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1.
Biotechnol Bioeng ; 117(2): 466-485, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31631325

RESUMO

The Chinese hamster ovary (CHO) cells used to produce biopharmaceutical proteins are known to contain type-C endogenous retrovirus (ERV) sequences in their genome and to release retroviral-like particles. Although evidence for their infectivity is missing, this has raised safety concerns. As the genomic origin of these particles remained unclear, we characterized type-C ERV elements at the genome, transcriptome, and viral particle RNA levels. We identified 173 type-C ERV sequences clustering into three functionally conserved groups. Transcripts from one type-C ERV group were full-length, with intact open reading frames, and cognate viral genome RNA was loaded into retroviral-like particles, suggesting that this ERV group may produce functional viruses. CRISPR-Cas9 genome editing was used to disrupt the gag gene of the expressed type-C ERV group. Comparison of CRISPR-derived mutations at the DNA and RNA level led to the identification of a single ERV as the main source of the release of RNA-loaded viral particles. Clones bearing a Gag loss-of-function mutation in this ERV showed a reduction of RNA-containing viral particle release down to detection limits, without compromising cell growth or therapeutic protein production. Overall, our study provides a strategy to mitigate potential viral particle contaminations resulting from ERVs during biopharmaceutical manufacturing.


Assuntos
Células CHO/virologia , Retrovirus Endógenos , Mutagênese Sítio-Dirigida/métodos , RNA Viral , Vírion/genética , Animais , Sistemas CRISPR-Cas , Cricetinae , Cricetulus , Contaminação de Medicamentos/prevenção & controle , Retrovirus Endógenos/genética , Retrovirus Endógenos/metabolismo , Edição de Genes , Genoma Viral/genética , Mutação com Perda de Função/genética , RNA Viral/genética , RNA Viral/metabolismo
2.
Sci Rep ; 9(1): 8827, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31222165

RESUMO

Viral contamination in biopharmaceutical manufacturing can lead to shortages in the supply of critical therapeutics. To facilitate the protection of bioprocesses, we explored the basis for the susceptibility of CHO cells to RNA virus infection. Upon infection with certain ssRNA and dsRNA viruses, CHO cells fail to generate a significant interferon (IFN) response. Nonetheless, the downstream machinery for generating IFN responses and its antiviral activity is intact in these cells: treatment of cells with exogenously-added type I IFN or poly I:C prior to infection limited the cytopathic effect from Vesicular stomatitis virus (VSV), Encephalomyocarditis virus (EMCV), and Reovirus-3 virus (Reo-3) in a STAT1-dependent manner. To harness the intrinsic antiviral mechanism, we used RNA-Seq to identify two upstream repressors of STAT1: Gfi1 and Trim24. By knocking out these genes, the engineered CHO cells exhibited activation of cellular immune responses and increased resistance to the RNA viruses tested. Thus, omics-guided engineering of mammalian cell culture can be deployed to increase safety in biotherapeutic protein production among many other biomedical applications.


Assuntos
Células CHO/virologia , Engenharia Genética , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Microbiologia Industrial , Animais , Biomarcadores , Cricetulus , Resistência a Medicamentos/imunologia , Engenharia Genética/métodos , Interferon Tipo I , Poli I-C/imunologia , Vírus de RNA/imunologia , Fator de Transcrição STAT1 , Transdução de Sinais , Replicação Viral
4.
Cell Host Microbe ; 15(6): 681-91, 2014 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-24922571

RESUMO

Neurotropic viruses, including mammalian reovirus, must disseminate from an initial site of replication to the central nervous system (CNS), often binding multiple receptors to facilitate systemic spread. Reovirus engages junctional adhesion molecule A (JAM-A) to disseminate hematogenously. However, JAM-A is dispensable for reovirus replication in the CNS. We demonstrate that reovirus binds Nogo receptor NgR1, a leucine-rich repeat protein expressed in the CNS, to infect neurons. Expression of NgR1 confers reovirus binding and infection of nonsusceptible cells. Incubating reovirus virions with soluble NgR1 neutralizes infectivity. Blocking NgR1 on transfected cells or primary cortical neurons abrogates reovirus infection. Concordantly, reovirus infection is ablated in primary cortical neurons derived from NgR1 null mice. Reovirus virions bind to soluble JAM-A and NgR1, while infectious disassembly intermediates (ISVPs) bind only to JAM-A. These results suggest that reovirus uses different capsid components to bind distinct cell-surface molecules, engaging independent receptors to facilitate spread and tropism.


