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1.
J Biotechnol ; 304: 1-9, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31404563

RESUMO

Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated proteins (Cas) 9 system is a powerful tool for genome editing and still being aggressively improved. Cas12a, a recently discovered Cas9 ortholog, is expected to become complementary to Cas9 due to its unique characteristics. Previously we attempted to establish an adenovirus (Ad) vector-mediated delivery of CRISPR-Cas12a system since Ad vector is widely used for gene transfer in basic researches and medical applications. However, we found difficulties preparing of Ad vectors at an adequate titer. In this study, we have developed Ad vectors that conditionally express Cas12a either by a tetracycline-controlled promoter or a hepatocyte specific promoter to avoid putative inhibitory effects of Cas12a. These vectors successfully proliferated in packaging cells, HEK293 cells, and were recovered at high titers. We have also developed packaging cells that express shRNA for Cas12a to suppress expression of Cas12a. Using the cells, the Ad vector directing constitutive expression of Cas12a proliferated efficiently and was successfully recovered at a high titer. Overall, we improved recovery of Ad vectors carrying CRISPR-Cas12a system, thus provided them as a tool in genome editing researches.


Assuntos
Adenoviridae/fisiologia , Proteínas Associadas a CRISPR/genética , RNA Guia/genética , Adenoviridae/genética , Sistemas CRISPR-Cas , Proliferação de Células , Edição de Genes , Vetores Genéticos/fisiologia , Células HEK293/citologia , Células HEK293/virologia , Humanos , Carga Viral
2.
Antiviral Res ; 167: 1-5, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30951731

RESUMO

The antiviral drug T-705 (favipiravir) and its non-fluorinated analogue T-1105 inhibit the polymerases of RNA viruses after being converted to their ribonucleoside triphosphate (RTP) metabolite. We here compared the activation efficiency of T-705 and T-1105 in four cell lines that are commonly used for their antiviral evaluation. In MDCK cells, the levels of T-705-RTP were markedly lower than those of T-1105-RTP, while the opposite was seen in A549, Vero and HEK293T cells. In the latter three cell lines, T-1105 activation was hindered by inefficient conversion of the ribonucleoside monophosphate to the ribonucleoside diphosphate en route to forming the active triphosphate. Accordingly, T-1105 had better anti-RNA virus activity in MDCK cells, while T-705 was more potent in the other three cell lines. Additionally, we identified a fourth metabolite, the NAD analogue of T-705/T-1105, and showed that it can be formed by nicotinamide mononucleotide adenylyltransferase.


Assuntos
Amidas/farmacologia , Antivirais/farmacologia , Linhagem Celular , Pirazinas/farmacologia , Vírus de RNA/efeitos dos fármacos , Animais , Linhagem Celular/efeitos dos fármacos , Linhagem Celular/metabolismo , Linhagem Celular/virologia , Chlorocebus aethiops , Cães , Células HEK293/efeitos dos fármacos , Células HEK293/metabolismo , Células HEK293/virologia , Humanos , Células Madin Darby de Rim Canino/efeitos dos fármacos , Células Madin Darby de Rim Canino/metabolismo , Células Madin Darby de Rim Canino/virologia , Ribonucleosídeos/metabolismo , Células Vero/efeitos dos fármacos , Células Vero/metabolismo , Células Vero/virologia
4.
Hereditas ; 156: 10, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30774581

RESUMO

Background: Influenza A virus (IAV) belongs to the Orthomyxoviridae family. IAV causes a highly contagious respiratory disease in humans that exacts severe economic losses globally. The virus uses strategies developed to exploit and subvert cellular proteins and pathways to increase its own replication and to inhibit antiviral immune response. Results: A/bar-headed goose/Qinghai/1/2005 (A/QH) was able to infect A549 and 293 T cells, with a high infection rate for A549 cells. To identify host cellular responses of human cells to influenza infection, differentially expressed genes (DEGs) between AIV-infected groups and uninfected controls were identified using RNA-sequencing. The DEGs were annotated by Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes pathway analyses, which revealed that the DEGs were mainly linked to cellular function and metabolic processes, while the cellular function that is probably associated with host cellular response of human cells, including defense response to virus and protein modification. All the DEGs and pathways were possibly involved in the response to IAV invasion. Conclusions: The global transcriptome analysis results revealed that sensitive genes and pathways of the cells were infected with the influenza virus and provided further evidence to investigate the complicated relationship between IAV and host cells.


