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1.
J Virol ; 95(19): e0068621, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34232709

RESUMO

During persistent human papillomavirus infection, the viral genome replicates as an extrachromosomal plasmid that is efficiently partitioned to daughter cells during cell division. We have previously shown that an element which overlaps the human papillomavirus 18 (HPV18) transcriptional enhancer promotes stable DNA replication of replicons containing the viral replication origin. Here, we perform comprehensive analyses to elucidate the function of this maintenance element. We conclude that no unique element or binding site in this region is absolutely required for persistent replication and partitioning and instead propose that the overall chromatin architecture of this region is important to promote efficient use of the replication origin. These results have important implications for the genome partitioning mechanism of papillomaviruses. IMPORTANCE Persistent infection with oncogenic human papillomaviruses (HPVs) is responsible for ∼5% of human cancers. The viral DNA replicates as an extrachromosomal plasmid and is partitioned to daughter cells in dividing keratinocytes. Using a complementation assay that allows us to separate viral transcription and replication, we provide insight into viral sequences that are required for long-term replication and persistence in keratinocytes. Understanding how viral genomes replicate persistently for such long periods of time will guide the development of antiviral therapies.


Assuntos
Genoma Viral , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/fisiologia , Sequências Reguladoras de Ácido Nucleico , Replicon/fisiologia , Replicação Viral , Sítios de Ligação , Cromatina/fisiologia , Replicação do DNA , Elementos Facilitadores Genéticos , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/fisiologia , Papillomavirus Humano 31/genética , Papillomavirus Humano 31/fisiologia , Queratinócitos/fisiologia , Queratinócitos/virologia , Plasmídeos , Regiões Promotoras Genéticas , Origem de Replicação , Fator de Transcrição AP-1/metabolismo , Transcrição Genética
2.
Viruses ; 12(6)2020 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-32486283

RESUMO

Single-stranded positive RNA ((+) ssRNA) viruses include several important human pathogens. Some members are responsible for large outbreaks, such as Zika virus, West Nile virus, SARS-CoV, and SARS-CoV-2, while others are endemic, causing an enormous global health burden. Since vaccines or specific treatments are not available for most viral infections, the discovery of direct-acting antivirals (DAA) is an urgent need. Still, the low-throughput nature of and biosafety concerns related to traditional antiviral assays hinders the discovery of new inhibitors. With the advances of reverse genetics, reporter replicon systems have become an alternative tool for the screening of DAAs. Herein, we review decades of the use of (+) ssRNA viruses replicon systems for the discovery of antiviral agents. We summarize different strategies used to develop those systems, as well as highlight some of the most promising inhibitors identified by the method. Despite the genetic alterations introduced, reporter replicons have been shown to be reliable systems for screening and identification of viral replication inhibitors and, therefore, an important tool for the discovery of new DAAs.


Assuntos
Antivirais/farmacologia , Descoberta de Drogas/métodos , Genes Reporter/fisiologia , Vírus de RNA/efeitos dos fármacos , Replicon/fisiologia , Animais , Antivirais/química , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Humanos , Vírus de RNA/genética , Transfecção , Células Vero
4.
Arch Virol ; 162(11): 3417-3423, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28779235

RESUMO

Japanese encephalitis virus (JEV), an important pathogen in Eastern and Southern Asia and the Pacific, has spread to Australia and other territories in recent years. Although the vaccine for JEV has been used in some countries, development of efficient antiviral drugs is still an urgent requirement. Replicon systems have been widely used in the research of viral replication and antiviral screening for West Nile virus (WNV), yellow fever virus (YFV) and dengue virus (DENV). Here, a novel JEV replicon harboring the Rluc and Pac gene (JEV-Pac-Rluc-Rep) was constructed. Furthermore, we established a BHK-21 cell line harboring JEV-Pac-Rluc-Rep (BHK-21/PAC/Rluc cell line) through continuous puromycin selection. Characterization of cell line stability showed that the replicon RNA could persistently replicate in this cell line for at least up to 10 rounds of passage. Using a known flavivirus inhibitor, the JEV replicon cell line was validated for antiviral screening. The JEV replicon cell line will be a valuable tool for both compound screening and viral replication studies.


