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1.
Emerg Microbes Infect ; 9(1): 20-31, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31859605

RESUMO

Porcine deltacoronavirus (PDCoV) is an emerging swine coronavirus that causes severe diarrhea, resulting in high mortality in neonatal piglets. Despite widespread outbreaks in many countries, no effective PDCoV vaccines are currently available. Here, we generated, for the first time, a full-length infectious cDNA clone of PDCoV. We further manipulated the infectious clone by replacing the NS6 gene with a green fluorescent protein (GFP) to generate rPDCoV-ΔNS6-GFP; likewise, rPDCoV-ΔNS7 was constructed by removing the ATG start codons of the NS7 gene. Growth kinetics studies suggest that rPDCoV-ΔNS7 could replicate similarly to that of the wild-type PDCoV, whereas rPDCoV-ΔNS6-GFP exhibited a substantial reduction of viral titer in vitro and in vivo. Piglets inoculated with rPDCoV-ΔNS7 or wild-type PDCoV showed similar diarrheic scores and pathological injury. In contrast, rPDCoV-ΔNS6-GFP-infected piglets did not show any clinical signs, indicating that the NS6 protein is an important virulence factor of PDCoV and that the NS6-deficient mutant virus might be a promising live-attenuated vaccine candidate. Taken together, the reverse genetics platform described here not only provides more insights into the role of PDCoV accessory proteins in viral replication and pathogenesis, but also allows the development of novel vaccines against PDCoV infection.


Assuntos
Infecções por Coronavirus/veterinária , Coronavirus/fisiologia , Doenças dos Suínos/virologia , Proteínas Virais Reguladoras e Acessórias/genética , Vacinas Virais/genética , Animais , Animais Recém-Nascidos , Doenças Transmissíveis Emergentes/imunologia , Doenças Transmissíveis Emergentes/patologia , Doenças Transmissíveis Emergentes/veterinária , Doenças Transmissíveis Emergentes/virologia , Coronavirus/classificação , Coronavirus/genética , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Citocinas/imunologia , DNA Complementar , Genoma Viral , Genética Reversa , Sus scrofa , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/patologia , Replicação Viral , Eliminação de Partículas Virais
2.
Vet Microbiol ; 239: 108464, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31767070

RESUMO

QX-like genotype infectious bronchitis virus (IBV) has become prevalent in recent years. Few studies have reported the effects of accessory proteins 3a and 3b on pathogenicity in vivo. We developed a reverse genetics system to manipulate the genome of a QX-like IBV strain IBYZ. Recombinant viruses rIBYZ-ScAUG3a, rIBYZ-ScAUG3b and rIBYZ-ScAUG3ab were generated. These viruses do not express the accessory proteins 3a, 3b, or 3ab due to a mutation in the AUG start codons. In SPF embryonated eggs, the recombinant viruses grew to the same viral load as parental strain rIBYZ. The pathogenicity of rIBYZ and recombinant viruses was examined in 1-day-old SPF chickens. In SPF chickens, rIBYZ-ScAUG3a had a lower mortality than rIBYZ. The clinical signs, gross lesions and histopathological changes of rIBYZ-ScAUG3a group were comparable to those of rIBYZ group. However, viral distribution and viral shedding showed that the viral loads of rIBYZ-ScAUG3a were lower than those of rIBYZ in tissue samples and swab specimens. The rIBYZ-ScAUG3b and rIBYZ-ScAUG3ab strains showed attenuated pathogenicity compared to rIBYZ, as no chickens died and all the parameters tested were considerably low. This study indicates that the absence of accessory proteins 3a and 3b in IBV lead to attenuated pathogenicity in chickens. Protein 3b has a greater effect on pathogenicity than protein 3a. These findings may be used in vaccination trials for the development of a new live-attenuated vaccine.


Assuntos
Infecções por Coronavirus/veterinária , Vírus da Bronquite Infecciosa/patogenicidade , Doenças das Aves Domésticas/virologia , Proteínas Virais Reguladoras e Acessórias/metabolismo , Animais , Galinhas , Infecções por Coronavirus/virologia , Vírus da Bronquite Infecciosa/genética , Proteínas Virais Reguladoras e Acessórias/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
3.
J Microbiol Biotechnol ; 29(11): 1817-1829, 2019 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-31546302