Assuntos
Células A549/metabolismo , Células HEK293/metabolismo , Virus da Influenza A Subtipo H5N1/fisiologia , Transcriptoma , Replicação Viral , Células A549/virologia , Perfilação da Expressão Gênica , Células HEK293/virologia , Humanos , Análise de Sequência de RNA
5.
PLoS Pathog ; 15(1): e1007569, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30677091

RESUMO

Human Cytomegalovirus (HCMV) infection induces several metabolic activities that are essential for viral replication. Despite the important role that this metabolic modulation plays during infection, the viral mechanisms involved are largely unclear. We find that the HCMV UL38 protein is responsible for many aspects of HCMV-mediated metabolic activation, with UL38 being necessary and sufficient to drive glycolytic activation and induce the catabolism of specific amino acids. UL38's metabolic reprogramming role is dependent on its interaction with TSC2, a tumor suppressor that inhibits mTOR signaling. Further, shRNA-mediated knockdown of TSC2 recapitulates the metabolic phenotypes associated with UL38 expression. Notably, we find that in many cases the metabolic flux activation associated with UL38 expression is largely independent of mTOR activity, as broad spectrum mTOR inhibition does not impact UL38-mediated induction of glycolysis, glutamine consumption, or the secretion of proline or alanine. In contrast, the induction of metabolite concentrations observed with UL38 expression are largely dependent on active mTOR. Collectively, our results indicate that the HCMV UL38 protein induces a pro-viral metabolic environment via inhibition of TSC2.


Assuntos
Proteínas do Capsídeo/metabolismo , Citomegalovirus/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Proteínas do Capsídeo/genética , Linhagem Celular , Citomegalovirus/genética , Infecções por Citomegalovirus/virologia , Fibroblastos/virologia , Glicólise , Células HEK293/virologia , Humanos , RNA Interferente Pequeno/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Replicação Viral
6.
Biotechnol Bioeng ; 114(11): 2539-2549, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28710851

RESUMO

Apoptosis has important functions during pathophysiologic processes. However, from a biopharmaceutical point of view, active apoptosis of host cells is undesirable during viral packaging or protein expression, because it decreases the efficiency of viral or protein production. Here we used the CRISPR/Cas technique to knock out four pro-apoptotic genes, Caspase3, Caspase6, Caspase7 and AIF1, in HEK293 cells, and successfully produced an apoptosis-resistant cell line. Furthermore, this cell line showed higher expression levels of pro-apoptotic proteins and higher packaging efficiency for the virus carrying these proteins than control HEK293 cells. This study not only produced an apoptosis-resistant cell line that is useful in producing apoptosis-inducing proteins or viruses expressing these proteins, but also provides a methodology to build other apoptosis-resistant cell lines.


Assuntos
Apoptose/genética , Sistemas CRISPR-Cas/genética , Melhoramento Genético/métodos , Células HEK293/fisiologia , Células HEK293/virologia , Lentivirus/crescimento & desenvolvimento , Proteínas Recombinantes/biossíntese , Técnicas de Inativação de Genes/métodos , Células HEK293/citologia , Humanos , Lentivirus/isolamento & purificação , Engenharia de Proteínas/métodos , Proteínas Recombinantes/isolamento & purificação
7.
Vaccine ; 35(26): 3423-3430, 2017 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-28495315

