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1.
Sci Immunol ; 5(47)2020 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-32404436

RESUMO

Gastrointestinal symptoms and fecal shedding of SARS-CoV-2 RNA are frequently observed in COVID-19 patients. However, it is unclear whether SARS-CoV-2 replicates in the human intestine and contributes to possible fecal-oral transmission. Here, we report productive infection of SARS-CoV-2 in ACE2+ mature enterocytes in human small intestinal enteroids. Expression of two mucosa-specific serine proteases, TMPRSS2 and TMPRSS4, facilitated SARS-CoV-2 spike fusogenic activity and promoted virus entry into host cells. We also demonstrate that viruses released into the intestinal lumen were inactivated by simulated human colonic fluid, and infectious virus was not recovered from the stool specimens of COVID-19 patients. Our results highlight the intestine as a potential site of SARS-CoV-2 replication, which may contribute to local and systemic illness and overall disease progression.


Assuntos
Betacoronavirus/fisiologia , Enterócitos/virologia , Proteínas de Membrana/metabolismo , Serina Endopeptidases/metabolismo , Internalização do Vírus , Animais , Linhagem Celular , Duodeno/citologia , Enterócitos/patologia , Humanos , Camundongos , Organoides/virologia , Peptidil Dipeptidase A/metabolismo , Rotavirus/fisiologia , Vesiculovirus/genética
2.
Arch Virol ; 165(5): 1089-1097, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32146506

RESUMO

Gibbon ape leukemia virus (GALV) can infect a wide variety of cells but fails to infect most cells derived from laboratory mice. Transduction of human hematopoietic stem cells with GALV retroviral vectors is more efficient than with amphotropic vectors. In this study, a Moloney murine leukemia virus-gibbon ape leukemia virus (MoMLV-GALV) vector was constructed by replacing the natural env gene of the full-length Moloney MLV genome with the GALV env gene. To monitor viral transmission by green fluorescent protein (GFP) expression, internal ribosomal entry site-enhanced GFP (IRES-EGFP) was positioned between the GALV env gene and the 3' untranslated region (3' UTR) to obtain pMoMLV-GALV-EGFP. The MoMLV-GALV-EGFP vector was able to replicate with high titer in TE671 human rhabdomyosarcoma cells and U-87 human glioma cells. To evaluate the potential of the MoMLV-GALV vector as a therapeutic agent, the gene for the fusogenic envelope G glycoprotein of vesicular stomatitis virus (VSV-G) was incorporated into the vector. Infection with the resulting MoMLV-GALV-VSV-G vector resulted in lysis of the U-87 cells due to syncytium formation. Syncytium formation was also observed in the transfected human prostate cancer cell line LNCaP after extended cultivation of cells. In addition, we deleted the GALV env gene from the MoMLV-GALV-VSV-G vector to improve viral genome stability. This MoMLV-VSV-G vector is also replication competent and induces syncytium formation in 293T, HT1080, TE671 and U-87 cells. These results suggest that replication of the MoMLV-GALV-VSV-G vector or MoMLV-VSV-G vector may directly lead to cytotoxicity. Therefore, the vectors developed in this study are potentially useful tools for cancer gene therapy.


Assuntos
Vetores Genéticos , Vírus da Leucemia do Macaco Gibão/crescimento & desenvolvimento , Vírus da Leucemia Murina/crescimento & desenvolvimento , Vesiculovirus/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Replicação Viral , Animais , Linhagem Celular , Terapia Genética/métodos , Humanos , Vírus da Leucemia do Macaco Gibão/genética , Vírus da Leucemia Murina/genética , Camundongos , Neoplasias/terapia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Recombinação Genética
3.
Cell ; 181(2): 271-280.e8, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32142651