Assuntos
Antivirais/uso terapêutico , Vírus da Encefalite Japonesa (Espécie)/fisiologia , Animais , Linhagem Celular , Cricetinae , Puromicina , Replicon/genética , Replicon/fisiologia , Replicação Viral
5.
Sci Rep ; 7(1): 3286, 2017 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-28607390

RESUMO

West Nile virus (WNV) is a neurotropic pathogen which causes zoonotic disease in humans. Recently, there have been an increasing number of infected cases and there are no clinically approved vaccines or effective drugs to treat WNV infections in humans. The purpose of this study was to facilitate vaccine and antiviral drug discovery by developing a packaging cell line-restricted WNV infectious replicon particle system. We constructed a DNA-based WNV replicon lacking the C-prM-E coding region and replaced it with a GFP coding sequence. To produce WNV replicon particles, cell lines stably-expressing prM-E and C-prM-E were constructed. When the WNV replicon plasmid was co-transfected with a WNV C-expressing plasmid into the prM-E-expressing cell line or directly transfected the C-prM-E expressing cell line, the replicon particle was able to replicate, form green fluorescence foci, and exhibit cytopathic plaques similar to that induced by the wild type virus. The infectious capacity of the replicon particles was restricted to the packaging cell line as the replicons demonstrated only one round of infection in other permissive cells. Thus, this system provides a safe and convenient reporter WNV manipulating tool which can be used to study WNV viral invasion mechanisms, neutralizing antibodies and antiviral efficacy.


Assuntos
Genes Reporter , Replicon/fisiologia , Montagem de Vírus/fisiologia , Febre do Nilo Ocidental/virologia , Vírus do Nilo Ocidental/fisiologia , Animais , Anticorpos Neutralizantes/imunologia , Antivirais/farmacologia , Linhagem Celular , DNA Viral/metabolismo , Descoberta de Drogas , Proteínas de Fluorescência Verde/metabolismo , Humanos , Cinética , Camundongos , Proteínas Virais/metabolismo , Replicação Viral
6.
Adv Exp Med Biol ; 1042: 229-257, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29357061

RESUMO

DNA replication occurs in a defined temporal order during S phase, known as the replication timing programme, which is regulated not only during the cell cycle but also during the process of development and differentiation. The units of replication timing regulation, known as replication domains (RDs), frequently comprise several nearly synchronously firing replication origins. Replication domains correspond to topologically associating domains (TADs) mapped by chromatin conformation capture methods and are likely to be the molecular equivalents of replication foci observed using cytogenetic methods. Both TAD and replication foci are considered to be stable structural units of chromosomes, conserved through the cell cycle and development, and accordingly, the boundaries of RDs also appear to be stable in different cell types. During both normal development and progression of disease, distinct cell states are characterized by unique replication timing signatures, with approximately half of genomic RDs switching replication timing between these cell states. Advances in functional genomics provide hope that we can soon gain an understanding of the cause and consequence of the replication timing programme and its myriad correlations with chromatin context and gene regulation.


Assuntos
Cromatina , Replicação do DNA/fisiologia , Genoma/genética , Replicon/fisiologia , Animais , Sítios de Ligação/genética , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Período de Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Genoma/fisiologia , Humanos
7.
J Virol ; 90(21): 9953-9966, 2016 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-27558430

