EBOV) screening data identified quinacrine as a potent (nanomolar) in vitro inhibitor. In the current study, quinacrine 25 mg/kg was shown to protect 70% of mice (statistically significant) from a lethal challenge with mouse-adapted EBOV with once-daily intraperitoneal dosing for 8 days.
Assuntos
Antimaláricos/farmacologia , Antivirais/farmacologia , Reposicionamento de Medicamentos , Ebolavirus/efeitos dos fármacos , Doença pelo Vírus Ebola/tratamento farmacológico , Quinacrina/farmacologia , Animais , Células CACO-2 , Chlorocebus aethiops , Modelos Animais de Doenças , Ebolavirus/crescimento & desenvolvimento , Células HeLa , Doença pelo Vírus Ebola/mortalidade , Doença pelo Vírus Ebola/patologia , Doença pelo Vírus Ebola/virologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Análise de Sobrevida , Tilorona/farmacologia , Células Vero , Carga Viral/efeitos dos fármacosRESUMO
The filoviruses Marburg virus (MARV) and Ebola virus (EBOV) cause hemorrhagic fever in humans and nonhuman primates, with high case fatality rates. MARV VP30 is known to be phosphorylated and to interact with nucleoprotein (NP), but its role in regulation of viral transcription is disputed. Here, we analyzed phosphorylation of VP30 by mass spectrometry, which resulted in identification of multiple phosphorylated amino acids. Modeling the full-length three-dimensional structure of VP30 and mapping the identified phosphorylation sites showed that all sites lie in disordered regions, mostly in the N-terminal domain of the protein. Minigenome analysis of the identified phosphorylation sites demonstrated that phosphorylation of a cluster of amino acids at positions 46 through 53 inhibits transcription. To test the effect of VP30 phosphorylation on its interaction with other MARV proteins, coimmunoprecipitation analyses were performed. They demonstrated the involvement of VP30 phosphorylation in interaction with two other proteins of the MARV ribonucleoprotein complex, NP and VP35. To identify the role of protein phosphatase 1 (PP1) in the identified effects, a small molecule, 1E7-03, targeting a noncatalytic site of the enzyme that previously was shown to increase EBOV VP30 phosphorylation was used. Treatment of cells with 1E7-03 increased phosphorylation of VP30 at a cluster of phosphorylated amino acids from Ser-46 to Thr-53, reduced transcription of MARV minigenome, enhanced binding to NP and VP35, and dramatically reduced replication of infectious MARV particles. Thus, MARV VP30 phosphorylation can be targeted for development of future antivirals such as PP1-targeting compounds. IMPORTANCE The largest outbreak of MARV occurred in Angola in 2004 to 2005 and had a 90% case fatality rate. There are no approved treatments available for MARV. Development of antivirals as therapeutics requires a fundamental understanding of the viral life cycle. Because of the close similarity of MARV to another member of Filoviridae family, EBOV, it was assumed that the two viruses have similar mechanisms of regulation of transcription and replication. Here, characterization of the role of VP30 and its phosphorylation sites in transcription of the MARV genome demonstrated differences from those of EBOV. The identified phosphorylation sites appeared to inhibit transcription and appeared to be involved in interaction with both NP and VP35 ribonucleoproteins. A small molecule targeting PP1 inhibited transcription of the MARV genome, effectively suppressing replication of the viral particles. These data demonstrate the possibility developing antivirals based on compounds targeting PP1.
Assuntos
Marburgvirus/crescimento & desenvolvimento , Nucleoproteínas/metabolismo , Proteínas Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Replicação Viral/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Chlorocebus aethiops , Genoma Viral/genética , Células HEK293 , Humanos , Indóis/farmacologia , Marburgvirus/genética , Espectrometria de Massas , Fosforilação , RNA Viral/genética , Transcrição Gênica/genética , Ureia/análogos & derivados , Ureia/farmacologia , Células Vero , Proteínas Virais/genéticaRESUMO
Old World (Africa) and New World (South America) arenaviruses are associated with human hemorrhagic fevers. Efforts to develop small molecule therapeutics have yielded several chemical series including the 4-acyl-1,6-dialkylpiperazin-2-ones. Herein, we describe an extensive exploration of this chemotype. In initial Phase I studies, R1 and R4 scanning libraries were assayed to identify potent substituents against Old World (Lassa) virus. In subsequent Phase II studies, R6 substituents and iterative R1, R4 and R6 substituent combinations were evaluated to obtain compounds with improved Lassa and New World (Machupo, Junin, and Tacaribe) arenavirus inhibitory activity, in vitro human liver microsome metabolic stability and aqueous solubility.
