Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 501
Filtrar
1.
FEBS J ; 288(17): 5071-5088, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34490733

RESUMO

While there is undeniable evidence to link endosomal acid-base homeostasis to viral pathogenesis, the lack of druggable molecular targets has hindered translation from bench to bedside. The recent identification of variants in the interferon-inducible endosomal Na+ /H+ exchanger 9 associated with severe coronavirus disease-19 (COVID-19) has brought a shift in the way we envision aberrant endosomal acidification. Is it linked to an increased susceptibility to viral infection or a propensity to develop critical illness? This review summarizes the genetic and cellular evidence linking endosomal Na+ /H+ exchangers and viral diseases to suggest how they can act as a broad-spectrum modulator of viral infection and downstream pathophysiology. The review also presents novel insights supporting the complex role of endosomal acid-base homeostasis in viral pathogenesis and discusses the potential causes for negative outcomes of clinical trials utilizing alkalinizing drugs as therapies for COVID-19. These findings lead to a pathogenic model of viral disease that predicts that nonspecific targeting of endosomal pH might fail, even if administered early on, and suggests that endosomal Na+ /H+ exchangers may regulate key host antiviral defence mechanisms and mediators that act to drive inflammatory organ injury.


Assuntos
COVID-19/terapia , SARS-CoV-2/patogenicidade , Trocadores de Sódio-Hidrogênio/genética , Viroses/terapia , COVID-19/genética , COVID-19/virologia , Endossomos/genética , Endossomos/virologia , Humanos , Prótons , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Viroses/genética , Viroses/virologia
2.
Antiviral Res ; 194: 105167, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34450201

RESUMO

Niemann-Pick type C1 (NPC1) receptor is an endosomal membrane protein that regulates intracellular cholesterol traffic. This protein has been shown to play an important role for several viruses. It has been reported that SARS-CoV-2 enters the cell through plasma membrane fusion and/or endosomal entry upon availability of proteases. However, the whole process is not fully understood yet and additional viral/host factors might be required for viral fusion and subsequent viral replication. Here, we report a novel interaction between the SARS-CoV-2 nucleoprotein (N) and the cholesterol transporter NPC1. Furthermore, we have found that some compounds reported to interact with NPC1, carbazole SC816 and sulfides SC198 and SC073, were able to reduce SARS-CoV-2 viral infection with a good selectivity index in human cell infection models. These findings suggest the importance of NPC1 for SARS-CoV-2 viral infection and a new possible potential therapeutic target to fight against COVID-19.


Assuntos
Transporte Biológico , COVID-19/tratamento farmacológico , Endossomos/virologia , Proteína C1 de Niemann-Pick/análise , SARS-CoV-2/fisiologia , Animais , Carbazóis/farmacologia , Chlorocebus aethiops , Endossomos/química , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Fusão de Membrana , Células Vero , Replicação Viral
3.
Viruses ; 13(6)2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-34064125

RESUMO

To initiate infection, a virus enters a host cell typically via receptor-dependent endocytosis. It then penetrates a subcellular membrane, reaching a destination that supports transcription, translation, and replication of the viral genome. These steps lead to assembly and morphogenesis of the new viral progeny. The mature virus finally exits the host cell to begin the next infection cycle. Strikingly, viruses hijack host molecular chaperones to accomplish these distinct entry steps. Here we highlight how DNA viruses, including polyomavirus and the human papillomavirus, exploit soluble and membrane-associated chaperones to enter a cell, penetrating and escaping an intracellular membrane en route for infection. We also describe the mechanism by which RNA viruses-including flavivirus and coronavirus-co-opt cytosolic and organelle-selective chaperones to promote viral endocytosis, protein biosynthesis, replication, and assembly. These examples underscore the importance of host chaperones during virus infection, potentially revealing novel antiviral strategies to combat virus-induced diseases.


Assuntos
Vírus de DNA/fisiologia , Chaperonas Moleculares/metabolismo , Vírus de RNA/fisiologia , Citosol/metabolismo , Vírus de DNA/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Endossomos/metabolismo , Endossomos/virologia , Interações Hospedeiro-Patógeno , Membranas Intracelulares/metabolismo , Vírus de RNA/metabolismo , Internalização do Vírus , Replicação Viral
4.
Cells ; 10(5)2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-34067054

RESUMO

The flavonoid naringenin (Nar), present in citrus fruits and tomatoes, has been identified as a blocker of an emerging class of human intracellular channels, namely the two-pore channel (TPC) family, whose role has been established in several diseases. Indeed, Nar was shown to be effective against neoangiogenesis, a process essential for solid tumor progression, by specifically impairing TPC activity. The goal of the present review is to illustrate the rationale that links TPC channels to the mechanism of coronavirus infection, and how their inhibition by Nar could be an efficient pharmacological strategy to fight the current pandemic plague COVID-19.