Assuntos
Proteínas da Mielina/metabolismo , Receptores de Superfície Celular/metabolismo , Reoviridae/metabolismo , Animais , Células CHO/virologia , Proteínas do Capsídeo/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Membrana Celular/metabolismo , Membrana Celular/virologia , Cricetulus , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Camundongos Mutantes , Proteínas da Mielina/genética , Neuraminidase/química , Neurônios/virologia , Receptor Nogo 1 , Receptores de Superfície Celular/genética , Reoviridae/patogenicidade , Infecções por Reoviridae/metabolismo , Infecções por Reoviridae/virologia , Vírion/metabolismo
5.
J Clin Invest ; 121(6): 2427-35, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21576824

RESUMO

Vectors based on adeno-associated virus (AAV) serotype 9 are candidates for in vivo gene delivery to many organs, but the receptor(s) mediating these tropisms have yet to be defined. We evaluated AAV9 uptake by glycans with terminal sialic acids (SAs), a common mode of cellular entry for viruses. We found, however, that AAV9 binding increased when terminal SA was enzymatically removed, suggesting that galactose, which is the most commonly observed penultimate monosaccharide to SA, may mediate AAV9 transduction. This was confirmed in mutant CHO Pro-5 cells deficient in the enzymes involved in glycoprotein biogenesis, as well as lectin interference studies. Binding of AAV9 to glycans with terminal galactose was demonstrated via glycan binding assays. Co-instillation of AAV9 vector with neuraminidase into mouse lung resulted in exposure of terminal galactose on the apical surface of conducting airway epithelial cells, as shown by lectin binding and increased transduction of these cells, demonstrating the possible utility of this vector in lung-directed gene transfer. Increasing the abundance of the receptor on target cells and improving vector efficacy may improve delivery of AAV vectors to their therapeutic targets.


Assuntos
Dependovirus/genética , Galactose/metabolismo , Terapia Genética/métodos , Vetores Genéticos/farmacocinética , Pulmão/metabolismo , Glicoproteínas de Membrana/metabolismo , Polissacarídeos/metabolismo , Receptores Virais/metabolismo , Animais , Células CHO/efeitos dos fármacos , Células CHO/metabolismo , Células CHO/virologia , Capsídeo/metabolismo , Cricetinae , Cricetulus , Dependovirus/classificação , Sistemas de Liberação de Medicamentos , Glicosilação , Pulmão/virologia , Masculino , Glicoproteínas de Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Neuraminidase/farmacologia , Ligação Proteica , Receptores Virais/efeitos dos fármacos , Transdução Genética/métodos
6.
Arch Virol ; 154(8): 1293-302, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19585075

RESUMO

Cowpox virus (CPXV), a member of the genus Orthopoxvirus (OPV), has reservoirs in small mammals and may cause disease in humans, felidae and other animals. In this study we compared CPXVs isolated from humans and cats in Fennoscandia by restriction enzyme and DNA sequence analysis. The HindIII restriction profiles clearly distinguished geographically distinct CPXV isolates, whereas only minor differences were found between the profiles of geographically linked isolates. The complete gene sequences encoding the cytokine response modifier B, the hemagglutinin and the Chinese hamster ovary host range protein were determined for the same isolates and included in phylogenetic analysis. By including representative OPV sequences from GenBank, detailed comparative analyses were performed showing pronounced heterogeneity among CPXVs compared to members of other OPV species. However, a close relationship between the Norwegian (3 of 4 isolates) and Swedish isolates was detected, whereas the isolate from Finland was more closely related to a Russian isolate for all three genes compared. We infer that the investigated CPXVs have distinct evolutionary histories in different rodent lineages.


Assuntos
Gatos/virologia , Vírus da Varíola Bovina/classificação , Varíola Bovina/veterinária , Animais , Células CHO/virologia , Varíola Bovina/virologia , Vírus da Varíola Bovina/genética , Vírus da Varíola Bovina/isolamento & purificação , Cricetinae , Cricetulus , Dinamarca , Finlândia , Genes Virais , Variação Genética , Hemaglutininas Virais/genética , Humanos , Dados de Sequência Molecular , Noruega , Filogenia , Receptores do Fator de Necrose Tumoral/genética , Suécia , Proteínas Virais/genética
7.
J Int Med Res ; 36(6): 1319-25, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-19094442