RESUMO

The recent emergence of the novel, pathogenic SARS-coronavirus 2 (SARS-CoV-2) in China and its rapid national and international spread pose a global health emergency. Cell entry of coronaviruses depends on binding of the viral spike (S) proteins to cellular receptors and on S protein priming by host cell proteases. Unravelling which cellular factors are used by SARS-CoV-2 for entry might provide insights into viral transmission and reveal therapeutic targets. Here, we demonstrate that SARS-CoV-2 uses the SARS-CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming. A TMPRSS2 inhibitor approved for clinical use blocked entry and might constitute a treatment option. Finally, we show that the sera from convalescent SARS patients cross-neutralized SARS-2-S-driven entry. Our results reveal important commonalities between SARS-CoV-2 and SARS-CoV infection and identify a potential target for antiviral intervention.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/tratamento farmacológico , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/tratamento farmacológico , Inibidores de Proteases/farmacologia , Serina Endopeptidases/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do Vírus/efeitos dos fármacos , Cloreto de Amônio/farmacologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/química , Betacoronavirus/genética , Linhagem Celular , Coronavirus/química , Coronavirus/genética , Coronavirus/fisiologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/terapia , Desenvolvimento de Medicamentos , Gabexato/análogos & derivados , Gabexato/farmacologia , Humanos , Imunização Passiva , Leucina/análogos & derivados , Leucina/farmacologia , Pandemias , Peptidil Dipeptidase A/química , Receptores Virais/química , Receptores Virais/metabolismo , Vírus da SARS/fisiologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Vesiculovirus/genética
4.
J Surg Res ; 245: 127-135, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31415934

RESUMO

BACKGROUND: The purpose of this study was to analyze the oncolytic and immunomodulatory functions of an M protein mutant of vesicular stomatitis virus (M51R VSV) in a murine model of peritoneal surface dissemination from colon cancer (PSD from CRC). METHODS: Luciferase-expressing CT26 peritoneal tumors were established in Balb/c mice to evaluate the impact of M51R VSV treatment on intraperitoneal tumor growth and overall survival. The mice were treated with either intraperitoneal phosphate buffered saline (n = 10) or 5 × 106 PFU M51R VSV (n = 10) at 5 d after tumor implantation. Tumor bioluminescence was measured every 3 d during the 60-day study period. The immunomodulatory effect of M51R VSV treatment was evaluated in mice treated with either intraperitoneal phosphate buffered saline (n = 21) or M51R VSV (n = 21). Peritoneal lavages were collected at days 1, 3, and 7 after M51R VSV treatment for flow cytometry and multiplex cytokine bead analysis. RESULTS: A single, intraperitoneal treatment with M51R VSV inhibited the growth of PSD from CRC as evidenced by decreased bioluminescence and improved survival. This treatment approach also resulted in significantly higher frequencies of peritoneal CD4+ T (10.95 ± 1.17 versus 6.19 ± 0.44, P = 0.004) and B1b cells (5.01 ± 0.97 versus 2.20 ± 0.2, P = 0.024). On the other hand, treatment with M51R VSV resulted in fewer myeloid-derived suppressor cells relative to controls (10.66 ± 1.48 versus 14.47 ± 1.06, P = 0.035). M51R-treated peritoneal cavities also contained lower concentrations of immunosuppressive monocyte chemoattractant protein-1 and interleukin 6 cytokines relative to controls. CONCLUSIONS: Our findings suggest that M51R VSV alters the innate and adaptive immune responses in PSD from CRC. Future studies will delineate specific components of antitumor immunity that result in its therapeutic effect.


Assuntos
Neoplasias do Colo/terapia , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/imunologia , Neoplasias Peritoneais/terapia , Vesiculovirus/imunologia , Imunidade Adaptativa , Animais , Linhagem Celular Tumoral/transplante , Neoplasias do Colo/patologia , Modelos Animais de Doenças , Feminino , Humanos , Imunidade Inata , Injeções Intraperitoneais , Camundongos , Mutação , Vírus Oncolíticos/genética , Neoplasias Peritoneais/secundário , Resultado do Tratamento , Vesiculovirus/genética , Proteínas da Matriz Viral/genética
5.
Nat Cell Biol ; 21(11): 1346-1356, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31685995

RESUMO

In the past two decades, emerging studies have suggested that DExD/H box helicases belonging to helicase superfamily 2 (SF2) play essential roles in antiviral innate immunity. However, the antiviral functions of helicase SF1, which shares a conserved helicase core with SF2, are little understood. Here we demonstrate that zinc finger NFX1-type containing 1 (ZNFX1), a helicase SF1, is an interferon (IFN)-stimulated, mitochondrial-localised dsRNA sensor that specifically restricts the replication of RNA viruses. Upon virus infection, ZNFX1 immediately recognizes viral RNA through its Armadillo-type fold and P-loop domain and then interacts with mitochondrial antiviral signalling protein to initiate the type I IFN response without depending on retinoic acid-inducible gene I-like receptors (RLRs). In short, as is the case with interferon-stimulated genes (ISGs) alone, ZNFX1 can induce IFN and ISG expression at an early stage of RNA virus infection to form a positively regulated loop of the well-known RLR signalling. This provides another layer of understanding of the complexity of antiviral immunity.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Antígenos de Neoplasias/genética , Mitocôndrias/imunologia , Fatores de Processamento de RNA/genética , RNA de Cadeia Dupla/genética , RNA Viral/genética , Vesiculovirus/genética , Células A549 , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/imunologia , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/imunologia , Regulação da Expressão Gênica , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Interferon Tipo I/genética , Interferon Tipo I/imunologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/virologia , Camundongos , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/virologia , Conformação de Ácido Nucleico , Poli I-C/farmacologia , Cultura Primária de Células , Ligação Proteica , Fatores de Processamento de RNA/imunologia , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/imunologia , RNA Viral/química , RNA Viral/imunologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Vesiculovirus/crescimento & desenvolvimento , Vesiculovirus/imunologia
6.
Nat Med ; 25(10): 1589-1600, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31591605

