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1.
The replication of mengovirus in HeLa cells preinfected with poliovirus in the presence of 10(-3) M guanidine was investigated. Although host cell protein synthesis is inhibited by the presence of nonreplicating poliovirus, it is found that mengovirus ribonucleic acid (RNA) and protein synthesis proceed normally under the same conditions. Furthermore, no effects on mengovirus growth by poliovirus can be detected either when Mengo protein synthesis is interrupted by Acti-Dione or when its RNA synthesis is reduced by incubation at 28 C. It is suggested that the poliovirus inhibitory factor may be able to distinguish between an RNA element required in the protein-synthesizing apparatus of the host cell and a comparable element in that of the heterologous virus. 相似文献
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Poliovirus Replication during HeLa Cell Life Cycle 总被引:2,自引:0,他引:2
VIRAL replication in infected animal cells is commonly investigated in asynchronized cultures. Since several synthetic processes of macromolecules occur at definite periods in the cell cycle1,2, the possibility existed that viral infection and replication might be also phase-linked. We have chosen to investigate this problem in HeLa S3 cells infected with type 1 Mahoney poliovirus3, since the system is well known4 and these cells can be easily synchronized. In addition, the replication of poliovirus RNA in asynchronized HeLa cells has been well characterized4,5. 相似文献
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Influence of Polycations on the Interaction Between Poliovirus Multistranded Ribonucleic Acid and HeLa Cells
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In contrast to single-stranded viral ribonucleic acid (RNA), poliovirus multistranded RNA (RI-RNA) is poorly adsorbed to suspended HeLa cells in the absence of polycations. In the presence of 20 mug of diethylaminoethyl (DEAE) dextran per ml and other polycations, 90% or more of the RI-RNA is adsorbed to HeLa cells within 2 min at 37 C. However, only 30% of the RI-RNA label penetrates into the cells independent of the concentration of DEAE dextran applied. DEAE dextran is adsorbed almost quantitatively to HeLa cells within 3 min at 37 C. Most of the DEAE dextran remains bound to the cell membrane and available for binding of RI-RNA for 15 min at 37 C. 相似文献
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Mechanism of Inhibition of Vaccinia Virus Replication in Adenovirus-Infected HeLa Cells 总被引:8,自引:7,他引:1
The ability of vaccinia virus to replicate in HeLa cells which had been previously infected with adenovirus type 2 (Ad2) was studied in order to gain insight into the mechanism by which adenovirus inhibits the expression of host cell functions. Vaccinia virus was employed in these studies because it replicates in the cytoplasm, whereas Ad2 replicates in the nucleus of the cell. It was found that vaccinia deoxyribonucleic acid (DNA) synthesis is greatly inhibited in adeno-preinfected HeLa cells provided that vaccinia superinfection does not occur before 18 hr after adeno infection. The inhibition of vaccinia DNA synthesis can be traced to an inhibition of vaccinia protein synthesis and viral uncoating. Vaccinia ribonucleic acid (RNA) synthesis is not inhibited in adeno-preinfected cells, but the vaccinia RNA does not become associated with polysomes. 相似文献
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Regulation of Protein Synthesis in HeLa Cells: III. Inhibition During Poliovirus Infection 总被引:1,自引:0,他引:1
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The mechanism of inhibition of host cell protein synthesis by poliovirus has been studied by examining the behavior of host polyribosomes and polyribosome-associated messenger ribonucleic acid (mRNA). Virus infection appears to result in a specific inhibition of the initiation of translation of host cell mRNA. The site of inhibition does not appear to be mRNA itself. Human cells respond to virus infection by producing a factor antagonistic to the virus inhibition which promotes the initiation of host mRNA translation. The production of the host factor is sensitive to actinomycin; however, the behavior of the host cell factor and that of host mRNA appear distinctly different. 相似文献