RESUMO

Despite major advances in developing capacities and alternative technologies to egg-based production of influenza vaccines, responsiveness to an influenza pandemic threat is limited by the time it takes to generate a Candidate Vaccine Virus (CVV) as reported by the 2015 WHO Informal Consultation report titled "Influenza Vaccine Response during the Start of a Pandemic". In previous work, we have shown that HEK-293 cell culture in suspension and serum free medium is an efficient production platform for cell culture manufacturing of influenza candidate vaccines. This report, took advantage of, recombinant DNA technology using Reverse Genetics of influenza strains, and advances in the large-scale transfection of suspension cultured HEK-293 cells. We demonstrate the efficient generation of H1N1 with the PR8 backbone reassortant under controlled bioreactor conditions in two sequential steps (transfection/rescue and infection/production). This approach could deliver a CVV for influenza vaccine manufacturing within two-weeks, starting from HA and NA pandemic sequences. Furthermore, the scalability of the transfection technology combined with the HEK-293 platform has been extensively demonstrated at >100L scale for several biologics, including recombinant viruses. Thus, this innovative approach is better suited to rationally engineer and mass produce influenza CVV within significantly shorter timelines to enable an effective global response in pandemic situations.


Assuntos
Células HEK293/virologia , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Genética Reversa , Cultura de Vírus , Reatores Biológicos , Testes de Inibição da Hemaglutinação , Humanos , Vacinas contra Influenza , Vírus Reordenados/crescimento & desenvolvimento , Transfecção
8.
J Mol Biol ; 429(8): 1171-1191, 2017 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-28315663

RESUMO

The retroviral restriction factors of the APOBEC3 (A3) cytidine deaminase family catalyze the deamination of cytidines in single-stranded viral DNA. APOBEC3C (A3C) is a strong antiviral factor against viral infectivity factor (vif)-deficient simian immunodeficiency virus Δvif, which is, however, a weak inhibitor against human immunodeficiency virus (HIV)-1 for reasons unknown. The precise link between the antiretroviral effect of A3C and its catalytic activity is incompletely understood. Here, we show that the S61P mutation in human A3C (A3C.S61P) boosted hypermutation in the viral genomes of simian immunodeficiency virus Δvif and murine leukemia virus but not in human immunodeficiency virus HIV-1Δvif. The enhanced antiviral activity of A3C.S61P correlated with enhanced in vitro cytidine deamination. Furthermore, the S61P mutation did not change the substrate specificity of A3C, ribonucleoprotein complex formation, self-association, Zinc coordination, or viral incorporation features. We propose that local structural changes induced by the serine-to-proline substitution are responsible for the gain of catalytic activity of A3C.S61P. Our results are a first step toward an understanding of A3C's DNA binding capacity, deamination-dependent editing, and antiviral functions at the molecular level. We conclude that the enhanced enzymatic activity of A3C is insufficient to restrict HIV-1, indicating an unknown escape mechanism of HIV-1.


Assuntos
Citidina Desaminase/química , Citidina Desaminase/metabolismo , HIV-1/patogenicidade , Substituição de Aminoácidos , Animais , Citidina Desaminase/genética , Citosina/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , DNA Viral/metabolismo , Células HEK293/virologia , HIV-1/genética , Interações Hospedeiro-Patógeno , Humanos , Vírus da Leucemia Murina/metabolismo , Vírus da Leucemia Murina/patogenicidade , Pan troglodytes , Conformação Proteica , Vírus da Imunodeficiência Símia/metabolismo , Vírus da Imunodeficiência Símia/patogenicidade , Zinco/metabolismo , Produtos do Gene vif do Vírus da Imunodeficiência Humana/genética , Produtos do Gene vif do Vírus da Imunodeficiência Humana/metabolismo
9.
Proteomics ; 17(5)2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28067018

RESUMO

Sendai virus (SeV) is an enveloped nonsegmented negative-strand RNA virus that belongs to the genus Respirovirus of the Paramyxoviridae family. As a model pathogen, SeV has been extensively studied to define the basic biochemical and molecular biologic properties of the paramyxoviruses. In addition, SeV-infected host cells were widely employed to uncover the mechanism of innate immune response. To identify proteins involved in the SeV infection process or the SeV-induced innate immune response process, system-wide evaluations of SeV-host interactions have been performed. cDNA microarray, siRNA screening and phosphoproteomic analysis suggested that multiple signaling pathways are involved in SeV infection process. Here, to study SeV-host interaction, a global quantitative proteomic analysis was performed on SeV-infected HEK 293T cells. A total of 4699 host proteins were quantified, with 742 proteins being differentially regulated. Bioinformatics analysis indicated that regulated proteins were mainly involved in "interferon type I (IFN-I) signaling pathway" and "defense response to virus," suggesting that these processes play roles in SeV infection. Further RNAi-based functional studies indicated that the regulated proteins, tripartite motif (TRIM24) and TRIM27, affect SeV-induced IFN-I production. Our data provided a comprehensive view of host cell response to SeV and identified host proteins involved in the SeV infection process or the SeV-induced innate immune response process.


Assuntos
Interações Hospedeiro-Patógeno/fisiologia , Proteoma/análise , Infecções por Respirovirus/metabolismo , Vírus Sendai/patogenicidade , Citoplasma/química , Citoplasma/metabolismo , Citoplasma/virologia , Células HEK293/virologia , Humanos , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Proteínas Nucleares/análise , Proteínas Nucleares/metabolismo , Reação em Cadeia da Polimerase/métodos , Proteoma/genética , Proteoma/metabolismo , Proteômica/métodos , Reprodutibilidade dos Testes , Infecções por Respirovirus/virologia , Fatores de Transcrição/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Replicação Viral
10.
Cell Host Microbe ; 20(6): 770-784, 2016 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-27866900

RESUMO

RIG-I detects double-stranded RNA (dsRNA) to trigger antiviral cytokine production. Protein deamidation is emerging as a post-translational modification that chiefly regulates protein function. We report here that UL37 of herpes simplex virus 1 (HSV-1) is a protein deamidase that targets RIG-I to block RNA-induced activation. Mass spectrometry analysis identified two asparagine residues in the helicase 2i domain of RIG-I that were deamidated upon UL37 expression or HSV-1 infection. Deamidation rendered RIG-I unable to sense viral dsRNA, thus blocking its ability to trigger antiviral immune responses and restrict viral replication. Purified full-length UL37 and its carboxyl-terminal fragment were sufficient to deamidate RIG-I in vitro. Uncoupling RIG-I deamidation from HSV-1 infection, by engineering deamidation-resistant RIG-I or introducing deamidase-deficient UL37 into the HSV-1 genome, restored RIG-I activation and antiviral immune signaling. Our work identifies a viral deamidase and extends the paradigm of deamidation-mediated suppression of innate immunity by microbial pathogens.


Assuntos
Proteína DEAD-box 58/metabolismo , DNA Helicases/metabolismo , Herpesvirus Humano 1/genética , Proteínas Virais/metabolismo , Adenosina Trifosfatases , Trifosfato de Adenosina/metabolismo , Antivirais/imunologia , Asparagina , Linhagem Celular/virologia , Citocinas/metabolismo , Proteína DEAD-box 58/efeitos dos fármacos , DNA Recombinante , Escherichia coli/genética , Células HEK293/virologia , Herpes Simples , Herpesvirus Humano 1/enzimologia , Humanos , Evasão da Resposta Imune , Imunidade Inata , Espectrometria de Massas , Mutação , Processamento de Proteína Pós-Traducional , RNA de Cadeia Dupla , RNA Viral/metabolismo , Transdução de Sinais , Proteínas Estruturais Virais/análise , Proteínas Estruturais Virais/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos
11.
Cell Host Microbe ; 20(6): 798-809, 2016 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-27866901

RESUMO

Chromosomal structure of nuclear DNA is usually maintained by insertion of nucleosomes into preexisting chromatin, both on newly synthesized DNA at replication forks and at sites of DNA damage. But during retrovirus infection, a histone-free DNA copy of the viral genome is synthesized that must be loaded with nucleosomes de novo. Here, we show that core histones are rapidly loaded onto unintegrated Moloney murine leukemia virus DNAs. Loading of nucleosomes requires nuclear entry, but does not require viral DNA integration. The histones associated with unintegrated DNAs become marked by covalent modifications, with a delay relative to the time of core histone loading. Expression from unintegrated DNA can be enhanced by modulation of the histone-modifying machinery. The data show that histone loading onto unintegrated DNAs occurs very rapidly after nuclear entry and does not require prior establishment of an integrated provirus.