RESUMO

Like almost all of the positive-strand RNA viruses, hepatitis C virus (HCV) induces host intracellular membrane modification to form the membrane-bound viral replication complex (RC), within which viral replicases amplify the viral RNA genome. Despite accumulated information about how HCV co-opts host factors for viral replication, our knowledge of the molecular mechanisms by which viral proteins hijack host factors for replicase assembly has only begun to emerge. Purification of the viral replicase and identification of the replicase-associated host factors to dissect their roles in RC biogenesis will shed light on the molecular mechanisms of RC assembly. To purify the viral replicase in the context of genuine viral replication, we developed an HCV subgenomic replicon system in which two different affinity tags were simultaneously inserted in frame into HCV NS5A and NS5B. After solubilizing the replicon cells, we purified the viral replicase by two-step affinity purification and identified the associated host factors by mass spectrometry. We identified valosin-containing protein (VCP), a member of the ATPases associated with diverse cellular activities (AAA+ATPase) family, as an active viral replication modulator whose ATPase activity is required for viral replication. A transient replication assay indicated that VCP is involved mainly in viral genome amplification. VCP associated with viral replicase and colocalized with a viral RC marker. Further, in an HCV replicase formation surrogate system, abolishing VCP function resulted in aberrant distribution of HCV NS5A. We propose that HCV may co-opt a host AAA+ATPase for its replicase assembly. IMPORTANCE: Almost all of the positive-strand RNA viruses share a replication strategy in which viral proteins modify host membranes to form the membrane-associated viral replicase. Viruses hijack host factors to facilitate this energy-unfavorable process. Understanding of this fundamental process is hampered by the challenges of purifying the replicase because of the technical difficulties involved. In this study, we developed an HCV subgenomic replicon system in which two different affinity tags were simultaneously inserted in frame into two replicase components. Using this dual-affinity-tagged replicon system, we purified the viral replicase and identified valosin-containing protein (VCP) AAA+ATPase as a pivotal viral replicase-associated host factor that is required for viral genome replication. Abolishing VCP function resulted in aberrant viral protein distribution. We propose that HCV hijacks a host AAA+ATPase for its replicase assembly. Understanding the molecular mechanism of VCP regulates viral replicase assembly may lead to novel antiviral strategies targeting the most conserved viral replication step.


Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Hepacivirus/metabolismo , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Replicação Viral/fisiologia , Cromatografia de Afinidade/métodos , Genoma Viral/genética , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/virologia , RNA Viral/genética , Replicon/fisiologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo
8.
Microbiol Immunol ; 60(6): 407-17, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27080060

RESUMO

Hepatitis C virus (HCV) non-structural protein 5A (NS5A) is a multifunctional protein that is involved in the HCV life cycle and pathogenesis. In this study, a host protein(s) interacting with NS5A by tandem affinity purification were searched for with the aim of elucidating the role of NS5A. An NS5A-interacting protein, SET and MYND domain-containing 3 (SMYD3), a lysine methyltransferase reportedly involved in the development of cancer, was identified. The interaction between NS5A and SMYD3 was confirmed in ectopically expressing, HCV RNA replicon-harboring and HCV-infected cells. The other HCV proteins did not bind to SMYD3. SMYD3 bound to NS5A of HCV genotypes 1b and 2a. Deletion mutational analysis revealed that domains II and III of NS5A (amino acids [aa] 250 to 447) and the MYND and N-SET domains of SMYD3 (aa 1 to 87) are involved in the full extent of NS5A-SMYD3 interaction. NS5A co-localized with SMYD3 exclusively in the cytoplasm, thereby inhibiting nuclear localization of SMYD3. Moreover, NS5A formed a complex with SMYD3 and heat shock protein 90 (HSP90), which is a positive regulator of SMYD3. The intensity of binding between SMYD3 and HSP90 was enhanced by NS5A. Luciferase reporter assay demonstrated that NS5A significantly induces activator protein 1 (AP-1) activity, this being potentiated by co-expression of SMYD3 with NS5A. Taken together, the present results suggest that NS5A interacts with SMYD3 and induces AP-1 activation, possibly by facilitating binding between HSP90 and SMYD3. This may be a novel mechanism of AP-1 activation in HCV-infected cells.


Assuntos
Hepacivirus/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Fator de Transcrição AP-1/biossíntese , Fator de Transcrição AP-1/metabolismo , Proteínas não Estruturais Virais/metabolismo , Linhagem Celular , Citoplasma/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Hepacivirus/genética , Hepatite C/virologia , Histona-Lisina N-Metiltransferase/biossíntese , Interações Hospedeiro-Patógeno , Humanos , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas , Replicon/fisiologia , Análise de Sequência de Proteína , Deleção de Sequência , Proteínas não Estruturais Virais/biossíntese , Proteínas não Estruturais Virais/genética , Replicação Viral/fisiologia
9.
Nat Rev Gastroenterol Hepatol ; 13(6): 362-74, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27075261

