Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 8 de 8
Filtrar
Mais filtros











Base de dados
Intervalo de ano de publicação
1.
J Virol ; 95(23): e0132321, 2021 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-34523971

RESUMO

Recently, a genome-wide association study using plasma HIV RNA from antiretroviral therapy-naive patients reported that 14 naturally occurring nonsynonymous single-nucleotide polymorphisms (SNPs) in HIV derived from antiretrovirus drug-naive patients were associated with virus load (VL). Those SNPs were detected in reverse transcriptase, RNase H, integrase, envelope, and Nef. However, the impact of each mutation on viral fitness was not investigated. Here, we constructed a series of HIV variants encoding each SNP and examined their replicative abilities. An HIV variant containing a Met-to-Ile change at codon 50 in integrase [HIV(IN:M50I)] was found as an impaired virus. Despite the mutation being in integrase, the virus release was significantly suppressed (P < 0.001). Transmission electron microscopy analysis revealed that abnormal bud accumulation on the plasma membrane and the released virus particles retained immature forms. Western blot analysis demonstrated a defect in autoprocessing of GagPol and Gag polyproteins' autoprocessing in the HIV(IN:M50I) particles, although Förster resonance energy transfer (FRET) assay displayed that GagPol containing IN:M50I forms a homodimer with a similar efficiency with GagPol (wild type). The impaired maturation and replication were rescued by two other VL-associated SNPs, Ser-to-Asn change at codon 17 of integrase and Asn-to-Ser change at codon 79 of RNase H. These data demonstrate that Gag and GagPol assembly, virus release, and autoprocessing are regulated by not only integrase but also RNase H. IMPORTANCE Nascent HIV-1 is a noninfectious viral particle. Cleaving Gag and GagPol polyproteins in the particle by mature HIV protease (PR), the nascent virus becomes an infectious virus. PR is initially translated as an inactive embedded enzyme in a GagPol polyprotein. The embedded PR in homodimerized GagPol polyproteins catalyzes a proteolytic reaction to release the mature PR. This excision step by self-cleavage is called autoprocessing. Here, during the evaluation of the roles of naturally emerging nonsynonymous SNPs in HIV RNA, we found that autoprocessing is inhibited by Met-to-Ile change at codon 50 in integrase GagPol. Other coexisting SNPs, Ser-to-Asn change at codon 17 in integrase or Asn-to-Ser mutation at codon 79 in RNase H, recovered this defect, suggesting that autoprocessing is regulated by not only integrase but also RNase H in GagPol polyprotein.


Assuntos
Integrase de HIV/metabolismo , HIV-1/fisiologia , Ribonuclease H/metabolismo , Liberação de Vírus/fisiologia , Antirretrovirais/farmacologia , Produtos do Gene gag/genética , Células HEK293 , Infecções por HIV , Integrase de HIV/genética , HIV-1/genética , Humanos , Mutação , Polimorfismo de Nucleotídeo Único , Proteólise , Ribonuclease H/genética , Vírion/metabolismo , Replicação Viral
2.
J Infect Dis ; 222(10): 1745-1755, 2020 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-32498080

RESUMO

Neurological signs and symptoms are the most common complications of Ebola virus disease. However, the mechanisms underlying the neurologic manifestations in Ebola patients are not known. In this study, peripheral ganglia were collected from 12 rhesus macaques that succumbed to Ebola virus (EBOV) disease from 5 to 8 days post exposure. Ganglionitis, characterized by neuronal degeneration, necrosis, and mononuclear leukocyte infiltrates, was observed in the dorsal root, autonomic, and enteric ganglia. By immunohistochemistry, RNAscope in situ hybridization, transmission electron microscopy, and confocal microscopy, we confirmed that CD68+ macrophages are the target cells for EBOV in affected ganglia. Further, we demonstrated that EBOV can induce satellite cell and neuronal apoptosis and microglial activation in infected ganglia. Our results demonstrate that EBOV can infect peripheral ganglia and results in ganglionopathy in rhesus macaques, which may contribute to the neurological signs and symptoms observed in acute and convalescent Ebola virus disease in human patients.