Assuntos
Antivirais/farmacologia , Arenavirus/efeitos dos fármacos , Piperazinas/farmacologia , Proteínas do Envelope Viral/antagonistas & inibidores , Antivirais/síntese química , Antivirais/química , Arenavirus/metabolismo , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Estrutura Molecular , Piperazinas/síntese química , Piperazinas/química , Relação Estrutura-Atividade , Proteínas do Envelope Viral/metabolismoRESUMO
Marburg virus (MARV; family Filoviridae) causes sporadic outbreaks of Marburg hemorrhagic fever in sub-Saharan Africa with case fatality rates reaching 90%. Wild-type filoviruses, including MARV and the closely related Ebola virus, are unable to suppress the type I interferon response in rodents, and therefore require adaptation of the viruses to cause disease in immunocompetent animals. In the current study, we demonstrate that STAT2 knockout Syrian hamsters are susceptible to infection with different wild-type MARV variants. MARV Musoke causes a robust and systemic infection resulting in lethal disease. Histopathological findings share features similar to those observed in human patients and other animal models of filovirus infection. Reverse-transcription polymerase chain reaction analysis of host transcripts shows a dysregulation of the innate immune response. Our results demonstrate that the STAT2 knockout hamster represents a novel small animal model of severe MARV infection and disease without the requirement for virus adaptation.
Assuntos
Doença do Vírus de Marburg/etiologia , Fator de Transcrição STAT2/fisiologia , Animais , Cricetinae , Citocinas/biossíntese , Modelos Animais de Doenças , Suscetibilidade a Doenças , Feminino , Masculino , Doença do Vírus de Marburg/imunologia , Doença do Vírus de Marburg/patologiaRESUMO
Background: Nipah virus (NiV) is a paramyxovirus (genus Henipavirus) that can cause severe respiratory illness and encephalitis in humans. Transmission occurs through consumption of NiV-contaminated foods, and contact with NiV-infected animals or human body fluids. However, it is unclear whether aerosols derived from aforesaid sources or others also contribute to transmission, and current knowledge on NiV-induced pathogenicity after small-particle aerosol exposure is still limited. Methods: Infectivity, pathogenicity, and real-time dissemination of aerosolized NiV in Syrian hamsters was evaluated using NiV-Malaysia (NiV-M) and/or its recombinant expressing firefly luciferase (rNiV-FlucNP). Results: Both viruses had an equivalent pathogenicity in hamsters, which developed respiratory and neurological symptoms of disease, similar to using intranasal route, with no direct correlations to the dose. We showed that virus replication was predominantly initiated in the lower respiratory tract and, although delayed, also intensely in the oronasal cavity and possibly the brain, with gradual increase of signal in these regions until at least day 5-6 postinfection. Conclusion: Hamsters infected with small-particle aerosolized NiV undergo similar clinical manifestations of the disease as previously described using liquid inoculum, and exhibit histopathological lesions consistent with NiV patient reports. NiV droplets could therefore play a role in transmission by close contact.
Assuntos
Aerossóis/administração & dosagem , Infecções por Henipavirus , Vírus Nipah/patogenicidade , Administração por Inalação , Animais , Cricetinae , Modelos Animais de Doenças , Infecções por Henipavirus/diagnóstico por imagem , Infecções por Henipavirus/patologia , Infecções por Henipavirus/transmissão , Infecções por Henipavirus/virologia , Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismo , Pulmão/diagnóstico por imagem , Pulmão/patologia , Pulmão/virologia , Mesocricetus , Imagem Óptica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Tilorone dihydrochloride (tilorone) is a small-molecule, orally bioavailable drug that is used clinically as an antiviral outside the United States. A machine-learning model trained on anti-Ebola virus (EBOV) screening data previously identified tilorone as a potent in vitro EBOV inhibitor, making it a candidate for the treatment of Ebola virus disease (EVD). In the present study, a series of in vitro ADMET (absorption, distribution, metabolism, excretion, toxicity) assays demonstrated the drug has excellent solubility, high Caco-2 permeability, was not a P-glycoprotein substrate, and had no inhibitory activity against five human CYP450 enzymes (3A4, 2D6, 2C19, 2C9, and 1A2). Tilorone was shown to have 52% human plasma protein binding with excellent plasma stability and a mouse liver microsome half-life of 48 min. Dose range-finding studies in mice demonstrated a maximum tolerated single dose of 100 mg/kg of body weight. A pharmacokinetics study in mice at 2- and 10-mg/kg dose levels showed that the drug is rapidly absorbed, has dose-dependent increases in maximum concentration of unbound drug in plasma and areas under the concentration-time curve, and has a half-life of approximately 18 h in both males and females, although the exposure was â¼2.5-fold higher in male mice. Tilorone doses of 25 and 50 mg/kg proved efficacious in protecting 90% of mice from a lethal challenge with mouse-adapted with once-daily intraperitoneal (i.p.) dosing for 8 days. A subsequent study showed that 30 mg/kg/day of tilorone given i.p. starting 2 or 24 h postchallenge and continuing through day 7 postinfection was fully protective, indicating promising activity for the treatment of EVD.