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Replication of Poliovirus in Phytohemagglutinin-stimulated Human Lymphocytes 总被引:19,自引:6,他引:13
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Poliovirus replication has been studied in human lymphocytes during the course of blastogenesis under phytohemagglutinin (PHA) stimulation. Enhancement of virus replication in PHA-stimulated leukocyte cultures was due to an increase in number of virus-producing cells. Virus yield was approximately 10 plaque-forming units (PFU) per producing cell, both in stimulated and in nonstimulated cultures. Adsorption and penetration studies showed that freshly drawn lymphocytes (unlike other leukocytes) were resistant to virus infection, but they became susceptible to the virus during PHA stimulation. Also, the eclipse of the virus after penetration was enhanced during blastogenesis of the lymphocytes. Our findings suggested that the monocytes in the leukocyte cultures were infected initially. In PHA-stimulated cultures, the virus then spread to lymphocytes which became susceptible to virus infection during blastogenesis. Polymorphonuclear cells died within 24 to 48 hr after initiation of the cultures and apparently could not support poliovirus replication. 相似文献
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Functional Coupling between Replication and Packaging of Poliovirus Replicon RNA 总被引:9,自引:7,他引:2
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Constance I. Nugent Kyle L. Johnson Peter Sarnow Karla Kirkegaard 《Journal of virology》1999,73(1):427-435
Poliovirus RNA genomes that contained deletions in the capsid-coding regions were synthesized in monkey kidney cells that constitutively expressed T7 RNA polymerase. These replication-competent subgenomic RNAs, or replicons (G. Kaplan and V. R. Racaniello, J. Virol. 62:1687–1696, 1988), were encapsidated in trans by superinfecting polioviruses. When superinfecting poliovirus resistant to the antiviral compound guanidine was used, the RNA replication of the replicon RNAs could be inhibited independently of the RNA replication of the guanidine-resistant helper virus. Inhibiting the replication of the replicon RNA also profoundly inhibited its trans-encapsidation, even though the capsid proteins present in the cells could efficiently encapsidate the helper virus. The observed coupling between RNA replication and RNA packaging could account for the specificity of poliovirus RNA packaging in infected cells and the observed effects of mutations in the coding regions of nonstructural proteins on virion morphogenesis. It is suggested that this coupling results from direct interactions between the RNA replication machinery and the capsid proteins. The coupling of RNA packaging to RNA replication and of RNA replication to translation (J. E. Novak and K. Kirkegaard, Genes Dev. 8:1726–1737, 1994) could serve as mechanisms for late proofreading of poliovirus RNA, allowing only those RNA genomes capable of translating a full complement of functional RNA replication proteins to be propagated. 相似文献
10.
Virus Replication, Cytopathology, and Lysosomal Enzyme Response of Mitotic and Interphase Hep-2 Cells Infected with Poliovirus 总被引:6,自引:3,他引:3
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Mitotic Hep-2 cells, selected by the PEL (colloidal silica) density gradient method and held in mitosis with Colcemid, are readily infected by poliovirus type I (Mahoney). They produce and release the same amount of virus as interphase, random-growing cells. In contrast to interphase cells, mitotic cells show no detectable virus-induced cytopathic effect at the light microscopy level and only slight alterations, consisting of small clusters of vacuoles, at the electron microscopy level. Mitotic cells contain the same total amount of lysosomal enzymes per cell as interphase cells, but they display no redistribution of lysosomal enzymes during the virus infection as interphase cells do. This supports the view that lysosomal enzyme redistribution is associated with the cytopathic effect in poliovirus infection but shows that virus synthesis and release is not dependent on either the cytopathic effect or lysosomal enzyme release. The possible reasons for the lack of cytopathic effect in mitotic cells are discussed. 相似文献