RESUMO

Porcine deltacoronavirus (PDCoV) is an emerging swine enteric coronavirus that causes diarrhea in neonatal piglets. Like other coronaviruses, PDCoV encodes at least three accessory or species-specific proteins; however, the biological roles of these proteins in PDCoV replication remain undetermined. As a first step toward understanding the biology of the PDCoV accessory proteins, we established a stable porcine cell line constitutively expressing the PDCoV NS7 protein in order to investigate the functional characteristics of NS7 for viral replication. Confocal microscopy and subcellular fractionation revealed that the NS7 protein was extensively distributed in the mitochondria. Proteomic analysis was then conducted to assess the expression dynamics of the host proteins in the PDCoV NS7-expressing cells. Highresolution two-dimensional gel electrophoresis initially identified 48 protein spots which were differentially expressed in the presence of NS7. Seven of these spots, including two upregulated and five down-regulated protein spots, showed statistically significant alterations, and were selected for subsequent protein identification. The affected cellular proteins identified in this study were classified into functional groups involved in various cellular processes such as cytoskeleton networks and cell communication, metabolism, and protein biosynthesis. A substantial down-regulation of α-actinin-4 was confirmed in NS7-expressing and PDCoV-infected cells. These proteomic data will provide insights into the understanding of specific cellular responses to the accessory protein during PDCoV infection.


Assuntos
Infecções por Coronaviridae/veterinária , Coronaviridae/fisiologia , Doenças dos Suínos/virologia , Proteínas Virais Reguladoras e Acessórias/metabolismo , Actinina/metabolismo , Animais , Linhagem Celular , Coronaviridae/genética , Coronaviridae/metabolismo , Infecções por Coronaviridae/virologia , Interações Hospedeiro-Patógeno , Mitocôndrias/metabolismo , Proteômica , Suínos , Proteínas Virais Reguladoras e Acessórias/genética
4.
Science ; 365(6457): 1025-1029, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31488688

RESUMO

Treatment of SIV-infected rhesus macaques with short-term antiretroviral therapy (ART) and partially overlapping infusions of antibody to integrin α4ß7 was reported to induce durable posttreatment viral suppression. In an attempt to replicate those observations, we treated macaques infected with the same virus and with the same ART and monoclonal antibody (mAb) regimens (anti-α4ß7 versus control mAb). Sequencing demonstrated that the virus used was actually SIVmac239-nef-stop, not wild-type SIVmac239. A positive correlation was found at 2 weeks after infection between the frequency of repair of attenuated Nef-STOP virus to pathogenic Nef-OPEN and plasma SIV RNA levels. Levels of plasma viremia before the first antibody infusion and preinfection levels of α4ß7 hi CD4+ T cells, but not treatment with antibody to α4ß7 , correlated with levels of viral replication upon discontinuation of all treatments. Follow-up plasma viremia, peripheral blood CD4+ T cell counts, and lymph node and rectal tissue viral load were not significantly different between anti-α4ß7 and control mAb groups.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Infecções por HIV/terapia , Integrina alfa4/imunologia , Cadeias beta de Integrinas/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/terapia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/imunologia , Animais , Antirretrovirais/uso terapêutico , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/imunologia , Códon de Terminação , Linfonodos/virologia , Macaca mulatta , RNA Viral/sangue , Reto/virologia , Síndrome de Imunodeficiência Adquirida dos Símios/sangue , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Vírus da Imunodeficiência Símia/fisiologia , Carga Viral , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/imunologia , Viremia/sangue , Viremia/imunologia , Viremia/terapia , Viremia/virologia , Replicação Viral
5.
Science ; 365(6457): 1029-1033, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31488689

RESUMO

Sustained virologic control of human immunodeficiency virus type 1 (HIV-1) infection after discontinuation of antiretroviral therapy (ART) is a major goal of the HIV-1 cure field. A recent study reported that administration of an antibody against α4ß7 induced durable virologic control after ART discontinuation in 100% of rhesus macaques infected with an attenuated strain of simian immunodeficiency virus (SIV) containing a stop codon in nef We performed similar studies in 50 rhesus macaques infected with wild-type, pathogenic SIVmac251. In animals that initiated ART during either acute or chronic infection, anti-α4ß7 antibody infusion had no detectable effect on the viral reservoir or viral rebound after ART discontinuation. These data demonstrate that anti-α4ß7 antibody administration did not provide therapeutic efficacy in the model of pathogenic SIVmac251 infection of rhesus macaques.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Anticorpos Antivirais/uso terapêutico , Integrina alfa4/imunologia , Cadeias beta de Integrinas/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/terapia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/imunologia , Animais , Antirretrovirais/uso terapêutico , Anticorpos Monoclonais/administração & dosagem , Anticorpos Antivirais/administração & dosagem , Códon de Terminação , DNA Viral/sangue , Infecções por HIV/terapia , Macaca mulatta , Síndrome de Imunodeficiência Adquirida dos Símios/sangue , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Vírus da Imunodeficiência Símia/fisiologia , Carga Viral , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/imunologia
6.
Science ; 365(6457): 1033-1036, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31488690