Assuntos
Ebolavirus/efeitos dos fármacos , Doença pelo Vírus Ebola/tratamento farmacológico , Tilorona/farmacologia , Animais , Antivirais/farmacologia , Células CACO-2 , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microssomos Hepáticos/efeitos dos fármacosRESUMO
BACKGROUND: La Crosse virus (LACV) causes pediatric encephalitis in the USA. LACV induces severe inflammation in the central nervous system, but the recruitment of inflammatory cells is poorly understood. A deeper understanding of LACV-induced neural pathology is needed in order to develop treatment options. However, there is a severe limitation of relevant human neuronal cell models of LACV infection. METHODS: We utilized human neural stem cell (hNSC)-derived neuron/astrocyte co-cultures to study LACV infection in disease-relevant primary cells. hNSCs were differentiated into neurons and astrocytes and infected with LACV. To characterize susceptibility and responses to infection, we measured viral titers and levels of viral RNA, performed immunofluorescence analysis to determine the cell types infected, performed apoptosis and cytotoxicity assays, and evaluated cellular responses to infection using qRT-PCR and Bioplex assays. RESULTS: hNSC-derived neuron/astrocyte co-cultures were susceptible to LACV infection and displayed apoptotic responses as reported in previous in vitro and in vivo studies. Neurons and astrocytes are both targets of LACV infection, with neurons becoming the predominant target later in infection possibly due to astrocytic responses to IFN. Additionally, neuron/astrocyte co-cultures responded to LACV infection with strong proinflammatory cytokine, chemokine, as well as MMP-2, MMP-7, and TIMP-1 responses. CONCLUSIONS: hNSC-derived neuron/astrocyte co-cultures reproduce key aspects of LACV infection in humans and mice and are useful models to study encephalitic viruses. Specifically, we show astrocytes to be susceptible to LACV infection and that neurons and astrocytes are important drivers of the inflammatory responses seen in LACV infection through the production of proinflammatory cytokines and chemokines.
Assuntos
Astrócitos/fisiologia , Citocinas/metabolismo , Vírus La Crosse/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/citologia , Neurônios/fisiologia , Neurônios/virologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Astrócitos/efeitos dos fármacos , Astrócitos/virologia , Células Cultivadas , Técnicas de Cocultura , Citocinas/genética , Regulação da Expressão Gênica/fisiologia , Humanos , Marcação In Situ das Extremidades Cortadas , Proteínas do Tecido Nervoso/genética , Neurônios/efeitos dos fármacos , Poli I-C/farmacologia , RNA Mensageiro , Estaurosporina/metabolismo , Fatores de Tempo , Replicação Viral/fisiologiaRESUMO
Ebola virus (EBOV), a member of the Filoviridae family, is a highly pathogenic virus that causes severe hemorrhagic fever in humans and is responsible for epidemics throughout sub-Saharan, central, and West Africa. The EBOV genome encodes VP35, an important viral protein involved in virus replication by acting as an essential cofactor of the viral polymerase as well as a potent antagonist of the host antiviral type I interferon (IFN-I) system. By using mass spectrometry analysis and coimmunoprecipitation assays, we show here that VP35 is ubiquitinated on lysine 309 (K309), a residue located on its IFN antagonist domain. We also found that VP35 interacts with TRIM6, a member of the E3-ubiquitin ligase tripartite motif (TRIM) family. We recently reported that TRIM6 promotes the synthesis of unanchored K48-linked polyubiquitin chains, which are not covalently attached to any protein, to induce efficient antiviral IFN-I-mediated responses. Consistent with this notion, VP35 also associated noncovalently with polyubiquitin chains and inhibited TRIM6-mediated IFN-I induction. Intriguingly, we also found that TRIM6 enhances EBOV polymerase activity in a minigenome assay and TRIM6 knockout cells have reduced replication of infectious EBOV, suggesting that VP35 hijacks TRIM6 to promote EBOV replication through ubiquitination. Our work provides evidence that TRIM6 is an important host cellular factor that promotes EBOV replication, and future studies will focus on whether TRIM6 could be targeted for therapeutic intervention against EBOV infection.IMPORTANCE EBOV belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans and other mammals with high mortality rates (40 to 90%). Because of its high pathogenicity and lack of licensed antivirals and vaccines, EBOV is listed as a tier 1 select-agent risk group 4 pathogen. An important mechanism for the severity of EBOV infection is its suppression of innate immune responses. The EBOV VP35 protein contributes to pathogenesis, because it serves as an essential cofactor of the viral polymerase as well as a potent antagonist of innate immunity. However, how VP35 function is regulated by host cellular factors is poorly understood. Here, we report that the host E3-ubiquitin ligase TRIM6 promotes VP35 ubiquitination and is important for efficient virus replication. Therefore, our study identifies a new host factor, TRIM6, as a potential target in the development of antiviral drugs against EBOV.