Assuntos
DNA Viral , Código das Histonas , Histonas/metabolismo , Retroviridae/genética , Internalização do Vírus , Animais , Ciclo Celular , Cromatina , Imunoprecipitação da Cromatina , Dano ao DNA , Replicação do DNA/genética , DNA Viral/análise , Epigenômica , Células HEK293/virologia , Humanos , Camundongos , Vírus da Leucemia Murina de Moloney/genética , Células NIH 3T3/virologia , Nucleossomos/genética , Infecções por Retroviridae/virologia , Integração Viral
12.
FEBS Lett ; 590(16): 2797-810, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27423063

RESUMO

NF90 is a novel host antiviral factor that regulates PKR activation and stress granule formation in influenza A virus (IAV)-infected cells, but the precise mechanisms by which it operates remain unclear. We identified NF90 as a novel interacting protein of IAV nonstructural protein 1 (NS1). The interaction was dependent on the RNA-binding properties of NS1. NS1 associated with NF90 and PKR simultaneously; however, the interaction between NF90 and PKR was restricted by NS1. Knockdown of NF90 promoted inhibition of PKR phosphorylation induced by NS1, while coexpression of NF90 impeded reduction of PKR phosphorylation and stress granule formation triggered by NS1. In summary, NF90 exerts its antiviral activity by antagonizing the inhibitory role of NS1 on PKR phosphorylation.


Assuntos
Vírus da Influenza A/metabolismo , Influenza Humana/virologia , Proteínas do Fator Nuclear 90/metabolismo , Proteínas não Estruturais Virais/metabolismo , eIF-2 Quinase/metabolismo , Células HEK293/virologia , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/patogenicidade , Influenza Humana/genética , Influenza Humana/metabolismo , Proteínas do Fator Nuclear 90/genética , Fosforilação/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteínas não Estruturais Virais/química , Replicação Viral/genética , eIF-2 Quinase/química
13.
J Virol ; 90(14): 6502-14, 2016 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-27147747

RESUMO

UNLABELLED: Enveloped viruses utilize transmembrane surface glycoproteins to gain entry into target cells. Glycoproteins from diverse viral families can be incorporated into nonnative viral particles in a process termed pseudotyping; however, the molecular mechanisms governing acquisition of these glycoproteins are poorly understood. For murine leukemia virus envelope (MLV Env) glycoprotein, incorporation into foreign viral particles has been shown to be an active process, but it does not appear to be caused by direct interactions among viral proteins. In this study, we coupled in vivo selection systems with Illumina next-generation sequencing (NGS) to test hundreds of thousands of MLV Env mutants for the ability to be enriched in viral particles and to perform other glycoprotein functions. NGS analyses on a subset of these mutants predicted that the residues important for incorporation are in the membrane-proximal external region (MPER), particularly W127 and W137, and the residues in the membrane-spanning domain (MSD) and also immediately flanking it (T140 to L163). These predictions were validated by directly measuring the impact of mutations in these regions on fusogenicity, infectivity, and incorporation. We suggest that these two regions dictate pseudotyping through interactions with specific lipid environments formed during viral assembly. IMPORTANCE: Researchers from numerous fields routinely exploit the ability to manipulate viral tropism by swapping viral surface proteins. However, this process, termed pseudotyping, is poorly understood at the molecular level. For murine leukemia virus envelope (MLV Env) glycoprotein, incorporation into foreign viral particles is an active process, but it does not appear to occur through direct viral protein-protein interactions. In this study, we tested hundreds of thousands of MLV Env mutants for the ability to be enriched in viral particles as well as perform other glycoprotein functions. Our analyses on a subset of these mutants predict that the glycoprotein regions embedded in and immediately flanking the viral membrane dictate active incorporation into viral particles. We suggest that pseudotyping occurs through specific lipid-protein interactions at the viral assembly site.