RESUMO

A study in nonhuman primates reported that infusions of an antibody against α4ß7 integrin, in combination with antiretroviral therapy, showed consistent, durable control of simian immunodeficiency virus (SIV) in rhesus macaques. The antibody used has pleiotropic effects, so we set out to gain insight into the underlying mechanism by comparing this treatment to treatment with non-neutralizing monoclonal antibodies against the SIV envelope glycoprotein that only block α4ß7 binding to SIV Env but have no other host-directed effects. Similar to the initial study, we used an attenuated strain of SIV containing a stop codon in nef. The study used 30 macaques that all began antiretroviral therapy and then were divided into five groups to receive different antibody treatments. Unlike the published report, we found no sustained virologic control by these treatments in vivo.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Anticorpos Antivirais/uso terapêutico , Integrina alfa4/imunologia , Cadeias beta de Integrinas/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/terapia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Antivirais/administração & dosagem , DNA Viral/sangue , Produtos do Gene env/imunologia , Infecções por HIV/terapia , Macaca mulatta , RNA Viral/sangue , Síndrome de Imunodeficiência Adquirida dos Símios/sangue , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Vírus da Imunodeficiência Símia/fisiologia , Linfócitos T/imunologia , Carga Viral , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/imunologia , Replicação Viral
7.
Nucleic Acids Res ; 47(17): 9368-9385, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31400113

RESUMO

Cellular non-membranous RNA-granules, P-bodies (RNA processing bodies, PB) and stress granules (SG), are important components of the innate immune response to virus invasion. Mechanisms governing how a virus modulates PB formation remain elusive. Here, we report the important roles of GW182 and DDX6, but not Dicer, Ago2 and DCP1A, in PB formation, and that Kaposi's sarcoma-associated herpesvirus (KSHV) lytic infection reduces PB formation through several specific interactions with viral RNA-binding protein ORF57. The wild-type ORF57, but not its N-terminal dysfunctional mutant, inhibits PB formation by interacting with the N-terminal GW-domain of GW182 and the N-terminal domain of Ago2, two major components of PB. KSHV ORF57 also induces nuclear Ago2 speckles. Homologous HSV-1 ICP27, but not EBV EB2, shares this conserved inhibitory function with KSHV ORF57. By using time-lapse confocal microscopy of HeLa cells co-expressing GFP-tagged GW182, we demonstrated that viral ORF57 inhibits primarily the scaffolding of GW182 at the initial stage of PB formation. Consistently, KSHV-infected iSLK/Bac16 cells with reduced GW182 expression produced far fewer PB and SG, but 100-fold higher titer of infectious KSHV virions when compared to cells with normal GW182 expression. Altogether, our data provide the first evidence that a DNA virus evades host innate immunity by encoding an RNA-binding protein that promotes its replication by blocking PB formation.


Assuntos
Autoantígenos/genética , RNA Helicases DEAD-box/genética , Herpesvirus Humano 8/genética , Proteínas Proto-Oncogênicas/genética , Proteínas de Ligação a RNA/genética , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Argonauta/genética , Regulação Viral da Expressão Gênica/genética , Células HeLa , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/virologia , Herpesvirus Humano 8/patogenicidade , Interações Hospedeiro-Patógeno/genética , Humanos , RNA Viral/genética , Replicação Viral/genética
8.
J Vet Med Sci ; 81(10): 1455-1460, 2019 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-31447457

RESUMO

The Feline coronavirus (FCoV) can lead to Feline infectious peritonitis (FIP), which the precise cause is still unknown. The theory of internal mutation suggests that a less virulent biotype of FCoV (FECV) would lead to another more pathogenic biotype (FIPV) capable of causing FIP. In this work, the 7b gene was amplified from 51 domestic cat plasma samples by semi-nested PCR and tested through phylogenetic and phylogeographical approaches. The 7b gene of Brazilian isolates displayed high conservation, a strong correlation between the geographic origin of the viral isolates and their genealogy, and its evolution was possibly shaped by a combination of high rates of nucleotide substitution and purifying selection.