Assuntos
Doença pelo Vírus Ebola/complicações , Doença pelo Vírus Ebola/patologia , Degeneração Neural/complicações , Degeneração Neural/patologia , Doenças do Sistema Nervoso Periférico/complicações , Doenças do Sistema Nervoso Periférico/patologia , Animais , Antígenos CD , Antígenos de Diferenciação Mielomonocítica , Modelos Animais de Doenças , Ebolavirus , Feminino , Gânglios , Gânglios Espinais/patologia , Gânglios Espinais/virologia , Cistos Glanglionares/patologia , Doença pelo Vírus Ebola/virologia , Humanos , Imuno-Histoquímica , Leucócitos Mononucleares , Macaca mulatta , Macrófagos/patologia , Masculino , Microglia/patologia , Microglia/virologia , Necrose , Sistema Nervoso Parassimpático/patologia , Doenças do Sistema Nervoso Periférico/virologia , Células Receptoras Sensoriais/patologia , Células Receptoras Sensoriais/virologia , Sistema Nervoso Simpático/patologia
3.
Viruses ; 11(1)2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30650570

RESUMO

Simian hemorrhagic fever virus (SHFV) causes a fulminant and typically lethal viral hemorrhagic fever (VHF) in macaques (Cercopithecinae: Macaca spp.) but causes subclinical infections in patas monkeys (Cercopithecinae: Erythrocebus patas). This difference in disease course offers a unique opportunity to compare host responses to infection by a VHF-causing virus in biologically similar susceptible and refractory animals. Patas and rhesus monkeys were inoculated side-by-side with SHFV. Unlike the severe disease observed in rhesus monkeys, patas monkeys developed a limited clinical disease characterized by changes in complete blood counts, serum chemistries, and development of lymphadenopathy. Viral RNA was measurable in circulating blood 2 days after exposure, and its duration varied by species. Infectious virus was detected in terminal tissues of both patas and rhesus monkeys. Varying degrees of overlap in changes in serum concentrations of interferon (IFN)-γ, monocyte chemoattractant protein (MCP)-1, and interleukin (IL)-6 were observed between patas and rhesus monkeys, suggesting the presence of common and species-specific cytokine responses to infection. Similarly, quantitative immunohistochemistry of livers from terminal monkeys and whole blood flow cytometry revealed varying degrees of overlap in changes in macrophages, natural killer cells, and T-cells. The unexpected degree of overlap in host response suggests that relatively small subsets of a host's response to infection may be responsible for driving hemorrhagic fever pathogenesis. Furthermore, comparative SHFV infection in patas and rhesus monkeys offers an experimental model to characterize host⁻response mechanisms associated with viral hemorrhagic fever and evaluate pan-viral hemorrhagic fever countermeasures.


Assuntos
Infecções por Arterivirus/veterinária , Arterivirus/patogenicidade , Febres Hemorrágicas Virais/veterinária , Interações Hospedeiro-Patógeno , Doenças dos Macacos/imunologia , Animais , Anticorpos Antivirais/sangue , Arterivirus/imunologia , Infecções por Arterivirus/imunologia , Citocinas/sangue , Erythrocebus , Feminino , Febres Hemorrágicas Virais/imunologia , Macaca , Macrófagos/virologia , Masculino , Doenças dos Macacos/virologia , RNA Viral , Replicação Viral
4.
J Virol ; 91(2)2017 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-27807241

RESUMO

Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in 2012 and is a highly pathogenic respiratory virus. There are no treatment options against MERS-CoV for humans or animals, and there are no large-scale clinical trials for therapies against MERS-CoV. To address this need, we developed an inactivated rabies virus (RABV) that contains the MERS-CoV spike (S) protein expressed on its surface. Our initial recombinant vaccine, BNSP333-S, expresses a full-length wild-type MERS-CoV S protein; however, it showed significantly reduced viral titers compared to those of the parental RABV strain and only low-level incorporation of full-length MERS-CoV S into RABV particles. Therefore, we developed a RABV-MERS vector that contained the MERS-CoV S1 domain of the MERS-CoV S protein fused to the RABV G protein C terminus (BNSP333-S1). BNSP333-S1 grew to titers similar to those of the parental vaccine vector BNSP333, and the RABV G-MERS-CoV S1 fusion protein was efficiently expressed and incorporated into RABV particles. When we vaccinated mice, chemically inactivated BNSP333-S1 induced high-titer neutralizing antibodies. Next, we challenged both vaccinated mice and control mice with MERS-CoV after adenovirus transduction of the human dipeptidyl peptidase 4 (hDPP4) receptor and then analyzed the ability of mice to control MERS-CoV infection. Our results demonstrated that vaccinated mice were fully protected from the MERS-CoV challenge, as indicated by the significantly lower MERS-CoV titers and MERS-CoV and mRNA levels in challenged mice than those in unvaccinated controls. These data establish that an inactivated RABV-MERS S-based vaccine may be effective for use in animals and humans in areas where MERS-CoV is endemic. IMPORTANCE: Rabies virus-based vectors have been proven to be efficient dual vaccines against rabies and emergent infectious diseases such as Ebola virus. Here we show that inactivated rabies virus particles containing the MERS-CoV S1 protein induce potent immune responses against MERS-CoV and RABV. This novel vaccine is easy to produce and may be useful to protect target animals, such as camels, as well as humans from deadly MERS-CoV and RABV infections. Our results indicate that this vaccine approach can prevent disease, and the RABV-based vaccine platform may be a valuable tool for timely vaccine development against emerging infectious diseases.