RESUMO

This study assessed the functional role of human scavenger receptor class B type I (SR-BI) as a putative hepatitis C virus (HCV) receptor using Chinese hamster ovary (CHO) cells transfected with human SR-BI (CHO-huSR-BI). The expression of SR-BI by primary Tupaia hepatocytes (PTHs), human hepatocarcinoma cell line (HepG2) cells, untransfected CHO cells and CHO-huSR-BI cells was analysed by Western blotting. Receptor competition assays showed that anti-SR-BI antibodies that block the binding of soluble envelope glycoprotein E2 could prevent HCV infection. Pre-incubation of CHO-huSR-BI and HepG2 cells with anti-SR-BI antibodies resulted in marked inhibition of E2 binding. After incubation with HCV RNA-positive serum from a patient with chronic HCV infection, however, HCV infection could not be detected in CHO-huSR-BI cells, but was detected in PTHs. These results demonstrate that, whilst SR-BI represents an important cell surface molecule for HCV infection, the presence of SR-BI alone is insufficient for HCV entry.


Assuntos
Células CHO/virologia , Carcinoma Hepatocelular/virologia , Hepacivirus/fisiologia , Hepatite C/virologia , Hepatócitos/virologia , Receptores Depuradores Classe B/fisiologia , Animais , Anticorpos Bloqueadores/farmacologia , Western Blotting , Células CHO/metabolismo , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Hepatócitos/metabolismo , Humanos , RNA Viral/farmacologia , Transfecção , Tupaia
8.
J Chromatogr A ; 1205(1-2): 17-25, 2008 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-18722622

RESUMO

The robustness of virus clearance with respect to protein A media reuse was demonstrated using media with four matrix chemistries: Protein A immobilized ProSep A, Poros A50, Protein A ceramic Hyper DF and MabSelect SuRe, an alkali resistant protein A ligand. Endogenous retrovirus clearance, step yield, impurity clearance and other performance parameters were evaluated periodically in media cycled up to 300 times. Media lifetime was generally limited by either declining step yield or media fouling. However, clearance of endogenous retrovirus remained in an acceptable range, either increasing or remaining constant. Multiply cycled media were tested for clearance of three viruses (SV40, X-MuLV, and MMV); clearance was comparable to naïve media. Overall, virus clearance by protein A chromatography appears to be extremely robust with respect to media age.


Assuntos
Cromatografia Líquida/métodos , Reutilização de Equipamento , Sefarose/análogos & derivados , Proteína Estafilocócica A/química , Animais , Células CHO/virologia , Cricetinae , Cricetulus , Microscopia Eletrônica de Varredura , Tamanho da Partícula , Retroviridae/isolamento & purificação , Sefarose/química
9.
Virology ; 373(1): 39-50, 2008 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-18155263

RESUMO

To identify genetic changes that lead to the attenuation of measles virus (MV), a strain of MV that is pathogenic in rhesus macaques was adapted to grow in Vero cells, Vero/hSLAM cells and, to simulate the process used to derive live attenuated vaccines, in primary chicken embryo fibroblasts (CEF). Comparison of the complete genomic sequences of the pathogenic wild-type (Davis87-wt) and four cell culture-adapted strains derived from it showed complete conservation of sequence in the Vero/hSLAM-passaged virus. Viruses adapted to Vero cells and CEF had predicted amino acid changes in the nucleocapsid protein, phosphoprotein, V protein, C protein, matrix protein, and the cytoplasmic tail of the hemagglutinin protein. All four cell culture-adapted strains, including the Vero/hSLAM cell-passaged virus, were able to productively infect Vero cells, but the peak viral titers differed. The Vero cell-adapted strains were unable to replicate in Chinese Hamster Ovary cells expressing CD46, indicating that they had not adapted to use the CD46 receptor. The Vero/hSLAM cell-passaged virus retained pathogenicity in rhesus macaques as measured by the appearance of a skin rash while the Vero cell-adapted and CEF-adapted strains had lost the ability to cause a rash. There were no significant differences in viral titers in peripheral blood mononuclear cells among monkeys infected with any of the viral stocks tested. These results identify a limited number of genetic changes in the genome of MV that lead to attenuation in vivo.