Assuntos
Coronavirus Felino/genética , Peritonite Infecciosa Felina/epidemiologia , Proteínas Virais Reguladoras e Acessórias/genética , Animais , Brasil , Gatos , Epidemiologia Molecular , Filogenia , Filogeografia , Virulência
9.
Nat Commun ; 10(1): 3886, 2019 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-31467279

RESUMO

Non-additive interactions between mutations occur extensively and also change across conditions, making genetic prediction a difficult challenge. To better understand the plasticity of genetic interactions (epistasis), we combine mutations in a single protein performing a single function (a transcriptional repressor inhibiting a target gene). Even in this minimal system, genetic interactions switch from positive (suppressive) to negative (enhancing) as the expression of the gene changes. These seemingly complicated changes can be predicted using a mathematical model that propagates the effects of mutations on protein folding to the cellular phenotype. More generally, changes in gene expression should be expected to alter the effects of mutations and how they interact whenever the relationship between expression and a phenotype is nonlinear, which is the case for most genes. These results have important implications for understanding genotype-phenotype maps and illustrate how changes in genetic interactions can often-but not always-be predicted by hierarchical mechanistic models.


Assuntos
Epistasia Genética , Expressão Gênica , Modelos Genéticos , Proteínas Repressoras/genética , Proteínas Virais Reguladoras e Acessórias/genética , Sequência de Bases , Genótipo , Modelos Teóricos , Mutagênese , Mutação , Fenótipo , Biologia de Sistemas , Termodinâmica
10.
PLoS Negl Trop Dis ; 13(7): e0007521, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31283766

RESUMO

BACKGROUND: Primate T-lymphotropic viruses type 1 (PTLV-1) are complex retroviruses infecting both human (HTLV-1) and simian (STLV-1) hosts. They share common epidemiological, clinical and molecular features. In addition to the canonical gag, pol, env retroviral genes, PTLV-1 purportedly encodes regulatory (i.e. Tax, Rex, and HBZ) and accessory proteins (i.e. P12/8, P13, P30). The latter have been found essential for viral persistence in vivo. METHODOLOGY/PRINCIPAL FINDINGS: We have isolated a STLV-1 virus from a bonnet macaque (Macaca radiata-Mra18C9), a monkey from India. The complete sequence was obtained and phylogenetic analyses were performed. The Mra18C9 strain is highly divergent from the known PTLV-1 strains. Intriguingly, the Mra18C9 lacks the 3 accessory open reading frames. In order to determine if the absence of accessory proteins is specific to this particular strain, a comprehensive analysis of the complete PTLV-1 genomes available in Genbank was performed and found that the lack of one or many accessory ORF is common among PTLV-1. CONCLUSION: This study raises many questions regarding the actual nature, role and importance of accessory proteins in the PTLV-1 biology.


Assuntos
Infecções por Deltaretrovirus/veterinária , Macaca radiata/virologia , Fases de Leitura Aberta , Vírus Linfotrópico T Tipo 1 de Símios/genética , Proteínas Virais Reguladoras e Acessórias/genética , Animais , Infecções por Deltaretrovirus/virologia , Índia , Filogenia , Análise de Sequência de DNA , Vírus Linfotrópico T Tipo 1 de Símios/isolamento & purificação
11.
mBio ; 10(3)2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31213558

RESUMO

The HIV-1 accessory protein Vpu enhances viral release by counteracting the restriction factor BST-2. Furthermore, Vpu promotes NK cell evasion by downmodulating cell surface NTB-A and PVR, known ligands of the NK cell receptors NTB-A and DNAM-1, respectively. While it has been established that Vpu's transmembrane domain (TMD) is required for the interaction and intracellular sequestration of BST-2, NTB-A, and PVR, it remains unclear how Vpu manages to target these proteins simultaneously. In this study, we show that upon upregulation, BST-2 is preferentially downregulated by Vpu over its other TMD substrates. We found that type I interferon (IFN)-mediated BST-2 upregulation greatly impairs the ability of Vpu to downregulate NTB-A and PVR. Our results suggest that occupation of Vpu by BST-2 affects its ability to downregulate other TMD substrates. Accordingly, knockdown of BST-2 increases Vpu's potency to downmodulate NTB-A and PVR in the presence of type I IFN treatment. Moreover, we show that expression of human BST-2, but not that of the macaque orthologue, decreases Vpu's capacity to downregulate NTB-A. Importantly, we show that type I IFNs efficiently sensitize HIV-1-infected cells to NTB-A- and DNAM-1-mediated direct and antibody-dependent NK cell responses. Altogether, our results reveal that type I IFNs decrease Vpu's polyfunctionality, thus reducing its capacity to protect HIV-1-infected cells from NK cell responses.IMPORTANCE The restriction factor BST-2 and the NK cell ligands NTB-A and PVR are among a growing list of membrane proteins found to be downregulated by HIV-1 Vpu. BST-2 antagonism enhances viral release, while NTB-A and PVR downmodulation contributes to NK cell evasion. However, it remains unclear how Vpu can target multiple cellular factors simultaneously. Here we provide evidence that under physiological conditions, BST-2 is preferentially targeted by Vpu over NTB-A and PVR. Specifically, we show that type I IFNs decrease Vpu's polyfunctionality by upregulating BST-2, thus reducing its capacity to protect HIV-1-infected cells from NK cell responses. This indicates that there is a hierarchy of Vpu substrates upon IFN treatment, revealing that for the virus, targeting BST-2 as part of its resistance to IFN takes precedence over evading NK cell responses. This reveals a potential weakness in HIV-1's immunoevasion mechanisms that may be exploited therapeutically to harness NK cell responses against HIV-1.


