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1.
PLoS Pathog ; 16(3): e1008392, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32150576

RESUMO

Coronaviruses recognize a variety of receptors using different domains of their envelope-anchored spike protein. How these diverse receptor recognition patterns affect viral entry is unknown. Mouse hepatitis coronavirus (MHV) is the only known coronavirus that uses the N-terminal domain (NTD) of its spike to recognize a protein receptor, CEACAM1a. Here we determined the cryo-EM structure of MHV spike complexed with mouse CEACAM1a. The trimeric spike contains three receptor-binding S1 heads sitting on top of a trimeric membrane-fusion S2 stalk. Three receptor molecules bind to the sides of the spike trimer, where three NTDs are located. Receptor binding induces structural changes in the spike, weakening the interactions between S1 and S2. Using protease sensitivity and negative-stain EM analyses, we further showed that after protease treatment of the spike, receptor binding facilitated the dissociation of S1 from S2, allowing S2 to transition from pre-fusion to post-fusion conformation. Together these results reveal a new role of receptor binding in MHV entry: in addition to its well-characterized role in viral attachment to host cells, receptor binding also induces the conformational change of the spike and hence the fusion of viral and host membranes. Our study provides new mechanistic insight into coronavirus entry and highlights the diverse entry mechanisms used by different viruses.


Assuntos
Antígeno Carcinoembrionário/química , Vírus da Hepatite Murina/química , Vírus da Hepatite Murina/fisiologia , Receptores Virais/química , Glicoproteína da Espícula de Coronavírus/química , Internalização do Vírus , Animais , Antígeno Carcinoembrionário/metabolismo , Antígeno Carcinoembrionário/ultraestrutura , Linhagem Celular Tumoral , Microscopia Crioeletrônica , Células HEK293 , Humanos , Fusão de Membrana , Camundongos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Multimerização Proteica , Proteólise , Receptores Virais/metabolismo , Receptores Virais/ultraestrutura , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Vírus da SARS/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/ultraestrutura , Ligação Viral
3.
Emerg Microbes Infect ; 9(1): 571-581, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32172658

RESUMO

Classical swine fever virus (CSFV) is a member of the genus Pestivirus in the Flaviviridae family. To date, the host factors required for CSFV entry remain poorly characterized. To identify the functional membrane protein(s) involved in CSFV infection, we analyzed the transcriptomic data from previous studies describing gene expression profiles for CSFV, and found twelve novel candidate proteins. One of these proteins, MERTK, significantly reduced CSFV protein expression by RNA interference screening using a recombinant CSFV that contains a luciferase reporter to measure CSFV protein expression. Furthermore, our results demonstrated that either anti-MERTK antibodies or soluble MERTK ectodomain could reduce CSFV infection in PK-15 cells in a dose-dependent manner. Mechanistically, MERTK interacted with the E2 protein of CSFV and facilitated virus entry. After virus entry, MERTK downregulates of mRNA expression of IFN-ß and promotes CSFV infection. Interestingly, the soluble MERTK ectodomain could also reduce the infection of bovine viral diarrhea virus (BVDV), another pestivirus. Taken together, our results suggested that MERTK is a CSFV entry factor that synergistically dampens innate immune responses in PK-15 cells and is also involved in BVDV infection.


Assuntos
Vírus da Febre Suína Clássica/fisiologia , Peste Suína Clássica/imunologia , Imunidade Inata , Internalização do Vírus , c-Mer Tirosina Quinase/metabolismo , Animais , Bovinos , Linhagem Celular , Humanos , Recombinação Genética , Suínos , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , c-Mer Tirosina Quinase/genética
4.
Biomolecules ; 10(2)2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-32092911

RESUMO

The world is currently witnessing an outbreak of a new coronavirus spreading quickly across China and affecting at least 24 other countries. With almost 65,000 infected, a worldwide death toll of at least 1370 (as of 14 February 2020), and with the potential to affect up to two-thirds of the world population, COVID-19 is considered by the World Health Organization (WHO) to be a global health emergency. The speed of spread and infectivity of COVID-19 (also known as Wuhan-2019-nCoV) are dramatically exceeding those of the Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV). In fact, since September 2012, the WHO has been notified of 2494 laboratory-confirmed cases of infection with MERS-CoV, whereas the 2002-2003 epidemic of SARS affected 26 countries and resulted in more than 8000 cases. Therefore, although SARS, MERS, and COVID-19 are all the result of coronaviral infections, the causes of the coronaviruses differ dramatically in their transmissibility. It is likely that these differences in infectivity of coronaviruses can be attributed to the differences in the rigidity of their shells which can be evaluated using computational tools for predicting intrinsic disorder predisposition of the corresponding viral proteins.


Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus/transmissão , Pneumonia Viral/transmissão , Proteínas Virais/metabolismo , Animais , Infecções por Coronavirus/epidemiologia , Humanos , Pandemias , Pneumonia Viral/epidemiologia , Proteínas Virais/genética , Internalização do Vírus
5.
Emerg Microbes Infect ; 9(1): 457-468, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32090689

RESUMO

Porcine deltacoronavirus (PDCoV) is a newly emerging threat to the global porcine industry. PDCoV has been successfully isolated using various medium additives including trypsin, and although we know it is important for viral replication, the mechanism has not been fully elucidated. Here, we systematically investigated the role of trypsin in PDCoV replication including cell entry, cell-to-cell membrane fusion and virus release. Using pseudovirus entry assays, we demonstrated that PDCoV entry is not trypsin dependent. Furthermore, unlike porcine epidemic diarrhea virus (PEDV), in which trypsin is important for the release of virus from infected cells, PDCoV release was not affected by trypsin. We also demonstrated that trypsin promotes PDCoV replication by enhancing cell-to-cell membrane fusion. Most importantly, our study illustrates two distinct spreading patterns from infected cells to uninfected cells during PDCoV transmission, and the role of trypsin in PDCoV replication in cells with different virus spreading types. Overall, these results clarify that trypsin promotes PDCoV replication by mediating cell-to-cell fusion transmission but is not crucial for viral entry. This knowledge can potentially contribute to improvement of virus production efficiency in culture, not only for vaccine preparation but also to develop antiviral treatments.


Assuntos
Fusão Celular , Coronavirus/fisiologia , Fusão de Membrana , Tripsina/metabolismo , Animais , Linhagem Celular , Humanos , Suínos , Internalização do Vírus , Replicação Viral
6.
Nat Commun ; 11(1): 164, 2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31919360

RESUMO

Host dependency factors that are required for influenza A virus infection may serve as therapeutic targets as the virus is less likely to bypass them under drug-mediated selection pressure. Previous attempts to identify host factors have produced largely divergent results, with few overlapping hits across different studies. Here, we perform a genome-wide CRISPR/Cas9 screen and devise a new approach, meta-analysis by information content (MAIC) to systematically combine our results with prior evidence for influenza host factors. MAIC out-performs other meta-analysis methods when using our CRISPR screen as validation data. We validate the host factors, WDR7, CCDC115 and TMEM199, demonstrating that these genes are essential for viral entry and regulation of V-type ATPase assembly. We also find that CMTR1, a human mRNA cap methyltransferase, is required for efficient viral cap snatching and regulation of a cell autonomous immune response, and provides synergistic protection with the influenza endonuclease inhibitor Xofluza.


Assuntos
Predisposição Genética para Doença/genética , Interações Hospedeiro-Patógeno/genética , Vírus da Influenza A/patogenicidade , Influenza Humana/genética , Influenza Humana/patologia , Células A549 , Proteínas Adaptadoras de Transdução de Sinal/genética , Antivirais/farmacologia , Sistemas CRISPR-Cas , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Estudo de Associação Genômica Ampla , Humanos , Proteínas de Membrana/genética , Metiltransferases/metabolismo , Proteínas do Tecido Nervoso/genética , Oxazinas/farmacologia , Piridinas/farmacologia , Tiepinas/farmacologia , Triazinas/farmacologia , ATPases Vacuolares Próton-Translocadoras/metabolismo , Internalização do Vírus
7.
Adv Exp Med Biol ; 1179: 17-37, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31741332