RESUMO

Recombinant vesicular stomatitis virus-Zaire Ebola virus (rVSV-ZEBOV) is the most advanced Ebola virus vaccine candidate and is currently being used to combat the outbreak of Ebola virus disease (EVD) in the Democratic Republic of the Congo (DRC). Here we examine the humoral immune response in a subset of human volunteers enrolled in a phase 1 rVSV-ZEBOV vaccination trial by performing comprehensive single B cell and electron microscopy structure analyses. Four studied vaccinees show polyclonal, yet reproducible and convergent B cell responses with shared sequence characteristics. EBOV-targeting antibodies cross-react with other Ebolavirus species, and detailed epitope mapping revealed overlapping target epitopes with antibodies isolated from EVD survivors. Moreover, in all vaccinees, we detected highly potent EBOV-neutralizing antibodies with activities comparable or superior to the monoclonal antibodies currently used in clinical trials. These include antibodies combining the IGHV3-15/IGLV1-40 immunoglobulin gene segments that were identified in all investigated individuals. Our findings will help to evaluate and direct current and future vaccination strategies and offer opportunities for novel EVD therapies.


Assuntos
Vacinas contra Ebola/administração & dosagem , Ebolavirus/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Imunidade Humoral/imunologia , Adulto , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Formação de Anticorpos/imunologia , Linfócitos B/imunologia , Linfócitos B/virologia , Vacinas contra Ebola/efeitos adversos , Vacinas contra Ebola/imunologia , Ebolavirus/patogenicidade , Feminino , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Vacinação/efeitos adversos , Vesiculovirus/genética , Voluntários
7.
J Biosci ; 44(2)2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31180044

RESUMO

ErbB-3 binding protein 1 (Ebp1) is a host protein which binds ErbB-3 receptor to induce signalling events for cell growth regulation. In addition, Ebp1 also interacts with ribonucleoprotein complexes. In recent times, Ebp1 was found to play an antagonistic role in viral infections caused by Influenza and Rinderpest viruses. In our present work we have tried to understand the role of Ebp1 in Chandipura virus (CHPV) infection. We have observed an induction in Ebp1 expression upon CHPV infection similar to other viruses. However, unlike other viruses an overexpressed Ebp1 only reduces viral protein expression, but does not affect its progeny formation. Additionally, this effect is being carried out in an indirect manner, as there is no interaction between Ebp1 and viral proteins. This is despite Ebp1's presence in viral inclusion bodies.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Interações Hospedeiro-Patógeno/genética , Neurônios/metabolismo , Proteínas de Ligação a RNA/genética , Vesiculovirus/genética , Replicação Viral , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Humanos , Corpos de Inclusão Viral/química , Neurônios/virologia , Plasmídeos/química , Plasmídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Transfecção , Células Vero , Vesiculovirus/crescimento & desenvolvimento , Vesiculovirus/metabolismo , Ensaio de Placa Viral
8.
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
9.
Emerg Infect Dis ; 25(8): 1563-1566, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31141474

RESUMO

Use of the vesicular stomatitis virus (VSV)-based Ebola virus vaccine during outbreaks and the potential use of a similar VSV-based Lassa virus vaccine has raised questions about the vaccines' stability should the cold chain fail. We demonstrated that current cold chain conditions might tolerate significant variances without affecting efficacy.