Assuntos
COVID-19/tratamento farmacológico , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Flavanonas/farmacologia , Neoplasias/tratamento farmacológico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Antivirais/farmacologia , Antivirais/uso terapêutico , Arabidopsis/metabolismo , COVID-19/epidemiologia , COVID-19/patologia , COVID-19/virologia , Bloqueadores dos Canais de Cálcio/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Endossomos/virologia , Flavanonas/uso terapêutico , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Lisossomos/virologia , Neoplasias/irrigação sanguínea , Neoplasias/patologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/patologia , Pandemias/prevenção & controle , SARS-CoV-2/patogenicidade , Vacúolos/metabolismo , Internalização do Vírus/efeitos dos fármacos
5.
mBio ; 12(3)2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33975940

RESUMO

Enveloped viruses exploit cellular trafficking pathways for their morphogenesis, providing potential scope for the development of new antiviral therapies. We have previously shown that herpes simplex virus 1 (HSV1) utilizes recycling endocytic membranes as the source of its envelope, in a process involving four Rab GTPases. To identify novel factors involved in HSV1 envelopment, we have screened a small interfering RNA (siRNA) library targeting over 80 human trafficking proteins, including coat proteins, adaptor proteins, fusion factors, fission factors, and Rab effectors. The depletion of 11 factors reduced virus yields by 20- to 100-fold, including three early secretory pathway proteins, four late secretory pathway proteins, and four endocytic pathway proteins, three of which are membrane fission factors. Five of the 11 targets were chosen for further analysis in virus infection, where it was found that the absence of only 1, the fission factor CHMP4C, but not the CHMP4A or CHMP4B paralogues, reduced virus production at the final stage of morphogenesis. Ultrastructural and confocal microscopy of CHMP4C-depleted, HSV1-infected cells showed an accumulation of endocytic membranes; extensive tubulation of recycling, transferrin receptor-positive endosomes indicative of aberrant fission; and a failure in virus envelopment. No effect on the late endocytic pathway was detected, while exogenous CHMP4C was shown to localize to recycling endosomes. Taken together, these data reveal a novel role for the CHMP4C fission factor in the integrity of the recycling endosomal network, which has been unveiled through the dependence of HSV1 on these membranes for the acquisition of their envelopes.IMPORTANCE Cellular transport pathways play a fundamental role in secretion and membrane biogenesis. Enveloped viruses exploit these pathways to direct their membrane proteins to sites of envelopment and, as such, are powerful tools for unraveling subtle activities of trafficking factors, potentially pinpointing therapeutic targets. Using the sensitive biological readout of virus production, over 80 trafficking factors involved in diverse and poorly defined cellular processes have been screened for involvement in the complex process of HSV1 envelopment. Out of 11 potential targets, CHMP4C, a key component in the cell cycle abscission checkpoint, stood out as being required for the process of virus wrapping in endocytic tubules, where it localized. In the absence of CHMP4C, recycling endocytic membranes failed to undergo scission in infected cells, causing transient tubulation and accumulation of membranes and unwrapped virus. These data reveal a new role for this important cellular factor in the biogenesis of recycling endocytic membranes.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/metabolismo , Montagem de Vírus/genética , Endocitose , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Endossomos/virologia , Células HeLa , Herpes Simples/virologia , Humanos
6.
Curr Opin Virol ; 47: 95-105, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33690104

RESUMO

Polyomaviruses are mostly non-pathogenic, yet some can cause human disease especially under conditions of immunosuppression, including JC, BK, and Merkel cell polyomaviruses. Direct interactions between viruses and the host early during infection dictate the outcome of disease, many of which remain enigmatic. However, significant work in recent years has contributed to our understanding of how this virus family establishes an infection, largely due to advances made for animal polyomaviruses murine and SV40. Here we summarize the major findings that have contributed to our understanding of polyomavirus entry, trafficking, disassembly, signaling, and immune evasion during the infectious process and highlight major unknowns in these processes that are open areas of study.