Assuntos
Células HEK293/virologia , Vírus da Leucemia Murina/genética , Infecções por Retroviridae/virologia , Proteínas do Envelope Viral/metabolismo , Montagem de Vírus , Internalização do Vírus , Sequência de Aminoácidos , Animais , Fusão Celular , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutagênese , Mutação/genética , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética
14.
Arch Virol ; 161(5): 1347-52, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26873814

RESUMO

The function of the hepatitis B virus X protein (HBx) has been investigated in hepatoma cell lines before; however, its function in the canonical HEK 293 cell line has not been addressed. In this study, we found that HBx increased cellular interaction by fusing the gap between HEK 293 cells, which is different from what has been reported previously. We also found that HBx enhanced the expression of E-cadherin in hepatoma cell lines instead of decreasing it as reported previously. The increase in E-cadherin was mediated by the enhanced levels of Src, which also differs from previous reports. Finally, we observed that HBx can accelerate cell growth by increasing the percentage of cells that are positioned at the division stage. Further analysis showed that the increased growth was caused by increased CDK4 expression and Ki67(+) populations. Additionally, reduced apoptosis was found in HEK 293 cells expressing HBx due to an increase in the anti-apoptotic protein-Bcl2. Collectively, the different functions of HBx in HEK 293 cells suggest that its role is cell dependent.


Assuntos
Células HEK293/virologia , Transativadores/fisiologia , Apoptose/fisiologia , Caderinas/metabolismo , Células HEK293/ultraestrutura , Vírus da Hepatite B/fisiologia , Humanos
15.
Arch Virol ; 161(5): 1151-8, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26831934

RESUMO

CCAAT/enhancer-binding protein (C/EBP) α, a member of the C/EBP family of transcription factors, is known to be involved in gene expression and DNA replication of human cytomegalovirus (HCMV). This study aimed to understand the regulation of endogenous C/EBPα during HCMV infection using an in vitro infection model. The expression and localization of C/EBPα were investigated in fibroblasts infected with HCMV. The overexpression of C/EBP homologous protein (CHOP), the endogenous inhibitor of C/EBP, was also employed to test the involvement of C/EBPα during HCMV infection. Our data showed that HCMV infection increases the expression of the full-length C/EBPα isoform (p42) especially during the late stage of infection at the transcriptional and post-translational levels. The increased p42 accumulated in the viral DNA replication compartment. p42 expression was not induced in cells treated with UV-irradiated virus or in cells infected with normal virus in the presence of ganciclovir. CHOP-mediated inhibition of C/EBP activity suppressed viral gene expression and DNA replication, which lowered the level of viral production. Together, our data suggest that HCMV-mediated C/EBPα regulation might play a beneficial role in the lytic cycle of HCMV.


Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/fisiologia , Citomegalovirus/fisiologia , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Citomegalovirus/genética , Infecções por Citomegalovirus/virologia , Fibroblastos/virologia , Imunofluorescência , Regulação Viral da Expressão Gênica/genética , Regulação Viral da Expressão Gênica/fisiologia , Células HEK293/virologia , Humanos , Immunoblotting , Reação em Cadeia da Polimerase em Tempo Real , Replicação Viral/fisiologia
16.
Sci Rep ; 5: 17680, 2015 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-26631448

RESUMO

Retroviral reverse transcription is accomplished by sequential strand-transfers of partial cDNA intermediates copied from viral genomic RNA. Here, we revealed an unprecedented role of 5'-end guanosine (G) of HIV-1 genomic RNA for reverse transcription. Based on current consensus for HIV-1 transcription initiation site, HIV-1 transcripts possess a single G at 5'-ends (G1-form). However, we found that HIV-1 transcripts with additional Gs at 5'-ends (G2- and G3-forms) were abundantly expressed in infected cells by using alternative transcription initiation sites. The G2- and G3-forms were also detected in the virus particle, although the G1-form predominated. To address biological impact of the 5'-G number, we generated HIV clone DNA to express the G1-form exclusively by deleting the alternative initiation sites. Virus produced from the clone showed significantly higher strand-transfer of minus strong-stop cDNA (-sscDNA). The in vitro assay using synthetic HIV-1 RNAs revealed that the abortive forms of -sscDNA were abundantly generated from the G3-form RNA, but dramatically reduced from the G1-form. Moreover, the strand-transfer of -sscDNA from the G1-form was prominently stimulated by HIV-1 nucleocapsid. Taken together, our results demonstrated that the 5'-G number that corresponds to HIV-1 transcription initiation site was critical for successful strand-transfer of -sscDNA during reverse transcription.