Assuntos
Vacinas contra Ebola/imunologia , Ebolavirus/imunologia , Vetores Genéticos , Doença pelo Vírus Ebola/prevenção & controle , Vesiculovirus , Animais , Modelos Animais de Doenças , Surtos de Doenças , Vacinas contra Ebola/administração & dosagem , Vacinas contra Ebola/genética , Feminino , Vetores Genéticos/genética , Cobaias , Doença pelo Vírus Ebola/epidemiologia , Doença pelo Vírus Ebola/mortalidade , Humanos , Imunização , Mortalidade , Potência de Vacina , Vesiculovirus/genética
10.
Neuroimage ; 197: 133-142, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31022567

RESUMO

The elucidation of neural networks is essential to understanding the mechanisms of brain functions and brain disorders. Neurotropic virus-based trans-synaptic tracing tools have become an effective method for dissecting the structure and analyzing the function of neural-circuitry. However, these tracing systems rely on fluorescent signals, making it hard to visualize the panorama of the labeled networks in mammalian brain in vivo. One MRI method, Diffusion Tensor Imaging (DTI), is capable of imaging the networks of the whole brain in live animals but without information of anatomical connections through synapses. In this report, a chimeric gene coding for ferritin and enhanced green fluorescent protein (EGFP) was integrated into Vesicular stomatitis virus (VSV), a neurotropic virus that is able to spread anterogradely in synaptically connected networks. After the animal was injected with the recombinant VSV (rVSV), rVSV-Ferritin-EGFP, into the somatosensory cortex (SC) for four days, the labeled neural-network was visualized in the postmortem whole brain with a T2-weighted MRI sequence. The modified virus transmitted from SC to synaptically connected downstream regions. The results demonstrate that rVSV-Ferritin-EGFP could be used as a bimodal imaging vector for detecting synaptically connected neural-network with both ex vivo MRI and fluorescent imaging. The strategy in the current study has the potential to longitudinally monitor the global structure of a given neural-network in living animals.


Assuntos
Mapeamento Encefálico/métodos , Imagem por Ressonância Magnética , Neurônios/citologia , Córtex Somatossensorial/citologia , Vesiculovirus/fisiologia , Animais , Ferritinas/genética , Vetores Genéticos/genética , Vetores Genéticos/fisiologia , Proteínas de Fluorescência Verde/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Vias Neurais/citologia , Vias Neurais/virologia , Neurônios/virologia , Córtex Somatossensorial/virologia , Vesiculovirus/genética
11.
J Biol Chem ; 294(16): 6430-6438, 2019 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-30804210

RESUMO

RIG-I senses viral RNA in the cytosol and initiates host innate immune response by triggering the production of type 1 interferon. A recent RNAi knockdown screen yielded close to hundred host genes whose products affected viral RNA-induced IFN-ß production and highlighted the complexity of the antiviral response. The stress granule protein G3BP1, known to arrest mRNA translation, was identified as a regulator of RIG-I-induced IFN-ß production. How G3BP1 functions in RIG-I signaling is not known, however. Here, we overexpress G3BP1 with RIG-I in HEK293T cells and found that G3BP1 significantly enhances RIG-I-induced ifn-b mRNA synthesis. More importantly, we demonstrate that G3BP1 binds RIG-I and that this interaction involves the C-terminal RGG domain of G3BP1. Confocal microscopy studies also show G3BP1 co-localization with RIG-I and with infecting vesicular stomatitis virus in Cos-7 cells. Interestingly, immunoprecipitation studies using biotin-labeled viral dsRNA or poly(I·C) and cell lysate-derived or in vitro translated G3BP1 indicated that G3BP1 could directly bind these substrates and again via its RGG domain. Computational modeling further revealed a juxtaposed interaction between G3BP1 RGG and RIG-I RNA-binding domains. Together, our data reveal G3BP1 as a critical component of RIG-I signaling and possibly acting as a co-sensor to promote RIG-I recognition of pathogenic RNA.


Assuntos
Proteína DEAD-box 58 , DNA Helicases , Interferon beta , Modelos Moleculares , Proteínas de Ligação a Poli-ADP-Ribose , Biossíntese de Proteínas , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , RNA de Cadeia Dupla , RNA Viral , Infecções por Rhabdoviridae , Vesiculovirus , Animais , Células COS , Proteína DEAD-box 58/química , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Células HEK293 , Humanos , Interferon beta/biossíntese , Interferon beta/genética , Camundongos , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Ligação Proteica , Células RAW 264.7 , RNA Helicases/genética , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , Infecções por Rhabdoviridae/genética , Infecções por Rhabdoviridae/metabolismo , Transdução de Sinais/genética , Vesiculovirus/química , Vesiculovirus/genética , Vesiculovirus/metabolismo
12.
J Gen Virol ; 100(2): 145-155, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30624178