Assuntos
Polyomavirus/fisiologia , Internalização do Vírus , Animais , Núcleo Celular/virologia , Retículo Endoplasmático/virologia , Endossomos/virologia , Humanos , Evasão da Resposta Imune , Transdução de Sinais , Ligação Viral
7.
Commun Biol ; 4(1): 229, 2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33603190

RESUMO

Human C-type lectin member 18A (CLEC18A) is ubiquitously expressed in human, and highest expression levels are found in human myeloid cells and liver. In contrast, mouse CLEC18A (mCLEC18A) is only expressed in brain, kidney and heart. However, the biological functions of CLEC18A are still unclear. We have shown that a single amino acid change (S339 →R339) in CTLD domain has profound effect in their binding to polysaccharides and house dust mite allergens. In this study, we further demonstrate that CLEC18A and its mutant CLEC18A(S339R) associate with TLR3 in endosome and bind poly (I:C) specifically. Compared to TLR3 alone, binding affinity to poly (I:C) is further increased in TLR3-CLEC18A and TLR3-CLEC18A(S339R) complexes. Moreover, CLEC18A and CLEC18A(S339R) enhance the production of type I and type III interferons (IFNs), but not proinflammatory cytokines, in response to poly (I:C) or H5N1 influenza A virus (IAV) infection. Compared to wild type (WT) mice, ROSA-CLEC18A and ROSA-CLEC18A(S339R) mice generate higher amounts of interferons and are more resistant to H5N1 IAV infection. Thus, CLEC18A is a TLR3 co-receptor, and may contribute to the differential immune responses to poly (I:C) and IAV infection between human and mouse.


Assuntos
Endossomos/metabolismo , Virus da Influenza A Subtipo H5N1/imunologia , Lectinas Tipo C/metabolismo , Infecções por Orthomyxoviridae/metabolismo , Receptor 3 Toll-Like/metabolismo , Animais , Animais Geneticamente Modificados , Citocinas/metabolismo , Modelos Animais de Doenças , Cães , Endossomos/efeitos dos fármacos , Endossomos/imunologia , Endossomos/virologia , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Mediadores da Inflamação/metabolismo , Virus da Influenza A Subtipo H5N1/patogenicidade , Lectinas Tipo C/agonistas , Lectinas Tipo C/genética , Células Madin Darby de Rim Canino , Camundongos Endogâmicos C57BL , Mutação , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Poli I-C/farmacologia , Transdução de Sinais , Especificidade da Espécie , Receptor 3 Toll-Like/agonistas
8.
mBio ; 12(1)2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33531399

RESUMO

Human bocavirus 1 (HBoV1), a nonenveloped single-stranded DNA parvovirus, causes mild to life-threatening respiratory tract infections, acute otitis media, and encephalitis in young children. HBoV1 often persists in nasopharyngeal secretions for months, hampering diagnosis. It has also been shown to persist in pediatric palatine and adenoid tonsils, which suggests that lymphoid organs are reservoirs for virus spread; however, the tissue site and host cells remain unknown. Our aim was to determine, in healthy nonviremic children with preexisting HBoV1 immunity, the adenotonsillar persistence site(s), host cell types, and virus activity. We discovered that HBoV1 DNA persists in lymphoid germinal centers (GCs), but not in the corresponding tonsillar epithelium, and that the cell types harboring the virus are mainly naive, activated, and memory B cells and monocytes. Both viral DNA strands and both sides of the genome were detected, as well as infrequent mRNA. Moreover, we showed, in B-cell and monocyte cultures and ex vivo tonsillar B cells, that the cellular uptake of HBoV1 occurs via the Fc receptor (FcγRII) through antibody-dependent enhancement (ADE). This resulted in viral mRNA transcription, known to occur exclusively from double-stranded DNA in the nucleus, however, with no detectable productive replication. Confocal imaging with fluorescent virus-like particles moreover disclosed endocytosis. To which extent the active HBoV1 GC persistence has a role in chronic inflammation or B-cell maturation disturbances, and whether the virus can be reactivated, will be interesting topics for forthcoming studies.IMPORTANCE Human bocavirus 1 (HBoV1), a common pediatric respiratory pathogen, can persist in airway secretions for months hampering diagnosis. It also persists in tonsils, providing potential reservoirs for airway shedding, with the exact location, host cell types, and virus activity unknown. Our study provides new insights into tonsillar HBoV1 persistence. We observed HBoV1 persistence exclusively in germinal centers where immune maturation occurs, and the main host cells were B cells and monocytes. In cultured cell lines and primary tonsillar B cells, we showed the virus uptake to be significantly enhanced by HBoV1-specific antibodies, mediated by the cellular IgG receptor, leading to viral mRNA synthesis, but without detectable productive replication. Possible implications of such active viral persistence could be tonsillar inflammation, disturbances in immune maturation, reactivation, or cell death with release of virus DNA, explaining the long-lasting HBoV1 airway shedding.