Assuntos
DNA Complementar/genética , HIV-1/genética , RNA Viral/genética , Transcrição Reversa , Sítio de Iniciação de Transcrição , Células HEK293/virologia , HIV-1/patogenicidade , Humanos , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/metabolismo
17.
Hepatobiliary Pancreat Dis Int ; 14(5): 492-501, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26459725

RESUMO

BACKGROUND: A novel hybrid bioartificial liver (HBAL) was constructed using an anionic resin adsorption column and a multi-layer flat-plate bioreactor containing porcine hepatocytes co-cultured with bone marrow mesenchymal stem cells (MSCs). This study aimed to evaluate the microbiological safety of the HBAL by detecting the transmission of porcine endogenous retroviruses (PERVs) into canines with acute liver failure (ALF) undergoing HBAL. METHODS: Eight dogs with ALF received a 6-hour HBAL treatment on the first day after the modeling by D-galactosamine administration. The plasma in the HBAL and the whole blood in the dogs were collected for PERV detection at regular intervals until one year later when the dogs were sacrificed to retrieve the tissues of several organs for immunohistochemistry and Western blotting for the investigation of PERV capsid protein gag p30 in the tissue. Furthermore, HEK293 cells were incubated to determine the in vitro infectivity. RESULTS: PERV RNA and reverse transcriptase activity were observed in the plasma of circuit 3, suggesting that PERV particles released in circuit 3. No positive PERV RNA and reverse transcriptase activity were detected in other plasma. No HEK293 cells were infected by the plasma in vitro. In addition, all PERV-related analyses in peripheral blood mononuclear cells and tissues were negative. CONCLUSION: No transmission of PERVs into ALF canines suggested a reliable microbiological safety of HBAL based on porcine hepatocytes.


Assuntos
Proteínas do Capsídeo/metabolismo , Retrovirus Endógenos/isolamento & purificação , Hepatócitos/virologia , Falência Hepática Aguda/terapia , Fígado Artificial/virologia , RNA Viral/análise , Proteínas dos Retroviridae/metabolismo , Proteínas do Envelope Viral/metabolismo , Animais , Modelos Animais de Doenças , Cães , Células HEK293/virologia , Humanos , Falência Hepática Aguda/sangue , Falência Hepática Aguda/metabolismo , DNA Polimerase Dirigida por RNA/análise , Suínos , Viroses/transmissão
18.
Sci Rep ; 5: 14794, 2015 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-26440769

RESUMO

Unlike other viral protease, Avibirnavirus infectious bursal disease virus (IBDV)-encoded viral protease VP4 forms unusual intracellular tubule-like structures during viral infection. However, the formation mechanism and potential biological functions of intracellular VP4 tubules remain largely elusive. Here, we show that VP4 can assemble into tubules in diverse IBDV-infected cells. Dynamic analysis show that VP4 initiates the assembly at early stage of IBDV infection, and gradually assembles into larger size of fibrils within the cytoplasm and nucleus. Intracellular assembly of VP4 doesn't involve the host cytoskeleton, other IBDV-encoded viral proteins or vital subcellular organelles. Interestingly, the last C-terminal hydrophobic and amyloidogenic stretch (238)YHLAMA(243) with two "aggregation-prone" alanine residues was found to be essential for its intracellular self-assembly. The assembled VP4 fibrils show significantly low solubility, subsequently, the deposition of highly assembled VP4 structures ultimately deformed the host cytoskeleton and nucleus, which was potentially associated with IBDV lytic infection. Importantly, the assembly of VP4 significantly reduced the cytotoxicity of protease activity in host cells which potentially prevent the premature cell death and facilitate viral replication. This study provides novel insights into the formation mechanism and biological functions of the Avibirnavirus protease-related fibrils.


