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1.
Emerg Microbes Infect ; 8(1): 1511-1523, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31631785

RESUMO

Interferons (IFNs) control viral infections by inducing expression of IFN-stimulated genes (ISGs) that restrict distinct steps of viral replication. We report herein that gamma-interferon-inducible lysosomal thiol reductase (GILT), a lysosome-associated ISG, restricts the infectious entry of selected enveloped RNA viruses. Specifically, we demonstrated that GILT was constitutively expressed in lung epithelial cells and fibroblasts and its expression could be further induced by type II interferon. While overexpression of GILT inhibited the entry mediated by envelope glycoproteins of SARS coronavirus (SARS-CoV), Ebola virus (EBOV) and Lassa fever virus (LASV), depletion of GILT enhanced the entry mediated by these viral envelope glycoproteins. Furthermore, mutations that impaired the thiol reductase activity or disrupted the N-linked glycosylation, a posttranslational modification essential for its lysosomal localization, largely compromised GILT restriction of viral entry. We also found that the induction of GILT expression reduced the level and activity of cathepsin L, which is required for the entry of these RNA viruses in lysosomes. Our data indicate that GILT is a novel antiviral ISG that specifically inhibits the entry of selected enveloped RNA viruses in lysosomes via disruption of cathepsin L metabolism and function and may play a role in immune control and pathogenesis of these viruses.


Assuntos
Ebolavirus/fisiologia , Doença pelo Vírus Ebola/imunologia , Febre Lassa/imunologia , Vírus Lassa/fisiologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/imunologia , Vírus da SARS/fisiologia , Síndrome Respiratória Aguda Grave/imunologia , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus , Catepsina L/genética , Catepsina L/imunologia , Linhagem Celular , Ebolavirus/genética , Doença pelo Vírus Ebola/genética , Doença pelo Vírus Ebola/virologia , Humanos , Febre Lassa/genética , Febre Lassa/virologia , Vírus Lassa/genética , Lisossomos/genética , Lisossomos/imunologia , Lisossomos/virologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Vírus da SARS/genética , Síndrome Respiratória Aguda Grave/genética , Síndrome Respiratória Aguda Grave/virologia , Proteínas do Envelope Viral/genética , Replicação Viral
3.
Int J Infect Dis ; 87: 15-20, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31357056

RESUMO

OBJECTIVES: Lassa fever (LF) causes annual outbreaks in endemic regions with high mortality of symptomatic patients. Ribavirin is recommended as standard treatment for LF in national and international guidelines but the evidence base for this recommendation has been questioned recently. METHODS: We conducted a systematic review and included 6 studies providing efficacy data of ribavirin treatment for LF (PROSPERO protocol CRD42018103994). RESULTS: Besides retrospective case series, the evidence mostly relies on a single prospective clinical trial with critical risk of bias. In this trial, LF associated mortality is reduced for patients with elevated aspartate aminotransferase (AST) when treated with ribavirin (OR 0.41, 95% CI 0.23-0.73), while mortality is higher for patients without elevated AST (OR 2.37, 95% CI 1.07-5.25). CONCLUSIONS: Based on the available data, current treatment guidelines may therefore put patients with mild LF at increased risk of death. The role of ribavirin in the treatment of LF requires urgent reassessment.


Assuntos
Febre Lassa/tratamento farmacológico , Vírus Lassa/efeitos dos fármacos , Ribavirina/uso terapêutico , Aspartato Aminotransferases/metabolismo , Ensaios Clínicos como Assunto , Humanos , Febre Lassa/enzimologia , Febre Lassa/mortalidade , Febre Lassa/virologia , Vírus Lassa/fisiologia , Estudos Prospectivos , Estudos Retrospectivos
4.
BMC Infect Dis ; 19(1): 606, 2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31291900

RESUMO

BACKGROUND: Infectious disease prevention and control strategies require a coordinated, transnational approach. To establish core capacities of the International Health Regulations (IHR), the World Health Organization (WHO) developed the Integrated Diseases Surveillance and Response (IDSR) strategy. Epidemic-prone Lassa fever, caused by Lassa virus, is an endemic disease in the West African countries of Ghana, Guinea, Mali, Benin, Liberia, Sierra Leone, Togo and Nigeria. It's one of the major public health threats in these countries. Here it is reported an epidemiological investigation of a cross-border case of Lassa fever, which demonstrated the importance of strengthened capacities of IHR and IDSR. CASE PRESENTATION: On January 9th, 2018 a 35-year-old Guinean woman with fever, neck pain, body pain, and vomiting went to a hospital in Ganta, Liberia. Over the course of her illness, the case visited various health care facilities in both Liberia and Guinea. A sample collected on January 10th was tested positive for Lassa virus by RT-PCR in a Liberian laboratory. The Guinean Ministry of Health (MoH) was officially informed by WHO Country Office for Guinea and for Liberia. CONCLUSION: This case report revealed how an epidemic-prone disease such as Lassa fever can rapidly spread across land borders and how such threat can be quickly controlled with communication and collaboration within the IHR framework.