RESUMO

Chronic hepatitis B virus (HBV) infection remains to be a serious threat to public health and is associated with many liver diseases including chronic hepatitis B (CHB), liver cirrhosis, and hepatocellular carcinoma. Although nucleos(t)ide analogues (NA) and pegylated interferon-α (Peg-IFNα) have been confirmed to be efficient in inhibiting HBV replication, it is difficult to eradicate HBV and achieve the clinical cure of CHB. Therefore, long-term therapy has been recommended to CHB treatment under the current antiviral therapy. In this context, the new antiviral therapy targeting one or multiple critical steps of viral life cycle may be an alternative approach in future. In the last decade, the functional receptor [sodium-taurocholate cotransporting polypeptide (NTCP)] of HBV entry into hepatocytes has been discovered, and the immature nucleocapsids containing the non- or partially reverse-transcribed pregenomic RNA, the nucleocapsids containing double-strand linear DNA (dslDNA), and the empty particles devoid of any HBV nucleic acid have been found to be released into circulation, which have supplemented the life cycle of HBV. The understanding of HBV life cycle may offer a new instruction for searching the potential antiviral targets, and the new viral markers used to monitor the efficacy of antiviral therapy for CHB patients in the future.


Assuntos
Antivirais , Vírus da Hepatite B , Hepatite B Crônica , Antivirais/farmacologia , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/genética , Vírus da Hepatite B/crescimento & desenvolvimento , Hepatite B Crônica/virologia , Hepatócitos/virologia , Humanos , Interferon-alfa/farmacologia , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
8.
PLoS Pathog ; 15(12): e1008209, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31790506

RESUMO

The processes of cell attachment and membrane fusion of Herpes Simplex Virus 1 involve many different envelope glycoproteins. Viral proteins gC and gD bind to cellular receptors. Upon binding, gD activates the gH/gL complex which in turn activates gB to trigger membrane fusion. Thus, these proteins must be located at the point of contact between cellular and viral envelopes to interact and allow fusion. Using super-resolution microscopy, we show that gB, gH/gL and most of gC are distributed evenly round purified virions. In contrast, gD localizes essentially as clusters which are distinct from gB and gH/gL. Upon cell binding, we observe that all glycoproteins, including gD, have a similar ring-like pattern, but the diameter of these rings was significantly smaller than those observed on cell-free viruses. We also observe that contrary to cell-free particles, gD mostly colocalizes with other glycoproteins on cell-bound particles. The differing patterns of localization of gD between cell-free and cell-bound viruses indicates that gD can be reorganized on the viral envelope following either a possible maturation of the viral particle or its adsorption to the cell. This redistribution of glycoproteins upon cell attachment could contribute to initiate the cascade of activations leading to membrane fusion.


Assuntos
Herpesvirus Humano 1/metabolismo , Proteínas do Envelope Viral/metabolismo , Vírion/metabolismo , Linhagem Celular , Glicoproteínas/metabolismo , Glicoproteínas/ultraestrutura , Herpesvirus Humano 1/ultraestrutura , Humanos , Microscopia/métodos , Proteínas do Envelope Viral/ultraestrutura , Vírion/ultraestrutura , Ligação Viral , Internalização do Vírus
9.
PLoS Negl Trop Dis ; 13(12): e0007819, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31825972

RESUMO

BACKGROUND: Ebolavirus (EBOV) outbreaks, while sporadic, cause tremendous morbidity and mortality. No therapeutics or vaccines are currently licensed; however, a vaccine has shown promise in clinical trials. A critical step towards development of effective therapeutics is a better understanding of factors that govern host susceptibility to this pathogen. As macrophages are an important cell population targeted during virus replication, we explore the effect of cytokine polarization on macrophage infection. METHODS/MAIN FINDINGS: We utilized a BSL2 EBOV model virus, infectious, recombinant vesicular stomatitis virus encoding EBOV glycoprotein (GP) (rVSV/EBOV GP) in place of its native glycoprotein. Macrophages polarized towards a M2-like anti-inflammatory state by combined IL-4 and IL-13 treatment were more susceptible to rVSV/EBOV GP, but not to wild-type VSV (rVSV/G), suggesting that EBOV GP-dependent entry events were enhanced by these cytokines. Examination of RNA expression of known surface receptors that bind and internalize filoviruses demonstrated that IL-4/IL-13 stimulated expression of the C-type lectin receptor DC-SIGN in human macrophages and addition of the competitive inhibitor mannan abrogated IL-4/IL-13 enhanced infection. Two murine DC-SIGN-like family members, SIGNR3 and SIGNR5, were upregulated by IL-4/IL-13 in murine macrophages, but only SIGNR3 enhanced virus infection in a mannan-inhibited manner, suggesting that murine SIGNR3 plays a similar role to human DC-SIGN. In vivo IL-4/IL-13 administration significantly increased virus-mediated mortality in a mouse model and transfer of ex vivo IL-4/IL-13-treated murine peritoneal macrophages into the peritoneal cavity of mice enhanced pathogenesis. SIGNIFICANCE: These studies highlight the ability of macrophage polarization to influence EBOV GP-dependent virus replication in vivo and ex vivo, with M2a polarization upregulating cell surface receptor expression and thereby enhancing virus replication. Our findings provide an increased understanding of the host factors in macrophages governing susceptibility to filoviruses and identify novel murine receptors mediating EBOV entry.


Assuntos
Ebolavirus/fisiologia , Interações Hospedeiro-Patógeno , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Macrófagos/imunologia , Macrófagos/virologia , Internalização do Vírus , Animais , Modelos Animais de Doenças , Feminino , Doença pelo Vírus Ebola/patologia , Doença pelo Vírus Ebola/virologia , Masculino , Camundongos Endogâmicos C57BL
10.
Vet Res ; 50(1): 110, 2019 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-31856906

RESUMO

Intestinal epithelium functions as a barrier to protect multicellular organisms from the outside world. It consists of epithelial cells closely connected by intercellular junctions, selective gates which control paracellular diffusion of solutes, ions and macromolecules across the epithelium and keep out pathogens. Rotavirus is one of the major enteric viruses causing severe diarrhea in humans and animals. It specifically infects the enterocytes on villi of small intestines. The polarity of rotavirus replication in their target enterocytes and the role of intestinal epithelial integrity were examined in the present study. Treatment with EGTA, a drug that chelates calcium and disrupts the intercellular junctions, (i) significantly enhanced the infection of rotavirus in primary enterocytes, (ii) increased the binding of rotavirus to enterocytes, but (iii) considerably blocked internalization of rotavirus. After internalization, rotavirus was resistant to EGTA treatment. To investigate the polarity of rotavirus infection, the primary enterocytes were cultured in a transwell system and infected with rotavirus at either the apical or the basolateral surface. Rotavirus preferentially infected enterocytes at the basolateral surface. Restriction of infection through apical inoculation was overcome by EGTA treatment. Overall, our findings demonstrate that integrity of the intestinal epithelium is crucial in the host's innate defense against rotavirus infection. In addition, the intercellular receptor is located basolaterally and disruption of intercellular junctions facilitates the binding of rotavirus to their receptor at the basolateral surface.


Assuntos
Enterócitos/virologia , Células Epiteliais/virologia , Mucosa Intestinal/citologia , Rotavirus/classificação , Rotavirus/fisiologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura/veterinária , Ácido Egtázico/farmacologia , Enterócitos/efeitos dos fármacos , Miofibroblastos/fisiologia , Suínos , Internalização do Vírus , Replicação Viral
11.
Emerg Microbes Infect ; 8(1): 1721-1733, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31769733

RESUMO

Human papillomavirus type 6 (HPV6) is the major etiologic agent of genital warts and recurrent respiratory papillomatosis. Although the commercial HPV vaccines cover HPV6, the neutralization sites and mode for HPV6 are poorly understood. Here, we identify the HPV6 neutralization sites and discriminate the inhibition of virus attachment and entry by three potent neutralizing antibodies (nAbs), 5D3, 17D5, and 15F7. Mutagenesis assays showed that these nAbs predominantly target surface loops BC, DE, and FG of HPV6 L1. Cryo-EM structures of the HPV6 pseudovirus (PsV) and its immune complexes revealed three distinct binding modalities - full-occupation-bound to capsid, top-center-bound-, and top-rim-bound to pentamers - and illustrated a structural atlas for three classes of antibody-bound footprints that are located at center-distal ring, center, and center-proximal ring of pentamer surface for 5D3, 17D5, and 15F7, respectively. Two modes of neutralization were identified: mAb 5D3 and 17D5 block HPV PsV from attaching to the extracellular matrix (ECM) and the cell surface, whereas 15F7 allows PsV attachment but prohibits PsV from entering the cell. These findings highlight three neutralization sites of HPV6 L1 and outline two antibody-mediated neutralization mechanisms against HPV6, which will be relevant for HPV virology and antiviral inhibitor design. HighlightsMajor neutralization sites of HPV6 were mapped on the pseudovirus cryo-EM structuremAb 15F7 binds HPV6 capsid with a novel top-rim binding modality and confers a post-attachment neutralizationmAb 17D5 binds capsid in top-centre manner but unexpectedly prevents virus from attachment to cell surface.


Assuntos
Papillomavirus Humano 6/fisiologia , Infecções por Papillomavirus/virologia , Ligação Viral , Internalização do Vírus , Animais , Anticorpos Neutralizantes/análise , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/análise , Anticorpos Antivirais/imunologia , Epitopos/genética , Epitopos/imunologia , Papillomavirus Humano 6/genética , Papillomavirus Humano 6/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Infecções por Papillomavirus/imunologia
12.
Vet Microbiol ; 239: 108455, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31767073

RESUMO

Clathrin-mediated endocytosis is a mechanism used for the invasion of cells by a variety of viruses. Mortalin protein is involved in a variety of cellular functions and plays a role in viral infection. In this study, we found that mortalin significantly inhibited the replication of porcine epidemic diarrhea virus (PEDV) through restricting virus entry. Mechanistically, a biochemical interaction between the carboxyl terminus of mortalin and clathrin heavy chain (CLTC) was been found, and mortalin could induce CLTC degradation through the proteasomal pathway, thereby inhibiting the clathrin-mediated endocytosis of PEDV into host cells. In addition, artificial changes in mortalin expression affected the cell entry of transferrin, further confirming the above results. Finally, we confirmed that this host-mounted antiviral mechanism was broadly applicable to other viruses, such as vesicular stomatitis virus (VSV), rotavirus (RV), and transmissible gastroenteritis virus (TGEV), which use the same clathrin-mediated endocytic to entry. These results reveal a new function of mortalin in inhibiting endocytosis, and provide a novel strategy for treating PEDV infections.


Assuntos
Clatrina/metabolismo , Endocitose/fisiologia , Proteínas de Choque Térmico HSP70/metabolismo , Interações entre Hospedeiro e Microrganismos/fisiologia , Vírus da Diarreia Epidêmica Suína/fisiologia , Internalização do Vírus , Replicação Viral/fisiologia , Animais , Linhagem Celular , Regulação para Baixo/fisiologia , Inativação Gênica , Proteínas de Choque Térmico HSP70/genética , Células HeLa , Humanos , RNA Interferente Pequeno/metabolismo , Células Vero
13.
Nat Struct Mol Biol ; 26(10): 980-987, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31570878

RESUMO

Nipah virus (NiV) and Hendra virus (HeV) are zoonotic henipaviruses (HNVs) responsible for outbreaks of encephalitis and respiratory illness with fatality rates of 50-100%. No vaccines or licensed therapeutics currently exist to protect humans against NiV or HeV. HNVs enter host cells by fusing the viral and cellular membranes via the concerted action of the attachment (G) and fusion (F) glycoproteins, the main targets of the humoral immune response. Here, we describe the isolation and humanization of a potent monoclonal antibody cross-neutralizing NiV and HeV. Cryo-electron microscopy, triggering and fusion studies show the antibody binds to a prefusion-specific quaternary epitope, conserved in NiV F and HeV F glycoproteins, and prevents membrane fusion and viral entry. This work supports the importance of the HNV prefusion F conformation for eliciting a robust immune response and paves the way for using this antibody for prophylaxis and post-exposure therapy with NiV- and HeV-infected individuals.