Assuntos
Substituição de Aminoácidos , Modelos Animais de Doenças , Vírus do Sarampo/genética , Vírus do Sarampo/patogenicidade , Sarampo/fisiopatologia , Adaptação Fisiológica , Animais , Antígenos CD/metabolismo , Células CHO/virologia , Embrião de Galinha , Chlorocebus aethiops , Cricetinae , Cricetulus , Feminino , Humanos , Macaca mulatta , Masculino , Sarampo/virologia , Vírus do Sarampo/fisiologia , Receptores de Superfície Celular/metabolismo , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária , Células Vero/virologia , Virulência , Replicação Viral
10.
J Virol ; 80(15): 7714-28, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16840350

RESUMO

Vaccinia virus does not grow in Chinese hamster ovary (CHO-K1) cells in the absence of a viral host range factor, cowpox protein CP77. In this study, CP77 was fused to the C terminus of green fluorescence protein (GFP-CP77) and a series of nested deletion mutants of GFP-CP77 was constructed for insertion into a vaccinia virus host range mutant, VV-hr, and expressed from a viral early promoter. Deletion mapping analyses demonstrated that the N-terminal 352 amino acids of CP77 were sufficient to support vaccinia virus growth in CHO-K1 cells, whereas the C-terminal residues 353 to 668 were dispensable. In yeast two-hybrid analyses, CP77 bound to a cellular protein, HMG20A, and GST pulldown analyses showed that residues 1 to 234 of CP77 were sufficient for this interaction. After VV-hr virus infection of CHO-K1 cells, HMG20A was translocated from the nucleus to viral factories and bound to the viral genome via the HMG box region. In control VV-hr-infected CHO-K1 cells, binding of HMG20A to the viral genome persisted from 2 to 8 h postinfection (h p.i.); in contrast, when CP77 was expressed, the association of HMG20A with viral genome was transient, with little HMG20A remaining bound at 8 h p.i. This indicates that dissociation of HMG20A from viral factories correlates well with CP77 host range activity in CHO-K1 cells. Finally, in cells expressing a CP77 deletion protein (amino acids 277 to 668) or a DeltaANK5 mutant that did not support vaccinia virus growth and did not contain the HMG20A binding site, HMG20A remained bound to viral DNA, demonstrating that the binding of CP77 to HMG20A is essential for its host range function. In summary, our data revealed that a novel cellular protein, HMG20A, the dissociation of which from viral DNA is regulated by CP77, providing the first cellular target regulated by viral host range CP77 protein.


Assuntos
Regulação Viral da Expressão Gênica , Genoma Viral , Proteínas de Grupo de Alta Mobilidade/metabolismo , Poxviridae/patogenicidade , Vaccinia virus/genética , Proteínas Virais/genética , Animais , Sítios de Ligação , Células CHO/virologia , Cricetinae , DNA Viral/genética , Humanos , Poxviridae/genética , Poxviridae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Deleção de Sequência , Técnicas do Sistema de Duplo-Híbrido , Vaccinia virus/metabolismo , Proteínas Virais/metabolismo
11.
Nat Genet ; 38(1): 38-46, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16369534

RESUMO

Severe acute respiratory syndrome (SARS) is caused by infection of a previously undescribed coronavirus (CoV). L-SIGN, encoded by CLEC4M (also known as CD209L), is a SARS-CoV binding receptor that has polymorphism in its extracellular neck region encoded by the tandem repeat domain in exon 4. Our genetic risk association study shows that individuals homozygous for CLEC4M tandem repeats are less susceptible to SARS infection. L-SIGN is expressed in both non-SARS and SARS-CoV-infected lung. Compared with cells heterozygous for L-SIGN, cells homozygous for L-SIGN show higher binding capacity for SARS-CoV, higher proteasome-dependent viral degradation and a lower capacity for trans infection. Thus, homozygosity for L-SIGN plays a protective role during SARS infection.


Assuntos
Moléculas de Adesão Celular/genética , Lectinas Tipo C/genética , Receptores de Superfície Celular/genética , Síndrome Respiratória Aguda Grave/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/patogenicidade , Animais , Células CHO/virologia , Moléculas de Adesão Celular/metabolismo , Chlorocebus aethiops , Estudos de Coortes , Cricetinae , Cricetulus , Predisposição Genética para Doença , Homozigoto , Hong Kong/epidemiologia , Humanos , Intestino Delgado/fisiologia , Lectinas Tipo C/metabolismo , Pulmão/fisiologia , Pulmão/virologia , Dados de Sequência Molecular , Complexo de Endopeptidases do Proteassoma/metabolismo , Receptores de Superfície Celular/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/metabolismo , Síndrome Respiratória Aguda Grave/epidemiologia , Sequências de Repetição em Tandem , Células Vero/virologia
12.
FEBS J ; 272(12): 3060-74, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15955065