Assuntos
Emigração e Imigração , Febre Lassa/diagnóstico , Vírus Lassa/fisiologia , Adulto , África Ocidental/epidemiologia , Monitoramento Epidemiológico , Feminino , Humanos , Regulamento Sanitário Internacional/normas , Febre Lassa/epidemiologia , Febre Lassa/patologia , Vírus Lassa/genética , Organização Mundial da Saúde
5.
Philos Trans R Soc Lond B Biol Sci ; 374(1775): 20180268, 2019 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-31056054

RESUMO

Lassa fever (LF) is a zoonotic disease that is widespread in West Africa and involves animal-to-human and human-to-human transmission. Animal-to-human transmission occurs upon exposure to rodent excreta and secretions, i.e. urine and saliva, and human-to-human transmission occurs via the bodily fluids of an infected person. To elucidate the seasonal drivers of LF epidemics, we employed a mathematical model to analyse the datasets of human infection, rodent population dynamics and climatological variations and capture the underlying transmission dynamics. The surveillance-based incidence data of human cases in Nigeria were explored, and moreover, a mathematical model was used for describing the transmission dynamics of LF in rodent populations. While quantifying the case fatality risk and the rate of exposure of humans to animals, we explicitly estimated the corresponding contact rate of humans with infected rodents, accounting for the seasonal population dynamics of rodents. Our findings reveal that seasonal migratory dynamics of rodents play a key role in regulating the cyclical pattern of LF epidemics. The estimated timing of high exposure of humans to animals coincides with the time shortly after the start of the dry season and can be associated with the breeding season of rodents in Nigeria. This article is part of the theme issue 'Modelling infectious disease outbreaks in humans, animals and plants: approaches and important themes'. This issue is linked with the subsequent theme issue 'Modelling infectious disease outbreaks in humans, animals and plants: epidemic forecasting and control'.


Assuntos
Febre Lassa/epidemiologia , Febre Lassa/veterinária , Doenças dos Roedores/epidemiologia , Roedores/virologia , Animais , Humanos , Febre Lassa/transmissão , Febre Lassa/virologia , Vírus Lassa/fisiologia , Modelos Teóricos , Nigéria/epidemiologia , Doenças dos Roedores/transmissão , Doenças dos Roedores/virologia , Roedores/fisiologia , Estações do Ano , Zoonoses/epidemiologia , Zoonoses/transmissão , Zoonoses/virologia
6.
J Mol Biol ; 431(11): 2095-2111, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31004664

RESUMO

Lassa virus (LASV) is a notorious human pathogen in West Africa. Its class I trimeric spike complex displays a distinct architecture, and its cell entry mechanism involves unique attributes not shared by other related viruses. We determined the crystal structure of the GP2 fusion glycoprotein from the spike complex of LASV (GP2LASV) in its post-fusion conformation. GP2LASV adopts a canonical helical bundle configuration similarly to other viruses in its family. The core packing of GP2LASV, however, is more organized compared to GP2 from other viruses reducing the formation of internal hydrophobic cavities. We demonstrate a link between the formation of such unfavorable hydrophobic cavities and the efficiencies of membrane fusion and cell entry. Our study suggests that LASV has evolved a more efficient membrane fusogen compared to other viruses from its family by optimizing the post-fusion configuration of its GP2 module.