Assuntos
Anticorpos Neutralizantes/farmacologia , Antivirais/farmacologia , Vírus Hendra/efeitos dos fármacos , Infecções por Henipavirus/tratamento farmacológico , Vírus Nipah/efeitos dos fármacos , Proteínas Virais de Fusão/antagonistas & inibidores , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Células HEK293 , Vírus Hendra/metabolismo , Infecções por Henipavirus/metabolismo , Infecções por Henipavirus/virologia , Humanos , Modelos Moleculares , Vírus Nipah/metabolismo , Proteínas Virais de Fusão/metabolismo , Internalização do Vírus/efeitos dos fármacos
14.
Nat Commun ; 10(1): 4460, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31575869

RESUMO

Viral infection is an intricate process that requires the concerted action of both viral and host cell components. Entry of viruses into cells is initiated by interactions between viral proteins and their cell surface receptors. Despite recent progress, the molecular mechanisms underlying the multistep reovirus entry process are poorly understood. Using atomic force microscopy, we investigated how the reovirus σ1 attachment protein binds to both α-linked sialic acid (α-SA) and JAM-A cell-surface receptors. We discovered that initial σ1 binding to α-SA favors a strong multivalent anchorage to JAM-A. The enhanced JAM-A binding by virions following α-SA engagement is comparable to JAM-A binding by infectious subvirion particles (ISVPs) in the absence of α-SA. Since ISVPs have an extended σ1 conformer, this finding suggests that α-SA binding triggers a conformational change in σ1. These results provide new insights into the function of viral attachment proteins in the initiation of infection and open new avenues for the use of reoviruses as oncolytic agents.


Assuntos
Polissacarídeos/metabolismo , Polissacarídeos/farmacologia , Ligação Proteica/efeitos dos fármacos , Receptores Virais/efeitos dos fármacos , Receptores Virais/metabolismo , Reoviridae/efeitos dos fármacos , Proteínas Virais/metabolismo , Ligação Viral/efeitos dos fármacos , Animais , Células CHO , Moléculas de Adesão Celular , Linhagem Celular , Cricetulus , Interações Hospedeiro-Patógeno , Modelos Moleculares , Ligação Proteica/fisiologia , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Superfície Celular/metabolismo , Proteínas Virais/química , Proteínas Virais/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos
15.
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
16.
PLoS Pathog ; 15(10): e1007956, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31589653

RESUMO

We report the analysis of a complex enveloped human virus, herpes simplex virus (HSV), assembled after in vivo incorporation of bio-orthogonal methionine analogues homopropargylglycine (HPG) or azidohomoalanine (AHA). We optimised protocols for the production of virions incorporating AHA (termed HSVAHA), identifying conditions which resulted in normal yields of HSV and normal particle/pfu ratios. Moreover we show that essentially every single HSVAHA capsid-containing particle was detectable at the individual particle level by chemical ligation of azide-linked fluorochromes to AHA-containing structural proteins. This was a completely specific chemical ligation, with no capsids assembled under normal methionine-containing conditions detected in parallel. We demonstrate by quantitative mass spectrometric analysis that HSVAHA virions exhibit no qualitative or quantitative differences in the repertoires of structural proteins compared to virions assembled under normal conditions. Individual proteins and AHA incorporation sites were identified in capsid, tegument and envelope compartments, including major essential structural proteins. Finally we reveal novel aspects of entry pathways using HSVAHA and chemical fluorochrome ligation that were not apparent from conventional immunofluorescence. Since ligation targets total AHA-containing protein and peptides, our results demonstrate the presence of abundant AHA-labelled products in cytoplasmic macrodomains and tubules which no longer contain intact particles detectable by immunofluorescence. Although these do not co-localise with lysosomal markers, we propose they may represent sites of proteolytic virion processing. Analysis of HSVAHA also enabled the discrimination from primary entering from secondary assembling virions, demonstrating assembly and second round infection within 6 hrs of initial infection and dual infections of primary and secondary virus in spatially restricted cytoplasmic areas of the same cell. Together with other demonstrated applications e.g., in genome biology, lipid and protein trafficking, this work further exemplifies the utility and potential of bio-orthogonal chemistry for studies in many aspects of virus-host interactions.


Assuntos
Aminoácidos/metabolismo , Herpes Simples/virologia , Herpesvirus Humano 1/fisiologia , Epitélio Pigmentado da Retina/virologia , Proteínas Estruturais Virais/metabolismo , Montagem de Vírus , Internalização do Vírus , Proliferação de Células , Células Cultivadas , Herpes Simples/metabolismo , Humanos , Epitélio Pigmentado da Retina/metabolismo
17.
Biochimie ; 166: 203-213, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31518617

RESUMO

Influenza A virus (IAV) is one of the most common infectious pathogen and associated with significant morbidity and mortality. Although processing the IAV hemagglutinin (HA) envelope glycoprotein precursor is a pre-requisite for viral membrane fusion activity, viral entry and transmission, HA-processing protease is not encoded in the IAV genome and thus the cellular trypsin-type serine HA-processing proteases determine viral infectious tropism and viral pathogenicity. The initial process of IAV infection of the airway is followed by marked upregulation of ectopic trypsin in various organs and endothelial cells through the induction of various proinflammatory cytokines, and this process has been termed the "influenza virus-cytokine-trypsin" cycle. In the advanced stage of IAV infection, the cytokine storm induces disorders of glucose and lipid metabolism and the "metabolic disorders-cytokine" cycle is then linked with the "influenza virus-cytokine-trypsin" cycle, to advance the pathogenic process into energy crisis and multiple organ failure. Application of protease inhibitors and treatment of metabolic disorders that break these cycles and their interconnection is therefore a promising therapeutic approach against influenza. This review discusses IAV pathogenicity on trypsin type serine HA-processing proteases, cytokines, metabolites and therapeutic options.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A , Influenza Humana , Serina Proteases/fisiologia , Internalização do Vírus/efeitos dos fármacos , Animais , Galinhas/virologia , Citocinas/metabolismo , Humanos , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/patogenicidade , Influenza Aviária/tratamento farmacológico , Influenza Aviária/virologia , Influenza Humana/tratamento farmacológico , Influenza Humana/virologia , Orthomyxoviridae/efeitos dos fármacos , Orthomyxoviridae/patogenicidade , Tripsina/metabolismo
18.
Int J Mol Sci ; 20(19)2019 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-31561440

RESUMO

Claudins regulate paracellular permeability in different tissues. The claudin-binding domain of Clostridium perfringens enterotoxin (cCPE) is a known modulator of a claudin subset. However, it does not efficiently bind to claudin-1 (Cldn1). Cldn1 is a pharmacological target since it is (i) an essential co-receptor for hepatitis C virus (HCV) infections and (ii) a key element of the epidermal barrier limiting drug delivery. In this study, we investigated the potential of a Cldn1-binding cCPE mutant (i) to inhibit HCV entry into hepatocytes and (ii) to open the epidermal barrier. Inhibition of HCV infection by blocking of Cldn1 with cCPE variants was analyzed in the Huh7.5 hepatoma cell line. A model of reconstructed human epidermis was used to investigate modulation of the epidermal barrier by cCPE variants. In contrast to cCPEwt, the Cldn1-binding cCPE-S305P/S307R/S313H inhibited infection of Huh7.5 cells with HCV in a dose-dependent manner. In addition, TJ modulation by cCPE variant-mediated targeting of Cldn1 and Cldn4 opened the epidermal barrier in reconstructed human epidermis. cCPE variants are potent claudin modulators. They can be applied for mechanistic in vitro studies and might also be used as biologics for therapeutic claudin targeting including HCV treatment (host-targeting antivirals) and improvement of drug delivery.


Assuntos
Claudinas/metabolismo , Enterotoxinas/metabolismo , Hepatócitos/metabolismo , Pele/metabolismo , Substituição de Aminoácidos , Linhagem Celular Tumoral , Claudinas/química , Enterotoxinas/química , Enterotoxinas/farmacologia , Epiderme/metabolismo , Hepacivirus/efeitos dos fármacos , Hepacivirus/fisiologia , Hepatite C/metabolismo , Hepatite C/virologia , Humanos , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Pele/citologia , Internalização do Vírus/efeitos dos fármacos , Replicação Viral
19.
Acta Virol ; 63(3): 316-321, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31507198