RESUMO

The mammalian members of the inorganic phosphate (P(i)) transporter (PiT) family, the type III sodium-dependent phosphate (NaP(i)) transporters PiT1 and PiT2, have been assigned housekeeping P(i) transport functions and are suggested to be involved in chondroblastic and osteoblastic mineralization and ectopic calcification. The PiT family members are conserved throughout all kingdoms and use either sodium (Na+) or proton (H+) gradients to transport P(i). Sequence logo analyses revealed that independent of their cation dependency these proteins harbor conserved signature sequences in their N- and C-terminal ends with the common core consensus sequence GANDVANA. With the exception of 10 proteins from extremophiles all 109 proteins analyzed carry an aspartic acid in one or both of the signature sequences. We changed either of the highly conserved aspartates, Asp28 and Asp506, in the N- and C-terminal signature sequences, respectively, of human PiT2 to asparagine and analyzed P(i) uptake function in Xenopus laevis oocytes. Both mutant proteins were expressed at the cell surface of the oocytes but exhibited knocked out NaP(i) transport function. Human PiT2 is also a retroviral receptor and we have previously shown that this function can be exploited as a control for proper processing and folding of mutant proteins. Both mutant transporters displayed wild-type receptor functions implying that their overall architecture is undisturbed. Thus the presence of an aspartic acid in either of the PiT family signature sequences is critical for the Na+-dependent P(i) transport function of human PiT2. The conservation of the aspartates among proteins using either Na+- or H+-gradients for P(i) transport suggests that they are involved in H+-dependent P(i) transport as well. Current results favor a membrane topology model in which the N- and C-terminal PiT family signature sequences are positioned in intra- and extracellular loops, respectively, suggesting that they are involved in related functions on either side of the membrane. The present data are in agreement with a possible role of the signature sequences in translocation of cations.


Assuntos
Ácido Aspártico/genética , Simportadores/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Asparagina/genética , Proteínas de Bactérias/genética , Transporte Biológico , Células CHO/virologia , Proteínas de Transporte/genética , Células Cultivadas , Sequência Conservada , Cricetinae , Cricetulus , Evolução Molecular , Feminino , Humanos , Vírus da Leucemia Murina/metabolismo , Vírus da Leucemia Murina/patogenicidade , Proteínas de Membrana/genética , Dados de Sequência Molecular , Família Multigênica , Mutação , Oócitos/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III , Simportadores/genética
13.
J Virol Methods ; 128(1-2): 61-6, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15904981

RESUMO

Magnetofection is a viral and non-viral gene delivery method using polyethyleneimine-conjugated super-paramagnetic nanoparticle under a magnetic field. Previous studies have indicated that magnetofection enhanced the infection of adenoviruses and retroviruses. It is shown that magnetofection enhances the infection of measles virus, a paramyxovirus. When cells expressing a measles virus receptor human SLAM were infected with a measles virus that encodes green fluorescent protein gene, magnetofection enhanced measles virus infection by 30- to 70-fold. The infection of SLAM-negative cells with measles virus was also enhanced by magnetofection, but to a lesser extent. These results indicate that magnetofection could be useful for isolation of measles virus from clinical specimens.


Assuntos
Magnetismo , Vírus do Sarampo/patogenicidade , Animais , Antígenos CD , Células CHO/virologia , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Glicoproteínas/genética , Glicoproteínas/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa/virologia , Humanos , Imunoglobulinas/genética , Imunoglobulinas/metabolismo , Vírus do Sarampo/genética , Vírus do Sarampo/metabolismo , Receptores de Superfície Celular , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária , Células Vero/virologia
14.
Biochem J ; 385(Pt 2): 451-9, 2005 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-15303968