Assuntos
Antígenos CD/genética , Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/imunologia , Proteínas do Vírus da Imunodeficiência Humana/genética , Interferon Tipo I/farmacologia , Células Matadoras Naturais/imunologia , Proteínas Virais Reguladoras e Acessórias/genética , Linfócitos T CD4-Positivos/virologia , Regulação para Baixo , Proteínas Ligadas por GPI/genética , Células HEK293 , HIV-1 , Proteínas do Vírus da Imunodeficiência Humana/imunologia , Humanos , Evasão da Resposta Imune , Receptores Virais/genética , Receptores Virais/imunologia , Família de Moléculas de Sinalização da Ativação Linfocitária/genética , Família de Moléculas de Sinalização da Ativação Linfocitária/imunologia , Ativação Transcricional , Regulação para Cima , Proteínas Virais Reguladoras e Acessórias/imunologia
12.
Nat Microbiol ; 4(5): 813-825, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30833724

RESUMO

Human immunodeficiency virus (HIV) actively modulates the protein stability of host cells to optimize viral replication. To systematically examine this modulation in HIV infection, we used isobaric tag-based mass spectrometry to quantify changes in the abundance of over 14,000 proteins during HIV-1 infection of human primary CD4+ T cells. We identified P-selectin glycoprotein ligand 1 (PSGL-1) as an HIV-1 restriction factor downregulated by HIV-1 Vpu, which binds to PSGL-1 and induces its ubiquitination and degradation through the ubiquitin ligase SCFß-TrCP2. PSGL-1 is induced by interferon-γ in activated CD4+ T cells to inhibit HIV-1 reverse transcription and potently block viral infectivity by incorporating in progeny virions. This infectivity block is antagonized by Vpu via PSGL-1 degradation. We further show that PSGL-1 knockdown can significantly abolish the anti-HIV activity of interferon-γ in primary CD4+ T cells. Our study identifies an HIV restriction factor and a key mediator of interferon-γ's anti-HIV activity.


Assuntos
Linfócitos T CD4-Positivos/virologia , Infecções por HIV/metabolismo , HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Glicoproteínas de Membrana/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Infecções por HIV/genética , Infecções por HIV/fisiopatologia , Infecções por HIV/virologia , HIV-1/genética , Proteínas do Vírus da Imunodeficiência Humana/genética , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Humanos , Interferon gama/genética , Interferon gama/metabolismo , Glicoproteínas de Membrana/genética , Proteólise , Proteômica , Proteínas Ligases SKP Culina F-Box/genética , Proteínas Ligases SKP Culina F-Box/metabolismo , Ubiquitinação , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/metabolismo
13.
mBio ; 10(2)2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30914508

RESUMO

Middle East respiratory syndrome coronavirus (MERS-CoV) was first identified in 2012 as a novel etiological agent of severe respiratory disease in humans. As during infection by other viruses, host sensing of viral double-stranded RNA (dsRNA) induces several antiviral pathways. These include interferon (IFN), oligoadenylate synthetase (OAS)-RNase L, and protein kinase R (PKR). Coronaviruses, including MERS-CoV, potently suppress the activation of these pathways, inducing only modest host responses. Our study describes the functions of two accessory proteins unique to MERS-CoV and related viruses, NS4a and NS4b, during infection in human airway epithelium-derived A549 cells. NS4a has been previously characterized as a dsRNA binding protein, while NS4b is a 2',5'-phosphodiesterase with structural and enzymatic similarity to NS2 encoded by mouse hepatitis virus (MHV). We found that deletion of NS4a results in increased interferon lambda (IFNL1) expression, as does mutation of either the catalytic site or nuclear localization sequence of NS4b. All of the mutant viruses we tested exhibited slight decreases in replication. We previously reported that, like MHV NS2, NS4b antagonizes OAS-RNase L, but suppression of IFN is a previously unidentified function for viral phosphodiesterases. Unexpectedly, deletion of NS4a does not result in robust activation of the PKR or OAS-RNase L pathways. Therefore, MERS-CoV likely encodes other proteins that contribute to suppression or evasion of these antiviral innate immune pathways that should be an important focus of future work. This study provides additional insight into the complex interactions between MERS-CoV and the host immune response.IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) is the second novel zoonotic coronavirus to emerge in the 21st century and cause outbreaks of severe respiratory disease. More than 2,200 cases and 800 deaths have been reported to date, yet there are no licensed vaccines or treatments. Coronaviruses encode unique accessory proteins that are not required for replication but most likely play roles in immune antagonism and/or pathogenesis. Our study describes the functions of MERS-CoV accessory proteins NS4a and NS4b during infection of a human airway-derived cell line. Loss of these accessory proteins during MERS-CoV infection leads to host antiviral activation and modestly attenuates replication. In the case of both NS4a and NS4b, we have identified roles during infection not previously described, yet the lack of robust activation suggests much remains to be learned about the interactions between MERS-CoV and the infected host.


Assuntos
Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Coronavírus da Síndrome Respiratória do Oriente Médio/patogenicidade , RNA de Cadeia Dupla/imunologia , RNA Viral/imunologia , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Células A549 , Células Epiteliais/virologia , Deleção de Genes , Humanos , Imunidade Inata , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Mutação , Proteínas não Estruturais Virais/genética , Proteínas Virais Reguladoras e Acessórias/genética , Replicação Viral
14.
Mol Cell ; 74(1): 143-157.e5, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30795892

RESUMO

Bacteriophage λN protein, a model anti-termination factor, binds nascent RNA and host Nus factors, rendering RNA polymerase resistant to all pause and termination signals. A 3.7-Å-resolution cryo-electron microscopy structure and structure-informed functional analyses reveal a multi-pronged strategy by which the intrinsically unstructured λN directly modifies RNA polymerase interactions with the nucleic acids and subverts essential functions of NusA, NusE, and NusG to reprogram the transcriptional apparatus. λN repositions NusA and remodels the ß subunit flap tip, which likely precludes folding of pause or termination RNA hairpins in the exit tunnel and disrupts termination-supporting interactions of the α subunit C-terminal domains. λN invades and traverses the RNA polymerase hybrid cavity, likely stabilizing the hybrid and impeding pause- or termination-related conformational changes of polymerase. λN also lines upstream DNA, seemingly reinforcing anti-backtracking and anti-swiveling by NusG. Moreover, λN-repositioned NusA and NusE sequester the NusG C-terminal domain, counteracting ρ-dependent termination. Other anti-terminators likely utilize similar mechanisms to enable processive transcription.


Assuntos
Bacteriófago lambda/metabolismo , Escherichia coli/metabolismo , RNA Bacteriano/biossíntese , Fatores de Transcrição/metabolismo , Terminação da Transcrição Genética , Proteínas Virais Reguladoras e Acessórias/metabolismo , Bacteriófago lambda/genética , Sítios de Ligação , Microscopia Crioeletrônica , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/virologia , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , RNA Bacteriano/química , RNA Bacteriano/genética , Relação Estrutura-Atividade , Fatores de Transcrição/química , Fatores de Transcrição/genética , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/genética
15.
Nucleic Acids Res ; 47(4): 1987-2001, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30462297

RESUMO

Kaposi's sarcoma-associated herpesvirus (KSHV) transcribes a long noncoding polyadenylated nuclear (PAN) RNA, which promotes the latent to lytic transition by repressing host genes involved in antiviral responses as well as viral proteins that support the latent state. KSHV also expresses several early proteins including ORF57 (Mta), a member of the conserved multifunctional ICP27 protein family, which is essential for productive replication. ORF57/Mta interacts with PAN RNA via a region termed the Mta responsive element (MRE), stabilizing the transcript and supporting nuclear accumulation. Here, using a close homolog of KSHV ORF57 from herpesvirus saimiri (HVS), we determined the crystal structure of the globular domain in complex with a PAN RNA MRE, revealing a uracil specific binding site that is also conserved in KSHV. Using solution NMR, RNA binding was also mapped within the disordered N-terminal domain of KSHV ORF57, and showed specificity for an RNA fragment containing a GAAGRG motif previously known to bind a homologous region in HVS ORF57. Together these data located novel differential RNA recognition sites within neighboring domains of herpesvirus ORF57 homologs, and revealed high-resolution details of their interactions with PAN RNA, thus providing insight into interactions crucial to viral function.