RESUMO

Earlier four monoclonal antibodies (MAbs) against surface glycoproteins Gn and Gc of puumala virus (PUUV, genus Orthohantavirus, family Hantaviridae, order Bunyavirales) were generated and for three MAbs with neutralizing capacity the localization of binding epitopes was predicted using pepscan and phage-display techniques. In this work, we produced vesicular stomatitis virus (VSV) particles pseudotyped with the Gn and Gc glycoproteins of PUUV and applied site-directed mutagenesis to dissect the structure of neutralizing epitopes. Replacement of cysteine amino acid (aa) residues with alanines resulted in pseudotype particles with diminished (16 to 18 %) neut-titres; double Cys→Ala mutants, as well as mutants with bulky aromatic and charged residues replaced with alanines, have shown even stronger reduction in neut-titres (from 25 % to the escape phenotype). In silico modelling of the neut-epitopes supported the hypothesis that these critical residues are located on the surface of viral glycoprotein molecules and thus can be recognized by the antibodies indeed. A similar pattern was observed in experiments with mutant pseudotypes and sera collected from patients suggesting that these neut-epitopes are utilized in a course of human PUUV infection. These data will help understanding the mechanisms of hantavirus neutralization and assist construction of vaccine candidates.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Epitopos de Linfócito B/imunologia , Hantavirus/imunologia , Glicoproteínas de Membrana/imunologia , Antígenos Virais/genética , Mapeamento de Epitopos , Epitopos de Linfócito B/genética , Vetores Genéticos , Humanos , Glicoproteínas de Membrana/genética , Mutagênese Sítio-Dirigida , Vesiculovirus/genética
13.
mBio ; 10(1)2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30622188

RESUMO

Rodent-to-human transmission of hantaviruses is associated with severe disease. Currently, no FDA-approved, specific antivirals or vaccines are available, and the requirement for high biocontainment (biosafety level 3 [BSL-3]) laboratories limits hantavirus research. To study hantavirus entry in a BSL-2 laboratory, we set out to generate replication-competent, recombinant vesicular stomatitis viruses (rVSVs) bearing the Gn and Gc (Gn/Gc) entry glycoproteins. As previously reported, rVSVs bearing New World hantavirus Gn/Gc were readily rescued from cDNAs, but their counterparts bearing Gn/Gc from the Old World hantaviruses, Hantaan virus (HTNV) or Dobrava-Belgrade virus (DOBV), were refractory to rescue. However, serial passage of the rescued rVSV-HTNV Gn/Gc virus markedly increased its infectivity and capacity for cell-to-cell spread. This gain in viral fitness was associated with the acquisition of two point mutations: I532K in the cytoplasmic tail of Gn and S1094L in the membrane-proximal stem of Gc. Follow-up experiments with rVSVs and single-cycle VSV pseudotypes confirmed these results. Mechanistic studies revealed that both mutations were determinative and contributed to viral infectivity in a synergistic manner. Our findings indicate that the primary mode of action of these mutations is to relocalize HTNV Gn/Gc from the Golgi complex to the cell surface, thereby affording significantly enhanced Gn/Gc incorporation into budding VSV particles. Finally, I532K/S1094L mutations in DOBV Gn/Gc permitted the rescue of rVSV-DOBV Gn/Gc, demonstrating that incorporation of cognate mutations into other hantaviral Gn/Gc proteins could afford the generation of rVSVs that are otherwise challenging to rescue. The robust replication-competent rVSVs, bearing HTNV and DOBV Gn/Gc, reported herein may also have utility as vaccines.IMPORTANCE Human hantavirus infections cause hantavirus pulmonary syndrome in the Americas and hemorrhagic fever with renal syndrome (HFRS) in Eurasia. No FDA-approved vaccines and therapeutics exist for these deadly viruses, and their development is limited by the requirement for high biocontainment. In this study, we identified and characterized key amino acid changes in the surface glycoproteins of HFRS-causing Hantaan virus that enhance their incorporation into recombinant vesicular stomatitis virus (rVSV) particles. The replication-competent rVSVs encoding Hantaan virus and Dobrava-Belgrade virus glycoproteins described in this work provide a powerful and facile system to study hantavirus entry under lower biocontainment and may have utility as hantavirus vaccines.