RESUMO

Viral hepatitis is a major cause of morbidity and mortality, affecting hundreds of millions of people worldwide. Hepatitis-causing viruses initiate disease by establishing both acute and chronic infections, and several of these viruses are specifically associated with the development of hepatocellular carcinoma. Consequently, intense research efforts have been focusing on increasing our understanding of hepatitis virus biology and on improving antiviral therapy and vaccination strategies. Although valuable information on viral hepatitis emerged from careful epidemiological studies on sporadic outbreaks in humans, experimental models using cell culture, rodent and non-human primates were essential in advancing the field. Through the use of these experimental models, improvement in both the treatment and prevention of viral hepatitis has progressed rapidly; however, agents of viral hepatitis are still among the most common pathogens infecting humans. In this Review, we describe the important part that these experimental models have played in the study of viral hepatitis and led to monumental advances in our understanding and treatment of these pathogens. Ongoing developments in experimental models are also described.


Assuntos
Antivirais/uso terapêutico , Modelos Animais de Doenças , Hepatite B Crônica/prevenção & controle , Hepatite C Crônica/prevenção & controle , Animais , Carcinoma Hepatocelular/virologia , Linhagem Celular , DNA Viral/fisiologia , Previsões , Hepacivirus/fisiologia , Vírus da Hepatite B/fisiologia , Hepatite B Crônica/virologia , Hepatite C Crônica/virologia , Hepatócitos/virologia , Humanos , Estágios do Ciclo de Vida/fisiologia , Neoplasias Hepáticas/virologia , Primatas , Replicon/fisiologia , Células-Tronco/virologia
10.
Antiviral Res ; 117: 1-9, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25666760

RESUMO

Previous studies have demonstrated that cyclopentenone prostaglandins (cyPGs) inhibit the replication of a wide variety of DNA and RNA viruses in different mammalian cell types. We investigated a new role for prostaglandin A1 (PGA1) in the inhibition of hepatitis C virus (HCV)-IRES-mediated translation. PGA1 exhibited dose-dependent inhibitory effects on HCV translation in HCV replicon cells. Furthermore, repetitive PGA1 treatment demonstrated the potential to safely induce the suppression of HCV translation. We also validated a new role for PGA1 in the inhibition of HCV-IRES-mediated translation by targeting cellular translation factors, including the small ribosomal subunit (40S) and eukaryotic initiation factors (eIFs). In pull-down assays, biotinylated PGA1 co-precipitated with the entire HCV IRES RNA/eIF3-40S subunit complex. Moreover, the interactions between PGA1 and the elongation factors and ribosomal subunit were dependent upon HCV IRES RNA binding, and the PGA1/HCV IRES RNA/eIF3-40S subunit complex inhibited HCV-IRES-mediated translation. The novel mechanism revealed in this study may aid in the search for more effective anti-HCV drugs.


Assuntos
Hepacivirus/crescimento & desenvolvimento , Hepacivirus/genética , Hepacivirus/metabolismo , Prostaglandinas A/metabolismo , Prostaglandinas A/farmacologia , Replicon/efeitos dos fármacos , Subunidades Ribossômicas Menores/efeitos dos fármacos , Linhagem Celular Tumoral , Fator de Iniciação 3 em Eucariotos/metabolismo , Humanos , Sítios Internos de Entrada Ribossomal , Biossíntese de Proteínas/efeitos dos fármacos , RNA Viral/genética , Replicon/fisiologia , Subunidades Ribossômicas Menores/metabolismo
11.
Genetika ; 49(5): 558-68, 2013 May.
Artigo em Russo | MEDLINE | ID: mdl-24159796

RESUMO

A basic replicon of the naphthalene degradation plasmid pFME5 (80 kb, IncP-7) has been constructed and sequenced. The nucleotide sequence of pFME5mini is almost identical to replicons of the pND6-1 subgroup, which was separated based on the reA-oriV homology in our previous work. The basic replicon of pFME5 is capable of replication and stable maintenance exclusively in Pseudomonas species. An analysis of the deletion mutation indicated that, in contrast to the parWAB region, the parC gene is not essential for the stability of pFME5mini and can be a common feature of IncP-7 replicons. We revealed that par-defective mutants of pFME5mini were slowly eliminated from the bacterial population in a nonselective medium compared to their pCAR1-based counterparts. Designed primers specific to the repA and parC genes can be used to detect IncP-7 plasmids, while primers specific to two variants of parA can be used for intragroup classification.