Assuntos
Herpesvirus Humano 8/genética , RNA Longo não Codificante/genética , Proteínas de Ligação a RNA/genética , Proteínas Virais Reguladoras e Acessórias/genética , Sítios de Ligação/genética , Regulação Viral da Expressão Gênica , Herpesvirus Saimiriíneo 2/genética , Humanos , Proteínas Imediatamente Precoces/genética , Motivos de Nucleotídeos/genética , RNA Mensageiro/genética
16.
Structure ; 27(1): 39-54.e6, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30482729

RESUMO

The multifunctional virion protein 35 (VP35) of ebolaviruses is a critical determinant of virulence and pathogenesis indispensable for viral replication and host innate immune evasion. Essential for VP35 function is homo-oligomerization via a coiled-coil motif. Here we report crystal structures of VP35 oligomerization domains from the prototypic Ebola virus (EBOV) and the non-pathogenic Reston virus (RESTV), together with a comparative biophysical characterization of the domains from all known species of the Ebolavirus genus. EBOV and RESTV VP35 oligomerization domains form bipartite parallel helix bundles with a canonical coiled coil in the N-terminal half and increased plasticity in the highly conserved C-terminal half. The domain assembles into trimers and tetramers in EBOV, whereas it exclusively forms tetramers in all other ebolavirus species. Substitution of coiled-coil leucine residues critical for immune antagonism leads to aberrant oligomerization. A conserved arginine involved in inter-chain salt bridges stabilizes the VP35 oligomerization domain and modulates between coiled-coil oligomeric states.


Assuntos
Ebolavirus/metabolismo , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/metabolismo , Arginina/genética , Arginina/metabolismo , Cristalografia por Raios X , Ebolavirus/química , Ebolavirus/classificação , Humanos , Modelos Moleculares , Mutação , Conformação Proteica , Domínios Proteicos , Multimerização Proteica , Proteínas Virais Reguladoras e Acessórias/genética
17.
mBio ; 9(5)2018 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-30301857

RESUMO

Ebola virus (EBOV) genome and mRNAs contain long, structured regions that could hijack host RNA-binding proteins to facilitate infection. We performed RNA affinity chromatography coupled with mass spectrometry to identify host proteins that bind to EBOV RNAs and identified four high-confidence proviral host factors, including Staufen1 (STAU1), which specifically binds both 3' and 5' extracistronic regions of the EBOV genome. We confirmed that EBOV infection rate and production of infectious particles were significantly reduced in STAU1-depleted cells. STAU1 was recruited to sites of EBOV RNA synthesis upon infection and enhanced viral RNA synthesis. Furthermore, STAU1 interacts with EBOV nucleoprotein (NP), virion protein 30 (VP30), and VP35; the latter two bridge the viral polymerase to the NP-coated genome, forming the viral ribonucleoprotein (RNP) complex. Our data indicate that STAU1 plays a critical role in EBOV replication by coordinating interactions between the viral genome and RNA synthesis machinery.IMPORTANCE Ebola virus (EBOV) is a negative-strand RNA virus with significant public health importance. Currently, no therapeutics are available for Ebola, which imposes an urgent need for a better understanding of EBOV biology. Here we dissected the virus-host interplay between EBOV and host RNA-binding proteins. We identified novel EBOV host factors, including Staufen1, which interacts with multiple viral factors and is required for efficient viral RNA synthesis.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Ebolavirus/genética , Interações Hospedeiro-Patógeno , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Linhagem Celular , Proteínas do Citoesqueleto/genética , Genoma Viral , Humanos , Ligação Proteica , RNA Viral/genética , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/metabolismo
18.
Sci Rep ; 8(1): 15005, 2018 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-30301920

RESUMO

The TREX complex mediates the passage of bulk cellular mRNA export to the nuclear export factor TAP/NXF1 via the export adaptors ALYREF or UIF, which appear to act in a redundant manner. TREX complex recruitment to nascent RNA is coupled with 5' capping, splicing and polyadenylation. Therefore to facilitate expression from their intronless genes, herpes viruses have evolved a mechanism to circumvent these cellular controls. Central to this process is a protein from the conserved ICP27 family, which binds viral transcripts and cellular TREX complex components including ALYREF. Here we have identified a novel interaction between HSV-1 ICP27 and an N-terminal domain of UIF in vivo, and used NMR spectroscopy to locate the UIF binding site within an intrinsically disordered region of ICP27. We also characterized the interaction sites of the ICP27 homolog ORF57 from KSHV with UIF and ALYREF using NMR, revealing previously unidentified binding motifs. In both ORF57 and ICP27 the interaction sites for ALYREF and UIF partially overlap, suggestive of mutually exclusive binding. The data provide a map of the binding sites responsible for promoting herpes virus mRNA export, enabling future studies to accurately probe these interactions and reveal the functional consequences for UIF and ALYREF redundancy.