Assuntos
Vetores Genéticos , Hantavirus/genética , Proteínas Mutantes/genética , Mutação Puntual , Proteínas Recombinantes/genética , Vesiculovirus/genética , Proteínas do Envelope Viral/genética , Linhagem Celular , Glicoproteínas/genética , Humanos , Genética Reversa , Inoculações Seriadas , Vesiculovirus/fisiologia , Liberação de Vírus , Replicação Viral
14.
Mol Ther ; 27(1): 151-163, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30389355

RESUMO

Investigators have utilized the CRISPR/Cas9 gene-editing system to specifically target well-conserved regions of HIV, leading to decreased infectivity and pathogenesis in vitro and ex vivo. We utilized a specialized extracellular vesicle termed a "gesicle" to efficiently, yet transiently, deliver Cas9 in a ribonucleoprotein form targeting the HIV long terminal repeat (LTR). Gesicles are produced through expression of vesicular stomatitis virus glycoprotein and package protein as their cargo, thus bypassing the need for transgene delivery, and allowing finer control of Cas9 expression. Using both NanoSight particle and western blot analysis, we verified production of Cas9-containing gesicles by HEK293FT cells. Application of gesicles to CHME-5 microglia resulted in rapid but transient transfer of Cas9 by western blot, which is present at 1 hr, but is undetectable by 24 hr post-treatment. Gesicle delivery of Cas9 protein preloaded with guide RNA targeting the HIV LTR to HIV-NanoLuc CHME-5 cells generated mutations within the LTR region and copy number loss. Finally, we demonstrated that this treatment resulted in reduced proviral activity under basal conditions and after stimulation with pro-inflammatory factors lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-α). These data suggest that gesicles are a viable alternative approach to deliver CRISPR/Cas9 technology.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/fisiologia , Edição de Genes/métodos , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/efeitos dos fármacos , Sistemas CRISPR-Cas/genética , Células HEK293 , Repetição Terminal Longa de HIV/genética , Repetição Terminal Longa de HIV/fisiologia , Ribonucleoproteínas Nucleares Heterogêneas/genética , Humanos , Lipopolissacarídeos/farmacologia , Mutação/genética , Provírus/genética , Provírus/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Vesiculovirus/genética , Vesiculovirus/metabolismo
15.
Curr Mol Pharmacol ; 12(2): 83-104, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30474542

RESUMO

BACKGROUND: Mycobacterium tuberculosis (Mtb) is considered as one of the most efficacious human pathogens. The global mortality rate of TB stands at approximately 2 million, while about 8 to 10 million active new cases are documented yearly. It is, therefore, a priority to develop vaccines that will prevent active TB. The vaccines currently used for the management of TB can only proffer a certain level of protection against meningitis, TB, and other forms of disseminated TB in children; however, their effectiveness against pulmonary TB varies and cannot provide life-long protective immunity. Based on these reasons, more efforts are channeled towards the development of new TB vaccines. During the development of TB vaccines, a major challenge has always been the lack of diversity in both the antigens contained in TB vaccines and the immune responses of the TB sufferers. Current efforts are channeled on widening both the range of antigens selection and the range of immune response elicited by the vaccines. The past two decades witnessed a significant progress in the development of TB vaccines; some of the discovered TB vaccines have recently even completed the third phase (phase III) of a clinical trial. OBJECTIVE: The objectives of this article are to discuss the recent progress in the development of new vaccines against TB; to provide an insight on the mechanism of vaccine-mediated specific immune response stimulation, and to debate on the interaction between vaccines and global interventions to end TB.


Assuntos
Vacinas Bacterianas/imunologia , Tuberculose/prevenção & controle , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Humanos , Sistema Imunitário/metabolismo , Mycobacterium tuberculosis/imunologia , Nanopartículas/química , Proteínas Recombinantes de Fusão/imunologia , Tuberculose/imunologia , Tuberculose/patologia , Vacinas de Subunidades/imunologia , Vesiculovirus/genética , Vesiculovirus/metabolismo
16.
Fish Shellfish Immunol ; 84: 299-303, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30308292

RESUMO

MicroRNAs (miRNAs) are small noncoding RNAs that have been reported to play important roles in virus replication. Snakehead vesiculovirus (SHVV) is a new rhabdovirus isolated from diseased hybrid snakehead and has caused heavy economical losses in cultured snakehead fish in China. Our previous study has revealed that miR-214 inhibited SHVV replication, but the underline mechanism was not completely understood. In this study, glycogen synthase (GS) gene was identified as a target gene of miR-214. Overexpression of miR-214 reduced cellular GS gene expression. Knockdown of GS by siRNA, similar to the overexpression of miR-214, inhibited SHVV replication. Moreover, we found that siGS-mediated inhibition of SHVV replication could be restored by reducing cellular miR-214 level via using miR-214 inhibitor, indicating that miR-214 inhibited SHVV replication at least partially via targeting GS. This study provided information for understanding the molecular mechanism of SHVV pathogenicity and a potential antiviral strategy against SHVV infection.