Assuntos
Naftalenos/metabolismo , Plasmídeos/metabolismo , Pseudomonas/metabolismo , Replicon/fisiologia , Biodegradação Ambiental , Plasmídeos/genética , Pseudomonas/genética
12.
J Gen Virol ; 94(Pt 12): 2657-2663, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24026670

RESUMO

The 5' untranslated region (5'UTR) of the recently described non-primate hepacivirus (NPHV) contains a region with sequence homology to the internal ribosomal entry site (IRES) of hepatitis C virus (HCV) and GB virus B (GBV-B). Here, we demonstrated internal translation initiation by the NPHV 5'UTR in a bicistronic vector. An RNA stem-loop upstream of the NPHV IRES was structurally distinct from corresponding regions in HCV and GBV-B, and was not required for IRES function. Insertion of the NPHV stem-loop into the corresponding region of the HCV 5'UTR within the HCV subgenomic replicon significantly impaired RNA replication, indicating that long-range interactions between the 5'UTR and cis-acting downstream elements within the NPHV genome are not interchangeable with those of HCV. Despite similarities in IRES structure and function between hepaciviruses, replication elements in the NPHV 5'UTR appear functionally distinct from those of HCV.


Assuntos
Regiões 5' não Traduzidas/genética , Hepacivirus/genética , Iniciação Traducional da Cadeia Peptídica , Ribossomos/metabolismo , Regiões 5' não Traduzidas/fisiologia , Animais , Linhagem Celular , Células HEK293 , Hepacivirus/metabolismo , Hepacivirus/fisiologia , Humanos , Conformação de Ácido Nucleico , Primatas/genética , Primatas/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Replicon/genética , Replicon/fisiologia , Replicação Viral/genética
13.
Extremophiles ; 17(1): 15-28, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23114983

RESUMO

Recently, the extremely thermophilic bacterium Thermus thermophilus HB8 has been demonstrated to harbor a circular plasmid designated by pVV8 in addition to two well-known plasmids, pTT8 and pTT27, and its entire sequence has been determined. The absence of any obvious replication initiation gene in the 81.2 kb plasmid prompted us to isolate its minimum replicon. By in vivo replication assays with fragments deleted in a stepwise manner, a minimum replicon containing a single ORF, TTHV001, was identified. A protein encoded by TTHV001 showed no amino acid sequence similarity to other function-known proteins. As the results of in vivo and in vitro experiments strongly suggested that the TTHV001 protein was involved in the replication initiation of pVV8, the protein and the gene were referred to as RepV and repV, respectively. The RepV protein binds to an inverted repeat sequence within its own repV gene and then triggers the unwinding of the DNA duplex in an A + T-rich region located just downstream from the inverted repeat. The in vivo replication assays with minimum replicon mutants in the RepV binding site or the unwinding region demonstrated that the unwinding in the region by the RepV binding was essential for pVV8 replication initiation.


Assuntos
Proteínas de Bactérias/metabolismo , DNA Helicases/metabolismo , Replicação do DNA/fisiologia , DNA Bacteriano/biossíntese , Plasmídeos/metabolismo , Thermus thermophilus/metabolismo , Transativadores/metabolismo , Proteínas de Bactérias/genética , DNA Helicases/genética , DNA Bacteriano/genética , Fases de Leitura Aberta/fisiologia , Plasmídeos/genética , Replicon/fisiologia , Thermus thermophilus/genética , Transativadores/genética
14.
Biochem Biophys Res Commun ; 414(4): 808-13, 2011 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-22008549