RESUMO

Heparan sulphate (HS) 3-O-sulphotransferase transfers sulphate to the 3-OH position of the glucosamine residue of HS to form 3-O-sulphated HS. The HS modified by 3-O-sulphotransferase isoform 3 binds to HSV-1 (herpes simplex virus type 1) gD (envelope glycoprotein D), and the resultant 3-O-sulphated HS serves as an entry receptor for HSV-1. In the present paper, we report the isolation and characterization of a novel HS 3-O-sulphotransferase isoform, designated HS 3-O-sulphotransferase isoform 6 (3-OST-6). Mouse 3-OST-6 gene was identified in the EST (expressed sequence tag) database and cloned into pcDNA3.1/Myc-His vector. A CHO (Chinese-hamster ovary) cell line that stably expresses 3-OST-6 (3OST6/CHO cells) was prepared. The disaccharide analysis of the HS isolated from 3OST6/CHO cells revealed that 3-OST-6 exhibits HS 3-O-sulphotransferase activity. Furthermore, 3OST6/CHO cells were susceptible to infection by HSV-1, but not by other alphaherpesviruses examined, suggesting that 3-OST-6 produces a specific entry receptor for HSV-1. Our results indicate that a new member of 3-OST family generates an entry receptor for HSV-1. The findings add to the growing body of evidence that HSV-1 entry is mediated by 3-O-sulphated HS generated by multiple members of 3-O-sulphotransferases.


Assuntos
Herpes Simples/enzimologia , Herpesvirus Humano 1/patogenicidade , Sulfotransferases/química , Sulfotransferases/fisiologia , Sequência de Aminoácidos/genética , Animais , Antitrombinas/metabolismo , Sequência de Bases/genética , Células CHO/química , Células CHO/enzimologia , Células CHO/metabolismo , Células CHO/virologia , Fusão Celular , Cricetinae , Cricetulus , DNA Complementar/genética , Bases de Dados Genéticas , Heparitina Sulfato/metabolismo , Humanos , Dados de Sequência Molecular , Especificidade por Substrato/genética , Especificidade por Substrato/fisiologia , Sulfotransferases/genética , Distribuição Tecidual
15.
Biotechnol Bioeng ; 85(6): 589-600, 2004 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-14966800

RESUMO

A balance between proliferation and cell death is critical for achieving desirable high cell densities in mammalian cell culture. In this study, we evaluate a recently discovered anti-apoptotic gene, aven, and examine its effectiveness alone and in combination with a member of the Bcl-2 family, bcl-xL. The commercially popular cell line, Chinese hamster ovary (CHO), was genetically modified to constitutively express aven, bcl-xL, and the two genes in combination. Cells were exposed to several model insults that simulate severe bioreactor environments, including serum deprivation, spent medium, and Sindbis virus infection, as well as staurosporine, a known chemical inducer of apoptosis. CHO cells exhibited DNA fragmentation, a hallmark of apoptosis, after exposure to these model insults. After exposure to serum deprivation, 4- and 5-day spent medium, and staurosporine, cells expressing Aven provided limited protection against cell death when compared with the protection afforded by cells expressing Bcl-xL alone. However, the highest survival levels for all insults were achieved when Aven was expressed in combination with Bcl-xL. In fact, Aven appeared to act synergistically to enhance the protective function of Bcl-xL for several insults, because the protective function of the two genes expressed together in one cell line often exceeded the additive protective levels of each anti-apoptosis gene expressed alone. Surprisingly, Aven expression provided a mildly pro-apoptotic response in CHO isolates infected with Sindbis virus. However, CHO cells expressing both Bcl-xL and Aven showed protection against Sindbis virus infection due to the inhibitory properties of the bcl-xL anti-apoptosis gene. This study shows that combinatorial anti-apoptosis cell engineering strategies may be the most effective mechanisms for providing extended protection against cell death in mammalian cell culture.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Apoptose , Reatores Biológicos , Células CHO/patologia , Células CHO/virologia , Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Estaurosporina/farmacologia , Infecções por Alphavirus/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Proteínas de Transporte/genética , Técnicas de Cultura de Células/métodos , Sobrevivência Celular , Cricetinae , Cricetulus , Meios de Cultura Livres de Soro , Citoproteção , Mamíferos , Proteínas de Membrana/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Recombinantes/metabolismo , Sindbis virus , Proteína bcl-X
16.
Proc Natl Acad Sci U S A ; 100(14): 8460-5, 2003 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-12824470

RESUMO

HIV buds from lipid rafts and requires cholesterol for its egress from and entry into cells. Viral accessory protein Nef plays a major role in this process. In this study, it not only increased the biosynthesis of lipid rafts and viral particles with newly synthesized cholesterol, but also enriched them. Furthermore, via the consensus cholesterol recognition motif at its C terminus, Nef bound cholesterol. When this sequence was mutated, Nef became unable to transport newly synthesized cholesterol into lipid rafts and viral particles. Interestingly, although its levels in lipid rafts were not affected, this mutant Nef protein was poorly incorporated into viral particles, and viral infectivity decreased dramatically. Thus, Nef also transports newly synthesized cholesterol to the site of viral budding. As such, it provides essential building blocks for the formation of viruses that replicate optimally in the host.