Assuntos
Anticorpos Facilitadores , Centro Germinativo/virologia , Bocavirus Humano/imunologia , Tonsila Palatina/virologia , Infecções por Parvoviridae/virologia , Adolescente , Adulto , Idoso , Linfócitos B/virologia , Criança , Pré-Escolar , DNA Viral/análise , Endossomos/virologia , Humanos , Lactente , Recém-Nascido , Pessoa de Meia-Idade , Monócitos/virologia , Infecções por Parvoviridae/imunologia , Adulto Jovem
9.
Nat Commun ; 12(1): 961, 2021 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-33574281

RESUMO

The global spread of SARS-CoV-2 is posing major public health challenges. One feature of SARS-CoV-2 spike protein is the insertion of multi-basic residues at the S1/S2 subunit cleavage site. Here, we find that the virus with intact spike (Sfull) preferentially enters cells via fusion at the plasma membrane, whereas a clone (Sdel) with deletion disrupting the multi-basic S1/S2 site utilizes an endosomal entry pathway. Using Sdel as model, we perform a genome-wide CRISPR screen and identify several endosomal entry-specific regulators. Experimental validation of hits from the CRISPR screen shows that host factors regulating the surface expression of angiotensin-converting enzyme 2 (ACE2) affect entry of Sfull virus. Animal-to-animal transmission with the Sdel virus is reduced compared to Sfull in the hamster model. These findings highlight the critical role of the S1/S2 boundary of SARS-CoV-2 spike protein in modulating virus entry and transmission and provide insights into entry of coronaviruses.


Assuntos
COVID-19/virologia , Sistemas CRISPR-Cas , Estudo de Associação Genômica Ampla , Interações Hospedeiro-Patógeno , SARS-CoV-2/fisiologia , Internalização do Vírus , Células A549 , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , COVID-19/genética , Chlorocebus aethiops , Modelos Animais de Doenças , Endossomos/virologia , Células HeLa , Humanos , Mesocricetus , Serina Endopeptidases , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero
10.
Int J Mol Sci ; 22(4)2021 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-33562748

RESUMO

Human adenoviruses (HAdVs) display a wide range of tissue tropism and can cause an array of symptoms from mild respiratory illnesses to disseminated and life-threatening infections in immunocompromised individuals. However, no antiviral drug has been approved specifically for the treatment of HAdV infections. Herein, we report our continued efforts to optimize salicylamide derivatives and discover compound 16 (JMX0493) as a potent inhibitor of HAdV infection. Compound 16 displays submicromolar IC50 values, a higher selectivity index (SI > 100) and 2.5-fold virus yield reduction compared to our hit compound niclosamide. Moreover, unlike niclosamide, our mechanistic studies suggest that the antiviral activity of compound 16 against HAdV is achieved through the inhibition of viral particle escape from the endosome, which bars subsequent uncoating and the presentation of lytic protein VI.


Assuntos
Adenovírus Humanos/fisiologia , Antivirais/farmacologia , Endossomos/virologia , Niclosamida/farmacologia , Salicilamidas/farmacologia , Células A549 , Adenovírus Humanos/efeitos dos fármacos , Descoberta de Drogas , Endossomos/efeitos dos fármacos , Células HEK293 , Humanos , Concentração Inibidora 50 , Niclosamida/química , Salicilamidas/química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Tropismo Viral , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
11.
PLoS Pathog ; 17(1): e1009275, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33513206

RESUMO

Filoviruses, such as the Ebola virus (EBOV) and Marburg virus (MARV), are causative agents of sporadic outbreaks of hemorrhagic fevers in humans. To infect cells, filoviruses are internalized via macropinocytosis and traffic through the endosomal pathway where host cathepsin-dependent cleavage of the viral glycoproteins occurs. Subsequently, the cleaved viral glycoprotein interacts with the late endosome/lysosome resident host protein, Niemann-Pick C1 (NPC1). This interaction is hypothesized to trigger viral and host membrane fusion, which results in the delivery of the viral genome into the cytoplasm and subsequent initiation of replication. Some studies suggest that EBOV viral particles activate signaling cascades and host-trafficking factors to promote their localization with host factors that are essential for entry. However, the mechanism through which these activating signals are initiated remains unknown. By screening a kinase inhibitor library, we found that receptor tyrosine kinase inhibitors potently block EBOV and MARV GP-dependent viral entry. Inhibitors of epidermal growth factor receptor (EGFR), tyrosine protein kinase Met (c-Met), and the insulin receptor (InsR)/insulin like growth factor 1 receptor (IGF1R) blocked filoviral GP-mediated entry and prevented growth of replicative EBOV in Vero cells. Furthermore, inhibitors of c-Met and InsR/IGF1R also blocked viral entry in macrophages, the primary targets of EBOV infection. Interestingly, while the c-Met and InsR/IGF1R inhibitors interfered with EBOV trafficking to NPC1, virus delivery to the receptor was not impaired in the presence of the EGFR inhibitor. Instead, we observed that the NPC1 positive compartments were phenotypically altered and rendered incompetent to permit viral entry. Despite their different mechanisms of action, all three RTK inhibitors tested inhibited virus-induced Akt activation, providing a possible explanation for how EBOV may activate signaling pathways during entry. In sum, these studies strongly suggest that receptor tyrosine kinases initiate signaling cascades essential for efficient post-internalization entry steps.