RESUMO

The advent of infectious molecular clones of Hepatitis C virus (HCV) has unlocked the understanding of HCV life cycle. However, packaging of the genomic RNA, which is crucial to generate infectious viral particles, remains poorly understood. Molecular interactions of the domain 1 (D1) of HCV Core protein and HCV RNA have been described in vitro. Since compaction of genetic information within HCV genome has hampered conventional mutational approach to study packaging in vivo, we developed a novel heterologous system to evaluate the interactions between HCV RNA and CoreD1. For this, we took advantage of the recruitment of Vpr fusion-proteins into HIV-1 particles. By fusing HCV Core D1 to Vpr we were able to package and transfer a HCV subgenomic replicon into a HIV-1 based lentiviral vector. We next examined how deletion mutants of basic sub-domains of Core D1 influenced HCV RNA recruitment. The results emphasized the crucial role of the first and third basic regions of D1 in packaging. Interestingly, the system described here allowed us to mobilise full-length JFH1 genome in CD81 defective cells, which are normally refractory to HCV infection. This finding paves the way to an evaluation of the replication capability of HCV in various cell types.


Assuntos
Hepacivirus/fisiologia , RNA Viral/fisiologia , Vírion/fisiologia , Montagem de Vírus , Replicação Viral , Linhagem Celular , Vetores Genéticos/genética , Vetores Genéticos/fisiologia , Hepacivirus/genética , Humanos , Lentivirus/genética , Lentivirus/fisiologia , RNA Viral/genética , Replicon/genética , Replicon/fisiologia
15.
J Microbiol ; 49(3): 516-23, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21717343

RESUMO

Porcine reproductive and respiratory syndrome virus (PRRSV), a member of the Arteriviridae family, is one of the most common and economically important swine pathogens. Although both live-attenuated and killed-inactivated vaccines against the virus have been available for a decade, PRRSV is still a major problem in the swine industry worldwide. To explore the possibility of producing single-round infectious PRRSV replicon particles as a potential vaccine strategy, we have now generated two necessary components: 1) a stable cell line (BHK/Sinrepl9/PRRSV-N) that constitutively expresses the viral nucleocapsid (N) protein localized to the cytoplasm and the nucleolus and 2) a PRRSV replicon vector (pBAC/PRRSV/Replicon-AN) with a 177-nucleotide deletion, removing the 3'-half portion of ORF7 in the viral genome, from which the self-replicating propagation-defective replicon RNAs were synthesized in vitro by SP6 polymerase run-off transcription. Transfection of this replicon RNA into N protein-expressing BHK-21 cells led to the secretion of infectious particles that packaged the replicon RNA, albeit with a low production efficiency of 0.4 × 10(2) to 1.1 × 10(2) infectious units/ml; the produced particles had only single-round infectivity with no cell-to-cell spread. This trans-complementation system for PRRSV provides a useful platform for studies to define the packaging signals and motifs present within the viral genome and N protein, respectively, and to develop viral replicon-based antiviral vaccines that will stop the infection and spread of this pathogen.


Assuntos
Proteínas do Nucleocapsídeo/metabolismo , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Montagem de Vírus , Animais , Linhagem Celular , Cricetinae , Proteínas do Nucleocapsídeo/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , RNA Viral/genética , RNA Viral/metabolismo , Replicon/genética , Replicon/fisiologia , Transfecção , Vacinas Virais , Vírion/metabolismo , Vírion/fisiologia
16.
Hepatology ; 54(5): 1580-90, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21793033