Assuntos
Interações Hospedeiro-Patógeno/genética , Proteínas Imediatamente Precoces/genética , Proteínas Nucleares/genética , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/genética , Proteínas Virais Reguladoras e Acessórias/genética , Transporte Ativo do Núcleo Celular/genética , Sítios de Ligação/genética , Núcleo Celular/genética , Exodesoxirribonucleases/genética , Regulação Viral da Expressão Gênica/genética , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/patogenicidade , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/patogenicidade , Humanos , Íntrons/genética , Ressonância Magnética Nuclear Biomolecular , Proteínas de Transporte Nucleocitoplasmático/genética , Fosfoproteínas/genética , Ligação Proteica/genética , Transporte de RNA/genética , RNA Mensageiro/genética
19.
Nat Microbiol ; 3(12): 1354-1361, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30297740

RESUMO

Host factors that silence provirus transcription in CD4+ memory T cells help HIV-1 escape eradication by the host immune system and by antiviral drugs1. These same factors, however, must be overcome for HIV-1 to propagate. Here we show that Vpx and Vpr encoded by diverse primate immunodeficiency viruses activate provirus transcription. Vpx and Vpr are adaptor proteins for the DCAF1-CUL4A/B E3 ubiquitin ligase that degrade SAMHD1 and increase reverse transcription2-4. Nonetheless, Vpx and Vpr have effects on reporter gene expression that are not explained by SAMHD1 degradation5-8. A screen for factors that mimic these effects identified the human silencing hub (HUSH) complex, FAM208A (TASOR/RAP140), MPHOSPH8 (MPP8), PPHLN1 (PERIPHILIN) and MORC29-13. Vpx associated with the HUSH complex and decreased steady-state level of these proteins in a DCAF1/CUL4A/B/proteasome-dependent manner14,15. Replication kinetics of HIV-1 and SIVMAC was accelerated to a similar extent by vpx or FAM208A knockdown. Finally, vpx increased steady-state levels of LINE-1 ORF1p, as previously described for FAM208A disruption11. These results demonstrate that the HUSH complex represses primate immunodeficiency virus transcription, and that, to counteract this restriction, viral Vpx or Vpr proteins degrade the HUSH complex.


Assuntos
Produtos do Gene vpr/metabolismo , Lentivirus de Primatas/metabolismo , Provírus/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Antígenos de Neoplasias , Proteínas de Transporte , Proteínas Culina , Produtos do Gene vpr/genética , Células HEK293 , Infecções por HIV/virologia , HIV-1/genética , Humanos , Lentivirus de Primatas/genética , Proteínas Nucleares , Fosfoproteínas , Proteínas Serina-Treonina Quinases , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases , Proteínas Virais Reguladoras e Acessórias/genética , Produtos do Gene vpr do Vírus da Imunodeficiência Humana
20.
Biochemistry ; 57(44): 6367-6378, 2018 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-30298725

RESUMO

Ebola virus (EBOV) is a filovirus that causes a severe and rapidly progressing hemorrhagic syndrome; a recent epidemic illustrated the urgent need for novel therapeutic agents because no drugs have been approved for treatment of Ebola virus. A key contribution to the high lethality observed during EBOV outbreaks comes from viral evasion of the host antiviral innate immune response in which viral protein VP35 plays a crucial role, blocking interferon type I production, first by masking the viral double-stranded RNA (dsRNA) and preventing its detection by the pattern recognition receptor RIG-I. Aiming to identify inhibitors of the interaction of VP35 with the viral dsRNA, counteracting the VP35 viral innate immune evasion, we established a new methodology for high-yield recombinant VP35 (rVP35) expression and purification and a novel and robust fluorescence-based rVP35-RNA interaction assay ( Z' factor of 0.69). Taking advantage of such newly established methods, we screened a small library of Sardinian natural extracts, identifying Limonium morisianum as the most potent inhibitor extract. A bioguided fractionation led to the identification of myricetin as the component that can inhibit rVP35-dsRNA interaction with an IC50 value of 2.7 µM. Molecular docking studies showed that myricetin interacts with the highly conserved region of the VP35 RNA binding domain, laying the basis for further structural optimization of potent inhibitors of VP35-dsRNA interaction.


Assuntos
Antivirais/farmacologia , Flavonoides/farmacologia , Fluorescência , Extratos Vegetais/farmacologia , RNA de Cadeia Dupla/antagonistas & inibidores , RNA Viral/antagonistas & inibidores , Proteínas Virais Reguladoras e Acessórias/antagonistas & inibidores , Ebolavirus/efeitos dos fármacos , Doença pelo Vírus Ebola/tratamento farmacológico , Doença pelo Vírus Ebola/virologia , Humanos , Simulação de Acoplamento Molecular , Plumbaginaceae/química , Conformação Proteica , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/metabolismo
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