Assuntos
Ebolavirus/fisiologia , Doença pelo Vírus Ebola/virologia , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Animais , Chlorocebus aethiops , Ebolavirus/genética , Endocitose , Endossomos/metabolismo , Endossomos/virologia , Interações Hospedeiro-Patógeno , Humanos , Espaço Intracelular/virologia , Lisossomos/metabolismo , Transporte Proteico , Proteínas Tirosina Quinases/genética , Células Vero , Vírion , Internalização do Vírus , Replicação Viral
12.
J Biol Chem ; 296: 100306, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33476648

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19, so understanding its biology and infection mechanisms is critical to facing this major medical challenge. SARS-CoV-2 is known to use its spike glycoprotein to interact with the cell surface as a first step in the infection process. As for other coronaviruses, it is likely that SARS-CoV-2 next undergoes endocytosis, but whether or not this is required for infectivity and the precise endocytic mechanism used are unknown. Using purified spike glycoprotein and lentivirus pseudotyped with spike glycoprotein, a common model of SARS-CoV-2 infectivity, we now demonstrate that after engagement with the plasma membrane, SARS-CoV-2 undergoes rapid, clathrin-mediated endocytosis. This suggests that transfer of viral RNA to the cell cytosol occurs from the lumen of the endosomal system. Importantly, we further demonstrate that knockdown of clathrin heavy chain, which blocks clathrin-mediated endocytosis, reduces viral infectivity. These discoveries reveal that SARS-CoV-2 uses clathrin-mediated endocytosis to gain access into cells and suggests that this process is a key aspect of virus infectivity.


Assuntos
Enzima de Conversão de Angiotensina 2/genética , Cadeias Pesadas de Clatrina/genética , Endocitose/genética , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Internalização do Vírus/efeitos dos fármacos , Células A549 , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Chlorocebus aethiops , Cadeias Pesadas de Clatrina/antagonistas & inibidores , Cadeias Pesadas de Clatrina/metabolismo , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Endossomos/virologia , Regulação da Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Interações Hospedeiro-Patógeno/genética , Humanos , Hidrazonas/farmacologia , Lentivirus/genética , Lentivirus/metabolismo , Ligação Proteica/efeitos dos fármacos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/metabolismo , Transdução de Sinais , Glicoproteína da Espícula de Coronavírus/metabolismo , Sulfonamidas/farmacologia , Tiazolidinas/farmacologia , Células Vero
14.
Cell Calcium ; 94: 102360, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33516131

RESUMO

Ion channels are necessary for correct lysosomal function including degradation of cargoes originating from endocytosis. Almost all enveloped viruses, including coronaviruses (CoVs), enter host cells via endocytosis, and do not escape endosomal compartments into the cytoplasm (via fusion with the endolysosomal membrane) unless the virus-encoded envelope proteins are cleaved by lysosomal proteases. With the ongoing outbreak of severe acute respiratory syndrome (SARS)-CoV-2, endolysosomal two-pore channels represent an exciting and emerging target for antiviral therapies. This review focuses on the latest knowledge of the effects of lysosomal ion channels on the cellular entry and uncoating of enveloped viruses, which may aid in development of novel therapies against emerging infectious diseases such as SARS-CoV-2.


Assuntos
Antivirais/uso terapêutico , COVID-19/virologia , Canais Iônicos/fisiologia , Lisossomos/virologia , SARS-CoV-2/fisiologia , Envelope Viral/fisiologia , Internalização do Vírus , Desenvelopamento do Vírus , Aminopiridinas/farmacologia , Aminopiridinas/uso terapêutico , Antivirais/farmacologia , Desenho de Fármacos , Endocitose , Endossomos/metabolismo , Endossomos/virologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Compostos Heterocíclicos com 3 Anéis/uso terapêutico , Humanos , Hidrazonas/farmacologia , Hidrazonas/uso terapêutico , Canais Iônicos/classificação , Lisossomos/enzimologia , Lisossomos/metabolismo , Modelos Biológicos , Morfolinas/farmacologia , Morfolinas/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , ATPases Vacuolares Próton-Translocadoras/fisiologia , Internalização do Vírus/efeitos dos fármacos , Desenvelopamento do Vírus/efeitos dos fármacos
15.
J Virol ; 95(3)2021 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-33177206