RESUMO

UNLABELLED: Studies of the hepatitis C virus (HCV) life-cycle rely heavily on Huh7.5 cells, but the reasons why these cells are exceptionally permissive for HCV replication are not clear. Based on recent clinical observations, we hypothesized that the Hedgehog (Hh) pathway, which has not been previously associated with HCV replication, may be involved in the Huh7.5 phenotype of increased permissiveness. We tested this hypothesis by comparing levels of a variety of Hh-related cellular markers in Huh7.5 cells with the parental Huh7 cells, which are far less permissive. Here we demonstrate that Huh7.5 cells, when compared with Huh7 cells, have substantially decreased expression of epithelial markers, increased levels of mesenchymal markers, and markedly up-regulated Hh pathway activity: Shh, >100-fold, Gli1, >30-fold, Ptc, 2-fold. In Huh7.5 cells, we found that cyclopamine, an Hh pathway antagonist, reduced HCV RNA levels by 50% compared with vehicle and inactive isomer controls. Moreover, in Huh7 cells treatment with recombinant Shh ligand and SAG, both Hh pathway agonists, stimulated HCV replication by 2-fold and 4-fold, respectively. These effects were observed with both viral infections and a subgenomic replicon. Finally, we demonstrated that GDC-0449 decreased HCV RNA levels in a dose-response manner. CONCLUSION: We have identified a relationship between HCV and Hh signaling where up-regulated pathway activity during infection promotes an environment conducive to replication. Given that Hh activity is very low in most hepatocytes, these findings may serve to further shift the model of HCV liver infection from modest widespread replication in hepatocytes to one where a subset of cells support high-level replication. These findings also introduce Hh pathway inhibitors as potential anti-HCV therapeutics.


Assuntos
Proteínas Hedgehog/metabolismo , Hepacivirus/crescimento & desenvolvimento , Hepatite C Crônica/metabolismo , Hepatite C Crônica/virologia , Transdução de Sinais/fisiologia , Anilidas/farmacologia , Antivirais/farmacologia , Biomarcadores/metabolismo , Carcinoma Hepatocelular , Linhagem Celular Tumoral , Hepacivirus/genética , Hepatite C Crônica/tratamento farmacológico , Humanos , Interferon-alfa/farmacologia , Neoplasias Hepáticas , Piridinas/farmacologia , Replicon/fisiologia , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/fisiologia , Replicação Viral/efeitos dos fármacos , Replicação Viral/fisiologia
17.
Gastroenterology ; 141(3): 1057-66, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21699799

RESUMO

BACKGROUND & AIMS: Hepatitis C virus (HCV) has a high propensity to establish persistence; better understanding of this process requires the development of a fully permissive and immunocompetent small animal model. Mouse cells can be engineered to express the human orthologs of the entry molecules CD81 and occludin to allow entry of HCV. However, RNA replication is poor in mouse cells, and it is not clear whether they support assembly and release of infectious HCV particles. We used a trans-complementation-based system to demonstrate HCV assembly competence of mouse liver cell lines. METHODS: A panel of 3 mouse hepatoma cell lines that contain a stable subgenomic HCV replicon was used for ectopic expression of the HCV structural proteins, p7, nonstructural protein 2, and/or apolipoprotein E (apoE). Assembly and release of infectious HCV particles was determined by measuring viral RNA, proteins, and infectivity of virus released into the culture supernatant. RESULTS: Mouse replicon cells released low amounts of HCV particles, but ectopic expression of apoE increased release of infectious HCV to levels observed in the human hepatoma cell line Huh7.5. Thus, apoE is the limiting factor for assembly of HCV in mouse hepatoma cells but probably not in primary mouse hepatocytes. Products of all 3 human alleles of apoE and mouse apoE support HCV assembly with comparable efficiency. Mouse and human cell-derived HCV particles have similar biophysical properties, dependency on entry factors, and levels of association with apoE. CONCLUSIONS: Mouse hepatic cells permit HCV assembly and might be developed to create an immunocompetent and fully permissive mouse model of HCV infection.


Assuntos
Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Hepacivirus/fisiologia , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Vírion/fisiologia , Montagem de Vírus/fisiologia , Alelos , Animais , Apolipoproteínas E/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Hepacivirus/genética , Hepatite C/fisiopatologia , Humanos , Camundongos , RNA Viral/genética , Replicon/fisiologia , Replicação Viral/fisiologia
18.
Sheng Wu Gong Cheng Xue Bao ; 26(8): 1088-94, 2010 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-21090113

RESUMO

To optimize a self-replicate Japanese enciphalitis virus (JEV) replicon, and to make it as an efficient vector to express the heterologous protein, we constructed three JEV replicons by PCR-based shortening the length of capsid genes. The vectors remained full or part of C gene, based on the JEV replicon pCTCJEV. Lac Z was selected as the reporter gene to verify the self-replicate ability of these DNA-based replicons. While transfected into the cell lines CME-4, which continuously expressing the JEV structure proteins C-prM-E, the JEV replicons pCMW-2M-1LACZ, pCMW-2M-3LACZ, which remained the first 23aa and 68aa of C protein, can express the reporter protein as the same level as pCMW-2M-LACZ with the full-length C protein. These results illustrated that the JEV replicon vector with 69-nt of the C gene can retain the self-replicate ability, and provide valuable tools to construct a possible vector for a long-lasting JEV RNA virus expression system.