Assuntos
Febre Lassa/virologia , Vírus Lassa/fisiologia , Internalização do Vírus , Animais , Linhagem Celular , Cristalografia por Raios X , Células HEK293 , Humanos , Febre Lassa/metabolismo , Vírus Lassa/química , Fusão de Membrana , Simulação de Dinâmica Molecular , Conformação Proteica
7.
Emerg Infect Dis ; 25(5): 865-874, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31002065

RESUMO

Lassa virus (LASV), a hemorrhagic fever virus endemic to West Africa, causes conjunctivitis in patients with acute disease. To examine ocular manifestations of LASV, we histologically examined eyes from infected guinea pigs. In fatal disease, LASV immunostaining was most prominent in the anterior uvea, especially in the filtration angle, ciliary body, and iris and in and around vessels in the bulbar conjunctiva and peripheral cornea, where it co-localized with an endothelial marker (platelet endothelial cell adhesion molecule). Antigen was primarily associated with infiltration of T-lymphocytes around vessels in the anterior uvea and with new vessel formation at the peripheral cornea. In animals that exhibited clinical signs but survived infection, eyes had little to no inflammation and no LASV immunostaining 6 weeks after infection. Overall, in this model, LASV antigen was restricted to the anterior uvea and was associated with mild chronic inflammation in animals with severe disease but was not detected in survivors.


Assuntos
Conjuntivite/virologia , Epitélio Posterior/virologia , Irite/virologia , Ceratite/virologia , Vírus Lassa/fisiologia , Animais , Biópsia , Conjuntivite/patologia , Modelos Animais de Doenças , Epitélio Posterior/patologia , Feminino , Cobaias , Imuno-Histoquímica , Irite/patologia , Ceratite/patologia , Masculino , Reação em Cadeia da Polimerase , RNA Viral
8.
Viruses ; 11(3)2019 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-30909570

RESUMO

Lassa virus (LASV) and Mopeia virus (MOPV) are two closely related Old-World mammarenaviruses. LASV causes severe hemorrhagic fever with high mortality in humans, whereas no case of MOPV infection has been reported. Comparing MOPV and LASV is a powerful strategy to unravel pathogenic mechanisms that occur during the course of pathogenic arenavirus infection. We used a yeast two-hybrid approach to identify cell partners of MOPV and LASV Z matrix protein in which two autophagy adaptors were identified, NDP52 and TAX1BP1. Autophagy has emerged as an important cellular defense mechanism against viral infections but its role during arenavirus infection has not been shown. Here, we demonstrate that autophagy is transiently induced by MOPV, but not LASV, in infected cells two days after infection. Impairment of the early steps of autophagy significantly decreased the production of MOPV and LASV infectious particles, whereas a blockade of the degradative steps impaired only MOPV infectious particle production. Our study provides insights into the role played by autophagy during MOPV and LASV infection and suggests that this process could partially explain their different pathogenicity.


Assuntos
Arenavirus/fisiologia , Autofagia , Vírus Lassa/fisiologia , Animais , Arenavirus/patogenicidade , Chlorocebus aethiops , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Vírus Lassa/patogenicidade , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Técnicas do Sistema de Duplo-Híbrido , Células Vero
9.
mBio ; 10(2)2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30914516

RESUMO

Recognition of functional receptors by viruses is a key determinant for their host range, tissue tropism, and disease potential. The highly pathogenic Lassa virus (LASV) currently represents one of the most important emerging pathogens. The major cellular receptor for LASV in human cells is the ubiquitously expressed and evolutionary highly conserved extracellular matrix receptor dystroglycan (DG). In the host, DG interacts with many cellular proteins in a tissue-specific manner. The resulting distinct supramolecular complexes likely represent the functional units for viral entry, and preexisting protein-protein interactions may critically influence DG's function in productive viral entry. Using an unbiased shotgun proteomic approach, we define the largely unknown molecular composition of DG complexes present in highly susceptible epithelial cells that represent important targets for LASV during viral transmission. We further show that the specific composition of cellular DG complexes can affect DG's function in receptor-mediated endocytosis of the virus. Under steady-state conditions, epithelial DG complexes underwent rapid turnover via an endocytic pathway that shared some characteristics with DG-mediated LASV entry. However, compared to steady-state uptake of DG, LASV entry via DG occurred faster and critically depended on additional signaling by receptor tyrosine kinases and the downstream effector p21-activating kinase. In sum, we show that the specific molecular composition of DG complexes in susceptible cells is a determinant for productive virus entry and that the pathogen can manipulate the existing DG-linked endocytic pathway. This highlights another level of complexity of virus-receptor interaction and provides possible cellular targets for therapeutic antiviral intervention.IMPORTANCE Recognition of cellular receptors allows emerging viruses to break species barriers and is an important determinant for their disease potential. Many virus receptors have complex tissue-specific interactomes, and preexisting protein-protein interactions may influence their function. Combining shotgun proteomics with a biochemical approach, we characterize the molecular composition of the functional receptor complexes used by the highly pathogenic Lassa virus (LASV) to invade susceptible human cells. We show that the specific composition of the receptor complexes affects productive entry of the virus, providing proof-of-concept. In uninfected cells, these functional receptor complexes undergo dynamic turnover involving an endocytic pathway that shares some characteristics with viral entry. However, steady-state receptor uptake and virus endocytosis critically differ in kinetics and underlying signaling, indicating that the pathogen can manipulate the receptor complex according to its needs. Our study highlights a remarkable complexity of LASV-receptor interaction and identifies possible targets for therapeutic antiviral intervention.