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Fran?ois Téoulé Cynthia Brisac Isabelle Pelletier Pierre-Olivier Vidalain Sophie Jégouic Carmen Mirabelli Ma?l Bessaud Nicolas Combelas Arnaud Autret Frédéric Tangy Francis Delpeyroux Bruno Blondel 《Journal of virology》2013,87(20):11031-11046
We have shown that the circulating vaccine-derived polioviruses responsible for poliomyelitis outbreaks in Madagascar have recombinant genomes composed of sequences encoding capsid proteins derived from poliovaccine Sabin, mostly type 2 (PVS2), and sequences encoding nonstructural proteins derived from other human enteroviruses. Interestingly, almost all of these recombinant genomes encode a nonstructural 3A protein related to that of field coxsackievirus A17 (CV-A17) strains. Here, we investigated the repercussions of this exchange, by assessing the role of the 3A proteins of PVS2 and CV-A17 and their putative cellular partners in viral replication. We found that the Golgi protein acyl-coenzyme A binding domain-containing 3 (ACBD3), recently identified as an interactor for the 3A proteins of several picornaviruses, interacts with the 3A proteins of PVS2 and CV-A17 at viral RNA replication sites, in human neuroblastoma cells infected with either PVS2 or a PVS2 recombinant encoding a 3A protein from CV-A17 [PVS2-3A(CV-A17)]. The small interfering RNA-mediated downregulation of ACBD3 significantly increased the growth of both viruses, suggesting that ACBD3 slowed viral replication. This was confirmed with replicons. Furthermore, PVS2-3A(CV-A17) was more resistant to the replication-inhibiting effect of ACBD3 than the PVS2 strain, and the amino acid in position 12 of 3A was involved in modulating the sensitivity of viral replication to ACBD3. Overall, our results indicate that exchanges of nonstructural proteins can modify the relationships between enterovirus recombinants and cellular interactors and may thus be one of the factors favoring their emergence. 相似文献
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Anticellular serum (ACS), in the absence of an active complement system, was shown to inhibit the attachment of poliovirus types 1 and 2, echovirus type 6, and coxsackievirus types A13, B1, and B3 to viral receptors of live HeLa cells. This is the first report to provide evidence that ACS has an inhibitory effect on the interaction between host cells and coxsackieviruses of group B. The titer of inhibitory activity of ACS varied inversely with the cell concentration used, the reaction being virtually completed after an incubation period of 30 min at 37 C. The inhibitory activity of ACS persisted for more than 4 hr at 37 C, and was shown to be reversible at pH 2.0, revealing that although the receptors for attaching virus were inactivated by ACS the inactivation was not permanent. These findings are consistent with the concept that antibodies in the ACS combine with and blockade viral receptors located at the cell surface. An antiserum with a specificity for inhibiting attachment of coxsackievirus B1 was obtained by dual absorption of ACS with cells saturated with coxsackievirus type B3 and chymotrypsin-treated cells. These findings offer an approach whereby the antigenic relationship of viral receptors to other constituents of the cell surface can be studied. 相似文献
15.
Rescue of Defective Poliovirus RNA Replication by 3AB-Containing Precursor Polyproteins 总被引:8,自引:7,他引:1
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This study demonstrates the in vitro complementation of an RNA replication-defective lesion in poliovirus RNA by providing a replicase/polymerase precursor polypeptide [P3(wt) {wild type}] in trans. The replication-defective mutation was a phenylalanine-to-histidine change (F69H) in the hydrophobic domain of the membrane-associated viral protein 3AB. RNAs encoding wild-type forms of protein 3AB or the P3 precursor polypeptide were cotranslated with full-length poliovirus RNAs containing the F69H mutation in a HeLa cell-free translation/replication assay in an attempt to trans complement the RNA replication defect exhibited by the 3AB(F69H) lesion. Unexpectedly, generation