Assuntos
Avibirnavirus/metabolismo , Interações Hospedeiro-Patógeno , Serina Endopeptidases/metabolismo , Proteínas Virais/metabolismo , Proteínas Estruturais Virais/metabolismo , Proteínas Amiloidogênicas/química , Proteínas Amiloidogênicas/metabolismo , Animais , Avibirnavirus/patogenicidade , Embrião de Galinha , Chlorocebus aethiops , Citoesqueleto/metabolismo , Células HEK293/virologia , Humanos , Vírus da Doença Infecciosa da Bursa/metabolismo , Vírus da Doença Infecciosa da Bursa/patogenicidade , Peptídeos/química , Peptídeos/metabolismo , Serina Endopeptidases/química , Solubilidade , Células Vero/virologia , Proteínas Virais/química , Proteínas Estruturais Virais/química
19.
Arch Virol ; 160(6): 1449-61, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25854689

RESUMO

Avian reovirus (ARV) causes viral arthritis, chronic respiratory diseases, retarded growth and malabsorption syndrome. It is well established that the ARV sigma-C protein induces apoptosis in host cells. However, the underlying molecular mechanism of this induction is still unclear. We report here the identification of eukaryotic elongation factor 1 alpha 1 (EEF1A1) as the interacting partner of σC. We found that σC-induced apoptosis in DF-1 cells could be completely abolished by knockdown of EEF1A1 by siRNA. Furthermore, knockdown of EEF1A1 markedly reduced ARV-induced apoptosis associated with decreased caspase-9 and -3 activation and cytochrome C release, leading to increased ARV growth in host cells. Thus, EEF1A1 plays a critical role in σC-induced apoptosis and inhibition of viral growth.


Assuntos
Apoptose , Proteínas do Capsídeo/fisiologia , Fator de Iniciação 1 em Eucariotos/fisiologia , Orthoreovirus Aviário/fisiologia , Infecções por Reoviridae/fisiopatologia , Animais , Apoptose/fisiologia , Western Blotting , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular , Embrião de Galinha/virologia , Imunofluorescência , Células HEK293/virologia , Humanos , Imunoprecipitação , Microscopia Confocal , Orthoreovirus Aviário/crescimento & desenvolvimento , Fator 1 de Elongação de Peptídeos/fisiologia , Infecções por Reoviridae/veterinária , Infecções por Reoviridae/virologia , Técnicas do Sistema de Duplo-Híbrido
20.
Antimicrob Agents Chemother ; 59(1): 590-8, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25385110

RESUMO

Doravirine (DOR, formerly known as MK-1439) is a human immunodeficiency type 1 virus (HIV-1) nonnucleoside reverse transcriptase inhibitor (NNRTI) that is currently in phase 2b clinical trials. In vitro resistance selection of subtype B virus (MT4-green fluorescent protein [GFP] cells), as well as subtype A and C viruses (MT4-GFP/CCR5 cells) was conducted with DOR, rilpivirine (RPV), and efavirine (EFV) under low-multiplicity-of-infection conditions in a 96-well format. Resistance selection was performed with escalating concentrations of the NNRTIs ranging from the 95% effective concentration (1 × EC(95)) to 1,000 × EC(95) in the presence of 10% fetal bovine serum. In the resistance selection of subtype B virus with DOR, a V106A mutant virus led to two mutation pathways, followed by the emergence separately of either F227L or L234I. In the resistance selection of subtype A and C viruses, similar mutation development pathways were detected, in which a V106A or V106M mutant was also the starting virus in the pathways. Mutations that are commonly associated with RPV and EFV in clinical settings were also identified in subtype B viruses such as the E138K and K103N mutants, respectively, in this in vitro resistance selection study. The susceptibility of subtype B mutant viruses selected by DOR, RPV, and EFV to NNRTIs was evaluated. Results suggest that mutant viruses selected by DOR are susceptible to RPV and EFV and mutants selected by RPV and EFV are susceptible to DOR. When the replication capacity of the V106A mutant was compared with that of the wild-type (WT) virus, the mutant virus was 4-fold less fit than the WT virus.


Assuntos
Farmacorresistência Viral/efeitos dos fármacos , HIV-1/efeitos dos fármacos , HIV-1/genética , Mutação , Piridonas/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Triazóis/farmacologia , Fármacos Anti-HIV/farmacologia , Farmacorresistência Viral/genética , Células HEK293/efeitos dos fármacos , Células HEK293/virologia , Humanos , Piridazinas/farmacologia , Rilpivirina/farmacologia , Seleção Genética
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