RESUMO

Previous studies have identified an interaction between the human papillomavirus (HPV) L2 minor capsid protein and sorting nexins 17 and 27 (SNX17 and SNX27) during virus infection. Further studies show the involvement of both retromer and retriever complexes in this process since knockdown of proteins from either complex impairs infection. In this study, we show that HPV L2 and 5-ethynyl-2'-deoxyuridine (EdU)-labeled pseudovirions colocalize with both retromer and retriever, with components of each complex being bound by L2 during infection. We also show that both sorting nexins may interact with either of the recycling complexes but that the interaction between SNX17 and HPV16 L2 is not responsible for retriever recruitment during infection, instead being required for retromer recruitment. Furthermore, we show that retriever recruitment most likely involves a direct interaction between L2 and the C16orf62 subunit of the retriever, in a manner similar to that of its interaction with the VPS35 subunit of retromer.IMPORTANCE Previous studies identified sorting nexins 17 and 27, as well as the retromer complex, as playing a role in HPV infection. This study shows that the newly identified retriever complex also plays an important role and begins to shed light on how both sorting nexins contribute to retromer and retriever recruitment during the infection process.


Assuntos
Proteínas do Capsídeo/metabolismo , Núcleo Celular/genética , Genoma Viral , Proteínas Oncogênicas Virais/metabolismo , Infecções por Papillomavirus/virologia , Nexinas de Classificação/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas do Capsídeo/genética , Membrana Celular/genética , Membrana Celular/virologia , Núcleo Celular/virologia , Endossomos/genética , Endossomos/virologia , Células HEK293 , Papillomavirus Humano 16/fisiologia , Humanos , Proteínas Oncogênicas Virais/genética , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/patologia , Transporte Proteico , Nexinas de Classificação/genética , Proteínas de Transporte Vesicular/genética
16.
J Virol ; 95(6)2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33328308

RESUMO

Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is a highly contagious disease of swine with high morbidity and mortality that negatively affects the pig industry worldwide, in particular in China. Soon after the endocytosis of CSFV, the virus makes full use of the components of host cells to complete its life cycle. The endocytosis sorting complex required for transport (ESCRT) system is a central molecular machine for membrane protein sorting and scission in eukaryotic cells that plays an essential role in many physiological metabolic processes, including invasion and egress of envelope viruses. However, the molecular mechanism that ESCRT uses to regulate the replication of CSFV is unknown. In this study, we demonstrated that the ESCRT-I complex Tsg101 protein participates in clathrin-mediated endocytosis of CSFV and is also involved in CSFV trafficking. Tsg101 assists the virus in entering the host cell through the late endosome (Rab7 and Rab9) and finally reaching the lysosome (Lamp-1). Interestingly, Tsg101 is also involved in the viral replication process by interacting with nonstructural proteins 4B and 5B of CSFV. Finally, confocal microscopy showed that the replication complex of Tsg101 and double-stranded RNA (dsRNA) or NS4B and NS5B protein was close to the endoplasmic reticulum (ER), not the Golgi, in the cytoplasm. Collectively, our finding highlights that Tsg101 regulates the process of CSFV entry and replication, indicating that the ESCRT plays an important role in the life cycle of CSFV. Thus, ESCRT molecules could serve as therapeutic targets to combat CSFV infection.IMPORTANCE CSF, caused by CSFV, is a World Organization for Animal Health (OIE) notifiable disease and causes significant financial losses to the pig industry globally. The ESCRT machinery plays an important regulatory role in several members of the genera Flavivirus and Hepacivirus within the family Flaviviridae, such as hepatitis C virus, Japanese encephalitis virus, and dengue virus. Previous reports have shown that assembling and budding of these viruses require ESCRT. However, the role of ESCRT in Pestivirus infection remains to be elucidated. We determined the molecular mechanisms of the regulation of CSFV infection by the major subunit Tsg101 of ESCRT-I. Interestingly, Tsg101 plays an essential regulatory role in both clathrin-mediated endocytosis and genome replication of CSFV. Overall, the results of this study provide further insights into the molecular function of ESCRT-I complex protein Tsg101 during CSFV infection, which may serve as a molecular target for pestivirus inhibitors.


Assuntos
Vírus da Febre Suína Clássica/fisiologia , Proteínas de Ligação a DNA/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Fatores de Transcrição/metabolismo , Internalização do Vírus , Replicação Viral , Animais , Linhagem Celular , Peste Suína Clássica/metabolismo , Peste Suína Clássica/virologia , Proteínas de Ligação a DNA/genética , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Endossomos/metabolismo , Endossomos/virologia , Interações Hospedeiro-Patógeno , Lisossomos/metabolismo , Lisossomos/virologia , RNA Viral/metabolismo , Suínos , Fatores de Transcrição/genética , Proteínas não Estruturais Virais/metabolismo , Compartimentos de Replicação Viral/metabolismo
17.
J Virol ; 95(6)2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33361427