Assuntos
Colesterol/metabolismo , Produtos do Gene nef/fisiologia , HIV-1/fisiologia , Microdomínios da Membrana/metabolismo , Vírion/metabolismo , Animais , Transporte Biológico , Células CHO/metabolismo , Células CHO/virologia , Linhagem Celular/metabolismo , Linhagem Celular/virologia , Colesterol/biossíntese , Cricetinae , Cricetulus , Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/genética , Indução Enzimática , Genes Reporter , Humanos , Células Jurkat/metabolismo , Células Jurkat/virologia , Rim/citologia , Rim/metabolismo , Lanosterol/metabolismo , Ácido Mevalônico/metabolismo , Modelos Biológicos , Oxirredutases/biossíntese , Oxirredutases/genética , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Recombinantes de Fusão/biossíntese , Esterol 14-Desmetilase , Replicação Viral , Produtos do Gene nef do Vírus da Imunodeficiência Humana
17.
J Virol ; 77(9): 5324-32, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12692234

RESUMO

Herpes simplex virus (HSV) infection of many cultured cells, e.g., Vero cells, can be initiated by receptor binding and pH-neutral fusion with the cell surface. Here we report that a major pathway for HSV entry into the HeLa and CHO-K1 cell lines is dependent on endocytosis and exposure to a low pH. Enveloped virions were readily detected in HeLa or receptor-expressing CHO cell vesicles by electron microscopy at <30 min postinfection. As expected, images of virus fusion with the Vero cell surface were prevalent. Treatment with energy depletion or hypertonic medium, which inhibits endocytosis, prevented uptake of HSV from the HeLa and CHO cell surface relative to uptake from the Vero cell surface. Incubation of HeLa and CHO cells with the weak base ammonium chloride or the ionophore monensin, which elevate the low pH of organelles, blocked HSV entry in a dose-dependent manner. Noncytotoxic concentrations of these agents acted at an early step during infection by HSV type 1 and 2 strains. Entry mediated by the HSV receptor HveA, nectin-1, or nectin-2 was also blocked. As analyzed by fluorescence microscopy, lysosomotropic agents such as the vacuolar H(+)-ATPase inhibitor bafilomycin A1 blocked the delivery of virus capsids to the nuclei of the HeLa and CHO cell lines but had no effect on capsid transport in Vero cells. The results suggest that HSV can utilize two distinct entry pathways, depending on the type of cell encountered.


Assuntos
Endocitose , Herpesvirus Humano 1/fisiologia , Herpesvirus Humano 1/patogenicidade , Animais , Células CHO/virologia , Chlorocebus aethiops , Cricetinae , Células HeLa/virologia , Humanos , Concentração de Íons de Hidrogênio , Microscopia Eletrônica , Receptores Virais/metabolismo , Células Vero/virologia
18.
Biotechnol Bioeng ; 82(3): 321-9, 2003 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-12599259

RESUMO

Viral safety is a predominant concern for monoclonal antibodies (mAbs) and other recombinant proteins (RPs) with pharmaceutical applications. Certain commercial purification modules, such as nanofiltration and low-pH inactivation, have been observed to reliably clear greater than 4 log(10) of large enveloped viruses, including endogenous retrovirus. The concept of "bracketed generic clearance" has been proposed for these steps if it could be prospectively demonstrated that viral log(10) reduction value (LRV) is not impacted by operating parameters that can vary, within a reasonable range, between commercial processes. In the case of low-pH inactivation, a common step in mAb purification processes employed after protein A affinity chromatography, these parameters would include pH, time and temperature of incubation, the content of salts, protein concentration, aggregates, impurities, model protein pI, and buffer composition. In this report, we define bracketed generic clearance conditions, using a prospectively defined bracket/matrix approach, where low-pH inactivation consistently achieves >or=4.6 log(10) clearance of xenotropic murine leukemia virus (X-MLV), a model for rodent endogenous retrovirus. The mechanism of retrovirus inactivation by low-pH treatment was also investigated.