Assuntos
Distroglicanas/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Vírus Lassa/fisiologia , Complexos Multiproteicos/metabolismo , Receptores Virais/metabolismo , Internalização do Vírus , Linhagem Celular , Humanos
11.
PLoS Negl Trop Dis ; 12(11): e0006829, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30399142

RESUMO

Lassa fever is a viral haemorrhagic fever caused by an arenavirus. The disease is endemic in West African countries, including Guinea. The rodents Mastomys natalensis and Mastomys erythroleucus have been identified as Lassa virus reservoirs in Guinea. In the absence of a vaccine, rodent control and human behavioural changes are the only options to prevent Lassa fever in highly endemic areas. We performed a 4 year intervention based on chemical rodent control, utilizing anticoagulant rodenticides in 3 villages and evaluating the rodent abundance before and after treatment. Three additional villages were investigated as controls. Analyses to assess the effectiveness of the intervention, bait consumption and rodent dynamics were performed. Anthropological investigations accompanied the intervention to integrate local understandings of human-rodent cohabitation and rodent control intervention. Patterns of bait consumption showed a peak at days 5-7 and no consumption at days 28-30. There was no difference between Bromadiolone and Difenacoum bait consumption. The main rodent species found in the houses was M. natalensis. The abundance of M. natalensis, as measured by the trapping success, varied between 3.6 and 16.7% before treatment and decreased significantly to 1-2% after treatment. Individuals in treated villages welcomed the intervention and trapping because mice are generally regarded as a nuisance. Immediate benefits from controlling rodents included protection of food and belongings. Before the intervention, local awareness of Lassa fever was non-existent. Despite their appreciation for the intervention, local individuals noted its limits and the need for complementary actions. Our results demonstrate that chemical treatment provides an effective tool to control local rodent populations and can serve as part of an effective, holistic approach combining rodent trapping, use of local rodenticides, environmental hygiene, house repairs and rodent-proof storage. These actions should be developed in collaboration with local stakeholders and communities.


Assuntos
Febre Lassa/transmissão , Murinae/fisiologia , Controle de Roedores/métodos , Rodenticidas/farmacologia , Animais , Reservatórios de Doenças/virologia , Guiné , Febre Lassa/epidemiologia , Febre Lassa/prevenção & controle , Febre Lassa/virologia , Vírus Lassa/fisiologia , Camundongos , Murinae/classificação , Murinae/virologia , Controle de Roedores/instrumentação , Saúde da População Rural
12.
PLoS Pathog ; 14(9): e1007322, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30265711

RESUMO

Lassa fever virus (LASV) is endemic in West Africa and causes severe hemorrhagic fever and sensorineural hearing loss. We identified a small molecule inhibitor of LASV and used it to analyze the mechanism of entry. Using a photo-reactive analog that retains antiviral activity as a probe, we identified the inhibitor target as lysosome-associated membrane protein 1 (LAMP1), a host factor that binds to the LASV glycoprotein (GP) during infection. We found that LAMP1 binding to LASV GP is cholesterol-dependent, and that the inhibitor blocks infection by competing with cholesterol in LAMP1. Mutational analysis of a docking-based model identified a putative inhibitor binding site in the cholesterol-binding pocket within the LAMP1 domain that binds GP. These findings identify a critical role for cholesterol in LASV entry and a potential target for therapeutic intervention.


Assuntos
Colesterol/metabolismo , Vírus Lassa/fisiologia , Vírus Lassa/patogenicidade , Glicoproteínas de Membrana Associadas ao Lisossomo/fisiologia , Receptores Virais/fisiologia , Adamantano/análogos & derivados , Adamantano/química , Adamantano/farmacologia , Animais , Antivirais/química , Antivirais/farmacologia , Linhagem Celular , Chlorocebus aethiops , Células HEK293 , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Febre Lassa/etiologia , Vírus Lassa/efeitos dos fármacos , Glicoproteínas de Membrana Associadas ao Lisossomo/antagonistas & inibidores , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Modelos Moleculares , Mutação , Estabilidade Proteica , Estrutura Terciária de Proteína , Receptores Virais/antagonistas & inibidores , Receptores Virais/genética , Células Vero , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/fisiologia , Internalização do Vírus/efeitos dos fármacos
13.
Parasit Vectors ; 11(1): 416, 2018 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-30005641

RESUMO

BACKGROUND: Lassa fever, killing thousands of people annually, is the most reported viral zoonotic disease in Nigeria. Recently, different rodent species carrying diverse lineages of the Lassa virus (LASV) in addition to a novel Mobala-like genetic sequence were detected within the country. Here, screening 906 small mammal specimens from 11 localities for IgG antibodies and incorporating previous PCR detection data involving the same populations, we further describe arenavirus prevalence across Nigeria in relation to host species and geographical location. METHODS: Small mammals were trapped during the period 2011-2015 according to geographical location (endemic and non-endemic zones for Lassa fever), season (rainy and dry seasons between 2011 and 2012 for certain localities) and habitat (indoors, peridomestic settings and sylvatic vegetation). Identification of animal specimens from genera such as Mastomys and Mus (Nannomys) was assisted by DNA sequencing. Small mammals were tested for LASV IgG antibody using an indirect immunofluorescence assay (IFA). RESULTS: Small mammals were infected in both the endemic and non-endemic zones for Lassa fever, with a wider range of species IgG-positive (n = 8) than those which had been previously detected to be PCR-positive (n = 3). IgG-positive species, according to number of infected individuals, were Mastomys natalensis (n = 40), Mastomys erythroleucus (n = 15), Praomys daltoni (n = 6), Mus baoulei (n = 5), Rattus rattus (n = 2), Crocidura spp. (n = 2), Mus minutoides (n = 1) and Praomys misonnei (n = 1). Multimammate mice (Mastomys natalensis and M. erythroleucus) were the most ubiquitously infected, with animals testing positive by either PCR or IgG in 7 out of the 11 localities sampled. IgG prevalence in M. natalensis ranged from 1% in Abagboro, 17-36 % in Eguare Egoro, Ekpoma and Ngel Nyaki, up to 52 % in Mayo Ranewo. Prevalence according to locality, season and age was not, however, statistically significant for M. natalensis in Eguare Egoro and Ekpoma, localities that were sampled longitudinally. CONCLUSIONS: Overall, our study demonstrates that arenavirus occurrence is probably more widely distributed geographically and in extent of host taxa than is currently realized. This expanded scope should be taken into consideration in Lassa fever control efforts. Further sampling should also be carried out to isolate and characterize potential arenaviruses present in small mammal populations we found to be seropositive.


Assuntos
Anticorpos Antivirais/sangue , Infecções por Arenaviridae/sangue , Infecções por Arenaviridae/veterinária , Arenavirus/fisiologia , Doenças dos Roedores/sangue , Doenças dos Roedores/epidemiologia , Animais , Infecções por Arenaviridae/epidemiologia , Infecções por Arenaviridae/virologia , Arenavirus/imunologia , Reservatórios de Doenças/virologia , Geografia , Insetívoros/virologia , Vírus Lassa/imunologia , Vírus Lassa/fisiologia , Camundongos , Nigéria/epidemiologia , Prevalência , RNA Viral/genética , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Doenças dos Roedores/virologia , Roedores/virologia , Estudos Soroepidemiológicos
14.
J Virol ; 92(16)2018 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-29875238

RESUMO

Lassa virus (LASV) is an Old World arenavirus responsible for hundreds of thousands of infections in West Africa every year. LASV entry into a variety of cell types is mediated by interactions with glycosyltransferase LARGE-modified O-linked glycans present on the ubiquitous receptor α-dystroglycan (αDG). However, cells lacking αDG are permissive to LASV infection, suggesting that alternative receptors exist. Previous studies demonstrated that the phosphatidylserine (PtdSer)-binding receptors Axl and Tyro3 along with C-type lectin receptors mediate αDG-independent entry. Here, we demonstrate that another PtdSer receptor, TIM-1, mediates LASV glycoprotein (GP)-pseudotyped virion entry into αDG-knocked-out HEK 293T and wild-type (WT) Vero cells, which express αDG lacking appropriate glycosylation. To investigate the mechanism by which TIM-1 mediates enhancement of entry, we demonstrate that mutagenesis of the TIM-1 IgV domain PtdSer-binding pocket abrogated transduction. Furthermore, the human TIM-1 IgV domain-binding monoclonal antibody ARD5 blocked transduction of pseudovirions bearing LASV GP in a dose-dependent manner. Finally, as we showed previously for other viruses that use TIM-1 for entry, a chimeric TIM-1 protein that substitutes the proline-rich region (PRR) from murine leukemia virus envelope (Env) for the mucin-like domain served as a competent receptor. These studies provide evidence that, in the absence of a functional αDG, TIM-1 mediates the entry of LASV pseudoviral particles through interactions of virions with the IgV PtdSer-binding pocket of TIM-1.IMPORTANCE PtdSer receptors, such as TIM-1, are emerging as critical entry factors for many enveloped viruses. Most recently, hepatitis C virus and Zika virus have been added to a growing list. PtdSer receptors engage with enveloped viruses through the binding of PtdSer embedded in the viral envelope, defining them as GP-independent receptors. This GP-independent entry mechanism should effectively mediate the entry of all enveloped viruses, yet LASV GP-pseudotyped viruses were previously found to be unresponsive to PtdSer receptor enhancement in HEK 293T cells. Here, we demonstrate that LASV pseudovirions can utilize the PtdSer receptor TIM-1 but only in the absence of appropriately glycosylated α-dystroglycan (αDG), the high-affinity cell surface receptor for LASV. Our studies shed light on LASV receptor utilization and explain why previous studies performed with α-DG-expressing cells did not find that LASV pseudovirions utilize PtdSer receptors for virus uptake.


Assuntos
Distroglicanas/deficiência , Receptor Celular 1 do Vírus da Hepatite A/metabolismo , Interações Hospedeiro-Patógeno , Vírus Lassa/fisiologia , Receptores Virais/metabolismo , Internalização do Vírus , Animais , Chlorocebus aethiops , Análise Mutacional de DNA , Células HEK293 , Receptor Celular 1 do Vírus da Hepatite A/genética , Humanos , Receptores Virais/genética , Células Vero
15.
J Virol ; 92(16)2018 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-29899092

RESUMO

Lassa virus (LASV) belongs to the Mammarenavirus genus (family Arenaviridae) and causes severe hemorrhagic fever in humans. At present, there are no Food and Drug Administration (FDA)-approved drugs or vaccines specific for LASV. Here, high-throughput screening of an FDA-approved drug library was performed against LASV entry by using pseudotype virus bearing LASV envelope glycoprotein (GPC). Two hit compounds, lacidipine and phenothrin, were identified as LASV entry inhibitors in the micromolar range. A mechanistic study revealed that both compounds inhibited LASV entry by blocking low-pH-induced membrane fusion. Accordingly, lacidipine showed virucidal effects on the pseudotype virus of LASV. Adaptive mutant analyses demonstrated that replacement of T40, located in the ectodomain of the stable-signal peptide (SSP), with lysine (K) conferred LASV resistance to lacidipine. Furthermore, lacidipine showed antiviral activity against LASV, the closely related Mopeia virus (MOPV), and the New World arenavirus Guanarito virus (GTOV). Drug-resistant variants indicated that V36M in the ectodomain of the SSP mutant and V436A in the transmembrane domain of the GP2 mutant conferred GTOV resistance to lacidipine, suggesting the interface between SSP and GP2 is the target of lacidipine. This study shows that lacidipine is a candidate for LASV therapy, reinforcing the notion that the SSP-GP2 interface provides an entry-targeted platform for arenavirus inhibitor design.IMPORTANCE Currently, there is no approved therapy to treat Lassa fever; therefore, repurposing of approved drugs will accelerate the development of a therapeutic stratagem. In this study, we screened an FDA-approved library of drugs and identified two compounds, lacidipine and phenothrin, which inhibited Lassa virus entry by blocking low-pH-induced membrane fusion. Additionally, both compounds extended their inhibition against the entry of Guanarito virus, and the viral targets were identified as the SSP-GP2 interface.


Assuntos
Antivirais/farmacologia , Di-Hidropiridinas/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Vírus Lassa/efeitos dos fármacos , Piretrinas/farmacologia , Internalização do Vírus/efeitos dos fármacos , Arenaviridae/efeitos dos fármacos , Arenavirus do Novo Mundo/efeitos dos fármacos , Análise Mutacional de DNA , Farmacorresistência Viral , Vírus Lassa/fisiologia
17.
mBio ; 9(1)2018 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-29295909

RESUMO

Lassa virus (LASV) is an arenavirus whose entry into host cells is mediated by a glycoprotein complex (GPC) comprised of a receptor binding subunit, GP1, a fusogenic transmembrane subunit, GP2, and a stable signal peptide. After receptor-mediated internalization, arenaviruses converge in the endocytic pathway, where they are thought to undergo low-pH-triggered, GPC-mediated fusion with a late endosome membrane. A unique feature of LASV entry is a pH-dependent switch from a primary cell surface receptor (α-dystroglycan) to an endosomal receptor, lysosomal-associated membrane protein (Lamp1). Despite evidence that the interaction between LASV GP1 and Lamp1 is critical, the function of Lamp1 in promoting LASV infection remains poorly characterized. Here we used wild-type (WT) and Lamp1 knockout (KO) cells to show that Lamp1 increases the efficiency of, but is not absolutely required for, LASV entry and infection. We then used cell-cell and pseudovirus-cell surface fusion assays to demonstrate that LASV GPC-mediated fusion occurs at a significantly higher pH when Lamp1 is present compared to when Lamp1 is missing. Correspondingly, we found that LASV entry occurs through less acidic endosomes in WT (Lamp1-positive) versus Lamp1 KO cells. We propose that, by elevating the pH threshold for fusion, Lamp1 allows LASV particles to exit the endocytic pathway before they encounter an increasingly acidic and harsh proteolytic environment, which could inactivate a significant percentage of incoming viruses. In this manner Lamp1 increases the overall efficiency of LASV entry and infection.IMPORTANCE Lassa virus is the most clinically important member of the Arenaviridae, a family that includes six additional biosafety level 4 (BSL4) hemorrhagic fever viruses. The lack of specific antiviral therapies for Lassa fever drives an urgent need to identify druggable targets, and interventions that block infection at the entry stage are particularly attractive. Lassa virus is only the second virus known to employ an intracellular receptor, the first being Ebola virus. Here we show that interaction with its intracellular receptor, Lamp1, enhances and upwardly shifts the pH dependence of fusion and consistently permits Lassa virus entry into cells through less acidic endosomes. We propose that in this manner, Lamp1 increases the overall efficiency of Lassa virus infection.


Assuntos
Endossomos/virologia , Interações Hospedeiro-Patógeno , Vírus Lassa/fisiologia , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Internalização do Vírus , Endossomos/química , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio
18.
J Virol ; 92(5)2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29237830

RESUMO

Fatal infection with the highly pathogenic Lassa virus (LASV) is characterized by extensive viral dissemination, indicating broad tissue tropism. The major cellular receptor for LASV is the highly conserved extracellular matrix receptor dystroglycan (DG). Binding of LASV depends on DG's tissue-specific posttranslational modification with the unusual O-linked polysaccharide matriglycan. Interestingly, functional glycosylation of DG does not always correlate with viral tropism observed in vivo The broadly expressed phosphatidylserine (PS) receptors Axl and Tyro3 were recently identified as alternative LASV receptor candidates. However, their role in LASV entry is not entirely understood. Here, we examine LASV receptor candidates in primary human cells and found coexpression of Axl with differentially glycosylated DG. To study LASV receptor use in the context of productive arenavirus infection, we employed recombinant lymphocytic choriomeningitis virus expressing LASV glycoprotein (rLCMV-LASV GP) as a validated biosafety level 2 (BSL2) model. We confirm and extend previous work showing that Axl can contribute to LASV entry in the absence of functional DG using "apoptotic mimicry" in a way similar to that of other enveloped viruses. We further show that Axl-dependent LASV entry requires receptor activation and involves a pathway resembling macropinocytosis. Axl-mediated LASV entry is facilitated by heparan sulfate and critically depends on the late endosomal protein LAMP-1 as an intracellular entry factor. In endothelial cells expressing low levels of functional DG, both receptors are engaged by the virus and can contribute to productive entry. In sum, we characterize the role of Axl in LASV entry and provide a rationale for targeting Axl in antiviral therapy.IMPORTANCE The highly pathogenic arenavirus Lassa virus (LASV) represents a serious public health problem in Africa. Although the principal LASV receptor, dystroglycan (DG), is ubiquitously expressed, virus binding critically depends on DG's posttranslational modification, which does not always correlate with tissue tropism. The broadly expressed phosphatidylserine receptor Axl was recently identified as an alternative LASV receptor candidate, but its role in LASV entry is unclear. Here, we investigate the exact role of Axl in LASV entry as a function of DG's posttranslational modification. We found that in the absence of functional DG, Axl can mediate LASV entry via apoptotic mimicry. Productive entry requires virus-induced receptor activation, involves macropinocytosis, and critically depends on LAMP-1. In endothelial cells that express low levels of glycosylated DG, both receptors can promote LASV entry. In sum, our study defines the roles of Axl in LASV entry and provides a rationale for targeting Axl in antiviral therapy.


Assuntos
Distroglicanas/metabolismo , Vírus Lassa/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Receptores Virais/metabolismo , Ligação Viral , Internalização do Vírus , Células A549 , Antivirais/farmacologia , Infecções por Arenaviridae/metabolismo , Linhagem Celular Tumoral , Distroglicanas/genética , Endossomos/metabolismo , Expressão Gênica , Glicosilação , Células HEK293 , Células HeLa , Heparitina Sulfato/farmacologia , Humanos , Vírus Lassa/efeitos dos fármacos , Vírus Lassa/patogenicidade , Vírus da Coriomeningite Linfocítica/genética , Vírus da Coriomeningite Linfocítica/metabolismo , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Pinocitose/fisiologia , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Proteínas Proto-Oncogênicas/genética , Interferência de RNA , Receptores Proteína Tirosina Quinases/efeitos dos fármacos , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Tropismo
19.
Methods Mol Biol ; 1604: 135-155, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-28986830

RESUMO

Host cell entry is the first and most fundamental step of every virus infection and represents a major barrier for zoonotic transmission and viral emergence. Targeting viral entry appears further as a promising strategy for therapeutic intervention. Several cellular receptors have been identified for Lassa virus, including dystroglycan, TAM receptor tyrosine kinases, and C-type lectins. Upon receptor binding, LASV enters the host cell via a largely unknown clathrin- and dynamin-independent endocytotic pathway that delivers the virus to late endosomes, where fusion occurs after engagement of a second, intracellular receptor, the late endosomal/lysosomal resident protein LAMP1. Here, we describe a series of experimental approaches to investigate LASV cell entry and to test candidate inhibitors for their action at this early and decisive step of infection.


Assuntos
Endocitose/fisiologia , Vírus Lassa/fisiologia , Animais , Clatrina/metabolismo , Dinaminas/metabolismo , Endocitose/genética , Endossomos/metabolismo , Humanos , Vírus Lassa/metabolismo , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Ligação Proteica , Internalização do Vírus
20.
Virology ; 512: 161-171, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28963882

RESUMO

Lassa virus (LASV) causes severe hemorrhagic fever with high mortality, yet no vaccine currently exists. Antibodies targeting viral attachment proteins are crucial for protection against many viral infections. However, the envelope glycoprotein (GP)-1 of LASV elicits weak antibody responses due to extensive glycan shielding. Here, we explored a novel vaccine strategy to enhance humoral immunity against LASV GP1. Using structural information, we designed a recombinant GP1 immunogen, and then encapsulated it into oxidation-sensitive polymersomes (PS) as nanocarriers that promote intracellular MHCII loading. Mice immunized with adjuvanted PS (LASV GP1) showed superior humoral responses than free LASV GP1, including antibodies with higher binding affinity to virion GP1, increased levels of polyfunctional anti-viral CD4 T cells, and IgG-secreting B cells. PS (LASV GP1) elicited a more diverse epitope repertoire of anti-viral IgG. Together, these data demonstrate the potential of our nanocarrier vaccine platform for generating virus-specific antibodies against weakly immunogenic viral antigens.


Assuntos
Glicoproteínas/metabolismo , Febre Lassa/prevenção & controle , Vírus Lassa/fisiologia , Nanoestruturas/química , Proteínas do Envelope Viral/metabolismo , Vacinas Virais/imunologia , Células A549 , Animais , Feminino , Regulação Viral da Expressão Gênica/imunologia , Glicoproteínas/genética , Células HEK293 , Humanos , Imunidade Humoral , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Envelope Viral/genética
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