RESUMO

The recent Zika virus (ZIKV) outbreaks and rapid spread in tropical Latin America since introduction to Brazil in 2014, and now appearing cases in the USA, are alarming. World Health Organization (WHO) has considered transmission of ZIKV, a serious public health problem because of the increasing number of outbreaks. There are currently no drugs approved for the treatment of ZIKV infection. Discovery of safe and effective drugs are hampered by the risk in treating pregnant woman and toxicity to the fetus. Sweet basil, known as Ocimum basilicum in the scientific community, is a very well-known medicinal herb. Numerous studies have documented its beneficial activity against a great variety of human pathogens ranging from bacteria and virus to fungus and protozoans. Although, basil extracts and oils have been tested successfully against other viruses, its application to tackle ZIKV infection has not been exploited at all. In this study, we report for the first time that highly diluted ethanol extracts prepared from basil leaves can effectively inhibit ZIKV replication in Vero E6 cells with a half maximal inhibitory concentration (IC50) value of 1:134. The diluted extract as well as the amount of ethanol that goes into its preparation have been found to be completely non-toxic to the above mentioned cell line. The extract seems to inhibit the virus at the step of attachment and entry into the host cell. The specific inhibition of ZIKV observed using the basil leaf extract suggests a new alternative mode of treatment against flavivirus. Keywords: Zika virus; basil extract; antiviral.


Assuntos
Ocimum basilicum , Extratos Vegetais , Internalização do Vírus , Infecção por Zika virus , Animais , Sobrevivência Celular/efeitos dos fármacos , Etanol/química , Ocimum basilicum/química , Extratos Vegetais/farmacologia , Folhas de Planta/química , Plantas Medicinais/química , Células Vero , Internalização do Vírus/efeitos dos fármacos , Zika virus/efeitos dos fármacos , Zika virus/fisiologia
20.
mSphere ; 4(5)2019 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-31533998

RESUMO

Dengue is caused by infection with any one of four dengue viruses (DENV); the risk of severe disease appears to be enhanced by the cross-reactive or subneutralizing levels of antibody from a prior DENV infection. These antibodies opsonize DENV entry through the activating Fc gamma receptors (FcγR), instead of infection through canonical receptor-mediated endocytosis, to result in higher levels of DENV replication. However, whether the enhanced replication is solely due to more efficient FcγR-mediated DENV entry or is also through FcγR-mediated alteration of the host transcriptome response to favor DENV infection remains unclear. Indeed, more efficient viral entry through activation of the FcγR can result in an increased viral antigenic load within target cells and confound direct comparisons of the host transcriptome response under antibody-dependent and antibody-independent conditions. Herein, we show that, despite controlling for the viral antigenic load in primary monocytes, the antibody-dependent and non-antibody-dependent routes of DENV entry induce transcriptome responses that are remarkably different. Notably, antibody-dependent DENV entry upregulated DENV host dependency factors associated with RNA splicing, mitochondrial respiratory chain complexes, and vesicle trafficking. Additionally, supporting findings from other studies, antibody-dependent DENV entry impeded the downregulation of ribosomal genes caused by canonical receptor-mediated endocytosis to increase viral translation. Collectively, our findings support the notion that antibody-dependent DENV entry alters host responses that support the viral life cycle and that host responses to DENV need to be defined in the context of its entry pathway.IMPORTANCE Dengue virus is the most prevalent mosquito-borne viral infection globally, resulting in variable manifestations ranging from asymptomatic viremia to life-threatening shock and multiorgan failure. Previous studies have indicated that the risk of severe dengue in humans can be increased by a specific range of preexisting anti-dengue virus antibody titers, a phenomenon termed antibody-dependent enhancement. There is hence a need to understand how antibodies augment dengue virus infection compared to the alternative canonical receptor-mediated viral entry route. Herein, we show that, besides facilitating viral uptake, antibody-mediated entry increases the expression of early host dependency factors to promote viral infection; these factors include RNA splicing, mitochondrial respiratory chain complexes, vesicle trafficking, and ribosomal genes. These findings will enhance our understanding of how differences in entry pathways can affect host responses and offer opportunities to design therapeutics that can specifically inhibit antibody-dependent enhancement of dengue virus infection.


Assuntos
Anticorpos Antivirais/imunologia , Vírus da Dengue/fisiologia , Interações entre Hospedeiro e Microrganismos , Receptores de IgG/imunologia , Internalização do Vírus , Anticorpos Facilitadores , Antígenos Virais/imunologia , Linhagem Celular , Células Cultivadas , Dengue/virologia , Humanos , Monócitos/imunologia , Monócitos/virologia , Análise de Sequência de RNA , Transcriptoma , Replicação Viral
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