Assuntos
Anticorpos Monoclonais/metabolismo , Técnicas de Cultura de Células/métodos , Vírus da Leucemia Murina/química , Vírus da Leucemia Murina/fisiologia , Proteínas Recombinantes/metabolismo , Inativação de Vírus , Animais , Anticorpos Monoclonais/química , Células CHO/metabolismo , Células CHO/virologia , Cricetinae , Concentração de Íons de Hidrogênio , Proteínas Recombinantes/química , Retroviridae/química , Retroviridae/fisiologia
19.
Biotechnol Prog ; 19(1): 137-43, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-12573016

RESUMO

Recombinant human adenovirus (rhAd) has been used extensively for functional protein expression in mammalian cells including those of human and nonhuman origin. High-level protein production by rhAd vectors is expected in their permissive host cells, such as the human embryonic kidney 293 (HEK293) cell line. This is attributed primarily to the permissiveness of HEK293 to rhAd infection and their ability to support viral DNA replication by providing the missing El proteins. However, the HEK293 cells tend to suffer from cytopathic effect (CPE) as a result of virus replication. Under these circumstances, the host cell function is compromised and the culture viability will be reduced. Consequently, newly synthesized polypeptides may not be processed properly at posttranslational levels. Therefore, the usefulness of HEK293 cells for the expression of complex targets such as secreted proteins could be limited. In the search for a more robust cell line as a production host for rhAd expression vectors, a series of screening experiments was performed to isolate clones from Chinese hamster ovary-K1 (CHO-K1) cells. First, multiple rounds of infection of CHO-K1 cells were performed utilizing an rhAd expressing GFP. After each cycle of infection, a small population of CHO cells with high GFP levels was enriched by FACS. Second, individual clones more permissive to human adenovirus infection were isolated from the highly enriched subpopulation by serial dilution. A single clone, designated CHO-K1-C5, was found to be particularly permissive to rhAd infection than the parental pool and has served as a production host in the successful expression of several secreted proteins.


Assuntos
Adenovírus Humanos/genética , Adenovírus Humanos/metabolismo , Células CHO/classificação , Células CHO/metabolismo , Clonagem Molecular/métodos , Animais , Células CHO/fisiologia , Células CHO/virologia , Linhagem Celular , Cricetinae , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Técnicas de Transferência de Genes , Engenharia Genética/métodos , Proteínas de Fluorescência Verde , Humanos , Rim/embriologia , Rim/metabolismo , Rim/fisiologia , Rim/virologia , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Biossíntese de Proteínas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética
20.
Biochemistry ; 41(51): 15376-87, 2002 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-12484777

RESUMO

Abnormally hyperphosphorylated tau polymers known as paired helical filaments constitute one of the major characteristic lesions that lead to the demise of neurons in Alzheimer's disease. Here, we demonstrate that the environmental toxin arsenite causes a significant increase in the phosphorylation of several amino acid residues (Thr-181, Ser-202, Thr-205, Thr-231, Ser-262, Ser-356, Ser-396, and Ser-404) in tau, which are also hyperphosphorylated under pathological conditions. Complementary phosphopeptide mapping revealed a dramatic increase in the (32)P-labeling of many peptides in tau following arsenite treatment. Although arsenite activates extracellular-signal regulated kinases-1/-2 and stress-activated protein kinases, these enzymes did not contribute to the arsenite-increased phosphorylation, nor did they appear to normally modify tau in vivo. Tau phosphorylation induced by arsenite did not involve glycogen synthase kinase-3 or protein phosphatase-1 or -2, but the activity responsible for tau hyperphosphorylation could be inhibited with the protein kinase inhibitor roscovitine. The effects of arsenite on the phosphorylation of some tau mutations (DeltaKappa280, V337M, and R406W) associated with frontal-temporal dementia with parkinsonism linked to chromosome 17 was analyzed. The unchallenged and arsenite-induced phosphorylation of some mutant proteins, especially R406W, was altered at several phosphorylation sites, indicating that these mutations can significantly affect the structure of tau in vivo. Although the major kinase(s) involved in aberrant tau phosphorylation remains elusive, these results indicate that environmental factors, such as arsenite, may be involved in the cascade leading to deregulation of tau function associated with neurodegeneration.


Assuntos
Arsenitos/toxicidade , Poluentes Ambientais/toxicidade , Proteínas tau/metabolismo , Animais , Células CHO/metabolismo , Células CHO/virologia , Linhagem Celular/metabolismo , Linhagem Celular/virologia , Cricetinae , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Proteína Quinase 8 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Vírus da Floresta de Semliki/genética , Proteínas tau/genética
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