Assuntos
Doenças dos Peixes/fisiopatologia , Proteínas de Peixes/genética , Glicogênio Sintase/genética , MicroRNAs/genética , Perciformes , RNA Viral/genética , Infecções por Rhabdoviridae/veterinária , Animais , Doenças dos Peixes/virologia , Proteínas de Peixes/metabolismo , Glicogênio Sintase/metabolismo , MicroRNAs/metabolismo , RNA Viral/metabolismo , Infecções por Rhabdoviridae/fisiopatologia , Infecções por Rhabdoviridae/virologia , Vesiculovirus/genética , Vesiculovirus/fisiologia
17.
J Virol ; 93(4)2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30487274

RESUMO

Vesicular stomatitis virus (VSV) (a rhabdovirus) and its variant VSV-ΔM51 are widely used model systems to study mechanisms of virus-host interactions. Here, we investigated how the cell cycle affects replication of these viruses using an array of cell lines with different levels of impairment of antiviral signaling and a panel of chemical compounds arresting the cell cycle at different phases. We observed that all compounds inducing cell cycle arrest in G2/M phase strongly enhanced the replication of VSV-ΔM51 in cells with functional antiviral signaling. G2/M arrest strongly inhibited type I and type III interferon (IFN) production as well as expression of IFN-stimulated genes in response to exogenously added IFN. Moreover, G2/M arrest enhanced the replication of Sendai virus (a paramyxovirus), which is also highly sensitive to the type I IFN response but did not stimulate the replication of a wild-type VSV that is more effective at evading antiviral responses. In contrast, the positive effect of G2/M arrest on virus replication was not observed in cells defective in IFN signaling. Altogether, our data show that replication of IFN-sensitive cytoplasmic viruses can be strongly stimulated during G2/M phase as a result of inhibition of antiviral gene expression, likely due to mitotic inhibition of transcription, a global repression of cellular transcription during G2/M phase. The G2/M phase thus could represent an "Achilles' heel" of the infected cell, a phase when the cell is inadequately protected. This model could explain at least one of the reasons why many viruses have been shown to induce G2/M arrest.IMPORTANCE Vesicular stomatitis virus (VSV) (a rhabdovirus) and its variant VSV-ΔM51 are widely used model systems to study mechanisms of virus-host interactions. Here, we investigated how the cell cycle affects replication of VSV and VSV-ΔM51. We show that G2/M cell cycle arrest strongly enhances the replication of VSV-ΔM51 (but not of wild-type VSV) and Sendai virus (a paramyxovirus) via inhibition of antiviral gene expression, likely due to mitotic inhibition of transcription, a global repression of cellular transcription during G2/M phase. Our data suggest that the G2/M phase could represent an "Achilles' heel" of the infected cell, a phase when the cell is inadequately protected. This model could explain at least one of the reasons why many viruses have been shown to induce G2/M arrest, and it has important implications for oncolytic virotherapy, suggesting that frequent cell cycle progression in cancer cells could make them more permissive to viruses.


Assuntos
Pontos de Checagem do Ciclo Celular/fisiologia , Vesiculovirus/genética , Replicação Viral/genética , Animais , Antivirais/farmacologia , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Citoplasma , Fase G2/fisiologia , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Expressão Gênica/genética , Humanos , Interferon Tipo I/metabolismo , Interferon gama/metabolismo , Interferons , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/genética , Vírus de RNA/imunologia , Vírus de RNA/metabolismo , Vírus Sendai/genética , Vírus Sendai/metabolismo , Transdução de Sinais , Vírus da Estomatite Vesicular Indiana/genética , Vesiculovirus/metabolismo , Proteínas da Matriz Viral/genética , Replicação Viral/imunologia
18.
Virology ; 526: 32-37, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30336336

RESUMO

Snakehead vesiculovirus (SHVV) is a new rhabdovirus isolated from diseased hybrid snakehead fish (Channa maculate ♀ x Channa argus ♂) and has caused serious economic losses in snakehead fish culture in China. To better understand the pathogenicity of SHVV, we developed a reverse genetics system for SHVV by using human and fish cells. In detail, human 293T cells were co-transfected with four plasmids encoding the full-length SHVV antigenomic RNA or the supporting proteins including nucleoprotein (N), phosphoprotein (P), and large polymerase (L), followed by the cultivation in Channel catfish ovary (CCO) cells. We also rescued a recombinant SHVV expressing enhanced green fluorescent protein (EGFP), which was inserted into the 3' non-coding region (NCR) of the glycoprotein (G) gene of SHVV. Our study provides a potential tool for unveiling the pathogenicity of SHVV and a template for the rescue of other fish viruses by using both human 293T and fish cells.