Assuntos
Proteínas do Capsídeo/fisiologia , Vírus da Encefalite Japonesa (Espécie)/genética , Replicon/genética , Replicação Viral/fisiologia , Proteínas do Capsídeo/genética , Linhagem Celular , Vírus da Encefalite Japonesa (Espécie)/fisiologia , Vetores Genéticos/genética , Humanos , Replicon/fisiologia , Transfecção , Replicação Viral/genética
19.
Plasmid ; 64(3): 177-85, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20621118

RESUMO

In several rhizobia, bacteria that inhabit the soil in free-living conditions and associate in symbiosis with the root of legumes as nitrogen-fixing organisms, plasmid DNA can constitute a high percentage of the genome. We have characterized acid-tolerant isolates of rhizobia-here represented by the strain Rhizobium sp. LPU83-that have an extended nodulation-host range including alfalfa, the common bean, and Leucena leucocephala. In this study we analyzed the plasmids of R. sp. LPU83 in order to characterize their role in the evolution of Medicago symbionts and their involvement in symbiotic behavior. The pLPU83a plasmid was found to be transmissible with no associated phenotypic traits. The symbiotic plasmid pLPU83b could be transferred at very low frequencies under laboratory conditions only when pLPU83a was present; could restore nodulation to a strain cured of its symbiotic plasmid, S. meliloti A818; but could not restore the full nitrogen fixation associated with alfalfa.


Assuntos
Fabaceae/microbiologia , Replicon/genética , Rhizobium/genética , Conjugação Genética/genética , DNA de Plantas/genética , Fabaceae/genética , Cinética , Mutagênese Sítio-Dirigida , Fixação de Nitrogênio/genética , Fixação de Nitrogênio/fisiologia , Raízes de Plantas/genética , Raízes de Plantas/microbiologia , Replicon/fisiologia , Rhizobium/fisiologia , Simbiose/genética
20.
Antimicrob Agents Chemother ; 54(10): 4168-77, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20660691

RESUMO

Acinetobacter baumannii is an opportunistic pathogen, especially in intensive care units, and multidrug-resistant isolates have increasingly been reported during the last decade. Despite recent progress in knowledge of antibiotic resistance mechanisms in A. baumannii, little is known about the genetic factors driving isolates toward multidrug resistance. In the present study, the A. baumannii plasmids were investigated through the analysis and classification of plasmid replication systems and the identification of A. baumannii-specific mobilization and addiction systems. Twenty-two replicons were identified by in silico analysis, and five other replicons were identified and cloned from previously uncharacterized A. baumannii resistance plasmids carrying the OXA-58 carbapenem-hydrolyzing oxacillinase. Replicons were classified into homology groups on the basis of their nucleotide homology. A novel PCR-based replicon typing scheme (the A. baumannii PCR-based replicon typing [AB-PBRT] method) was devised to categorize the A. baumannii plasmids into homogeneous groups on the basis of the nucleotide homology of their respective replicase genes. The AB-PBRT technique was applied to a collection of multidrug-resistant A. baumannii clinical isolates carrying the bla(OXA-58) or bla(OXA-23) carbapenemase gene. A putative complete conjugative apparatus was identified on one plasmid whose self-conjugative ability was demonstrated in vitro. We showed that this conjugative plasmid type was widely diffused in our collection, likely representing the most important vehicle promoting the horizontal transmission of A. baumannii resistance plasmids.


Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/genética , Carbapenêmicos/metabolismo , Plasmídeos/genética , Reação em Cadeia da Polimerase/métodos , Replicon/fisiologia , Acinetobacter baumannii/metabolismo , Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Replicon/genética , beta-Lactamases/genética
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