of 3AB(wt) in trans was not able to efficiently complement the defective replication complex; however, cotranslation of the large P3(wt) precursor protein allowed rescue of RNA replication. Furthermore, P3 proteins harboring mutations that resulted in either an inactive polymerase or an inactive proteinase domain displayed differential abilities to trans complement the RNA replication defect. Our results indicate that replication proteins like 3AB may need to be delivered to the poliovirus replication complex in the form of a larger 3AB-containing protein precursor prior to complex assembly rather than as the mature viral cleavage product. 相似文献
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S. V. Ugraitskaya N. V. Shishova E. L. Gagarinskiy N. E. Shvirst S. A. Kaurova M. V. Goltyaev L. V. Zalomova A. L. Kovtun E. E. FesenkoJr. 《Biophysics》2018,63(3):387-392
The effect of helium on cell survival during cryopreservation was studied using the HeLa and L929 cell lines. Cell suspensions were incubated in an atmosphere of helium, nitrogen, or air and frozen in the presence or absence of glycerol as a cryoprotectant. After thawing, the cell viability was evaluated by the Trypan Blue exclusion test and culture development for 18 h. Helium was found to provide better preservation of cell suspensions compared with nitrogen and air. The positive effect of helium was the greatest in the case of freezing without cryoprotectants (the HeLa cell survival increased by a factor of 1.5–2) and somewhat lower in the case of freezing in the presence of low glycerol concentrations (the L929 cell survival increased by a factor of 1.2–1.5 in the presence of 3% glycerol). Use of helium for cell suspensions may improve cryopreservation methods by making it possible to reduce the concentrations of conventional cryoprotectants, which are generally highly toxic and undesirable to use for cryopreservation of biological material for medical applications. 相似文献
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Rent1是无义介导的mRNA降解(NMD)通路中的关键因子之一,通过引导含有提前出现的终止密码子(PTC)的mRNA至P-body,从而引发mRNA降解.为了进一步研究Rent1的生理功能,应用RNA干扰(RNAi)技术抑制Rent1的表达.试验发现,抑制Rent1的表达能够增强HeLa细胞对纤维粘连蛋白的黏附能力,另外,抑制Rent1的表达能够增加整合素基因ITGA2、ITGA3、ITGA6、ITGB5的表达. 相似文献
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具癌基因特性的Skp2在大多数肿瘤组织和肿瘤细胞中异常高表达,它作为SCFSkp2复合物的底物识别亚基调控p27KIP蛋白的稳定性而促进细胞G1/S期转换.为进一步明确Skp2与G2/M周期检查点的关系,在HeLa细胞中过表达Skp2以及通过反义寡核苷酸抑制Skp2表达.结果发现:Skp2能促进细胞周期运转,表现为S期细胞增多和G2/M期细胞减少,其中F-box结构域具有重要的功能意义;反义寡核苷酸抑制Skp2表达后,HeLa细胞发生显著的G2/M期阻滞;MTT检测结果表明,400nmol/L的Skp2的反义寡核苷酸能明显抑制HeLa细胞的增殖活性;Western印迹结果表明,HeLa细胞中Skp2可能通过负调控p21WAF的稳定性来参与G2/M检查点调控,这在用放线菌素D处理HeLa细胞的实验中得到验证.这些结果初步揭示了Skp2参与HeLa细胞G2/M周期检查点调控的分子机制. 相似文献
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George A. Belov Qian Feng Krisztina Nikovics Catherine L. Jackson Ellie Ehrenfeld 《PLoS pathogens》2008,4(11)
Replication of many RNA viruses is accompanied by extensive remodeling of intracellular membranes. In poliovirus-infected cells, ER and Golgi stacks disappear, while new clusters of vesicle-like structures form sites for viral RNA synthesis. Virus replication is inhibited by brefeldin A (BFA), implicating some components(s) of the cellular secretory pathway in virus growth. Formation of characteristic vesicles induced by expression of viral proteins was not inhibited by BFA, but they were functionally deficient. GBF1, a guanine nucleotide exchange factor for the small cellular GTPases, Arf, is responsible for the sensitivity of virus infection to BFA, and is required for virus replication. Knockdown of GBF1 expression inhibited virus replication, which was rescued by catalytically active protein with an intact N-terminal sequence. We identified a mutation in GBF1 that allows growth of poliovirus in the presence of BFA. Interaction between GBF1 and viral protein 3A determined the outcome of infection in the presence of BFA. 相似文献
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通过使用噬斑形成试验、透射电镜及检测志贺菌蛋白表达等方法,研究志贺菌对Hela细胞的侵袭能力。研究发现:37℃培养条件下,胞质内出现成堆的志贺菌;30℃培养条件下,细菌主要分布在细胞外。SDS-PAGE显示,与30℃培养条件相比,在37℃培养下,志贺菌表达蛋白质的种类和数量明显增加。噬斑形成发现,12株福氏志贺菌强毒株,有9株噬斑数>1000个/ml,而2株弱毒株噬斑数则在50个/ml以下。透射电镜证实了志贺菌对细胞的黏附、侵入和释放过程。结果表明:志贺菌对细胞的侵袭能力受温度的影响;不同志贺菌流行株对细胞的侵袭力存在着差异:实验也显示,应用HeLa细胞研究志贺菌侵袭力是一种简便易行且价廉的方法。 相似文献