Assuntos
Doenças dos Peixes/virologia , Genética Reversa , Infecções por Rhabdoviridae/virologia , Vesiculovirus/genética , Animais , Células Cultivadas , DNA Complementar/genética , Feminino , Peixes , Genes Virais/genética , Genoma Viral/genética , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Ovário/citologia , RNA Viral/biossíntese , RNA Viral/genética , Vesiculovirus/crescimento & desenvolvimento
19.
J Gen Virol ; 100(2): 278-288, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30566072

RESUMO

A first step towards the development of a human immunodeficiency virus (HIV) animal model has been the identification and surmounting of species-specific barriers encountered by HIV along its replication cycle in cells from small animals. Serine incorporator proteins 3 (SERINC3) and 5 (SERINC5) were recently identified as restriction factors that reduce HIV-1 infectivity. Here, we compared the antiviral activity of SERINC3 and SERINC5 among mice, rats and rabbits, and their susceptibility to viral counteraction to their human counterparts. In the absence of viral antagonists, rodent and lagomorph SERINC3 and SERINC5 displayed anti-HIV activity in a similar range to human controls. Vesicular stomatitis virus G protein (VSV-G) pseudotyped virions were considerably less sensitive to restriction by all SERINC3/5 orthologs. Interestingly, HIV-1 Nef, murine leukemia virus (MLV) GlycoGag and equine infectious anemia virus (EIAV) S2 counteracted the antiviral activity of all SERINC3/5 orthologs with similar efficiency. Our results demonstrate that the antiviral activity of SERINC3/5 proteins is conserved in rodents and rabbits, and can be overcome by all three previously reported viral antagonists.


Assuntos
HIV-1/crescimento & desenvolvimento , HIV-1/imunologia , Interações Hospedeiro-Patógeno , Fatores Imunológicos/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Animais , Vetores Genéticos , Camundongos , Coelhos , Ratos , Vesiculovirus/genética , Vesiculovirus/crescimento & desenvolvimento
20.
Antiviral Res ; 160: 143-150, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30393014

RESUMO

Organic compounds with a caffeoyl moiety (e.g. caffeic acid, rosmarinic acid, chicoric acid, etc.) have antiviral properties towards herpes simplex (HSV), influenza and immunodeficiency viruses (HIV). This study evaluated the HSV antiviral properties of caffeic acid when paired with a variety of metal and other inorganic ions. The results demonstrated that the antiviral activity of caffeic acid increased upwards of 100-fold by the addition of cations, such as Fe3+, and anionic molecules, such as molybdate and phosphate. Cellular toxicity tests of the caffeic acid chelates showed that they have low toxicities with selectivity indices (TD50/EC50) for Fe3+, MoO42-, and PO43- chelates being 1700, >540, and >30, respectively. Caffeic acid paired with Fe3+ was tested against eight strains of viruses, including those from different families. The caffeic acid chelates were mostly effective against HSV1 and HSV2, but they also had moderate activity against vaccinia virus and a VSV-Ebola pseudotyped virus. All the viruses that were strongly impacted by the caffeic chelates require heparan sulfate proteoglycans for cellular attachment, so it is likely that caffeic chelates target and interfere with this mechanism. Since the caffeic acid chelates target an extra-cellular process, they might be able to be combined with existing medications, such as acyclovir, that target an intracellular process to achieve greater viral control.


Assuntos
Antivirais/farmacologia , Ácidos Cafeicos/farmacologia , Quelantes/farmacologia , Ebolavirus/efeitos dos fármacos , Ferro/farmacologia , Simplexvirus/efeitos dos fármacos , Vírus Vaccinia/efeitos dos fármacos , Antivirais/química , Antivirais/toxicidade , Ácidos Cafeicos/química , Ácidos Cafeicos/toxicidade , Quelantes/química , Quelantes/toxicidade , Ebolavirus/genética , Proteoglicanas de Heparan Sulfato/metabolismo , Ferro/química , Ferro/toxicidade , Testes de Sensibilidade Microbiana , Fosfatos/química , Fosfatos/farmacologia , Fosfatos/toxicidade , Receptores Virais/metabolismo , Vesiculovirus/efeitos dos fármacos , Vesiculovirus/genética , Ligação Viral/efeitos dos fármacos
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