Assuntos
Infecções por Coronavirus/imunologia , Proteção Cruzada/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Vírus da Raiva/imunologia , Raiva/imunologia , Vacinas Virais/imunologia , Animais , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/virologia , Modelos Animais de Doenças , Regulação Viral da Expressão Gênica , Humanos , Imunização , Camundongos , Interações Microbianas , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Raiva/prevenção & controle , Raiva/virologia , Vírus da Raiva/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Atenuadas , Vacinas Sintéticas , Proteínas Virais/genética , Proteínas Virais/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/efeitos adversos , Vacinas Virais/genética , Montagem de Vírus
5.
J Virol ; 89(23): 11820-33, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26378163

RESUMO

UNLABELLED: Severe acute respiratory syndrome (SARS) emerged in November 2002 as a case of atypical pneumonia in China, and the causative agent of SARS was identified to be a novel coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV). Bone marrow stromal antigen 2 (BST-2; also known as CD317 or tetherin) was initially identified to be a pre-B-cell growth promoter, but it also inhibits the release of virions of the retrovirus human immunodeficiency virus type 1 (HIV-1) by tethering budding virions to the host cell membrane. Further work has shown that BST-2 restricts the release of many other viruses, including the human coronavirus 229E (hCoV-229E), and the genomes of many of these viruses encode BST-2 antagonists to overcome BST-2 restriction. Given the previous studies on BST-2, we aimed to determine if BST-2 has the ability to restrict SARS-CoV and if the SARS-CoV genome encodes any proteins that modulate BST-2's antiviral function. Through an in vitro screen, we identified four potential BST-2 modulators encoded by the SARS-CoV genome: the papain-like protease (PLPro), nonstructural protein 1 (nsp1), ORF6, and ORF7a. As the function of ORF7a in SARS-CoV replication was previously unknown, we focused our study on ORF7a. We found that BST-2 does restrict SARS-CoV, but the loss of ORF7a leads to a much greater restriction, confirming the role of ORF7a as an inhibitor of BST-2. We further characterized the mechanism of BST-2 inhibition by ORF7a and found that ORF7a localization changes when BST-2 is overexpressed and ORF7a binds directly to BST-2. Finally, we also show that SARS-CoV ORF7a blocks the restriction activity of BST-2 by blocking the glycosylation of BST-2. IMPORTANCE: The severe acute respiratory syndrome coronavirus (SARS-CoV) emerged from zoonotic sources in 2002 and caused over 8,000 infections and 800 deaths in 37 countries around the world. Identifying host factors that regulate SARS-CoV pathogenesis is critical to understanding how this lethal virus causes disease. We have found that BST-2 is capable of restricting SARS-CoV release from cells; however, we also identified a SARS-CoV protein that inhibits BST-2 function. We show that the SARS-CoV protein ORF7a inhibits BST-2 glycosylation, leading to a loss of BST-2's antiviral function.


Assuntos
Antígenos CD/fisiologia , Glicosilação , Fases de Leitura Aberta/genética , Síndrome Respiratória Aguda Grave/virologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/fisiologia , Vírion/fisiologia , Ligação Viral , Animais , Chlorocebus aethiops , Cromatografia de Afinidade , Clonagem Molecular , Proteases 3C de Coronavírus , Cisteína Endopeptidases/genética , Primers do DNA/genética , Citometria de Fluxo , Proteínas Ligadas por GPI/fisiologia , Células HEK293 , Humanos , Imunoprecipitação , Microscopia Confocal , Microscopia Eletrônica , Fases de Leitura Aberta/fisiologia , RNA Polimerase Dependente de RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Vero , Proteínas não Estruturais Virais/genética , Proteínas Virais/genética
6.
Virus Res ; 197: 54-8, 2015 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-25481284

RESUMO

Using a recombinant rabies (RABV) vaccine platform, we have developed several safe and effective vaccines. Most recently, we have developed a RABV-based ebolavirus (EBOV) vaccine that is efficacious in nonhuman primates. One safety feature of this vaccine is the utilization of a live but replication-deficient RABV construct. In this construct, the RABV glycoprotein (G) has been deleted from the genome, requiring G trans complementation in order for new infectious viruses to be released from the initial infected cell. Here we analyze this safety feature of the bivalent RABV-based EBOV vaccine comprised of the G-deleted RABV backbone expressing EBOV glycoprotein (GP). We found that, while the level of RABV genome in infected cells is equivalent regardless of G supplementation, the production of infectious virus is indeed restricted by the lack of G, and most importantly, that the presence of EBOV GP does not substitute for G. These findings further support the safety profile of this replication-deficient RABV-EBOV bivalent vaccine.


