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1.
Arch Virol ; 165(5): 1079-1087, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32144546

RESUMO

Epizootic hemorrhagic disease virus (EHDV) is a member of the genus Orbivirus, family Reoviridae, and has a genome consisting of 10 linear double-stranded (ds) RNA segments. The current reverse genetics system (RGS) for engineering the EHDV genome relies on the use of in vitro-synthesized capped viral RNA transcripts. To obtain more-efficient and simpler RGSs for EHDV, we developed an entirely DNA (plasmid or PCR amplicon)-based RGS for viral rescue. This RGS enabled the rescue of infectious EHDV from BSR-T7 cells following co-transfection with seven helper viral protein expression plasmids and 10 cDNA rescue plasmids or PCR amplicons representing the EHDV genome. Furthermore, we optimized the DNA-based systems and confirmed that some of the helper expression plasmids were not essential for the recovery of infectious EHDV. Thus, DNA-based RGSs may offer a more efficient method of recombinant virus recovery and accelerate the study of the biological characteristics of EHDV and the development of novel vaccines.


Assuntos
Vírus da Doença Hemorrágica Epizoótica/genética , Genética Reversa/métodos , Virologia/métodos , Animais , Linhagem Celular , DNA Complementar/genética , Vírus da Doença Hemorrágica Epizoótica/crescimento & desenvolvimento , Mesocricetus , Plasmídeos , RNA Viral/genética , Recombinação Genética , Infecções por Reoviridae/virologia
2.
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
3.
Arch Virol ; 165(2): 367-375, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31845151

RESUMO

The genus Tobravirus comprises three species: Tobacco rattle virus, Pea early-browning virus and Pepper ringspot virus. The genomes of tobraviruses consist of two positive-sense single-stranded RNA segments (RNA1 and RNA2). Infectious clones of TRV are extensively used as virus-induced gene-silencing (VIGS) vectors for studies of virus-host interactions and functions of plant genes. Complete infectious clones of pepper ringspot virus (PepRSV), the only tobravirus present in Brazil, however, have not yet been reported. Infectious clones will help to identify unique features of PepRSV RNA2 and provide another option for development of VIGS vectors. We constructed infectious clones based on two PepRSV isolates, CAM (RNA1 and RNA2) and LAV (RNA2). The cDNA constructs for both homologous (RNA1 and RNA2 of the CAM isolate) and heterologous (RNA1/CAM and RNA2/LAV) combinations were infectious in Nicotiana benthamiana plants. VIGS vector constructs with green fluorescent protein or phytoene desaturase genes inserted in RNA2 silenced the target genes. The systemic translocation of the PepRSV RNA1 construct alone (nonmultiple infection) was also confirmed in an N. benthamiana plant. These results are similar to those reported for tobacco rattle virus.


Assuntos
Vetores Genéticos , Doenças das Plantas/virologia , Vírus de RNA/crescimento & desenvolvimento , Vírus de RNA/genética , Brasil , Genes Reporter , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Oxirredutases/análise , Oxirredutases/genética , Vírus de RNA/isolamento & purificação , Genética Reversa , Coloração e Rotulagem , Tabaco/virologia
4.
Nucleic Acids Res ; 48(4): 1985-1999, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31875230

RESUMO

A number of regulatory nascent peptides have been shown to regulate gene expression by causing programmed ribosome stalling during translation. Nascent peptide emerges from the ribosome through the exit tunnel, and one-third of the way along which ß-loop structures of ribosomal proteins uL4 and uL22 protrude into the tunnel to form the constriction region. Structural studies have shown interactions between nascent peptides and the exit tunnel components including the constriction region. In eukaryotes, however, there is a lack of genetic studies for the involvement of the constriction region in ribosome stalling. Here, we established transgenic Arabidopsis lines that carry mutations in the ß-loop structure of uL4. Translation analyses using a cell-free translation system derived from the transgenic Arabidopsis carrying the mutant ribosome showed that the uL4 mutations reduced the ribosome stalling of four eukaryotic stalling systems, including those for which stalled structures have been solved. Our data, which showed differential effects of the uL4 mutations depending on the stalling systems, explained the spatial allocations of the nascent peptides at the constriction that were deduced by structural studies. Conversely, our data may predict allocation of the nascent peptide at the constriction of stalling systems for which structural studies are not done.


Assuntos
Peptídeos/genética , Biossíntese de Proteínas/genética , Proteínas Ribossômicas/química , Ribossomos/genética , Sequência de Aminoácidos/genética , Arabidopsis/química , Arabidopsis/genética , Sistema Livre de Células , Células Eucarióticas/química , Células Eucarióticas/metabolismo , Peptídeos/química , Genética Reversa , Proteínas Ribossômicas/genética , Ribossomos/química
5.
Virol J ; 16(1): 162, 2019 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-31864377

RESUMO

RNA secondary structures play a key role in splicing, gene expression, microRNA biogenesis, RNA editing, and other biological processes. The importance of RNA structures has been demonstrated in the life cycle of RNA-containing viruses, including the influenza virus. At least two regions of conserved secondary structure in NS segment (+) RNA are predicted to vary among influenza virus strains with respect to thermodynamic stability; both fall in the NS1 open reading frame. The NS1 protein is involved in multiple virus-host interaction processes, and its main function is to inhibit the cellular immune response to viral infection. Using a reverse genetics approach, four influenza virus strains were constructed featuring mutations that have different effects on RNA secondary structure. Growth curve experiments and ELISA data show that, at least in the first viral replication cycle, mutations G123A and A132G affecting RNA structure in the (82-148) NS RNA region influence NS1 protein expression.


Assuntos
Expressão Gênica , Conformação de Ácido Nucleico , Orthomyxoviridae/crescimento & desenvolvimento , RNA Viral/química , Proteínas não Estruturais Virais/biossíntese , Animais , Cães , Células Madin Darby de Rim Canino , Mutagênese Sítio-Dirigida , Orthomyxoviridae/genética , Mutação Puntual , RNA Viral/metabolismo , Genética Reversa , Replicação Viral
6.
PLoS Negl Trop Dis ; 13(11): e0007904, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31751340

RESUMO

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease endemic to Africa and the Middle East, affecting both humans and ruminants. There are no licensed vaccines or antivirals available for humans, whereas research using RVF virus (RVFV) is strictly regulated in many countries with safety concerns. Nonpathogenic Arumowot virus (AMTV), a mosquito-borne phlebovirus in Africa, is likely useful for the screening of broad-acting antiviral candidates for phleboviruses including RVFV, as well as a potential vaccine vector for RVF. In this study, we aimed to generate T7 RNA polymerase-driven reverse genetics system for AMTV. We hypothesized that recombinant AMTV (rAMTV) is viable, and AMTV NSs protein is dispensable for efficient replication of rAMTV in type-I interferon (IFN)-incompetent cells, whereas AMTV NSs proteins support robust viral replication in type-I IFN-competent cells. The study demonstrated the rescue of rAMTV and that lacking the NSs gene (rAMTVΔNSs), that expressing green fluorescent protein (GFP) (rAMTV-GFP) or that expressing Renilla luciferase (rAMTV-rLuc) from cloned cDNA. The rAMTV-rLuc and the RVFV rMP12-rLuc showed a similar susceptibility to favipiravir or ribavirin. Interestingly, neither of rAMTV nor rAMTVΔNSs replicated efficiently in human MRC-5 or A549 cells, regardless of the presence of NSs gene. Little accumulation of AMTV NSs protein occurred in those cells, which was restored via treatment with proteasomal inhibitor MG132. In murine MEF or Hepa1-6 cells, rAMTV, but not rAMTVΔNSs, replicated efficiently, with an inhibition of IFN-ß gene upregulation. This study showed an establishment of the first reverse genetics for AMTV, a lack of stability of AMTV NSs proteins in human cells, and an IFN-ß gene antagonist function of AMTV NSs proteins in murine cells. The AMTV can be a nonpathogenic surrogate model for studying phleboviruses including RVFV.


Assuntos
DNA Complementar/genética , Phlebovirus/crescimento & desenvolvimento , Phlebovirus/genética , Proteólise , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Animais , Linhagem Celular , Humanos , Camundongos , Phlebovirus/isolamento & purificação , Genética Reversa
7.
PLoS Biol ; 17(10): e3000502, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31600204

RESUMO

The impacts of invertebrate RNA virus population dynamics on virulence and infection outcomes are poorly understood. Deformed wing virus (DWV), the main viral pathogen of honey bees, negatively impacts bee health, which can lead to colony death. Despite previous reports on the reduction of DWV diversity following the arrival of the parasitic mite Varroa destructor, the key DWV vector, we found high genetic diversity of DWV in infested United States honey bee colonies. Phylogenetic analysis showed that divergent US DWV genotypes are of monophyletic origin and were likely generated as a result of diversification after a genetic bottleneck. To investigate the population dynamics of this divergent DWV, we designed a series of novel infectious cDNA clones corresponding to coexisting DWV genotypes, thereby devising a reverse-genetics system for an invertebrate RNA virus quasispecies. Equal replication rates were observed for all clone-derived DWV variants in single infections. Surprisingly, individual clones replicated to the same high levels as their mixtures and even the parental highly diverse natural DWV population, suggesting that complementation between genotypes was not required to replicate to high levels. Mixed clone-derived infections showed a lack of strong competitive exclusion, suggesting that the DWV genotypes were adapted to coexist. Mutational and recombination events were observed across clone progeny, providing new insights into the forces that drive and constrain virus diversification. Accordingly, our results suggest that Varroa influences DWV dynamics by causing an initial selective sweep, which is followed by virus diversification fueled by negative frequency-dependent selection for new genotypes. We suggest that this selection might reflect the ability of rare lineages to evade host defenses, specifically antiviral RNA interference (RNAi). In support of this hypothesis, we show that RNAi induced against one DWV strain is less effective against an alternate strain from the same population.


Assuntos
Vetores Aracnídeos/virologia , Abelhas/virologia , Evasão da Resposta Imune/genética , Vírus de RNA/genética , Varroidae/virologia , Animais , Abelhas/genética , Abelhas/imunologia , Abelhas/parasitologia , Células Clonais , Biblioteca Gênica , Variação Genética , Genótipo , Mutação , Filogenia , Interferência de RNA/imunologia , Vírus de RNA/classificação , Vírus de RNA/imunologia , Vírus de RNA/patogenicidade , Recombinação Genética , Genética Reversa/métodos , Seleção Genética , Virulência , Replicação Viral
8.
Parasit Vectors ; 12(1): 470, 2019 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-31604476

RESUMO

BACKGROUND: Transmission of vector-borne virus by insects is a complex mechanism consisting of many different processes; viremia in the host, uptake, infection and dissemination in the vector, and delivery of virus during blood-feeding leading to infection of the susceptible host. Bluetongue virus (BTV) is the prototype vector-borne orbivirus (family Reoviridae). BTV serotypes 1-24 (typical BTVs) are transmitted by competent biting Culicoides midges and replicate in mammalian (BSR) and midge (KC) cells. Previously, we showed that genome segment 10 (S10) encoding NS3/NS3a protein is required for virus propagation in midges. BTV serotypes 25-27 (atypical BTVs) do not replicate in KC cells. Several distinct BTV26 genome segments cause this so-called 'differential virus replication' in vitro. METHODS: Virus strains were generated using reverse genetics and their growth was examined in vitro. The midge feeding model has been developed to study infection, replication and disseminations of virus in vivo. A laboratory colony of C. sonorensis, a known competent BTV vector, was fed or injected with BTV variants and propagation in the midge was examined using PCR testing. Crossing of the midgut infection barrier was examined by separate testing of midge heads and bodies. RESULTS: A 100 nl blood meal containing ±105.3 TCID50/ml of BTV11 which corresponds to ±20 TCID50 infected 50% of fully engorged midges, and is named one Midge Alimentary Infective Dose (MAID50). BTV11 with a small in-frame deletion in S10 infected blood-fed midge midguts but virus release from the midgut into the haemolymph was blocked. BTV11 with S1[VP1] of BTV26 could be adapted to virus growth in KC cells, and contained mutations subdivided into 'corrections' of the chimeric genome constellation and mutations associated with adaptation to KC cells. In particular one amino acid mutation in outer shell protein VP2 overcomes differential virus replication in vitro and in vivo. CONCLUSION: Small changes in NS3/NS3a or in the outer shell protein VP2 strongly affect virus propagation in midges and thus vector competence. Therefore, spread of disease by competent Culicoides midges can strongly differ for very closely related viruses.


Assuntos
Vírus Bluetongue/fisiologia , Ceratopogonidae/virologia , Deleção de Genes , Insetos Vetores/virologia , Mutação Puntual , Animais , Vírus Bluetongue/genética , Linhagem Celular , Embrião de Galinha , Cricetinae , Cervos , Feminino , Técnicas Imunoenzimáticas , Genética Reversa , Replicação Viral , Sequenciamento Completo do Genoma
9.
Nat Biotechnol ; 37(11): 1314-1321, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31570900

RESUMO

Most microorganisms from all taxonomic levels are uncultured. Single-cell genomes and metagenomes continue to increase the known diversity of Bacteria and Archaea; however, while 'omics can be used to infer physiological or ecological roles for species in a community, most of these hypothetical roles remain unvalidated. Here, we report an approach to capture specific microorganisms from complex communities into pure cultures using genome-informed antibody engineering. We apply our reverse genomics approach to isolate and sequence single cells and to cultivate three different species-level lineages of human oral Saccharibacteria (TM7). Using our pure cultures, we show that all three Saccharibacteria species are epibionts of diverse Actinobacteria. We also isolate and cultivate human oral SR1 bacteria, which are members of a lineage of previously uncultured bacteria. Reverse-genomics-enabled cultivation of microorganisms can be applied to any species from any environment and has the potential to unlock the isolation, cultivation and characterization of species from as-yet-uncultured branches of the microbial tree of life.


Assuntos
Actinobacteria/metabolismo , Anticorpos/metabolismo , Proteínas de Membrana/imunologia , Boca/microbiologia , Análise de Célula Única/métodos , Actinobacteria/classificação , Actinobacteria/genética , Actinobacteria/isolamento & purificação , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Genômica , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Moleculares , Filogenia , Conformação Proteica , Genética Reversa , Análise de Sequência de DNA
10.
Virol J ; 16(1): 112, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31488178

RESUMO

BACKGROUND: Reverse genetics systems enable the manipulation of viral genomes and therefore serve as robust reverse genetic tools to study RNA viruses. A DNA-launched rescue system initiates the transcription of viral genomic cDNA from eukaryotic promoter in transfected cells, generating homogenous RNA transcripts in vitro and thus enhancing virus rescue efficiency. As one of the hazardous pathogens to ducklings, the current knowledge of the pathogenesis of duck astrovirus type 1 (DAstV-1) is limited. The construction of a DNA-launched rescue system can help to accelerate the study of the virus pathogenesis. However, there is no report of such a system for DAstV-1. METHODS: In this study, a DNA-launched infectious clone of DAstV-1 was constructed from a cDNA plasmid, which contains a viral cDNA sequence flanked by hammerhead ribozyme (HamRz) and a hepatitis delta virus ribozyme (HdvRz) sequence at both terminals of the viral genome. A silent nucleotide mutation creating a Bgl II site in the ORF2 gene was made to distinguish the rescued virus (rDAstV-1) from the parental virus (pDAstV-1). Immunofluorescence assay (IFA) and western blot were conducted for rescued virus identification in duck embryo fibroblast (DEF) cells pre-treated with trypsin. The growth characteristics of rDAstV-1 and pDAstV-1 in DEF cells and the tissue tropism in 2-day-old ducklings of rDAstV-1 and pDAstV-1 were determined. RESULTS: The infectious DAstV-1 was successfully rescued from baby hamster kidney (BHK-21) cells and could propagate in DEF cells pre-treated with 1 µg/ml trypsin. Upon infection of DEF cells pre-treated with trypsin, DAstV-1 mRNA copies were identified after serial passaging, and the result showed that rDAstV-1 and pDAstV-1 shared similar replication kinetics. Animal experiment showed that the rDAstV-1 had an extensive tissue tropism, and the virus was capable of invading both the central and the peripheral immune organs in infected ducklings. CONCLUSIONS: An improved DNA-launched reverse genetics system for DAstV-1 was firstly constructed. Infectious virus recovered from BHK-21 cells could propagate in DEF cells pre-treated with trypsin. This is the first report of the successful in vitro cultivation of DAstV-1. We believe this valuable experimental system will contribute to the further study of DAstV-1 genome function and pathogenesis.


Assuntos
Infecções por Astroviridae/veterinária , Avastrovirus/genética , Avastrovirus/isolamento & purificação , Patos/virologia , Genética Reversa/métodos , Cultura de Vírus/métodos , Animais , Infecções por Astroviridae/virologia , Avastrovirus/crescimento & desenvolvimento , Linhagem Celular , Clonagem Molecular , DNA Complementar/genética , Genoma Viral , Plasmídeos , RNA Viral/genética , Transfecção , Tropismo Viral , Vírion/genética
11.
PLoS Genet ; 15(8): e1008288, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31393878

RESUMO

Neuronal physiology is particularly sensitive to acute stressors that affect excitability, many of which can trigger seizures and epilepsies. Although intrinsic neuronal homeostasis plays an important role in maintaining overall nervous system robustness and its resistance to stressors, the specific genetic and molecular mechanisms that underlie these processes are not well understood. Here we used a reverse genetic approach in Drosophila to test the hypothesis that specific voltage-gated ion channels contribute to neuronal homeostasis, robustness, and stress resistance. We found that the activity of the voltage-gated potassium channel seizure (sei), an ortholog of the mammalian ERG channel family, is essential for protecting flies from acute heat-induced seizures. Although sei is broadly expressed in the nervous system, our data indicate that its impact on the organismal robustness to acute environmental stress is primarily mediated via its action in excitatory neurons, the octopaminergic system, as well as neuropile ensheathing and perineurial glia. Furthermore, our studies suggest that human mutations in the human ERG channel (hERG), which have been primarily implicated in the cardiac Long QT Syndrome (LQTS), may also contribute to the high incidence of seizures in LQTS patients via a cardiovascular-independent neurogenic pathway.


Assuntos
Proteínas de Drosophila/genética , Resposta ao Choque Térmico/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Convulsões/genética , Regulador Transcricional ERG/genética , Animais , Animais Geneticamente Modificados , Drosophila , Proteínas de Drosophila/metabolismo , Técnicas de Silenciamento de Genes , Incidência , Síndrome do QT Longo/complicações , Síndrome do QT Longo/genética , Neurônios/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Genética Reversa , Convulsões/epidemiologia , Regulador Transcricional ERG/metabolismo
12.
Arch Virol ; 164(10): 2505-2513, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31377888

RESUMO

Infectious hematopoietic necrosis virus (IHNV) was developed as a vector to aid the construction of vaccines against viral diseases such as viral hemorrhagic septicemia virus, spring viremia of carp virus, and influenza virus H1N1. However, the optimal site for foreign gene expression in the IHNV vector has not been determined. In the present study, five recombinant viruses with the green fluorescence protein (GFP) gene inserted into different genomic junction regions of the IHNV genomic sequence were generated using reverse genetics technology. Viral growth was severely delayed when the GFP gene was inserted into the intergenic region between the N and P genes. Real-time fluorescence quantitative PCR assays showed that the closer the GFP gene was inserted towards the 3' end, the higher the GFP mRNA levels. Measurement of the GFP fluorescence intensity, which is the most direct method to determine the GFP protein expression level, showed that the highest GFP protein level was obtained when the gene was inserted into the intergenic region between the P and M genes. The results of this study suggest that the P and M gene junction region is the optimal site within the IHNV vector to express foreign genes, providing valuable information for the future development of live vector vaccines.


Assuntos
Expressão Gênica , Vetores Genéticos , Vírus da Necrose Hematopoética Infecciosa/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Fluorometria , Genes Reporter , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Reação em Cadeia da Polimerase em Tempo Real , Genética Reversa
13.
Virol J ; 16(1): 90, 2019 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-31319897

RESUMO

BACKGROUND: Nelson Bay orthoreovirus (NBV) was first isolated over 40 years ago from a fruit bat in Australia. Normally, NBV does not cause human diseases, but recently several NBV strains have been associated with human respiratory tract infections, thus attracting clinical attention. Autophagy, an evolutionarily conserved process in eukaryotic cells, degrades intracellular substrates, participates in multiple physiological processes, and maintains cellular homeostasis. In addition, autophagy is intimately involved in viral infection. METHODS: A new strain of NBV, isolated from a patient with a respiratory tract infection who returned to Japan from Bali, Indonesia, in 2007, was used in this study. NBV was rescued using a reverse genetics system involving cotransfection of BHK cells with 11 plasmids (pT7-L1 MB, pT7-L2 MB, pT7-L3 MB, pT7-M1 MB, pT7-M2 MB, pT7-M3 MB, pT7-S1 MB, pT7-S2 MB, pT7-S3 MB, pT7-S4 MB, and pcDNA3.1-T7), yielding NBV-MB. Recovered viruses were confirmed by immunofluorescence. The effect of NBV-MB on autophagy was evaluated by measuring the LC3-I/II proteins by immunoblot analysis after infection of BHK cells. Furthermore, after treatment with rapamycin (RAPA), 3-methyladenine (3-MA), chloroquine (CQ), or plasmid (GFP-LC3) transfection, the changes in expression of the LC3 gene and the amount of LC3-I/II protein were examined. In addition, variations in viral titer were assayed after treatment of BHK cells with drugs or after transfection with plasmids pCAGM3 and pCAGS3, which encode virus nonstructural proteins µNS and σNS, respectively. RESULTS: NBV-MB infection induced autophagy in host cells; however, the level of induction was dependent on viral replication. Induction of autophagy increased viral replication. By contrast, inhibiting autophagy suppressed NBV replication, albeit not significantly. The NBV-MB nonstructural protein µNS was involved in the induction of autophagy with viral infection. CONCLUSIONS: NBV-MB infection triggered autophagy. Also, the NBV nonstructural protein µNS may contribute to augmentation of autophagy upon viral infection.


Assuntos
Autofagia , Interações entre Hospedeiro e Microrganismos , Orthoreovirus/fisiologia , Replicação Viral , Linhagem Celular , Células HEK293 , Humanos , Infecções por Reoviridae/virologia , Genética Reversa , Carga Viral , Proteínas Virais/genética
14.
Plant Cell Physiol ; 60(10): 2293-2306, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31268148

RESUMO

ROOT UV-B SENSITIVE4 (RUS4) encodes a protein with no known function that contains a conserved Domain of Unknown Function 647 (DUF647). The DUF647-containing proteins RUS1 and RUS2 have previously been associated with root UV-B-sensing pathway that plays a major role in Arabidopsis early seedling morphogenesis and development. Here, we show that RUS4 knockdown Arabidopsis plants, referred to as amiR-RUS4, were severely reduced in male fertility with indehiscent anthers. Light microscopy of anther sections revealed a significantly reduced secondary wall thickening in the endothecium of amiR-RUS4 anthers. We further show that the transcript abundance of the NAC domain genes NAC SECONDARY WALL THICKENING PROMOTING FACTOR1 (NST1) and NST2, which have been shown to regulate the secondary cell wall thickenings in the anther endothecium, were dramatically reduced in the amiR-RUS4 floral buds. Expression of the secondary cell wall-associated MYB transcription factor genes MYB103 and MYB85 were also strongly reduced in floral buds of the amiR-RUS4 plants. Overexpression of RUS4 led to increased secondary thickening in the endothecium. However, the rus4-2 mutant exhibited no obvious phenotype. Promoter-GUS analysis revealed that the RUS4 promoter was highly active in the anthers, supporting its role in anther development. Taken together, these results suggest that RUS4, probably functions redundantly with other genes, may play an important role in the secondary thickening formation in the anther endothecium by indirectly affecting the expression of secondary cell wall biosynthetic genes.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Infertilidade das Plantas/genética , Fatores de Transcrição/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Parede Celular/metabolismo , Flores/genética , Flores/crescimento & desenvolvimento , Flores/fisiologia , Expressão Gênica , Técnicas de Silenciamento de Genes , Genes Reporter , Fenótipo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Genética Reversa , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/fisiologia , Fatores de Transcrição/genética
15.
PLoS One ; 14(7): e0219168, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31276568

RESUMO

In reverse genetic experiments we have isolated recombinant mumps viruses (rMuV) that carry large numbers of mutations clustered in small parts of their genome, which are not caused by biased hyper-mutation. In two separate experiments we obtained such recombinant viruses: one virus had 11 mutations in the V/P region of the genome; the other, which also contained an extra transcription unit encoding green fluorescent protein (EGFP), had 32 mutations in the N gene. These specific sets of mutations have not been observed in naturally occurring MuV isolates. Unusually, the vast majority of the mutations (48/51) were synonymous. On passage in Vero cells and human B-LCL cells, a B lymphocyte-like cell line, these mutations appear stable as no reversion occurred to the original consensus sequence, although mutations in other parts of the genome occurred and changed in frequency during passage. Defective interfering RNAs accumulate in passage in Vero cells but not in B-LCL cells. Interestingly, in all passaged samples the level of variation in the EGFP gene is the same as in the viral genes, though it is unlikely that this gene is under any functionality constraint. What mechanism gave rise to these viruses with clustered mutations and their stability remains an open question, which is likely of interest to a wider field than mumps reverse genetics.


Assuntos
DNA Complementar/genética , Vírus da Caxumba/fisiologia , Mutação , Proteínas Virais/genética , Animais , Linhagem Celular Tumoral , Proteínas de Fluorescência Verde/genética , Humanos , Vírus da Caxumba/genética , Genética Reversa/métodos , Inoculações Seriadas , Células Vero , Replicação Viral
16.
Appl Microbiol Biotechnol ; 103(18): 7597-7615, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31346683

RESUMO

Diastatic strains of Saccharomyces cerevisiae are common contaminants in beer fermentations and are capable of producing an extracellular STA1-encoded glucoamylase. Recent studies have revealed variable diastatic ability in strains tested positive for STA1, and here, we elucidate genetic determinants behind this variation. We show that poorly diastatic strains have a 1162-bp deletion in the promoter of STA1. With CRISPR/Cas9-aided reverse engineering, we show that this deletion greatly decreases the ability to grow in beer and consume dextrin, and the expression of STA1. New PCR primers were designed for differentiation of highly and poorly diastatic strains based on the presence of the deletion in the STA1 promoter. In addition, using publically available whole genome sequence data, we show that the STA1 gene is prevalent among the 'Beer 2'/'Mosaic Beer' brewing strains. These strains utilize maltotriose efficiently, but the mechanisms for this have been unknown. By deleting STA1 from a number of highly diastatic strains, we show here that extracellular hydrolysis of maltotriose through STA1 appears to be the dominant mechanism enabling maltotriose use during wort fermentation in STA1+ strains. The formation and retention of STA1 seems to be an alternative evolutionary strategy for efficient utilization of sugars present in brewer's wort. The results of this study allow for the improved reliability of molecular detection methods for diastatic contaminants in beer and can be exploited for strain development where maltotriose use is desired.


Assuntos
Proteínas Fúngicas/genética , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Amido/metabolismo , Trissacarídeos/metabolismo , Cerveja/microbiologia , Sistemas CRISPR-Cas , Dextrinas/metabolismo , Fermentação , Reprodutibilidade dos Testes , Genética Reversa , Proteínas de Saccharomyces cerevisiae/genética , Deleção de Sequência
17.
Virus Genes ; 55(4): 550-556, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31161411

RESUMO

Japanese encephalitis virus SA14-14-2 (JEV SA14-14-2) is a widely used vaccine in China and other southeastern countries to prevent Japanese encephalitis in children. In this study, a stable infectious cDNA clone of JEV SA14-14-2 with a low copy number pACYC177 vector dependent on the T7 promoter and T7 terminator was developed. Two introns were inserted into the capsid gene and envelope gene of JEV cDNA for gene stability. Hepatitis delta virus ribozyme (HDVr) was engineered into the 3' UTR cDNA of JEV for authentic 3' UTR transcription. The rescued virus showed biological properties indistinguishable from those of the parent strain (JEV SA14-14-2). The establishment of a JEV SA14-14-2 reverse genetics system lays the foundation for the further development of other flavivirus vaccines and viral pathogenesis studies.


Assuntos
Vírus da Encefalite Japonesa (Espécie)/genética , Genética Reversa/métodos , Linhagem Celular , DNA Complementar , DNA Viral , Vírus da Encefalite Japonesa (Espécie)/ultraestrutura , Vetores Genéticos , Genoma Viral , Regiões Promotoras Genéticas , Sequenciamento Completo do Exoma
18.
J Microbiol Biotechnol ; 29(6): 999-1007, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31154749

RESUMO

Middle East respiratory syndrome coronavirus (MERS-CoV) was first identified in Saudi Arabia in 2012 and related infection cases have been reported in over 20 countries. Roughly 10,000 human cases have so far been reported in total with fatality rates at up to 40%. The majority of cases have occurred in Saudi Arabia with mostly sporadic outbreaks outside the country except for the one in South Korea in 2015. The Korean MERS-CoV strain was isolated from the second Korean patient and its genome was fully sequenced and deposited. To develop virusspecific protective and therapeutic agents against the Korean isolate and to investigate molecular determinants of virus-host interactions, it is of paramount importance to generate its full-length cDNA. Here we report that two full-length cDNAs from a Korean patientisolated MERS-CoV strain were generated by a combination of conventional cloning techniques and efficient Gibson assembly reactions. The full-length cDNAs were validated by restriction analysis and their sequence was verified by Sanger method. The resulting cDNA was efficiently transcribed in vitro and the T7 promoter-driven expression was robust. The resulting reverse genetic system will add to the published list of MERS-CoV cDNAs and facilitate the development of Korean isolate-specific antiviral measures.


Assuntos
DNA Complementar/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Genética Reversa , Infecções por Coronavirus/virologia , Genes Virais/genética , Engenharia Genética , Genoma Viral/genética , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/isolamento & purificação , RNA Viral/genética , Transcrição Genética
19.
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
20.
Genetics ; 212(4): 1163-1179, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31243056

RESUMO

Malassezia encompasses a monophyletic group of basidiomycetous yeasts naturally found on the skin of humans and other animals. Malassezia species have lost genes for lipid biosynthesis, and are therefore lipid-dependent and difficult to manipulate under laboratory conditions. In this study, we applied a recently-developed Agrobacterium tumefaciens-mediated transformation protocol to perform transfer (T)-DNA random insertional mutagenesis in Malassezia furfur A total of 767 transformants were screened for sensitivity to 10 different stresses, and 19 mutants that exhibited a phenotype different from the wild type were further characterized. The majority of these strains had single T-DNA insertions, which were identified within open reading frames of genes, untranslated regions, and intergenic regions. Some T-DNA insertions generated chromosomal rearrangements while others could not be characterized. To validate the findings of our forward genetic screen, a novel clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system was developed to generate targeted deletion mutants for two genes identified in the screen: CDC55 and PDR10 This system is based on cotransformation of M. furfur mediated by A. tumefaciens, to deliver both a CAS9-gRNA construct that induces double-strand DNA breaks and a gene replacement allele that serves as a homology-directed repair template. Targeted deletion mutants for both CDC55 and PDR10 were readily generated with this method. This study demonstrates the feasibility and reliability of A. tumefaciens-mediated transformation to aid in the identification of gene functions in M. furfur, through both insertional mutagenesis and CRISPR/Cas9-mediated targeted gene deletion.


Assuntos
Sistemas CRISPR-Cas , Malassezia/genética , Mutagênese , Genética Reversa/métodos , Agrobacterium/genética , Farmacorresistência Fúngica/genética , Deleção de Genes , Mutagênese Insercional , Transformação Genética
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