RESUMO

Infectious bursal disease virus (IBDV) is the archetypal member of the family Birnaviridae and the etiological agent of Gumboro disease, a highly contagious immunosuppressive infection of concern to the global poultry sector for its adverse health effects in chicks. Unlike most double-stranded RNA (dsRNA) viruses, which enclose their genomes within specialized cores throughout their viral replication cycle, birnaviruses organize their bisegmented dsRNA genome in ribonucleoprotein (RNP) structures. Recently, we demonstrated that IBDV exploits endosomal membranes for replication. The establishment of IBDV replication machinery on the cytosolic leaflet of endosomal compartments is mediated by the viral protein VP3 and its intrinsic ability to target endosomes. In this study, we identified the early endosomal phosphatidylinositol 3-phosphate [PtdIns(3)P] as a key host factor of VP3 association with endosomal membranes and consequent establishment of IBDV replication complexes in early endosomes. Indeed, our data reveal a crucial role for PtdIns(3)P in IBDV replication. Overall, our findings provide new insights into the replicative strategy of birnaviruses and strongly suggest that it resembles those of positive-strand RNA (+ssRNA) viruses, which replicate in association with host membranes. Furthermore, our findings support the role of birnaviruses as evolutionary intermediaries between +ssRNA and dsRNA viruses and, importantly, demonstrate a novel role for PtdIns(3)P in the replication of a dsRNA virus.IMPORTANCE Infectious bursal disease virus (IBDV) infects chicks and is the causative agent of Gumboro disease. During IBDV outbreaks in recent decades, the emergence of very virulent variants and the lack of effective prevention/treatment strategies to fight this disease have had devastating consequences for the poultry industry. IBDV belongs to the peculiar family Birnaviridae Unlike most dsRNA viruses, birnaviruses organize their genomes in ribonucleoprotein complexes and replicate in a core-independent manner. We recently demonstrated that IBDV exploits host cell endosomes as platforms for viral replication, a process that depends on the VP3 viral protein. In this study, we delved deeper into the molecular characterization of IBDV-endosome association and investigated the role of host cell phosphatidylinositide lipids in VP3 protein localization and IBDV infection. Together, our findings demonstrate that PtdIns(3)P serves as a scaffold for the association of VP3 to endosomes and reveal its essential role for IBDV replication.


Assuntos
Endossomos/metabolismo , Vírus da Doença Infecciosa da Bursa/fisiologia , Fosfatos de Fosfatidilinositol/metabolismo , Compartimentos de Replicação Viral/metabolismo , Animais , Linhagem Celular , Endossomos/virologia , Membranas Intracelulares/metabolismo , Codorniz , Proteínas Estruturais Virais/metabolismo , Replicação Viral
18.
PLoS Pathog ; 16(11): e1009028, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33253291

RESUMO

Oncogenic human papillomaviruses (HPVs) replicate in differentiating epithelium, causing 5% of cancers worldwide. Like most other DNA viruses, HPV infection initiates after trafficking viral genome (vDNA) to host cell nuclei. Cells possess innate surveillance pathways to detect microbial components or physiological stresses often associated with microbial infections. One of these pathways, cGAS/STING, induces IRF3-dependent antiviral interferon (IFN) responses upon detection of cytosolic DNA. Virion-associated vDNA can activate cGAS/STING during initial viral entry and uncoating/trafficking, and thus cGAS/STING is an obstacle to many DNA viruses. HPV has a unique vesicular trafficking pathway compared to many other DNA viruses. As the capsid uncoats within acidic endosomal compartments, minor capsid protein L2 protrudes across vesicular membranes to facilitate transport of vDNA to the Golgi. L2/vDNA resides within the Golgi lumen until G2/M, whereupon vesicular L2/vDNA traffics along spindle microtubules, tethering to chromosomes to access daughter cell nuclei. L2/vDNA-containing vesicles likely remain intact until G1, following nuclear envelope reformation. We hypothesize that this unique vesicular trafficking protects HPV from cGAS/STING surveillance. Here, we investigate cGAS/STING responses to HPV infection. DNA transfection resulted in acute cGAS/STING activation and downstream IFN responses. In contrast, HPV infection elicited minimal cGAS/STING and IFN responses. To determine the role of vesicular trafficking in cGAS/STING evasion, we forced premature viral penetration of vesicular membranes with membrane-perturbing cationic lipids. Such treatment renders a non-infectious trafficking-defective mutant HPV infectious, yet susceptible to cGAS/STING detection. Overall, HPV evades cGAS/STING by its unique subcellular trafficking, a property that may contribute to establishment of infection.


Assuntos
Alphapapillomavirus/fisiologia , Genoma Viral/genética , Fator Regulador 3 de Interferon/metabolismo , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/metabolismo , Infecções por Papillomavirus/virologia , Alphapapillomavirus/genética , Alphapapillomavirus/imunologia , Transporte Biológico , Capsídeo/metabolismo , Endossomos/virologia , Humanos , Mutação , Vírion , Internalização do Vírus
19.
J Virol ; 95(1)2020 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-33115879

RESUMO

Many enveloped viruses infect cells within endocytic compartments. The pH drop that accompanies endosomal maturation, often in conjunction with proteolysis, triggers viral proteins to insert into the endosomal membrane and drive fusion. Fusion dynamics have been studied by tracking viruses within living cells, which limits the precision with which fusion can be synchronized and controlled, and reconstituting viral fusion to synthetic membranes, which introduces nonphysiological membrane curvature and composition. To overcome these limitations, we report chemically controllable triggering of single-virus fusion within endosomes. We isolated influenza (A/Aichi/68; H3N2) virus:endosome conjugates from cells, immobilized them in a microfluidic flow cell, and rapidly and controllably triggered fusion. Observed lipid-mixing kinetics were surprisingly similar to those of influenza virus fusion with model membranes of opposite curvature: 80% of single-virus events had indistinguishable kinetics. This result suggests that endosomal membrane curvature is not a key permissive feature for viral entry, at least lipid mixing. The assay preserved endosomal membrane asymmetry and protein composition, providing a platform to test how cellular restriction factors and altered endosomal trafficking affect viral membrane fusion.IMPORTANCE Many enveloped viruses infect cells via fusion to endosomes, but controlling this process within living cells has been challenging. We studied the fusion of influenza virus virions to endosomes in a chemically controllable manner. Extracting virus:endosome conjugates from cells and exogenously triggering fusion permits precise study of virus:endosome fusion kinetics. Surprisingly, endosomal curvature does not grossly alter fusion kinetics, although membrane deformability does. This supports a model for influenza virus entry where cells restrict or permit membrane fusion by changing deformability, for instance, using interferon-induced proteins.


Assuntos
Endossomos/virologia , Vírus da Influenza A Subtipo H3N2/fisiologia , Internalização do Vírus , Animais , Linhagem Celular , Cricetinae , Endossomos/metabolismo , Membranas Intracelulares/metabolismo , Cinética , Lipossomos/metabolismo , Lipídeos de Membrana/metabolismo
20.
Cells ; 9(9)2020 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-32887393

RESUMO

The human hepatitis B virus (HBV), that is causative for more than 240 million cases of chronic liver inflammation (hepatitis), is an enveloped virus with a partially double-stranded DNA genome. After virion uptake by receptor-mediated endocytosis, the viral nucleocapsid is transported towards the nuclear pore complex. In the nuclear basket, the nucleocapsid disassembles. The viral genome that is covalently linked to the viral polymerase, which harbors a bipartite NLS, is imported into the nucleus. Here, the partially double-stranded DNA genome is converted in a minichromosome-like structure, the covalently closed circular DNA (cccDNA). The DNA virus HBV replicates via a pregenomic RNA (pgRNA)-intermediate that is reverse transcribed into DNA. HBV-infected cells release apart from the infectious viral parrticle two forms of non-infectious subviral particles (spheres and filaments), which are assembled by the surface proteins but lack any capsid and nucleic acid. In addition, naked capsids are released by HBV replicating cells. Infectious viral particles and filaments are released via multivesicular bodies; spheres are secreted by the classic constitutive secretory pathway. The release of naked capsids is still not fully understood, autophagosomal processes are discussed. This review describes intracellular trafficking pathways involved in virus entry, morphogenesis and release of (sub)viral particles.


Assuntos
DNA Viral/genética , Genoma Viral , Vírus da Hepatite B/genética , Hepatite B Crônica/virologia , Vírion/genética , Liberação de Vírus , Transporte Biológico , DNA Circular/genética , DNA Circular/metabolismo , DNA Viral/metabolismo , Endossomos/metabolismo , Endossomos/virologia , Antígenos do Núcleo do Vírus da Hepatite B/genética , Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Antígenos de Superfície da Hepatite B/genética , Antígenos de Superfície da Hepatite B/metabolismo , Antígenos E da Hepatite B/genética , Antígenos E da Hepatite B/metabolismo , Vírus da Hepatite B/crescimento & desenvolvimento , Vírus da Hepatite B/metabolismo , Hepatite B Crônica/genética , Hepatite B Crônica/patologia , Interações Hospedeiro-Patógeno/genética , Humanos , Nucleocapsídeo/genética , Nucleocapsídeo/metabolismo , Nucleocapsídeo/ultraestrutura , Transcrição Reversa , Vírion/crescimento & desenvolvimento , Vírion/metabolismo , Replicação Viral
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...