Assuntos
Antígenos Virais/biossíntese , Vacinas contra Ebola/imunologia , Expressão Gênica , Glicoproteínas/biossíntese , Vacina Antirrábica/imunologia , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/imunologia , Animais , Antígenos Virais/genética , Vacinas contra Ebola/genética , Deleção de Genes , Teste de Complementação Genética , Glicoproteínas/genética , Doença pelo Vírus Ebola/prevenção & controle , Primatas , Raiva/prevenção & controle , Vacina Antirrábica/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Proteínas do Envelope Viral/genética
7.
PLoS One ; 9(11): e112060, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25409519

RESUMO

Middle East respiratory syndrome coronavirus (MERS-CoV) is a recently isolated betacoronavirus identified as the etiologic agent of a frequently fatal disease in Western Asia, Middle East respiratory syndrome. Attempts to identify the natural reservoirs of MERS-CoV have focused in part on dromedaries. Bats are also suspected to be reservoirs based on frequent detection of other betacoronaviruses in these mammals. For this study, ten distinct cell lines derived from bats of divergent species were exposed to MERS-CoV. Plaque assays, immunofluorescence assays, and transmission electron microscopy confirmed that six bat cell lines can be productively infected. We found that the susceptibility or resistance of these bat cell lines directly correlates with the presence or absence of cell surface-expressed CD26/DPP4, the functional human receptor for MERS-CoV. Human anti-CD26/DPP4 antibodies inhibited infection of susceptible bat cells in a dose-dependent manner. Overexpression of human CD26/DPP4 receptor conferred MERS-CoV susceptibility to resistant bat cell lines. Finally, sequential passage of MERS-CoV in permissive bat cells established persistent infection with concomitant downregulation of CD26/DPP4 surface expression. Together, these results imply that bats indeed could be among the MERS-CoV host spectrum, and that cellular restriction of MERS-CoV is determined by CD26/DPP4 expression rather than by downstream restriction factors.


Assuntos
Anticorpos Monoclonais/imunologia , Quirópteros/virologia , Dipeptidil Peptidase 4/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/fisiologia , Animais , Linhagem Celular , Chlorocebus aethiops , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/virologia , Reservatórios de Doenças/virologia , Suscetibilidade a Doenças , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/patogenicidade , Receptores Virais/metabolismo , Células Vero , Tropismo Viral
8.
FASEB J ; 28(1): 106-16, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24097312

RESUMO

A 27-aa peptide (P27) was previously shown to decrease the accumulation of human immunodeficiency virus type 1 (HIV-1) in the supernatant of chronically infected cells; however, the mechanism was not understood. Here, we show that P27 prevents virus accumulation by inducing macropinocytosis (MPC). Treatment of HIV-1- and human T-cell lymphotropic virus type 1 (HTLV-1)-infected cells with 2-10 µM P27 caused cell membrane ruffling and uptake of virus and polymerized forms of the peptide into large vacuoles. As demonstrated by electron microscopy, activation of MPC did not require virus or cells infected with virus, as P27 initiated its own uptake in the absence of virus. Inhibitors of MPC, Cytochalasin D and amiloride, decreased P27-mediated uptake of soluble dextran and inhibited P27-induced virus uptake by >60%, which provides further evidence that P27 induces MPC. In CD4(+) HeLa cells, HIV-1 infection was enhanced by P27 up to 4-fold, and P27 increased infection at concentrations as low as 20 nM. The 5-aa C-terminal domain of P27 was necessary for virus uptake and may be responsible for the polymerization of P27 into fibrils. These forms of P27 may play a key role in triggering MPC, making this peptide a useful tool for studying virus uptake and infection, as well as MPC of other macromolecules.


Assuntos
Endocitose/efeitos dos fármacos , Peptídeos/farmacologia , Pinocitose/efeitos dos fármacos , Amilorida/farmacologia , Linhagem Celular , Citocalasina D/farmacologia , Humanos , Retroviridae/fisiologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA