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1.
PLoS Negl Trop Dis ; 13(6): e0006983, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31242184

RESUMO

BACKGROUND: T cell immunoglobulin mucin domain-1 (TIM-1) is a phosphatidylserine (PS) receptor, mediating filovirus entry into cells through interactions with PS on virions. TIM-1 expression has been implicated in Ebola virus (EBOV) pathogenesis; however, it remains unclear whether this is due to TIM-1 serving as a filovirus receptor in vivo or, as others have suggested, TIM-1 induces a cytokine storm elicited by T cell/virion interactions. Here, we use a BSL2 model virus that expresses EBOV glycoprotein to demonstrate the importance of TIM-1 as a virus receptor late during in vivo infection. METHODOLOGY/PRINCIPAL FINDINGS: Infectious, GFP-expressing recombinant vesicular stomatitis virus encoding either full length EBOV glycoprotein (EBOV GP/rVSV) or mucin domain deleted EBOV glycoprotein (EBOV GPΔO/rVSV) was used to assess the role of TIM-1 during in vivo infection. GFP-expressing rVSV encoding its native glycoprotein G (G/rVSV) served as a control. TIM-1-sufficient or TIM-1-deficient BALB/c interferon α/ß receptor-/- mice were challenged with these viruses. While G/rVSV caused profound morbidity and mortality in both mouse strains, TIM-1-deficient mice had significantly better survival than TIM-1-expressing mice following EBOV GP/rVSV or EBOV GPΔO/rVSV challenge. EBOV GP/rVSV or EBOV GPΔO/rVSV in spleen of infected animals was high and unaffected by expression of TIM-1. However, infectious virus in serum, liver, kidney and adrenal gland was reduced late in infection in the TIM-1-deficient mice, suggesting that virus entry via this receptor contributes to virus load. Consistent with higher virus loads, proinflammatory chemokines trended higher in organs from infected TIM-1-sufficient mice compared to the TIM-1-deficient mice, but proinflammatory cytokines were more modestly affected. To assess the role of T cells in EBOV GP/rVSV pathogenesis, T cells were depleted in TIM-1-sufficient and -deficient mice and the mice were challenged with virus. Depletion of T cells did not alter the pathogenic consequences of virus infection. CONCLUSIONS: Our studies provide evidence that at late times during EBOV GP/rVSV infection, TIM-1 increased virus load and associated mortality, consistent with an important role of this receptor in virus entry. This work suggests that inhibitors which block TIM-1/virus interaction may serve as effective antivirals, reducing virus load at late times during EBOV infection.


Assuntos
Ebolavirus/fisiologia , Doença pelo Vírus Ebola/virologia , Receptor Celular 1 do Vírus da Hepatite A/metabolismo , Receptores Virais/metabolismo , Internalização do Vírus , Animais , Ebolavirus/genética , Feminino , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Receptor Celular 1 do Vírus da Hepatite A/deficiência , Camundongos Endogâmicos BALB C , Camundongos Knockout , Receptores Virais/deficiência , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Genética Reversa , Vesiculovirus/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo
2.
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
3.
Genetics ; 208(1): 283-296, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29162626

RESUMO

Orofacial clefts are one of the most common birth defects, affecting 1-2 per 1000 births, and have a complex etiology. High-resolution array-based comparative genomic hybridization has increased the ability to detect copy number variants (CNVs) that can be causative for complex diseases such as cleft lip and/or palate. Utilizing this technique on 97 nonsyndromic cleft lip and palate cases and 43 cases with cleft palate only, we identified a heterozygous deletion of Isthmin 1 in one affected case, as well as a deletion in a second case that removes putative 3' regulatory information. Isthmin 1 is a strong candidate for clefting, as it is expressed in orofacial structures derived from the first branchial arch and is also in the same "synexpression group" as fibroblast growth factor 8 and sprouty RTK signaling antagonist 1a and 2, all of which have been associated with clefting. CNVs affecting Isthmin 1 are exceedingly rare in control populations, and Isthmin 1 scores as a likely haploinsufficiency locus. Confirming its role in craniofacial development, knockdown or clustered randomly interspaced short palindromic repeats/Cas9-generated mutation of isthmin 1 in Xenopus laevis resulted in mild to severe craniofacial dysmorphologies, with several individuals presenting with median clefts. Moreover, knockdown of isthmin 1 produced decreased expression of LIM homeobox 8, itself a gene associated with clefting, in regions of the face that pattern the maxilla. Our study demonstrates a successful pipeline from CNV identification of a candidate gene to functional validation in a vertebrate model system, and reveals Isthmin 1 as both a new human clefting locus as well as a key craniofacial patterning gene.


Assuntos
Fenda Labial/genética , Fissura Palatina/genética , Anormalidades Craniofaciais/genética , Morfogênese/genética , Organogênese/genética , Trombospondinas/genética , Sistemas CRISPR-Cas , Estudos de Casos e Controles , Hibridização Genômica Comparativa , Anormalidades Craniofaciais/embriologia , Variações do Número de Cópias de DNA , Deleção de Genes , Haploinsuficiência , Humanos , Locos de Características Quantitativas
4.
mBio ; 8(5)2017 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-28874468

RESUMO

Receptor molecules play key roles in the cellular entry of picornaviruses, and TIM1 (HAVCR1) is widely accepted to be the receptor for hepatitis A virus (HAV), an unusual, hepatotropic human picornavirus. However, its identification as the hepatovirus receptor predated the discovery that hepatoviruses undergo nonlytic release from infected cells as membrane-cloaked, quasi-enveloped HAV (eHAV) virions that enter cells via a pathway distinct from naked, nonenveloped virions. We thus revisited the role of TIM1 in hepatovirus entry, examining both adherence and infection/replication in cells with clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-engineered TIM1 knockout. Cell culture-derived, gradient-purified eHAV bound Huh-7.5 human hepatoma cells less efficiently than naked HAV at 4°C, but eliminating TIM1 expression caused no difference in adherence of either form of HAV, nor any impact on infection and replication in these cells. In contrast, TIM1-deficient Vero cells showed a modest reduction in quasi-enveloped eHAV (but not naked HAV) attachment and replication. Thus, TIM1 facilitates quasi-enveloped eHAV entry in Vero cells, most likely by binding phosphatidylserine (PtdSer) residues on the eHAV membrane. Both Tim1-/-Ifnar1-/- and Tim4-/-Ifnar1-/- double-knockout mice were susceptible to infection upon intravenous challenge with infected liver homogenate, with fecal HAV shedding and serum alanine aminotransferase (ALT) elevations similar to those in Ifnar1-/- mice. However, intrahepatic HAV RNA and ALT elevations were modestly reduced in Tim1-/-Ifnar1-/- mice compared to Ifnar1-/- mice challenged with a lower titer of gradient-purified HAV or eHAV. We conclude that TIM1 is not an essential hepatovirus entry factor, although its PtdSer-binding activity may contribute to the spread of quasi-enveloped virus and liver injury in mice.IMPORTANCE T cell immunoglobulin and mucin-containing domain protein 1 (TIM1) was reported more than 2 decades ago to be an essential cellular receptor for hepatitis A virus (HAV), a picornavirus in the Hepatovirus genus, resulting in its designation as "hepatitis A virus cellular receptor 1" (HAVCR1) by the Human Genome Organization Gene Nomenclature Committee. However, recent studies have shown that HAV exists in nature as both naked, nonenveloped (HAV) virions and membrane-cloaked, quasi-enveloped infectious virus (eHAV), prompting us to revisit the role of TIM1 in viral entry. We show here that TIM1 (HAVCR1) is not an essential cellular receptor for HAV entry into cultured cells or required for viral replication and pathogenesis in permissive strains of mice, although it may facilitate early stages of infection by binding phosphatidylserine on the eHAV surface. This work thus corrects the published record and sets the stage for future efforts to identify specific hepatovirus entry factors.


Assuntos
Receptor Celular 1 do Vírus da Hepatite A/metabolismo , Vírus da Hepatite A/fisiologia , Hepatite A/virologia , Interações Hospedeiro-Patógeno , Internalização do Vírus , Animais , Sistemas CRISPR-Cas , Carcinoma Hepatocelular , Linhagem Celular Tumoral , Chlorocebus aethiops , Receptor Celular 1 do Vírus da Hepatite A/deficiência , Receptor Celular 1 do Vírus da Hepatite A/genética , Vírus da Hepatite A/metabolismo , Vírus da Hepatite A/patogenicidade , Humanos , Fígado/patologia , Fígado/fisiopatologia , Fígado/virologia , Camundongos , Receptor de Interferon alfa e beta/deficiência , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/metabolismo , Células Vero , Vírion/metabolismo , Vírion/patogenicidade , Vírion/fisiologia , Ligação Viral , Replicação Viral
5.
J Virol ; 91(17)2017 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-28615211

RESUMO

The recent Ebola virus (EBOV) epidemic in West Africa demonstrates the potential for a significant public health burden caused by filoviral infections. No vaccine or antiviral is currently FDA approved. To expand the vaccine options potentially available, we assessed protection conferred by an EBOV vaccine composed of vesicular stomatitis virus pseudovirions that lack native G glycoprotein (VSVΔG) and bear EBOV glycoprotein (GP). These pseudovirions mediate a single round of infection. Both single-dose and prime/boost vaccination regimens protected mice against lethal challenge with mouse-adapted Ebola virus (ma-EBOV) in a dose-dependent manner. The prime/boost regimen provided significantly better protection than a single dose. As N-linked glycans are thought to shield conserved regions of the EBOV GP receptor-binding domain (RBD), thereby blocking epitopes within the RBD, we also tested whether VSVΔG bearing EBOV GPs that lack GP1 N-linked glycans provided effective immunity against challenge with ma-EBOV or a more distantly related virus, Sudan virus. Using a prime/boost strategy, high doses of GP/VSVΔG partially or fully denuded of N-linked glycans on GP1 protected mice against ma-EBOV challenge, but these mutants were no more effective than wild-type (WT) GP/VSVΔG and did not provide cross protection against Sudan virus. As reported for other EBOV vaccine platforms, the protection conferred correlated with the quantity of EBOV GP-specific Ig produced but not with the production of neutralizing antibodies. Our results show that EBOV GP/VSVΔG pseudovirions serve as a successful vaccination platform in a rodent model of Ebola virus disease and that GP1 N-glycan loss does not influence immunogenicity or vaccination success.IMPORTANCE The West African Ebola virus epidemic was the largest to date, with more than 28,000 people infected. No FDA-approved vaccines are yet available, but in a trial vaccination strategy in West Africa, recombinant, infectious VSV encoding the Ebola virus glycoprotein effectively prevented virus-associated disease. VSVΔG pseudovirion vaccines may prove as efficacious and have better safety, but they have not been tested to date. Thus, we tested the efficacy of VSVΔG pseudovirions bearing Ebola virus glycoprotein as a vaccine platform. We found that wild-type Ebola virus glycoprotein, in the context of this platform, provides robust protection of EBOV-challenged mice. Further, we found that removal of the heavy glycan shield surrounding conserved regions of the glycoprotein does not enhance vaccine efficacy.

6.
Methods Mol Biol ; 1628: 53-63, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28573610

RESUMO

Members of the family Filoviridae are filamentous, enveloped, and nonsegmented negative-stranded RNA viruses that can cause severe hemorrhagic disease in humans and nonhuman primates with high mortality rates. Current efforts to analyze the structure and biology of these viruses as well as the development of antivirals have been hindered by the necessity of biosafety level 4 containment (BSL4). Here, we outline how to produce and work with Ebola virus glycoprotein bearing vesicular stomatitis virus (VSV) pseudovirions. These pseudovirions can be safely used to evaluate early steps of the filovirus life cycle without need for BSL4 containment. Virus gene expression in the transduced cells is easy to assess since the pseudovirions encode a reporter gene in place of the VSV G glycoprotein gene. Adoption of VSV for use as a pseudovirion system for filovirus GP has significantly expanded access for researchers to study specific aspects of the viral life cycle outside of BSL4 containment and has allowed substantial growth of filovirus research.


Assuntos
Ebolavirus/patogenicidade , Glicoproteínas de Membrana/genética , Estomatite Vesicular/virologia , Vesiculovirus/patogenicidade , Proteínas do Envelope Viral/genética , Contenção de Riscos Biológicos , Ebolavirus/genética , Genes Reporter/genética , Humanos , Vírus de RNA/genética , Vírus de RNA/patogenicidade , Estomatite Vesicular/patologia , Vesiculovirus/genética , Vírion/genética , Internalização do Vírus
7.
J Virol ; 91(7)2017 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-28100617

RESUMO

The glycoprotein complex (GPC) of arenaviruses, composed of stable signal peptide, GP1, and GP2, is the only antigen correlated with antibody-mediated neutralization. However, despite strong cross-reactivity of convalescent antisera between related arenavirus species, weak or no cross-neutralization occurs. Two closely related clade B viruses, Machupo virus (MACV) and Junín virus (JUNV), have nearly identical overall GPC architecture and share a host receptor, transferrin receptor 1 (TfR1). Given structural and functional similarities of the GP1 receptor binding site (RBS) of these viruses and the recent demonstration that the RBS is an important target for neutralizing antibodies, it is not clear how these viruses avoid cross-neutralization. To address this, MACV/JUNV chimeric GPCs were assessed for interaction with a group of α-JUNV GPC monoclonal antibodies (MAbs) and mouse antisera against JUNV or MACV GPC. All six MAbs targeted GP1, with those that neutralized JUNV GPC-pseudovirions competing with each other for RBS binding. However, these MAbs were unable to bind to a chimeric GPC composed of JUNV GP1 containing a small disulfide bonded loop (loop 10) unique to MACV GPC, suggesting that this loop may block MAbs interaction with the GP1 RBS. Consistent with this loop causing interference, mouse anti-JUNV GPC antisera that solely neutralized pseudovirions bearing autologous GP1 provided enhanced neutralization of MACV GPC when this loop was removed. Our studies provide evidence that loop 10, which is unique to MACV GP1, is an important impediment to binding of neutralizing antibodies and contributes to the poor cross-neutralization of α-JUNV antisera against MACV.IMPORTANCE Multiple New World arenaviruses can cause severe disease in humans, and some geographic overlap exists among these viruses. A vaccine that protects against a broad range of New World arenaviruses is desirable for purposes of simplicity, cost, and broad protection against multiple National Institute of Allergy and Infectious Disease-assigned category A priority pathogens. In this study, we sought to better understand how closely related arenaviruses elude cross-species neutralization by investigating the structural bases of antibody binding and avoidance. In our studies, we found that neutralizing antibodies against two New World arenaviruses, Machupo virus (MACV) and Junín virus (JUNV), bound to the envelope glycoprotein 1 (GP1) with JUNV monoclonal antibodies targeting the receptor binding site (RBS). We further show that altered structures surrounding the RBS pocket in MACV GP1 impede access of JUNV-elicited antibodies.


Assuntos
Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Antígenos Virais/imunologia , Vírus Junin/imunologia , Proteínas Virais de Fusão/imunologia , Sequência de Aminoácidos , Animais , Antígenos Virais/química , Sítios de Ligação , Chlorocebus aethiops , Reações Cruzadas , Células HEK293 , Humanos , Camundongos , Ligação Proteica , Especificidade da Espécie , Células Vero , Proteínas Virais de Fusão/química
8.
J Virol ; 90(13): 6097-6111, 2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-27122575

RESUMO

UNLABELLED: Phosphatidylserine (PtdSer) receptors that are responsible for the clearance of dying cells have recently been found to mediate enveloped virus entry. Ebola virus (EBOV), a member of the Filoviridae family of viruses, utilizes PtdSer receptors for entry into target cells. The PtdSer receptors human and murine T-cell immunoglobulin mucin (TIM) domain proteins TIM-1 and TIM-4 mediate filovirus entry by binding to PtdSer on the virion surface via a conserved PtdSer binding pocket within the amino-terminal IgV domain. While the residues within the TIM-1 IgV domain that are important for EBOV entry are characterized, the molecular details of virion-TIM-4 interactions have yet to be investigated. As sequences and structural alignments of the TIM proteins suggest distinct differences in the TIM-1 and TIM-4 IgV domain structures, we sought to characterize TIM-4 IgV domain residues required for EBOV entry. Using vesicular stomatitis virus pseudovirions bearing EBOV glycoprotein (EBOV GP/VSVΔG), we evaluated virus binding and entry into cells expressing TIM-4 molecules mutated within the IgV domain, allowing us to identify residues important for entry. Similar to TIM-1, residues in the PtdSer binding pocket of murine and human TIM-4 (mTIM-4 and hTIM-4) were found to be important for EBOV entry. However, additional TIM-4-specific residues were also found to impact EBOV entry, with a total of 8 mTIM-4 and 14 hTIM-4 IgV domain residues being critical for virion binding and internalization. Together, these findings provide a greater understanding of the interaction of TIM-4 with EBOV virions. IMPORTANCE: With more than 28,000 cases and over 11,000 deaths during the largest and most recent Ebola virus (EBOV) outbreak, there has been increased emphasis on the development of therapeutics against filoviruses. Many therapies under investigation target EBOV cell entry. T-cell immunoglobulin mucin (TIM) domain proteins are cell surface factors important for the entry of many enveloped viruses, including EBOV. TIM family member TIM-4 is expressed on macrophages and dendritic cells, which are early cellular targets during EBOV infection. Here, we performed a mutagenesis screening of the IgV domain of murine and human TIM-4 to identify residues that are critical for EBOV entry. Surprisingly, we identified more human than murine TIM-4 IgV domain residues that are required for EBOV entry. Defining the TIM IgV residues needed for EBOV entry clarifies the virus-receptor interactions and paves the way for the development of novel therapeutics targeting virus binding to this cell surface receptor.


Assuntos
Ebolavirus/fisiologia , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Internalização do Vírus , Animais , Linhagem Celular , Ebolavirus/genética , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Camundongos , Modelos Moleculares , Mutagênese , Receptores de Superfície Celular/genética , Receptores Virais/genética , Receptores Virais/metabolismo , Ligação Viral
9.
Hum Mol Genet ; 22(6): 1097-111, 2013 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-23223018

RESUMO

Neural tube defects (NTDs) are common birth defects of complex etiology. Family and population-based studies have confirmed a genetic component to NTDs. However, despite more than three decades of research, the genes involved in human NTDs remain largely unknown. We tested the hypothesis that rare copy number variants (CNVs), especially de novo germline CNVs, are a significant risk factor for NTDs. We used array-based comparative genomic hybridization (aCGH) to identify rare CNVs in 128 Caucasian and 61 Hispanic patients with non-syndromic lumbar-sacral myelomeningocele. We also performed aCGH analysis on the parents of affected individuals with rare CNVs where parental DNA was available (42 sets). Among the eight de novo CNVs that we identified, three generated copy number changes of entire genes. One large heterozygous deletion removed 27 genes, including PAX3, a known spina bifida-associated gene. A second CNV altered genes (PGPD8, ZC3H6) for which little is known regarding function or expression. A third heterozygous deletion removed GPC5 and part of GPC6, genes encoding glypicans. Glypicans are proteoglycans that modulate the activity of morphogens such as Sonic Hedgehog (SHH) and bone morphogenetic proteins (BMPs), both of which have been implicated in NTDs. Additionally, glypicans function in the planar cell polarity (PCP) pathway, and several PCP genes have been associated with NTDs. Here, we show that GPC5 orthologs are expressed in the neural tube, and that inhibiting their expression in frog and fish embryos results in NTDs. These results implicate GPC5 as a gene required for normal neural tube development.


Assuntos
Polaridade Celular , Variações do Número de Cópias de DNA , Glipicanas/genética , Disrafismo Espinal/genética , Animais , Estudos de Coortes , Feminino , Predisposição Genética para Doença , Hispânico ou Latino/genética , Humanos , Masculino , Tubo Neural/embriologia , Tubo Neural/metabolismo , Disrafismo Espinal/embriologia , Disrafismo Espinal/fisiopatologia , População Branca/genética , Peixe-Zebra
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