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991.
Extent of measles virus spread and immune suppression differentiates between wild-type and vaccine strains in the cotton rat model (Sigmodon hispidus) 下载免费PDF全文
Pfeuffer J Püschel K Meulen Vt Schneider-Schaulies J Niewiesk S 《Journal of virology》2003,77(1):150-158
Infection of humans with wild-type measles virus leads to strong immune suppression and secondary infections, whereas immunization with an attenuated vaccine strain does not. Using the cotton rat model (Sigmodon hispidus), we investigated whether vaccine and wild-type viruses differ in viral spread and whether this is correlated with inhibition of of proliferation of spleen cells ex vivo after mitogen stimulation. After intranasal infection of cotton rats with wild-type and vaccine strains, it was found that wild-type virus replicates better in lung tissue, spreads to the mediastinal lymph nodes, and induces a more pronounced and longer-lasting inhibition of proliferation of spleen cells ex vivo after mitogen stimulation than does vaccine virus. To induce the same degree of proliferation inhibition, 1,000-fold less wild-type virus was required than vaccine virus. With this system, the virulence of various measles virus isolates and recombinant viruses was tested. Four (in humans and/or monkeys) highly pathogenic virus strains were immunosuppressive, whereas viruses of vaccine virus genotype A were not. Using virus pairs which, due to passage on fibroblasts versus lymphoid cells or due to a point mutation in the hemagglutinin (N481 --> Y), differed in their usage of the two receptor molecules CD46 and CD150 on human cells, it was found that viruses using exclusively CD150 in vitro spread to mediastinal lymph nodes and induced strong immune suppression. These data demonstrate that important parameters of virulence seen in humans, such as viral spread and immune suppression, are reflected in the cotton rat model. 相似文献
992.
Quantitative role of the human papillomavirus type 16 E5 gene during the productive stage of the viral life cycle 总被引:6,自引:0,他引:6 下载免费PDF全文
Genther SM Sterling S Duensing S Münger K Sattler C Lambert PF 《Journal of virology》2003,77(5):2832-2842
Human papillomaviruses (HPVs) are small circular DNA viruses that cause warts. Infection with high-risk anogenital HPVs, such as HPV type 16 (HPV16), is associated with human cancers, specifically cervical cancer. The life cycle of HPVs is intimately tied to the differentiation status of the host epithelium and has two distinct stages: the nonproductive stage and the productive stage. In the nonproductive stage, which arises in the poorly differentiated basal epithelial compartment of a wart, the virus maintains itself as a low-copy-number nuclear plasmid. In the productive stage, which arises as the host cell undergoes terminal differentiation, viral DNA is amplified; the capsid genes, L1 and L2, are expressed; and progeny virions are produced. This stage of the viral life cycle relies on the ability of the virus to reprogram the differentiated cells to support DNA synthesis. Papillomaviruses encode multiple oncoproteins, E5, E6, and E7. In the present study, we analyze the role of one of these viral oncogenes, E5, in the viral life cycle. To assess the role of E5 in the HPV16 life cycle, we introduced wild-type (WT) or E5 mutant HPV16 genomes into NIKS, a keratinocyte cell line that supports the papillomavirus life cycle. By culturing these cells under conditions that allow them to remain undifferentiated, a state similar to that of basal epithelial cells, we determined that E5 does not play an essential role in the nonproductive stage of the HPV16 life cycle. To determine if E5 plays a role in the productive stage of the viral life cycle, we cultured keratinocyte populations in organotypic raft cultures, which promote the differentiation and stratification of epithelial cells. We found that cells harboring E5 mutant genomes displayed a quantitative reduction in the percentage of suprabasal cells undergoing DNA synthesis, compared to cells containing WT HPV16 DNA. This reduction in DNA synthesis, however, did not prevent amplification of viral DNA in the differentiated cellular compartment. Likewise, late viral gene expression and the perturbation of normal keratinocyte differentiation were retained in cells harboring E5 mutant genomes. These data demonstrate that E5 plays a subtle role during the productive stage of the HPV16 life cycle. 相似文献
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995.
The latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus permits replication of terminal repeat-containing plasmids 下载免费PDF全文
The latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus can associate with mitotic chromosomes and promote latent episome maintenance and segregation. Here we report that LANA also mediates the replication of plasmid DNAs bearing viral terminal repeats. The predicted secondary structure of LANA's C terminus reveals striking similarity to the known structure of the DNA-binding domain of Epstein-Barr virus EBNA1, despite the absence of primary sequence homology between these proteins, suggesting conservation of the key mechanistic features of latent gammaherpesvirus DNA replication. 相似文献
996.
Pöhlmann S Zhang J Baribaud F Chen Z Leslie GJ Lin G Granelli-Piperno A Doms RW Rice CM McKeating JA 《Journal of virology》2003,77(7):4070-4080
DC-SIGN and DC-SIGNR are two closely related membrane-associated C-type lectins that bind human immunodeficiency virus (HIV) envelope glycoprotein with high affinity. Binding of HIV to cells expressing DC-SIGN or DC-SIGNR can enhance the efficiency of infection of cells coexpressing the specific HIV receptors. DC-SIGN is expressed on some dendritic cells, while DC-SIGNR is localized to certain endothelial cell populations, including hepatic sinusoidal endothelial cells. We found that soluble versions of the hepatitis C virus (HCV) E2 glycoprotein and retrovirus pseudotypes expressing chimeric forms of both HCV E1 and E2 glycoproteins bound efficiently to DC-SIGN and DC-SIGNR expressed on cell lines and primary human endothelial cells but not to other C-type lectins tested. Soluble E2 bound to immature and mature human monocyte-derived dendritic cells (MDDCs). Binding of E2 to immature MDDCs was dependent on DC-SIGN interactions, while binding to mature MDDCs was partly independent of DC-SIGN, suggesting that other cell surface molecules may mediate HCV glycoprotein interactions. HCV interactions with DC-SIGN and DC-SIGNR may contribute to the establishment or persistence of infection both by the capture and delivery of virus to the liver and by modulating dendritic cell function. 相似文献
997.
Persistent replication of hepatitis C virus replicons expressing the beta-lactamase reporter in subpopulations of highly permissive Huh7 cells 总被引:2,自引:0,他引:2
Murray EM Grobler JA Markel EJ Pagnoni MF Paonessa G Simon AJ Flores OA 《Journal of virology》2003,77(5):2928-2935
Progress toward development of better therapies for the treatment of hepatitis C virus (HCV) infection has been hampered by poor understanding of HCV biology and the lack of biological assays suitable for drug screening. Here we describe a powerful HCV replication system that employs HCV replicons expressing the beta-lactamase reporter (bla replicons) and subpopulations of Huh7 cells that are more permissive (or "enhanced") to HCV replication than na?ve Huh7 cells. Enhanced cells represent a small fraction of permissive cells present among na?ve Huh7 cells that is enriched during selection with replicons expressing the neomycin phosphotransferase gene (neo replicons). The level of permissiveness of cell lines harboring neo replicons can vary greatly, and the enhanced phenotype is usually revealed upon removal of the neo replicon with inhibitors of HCV replication. Replicon removal is responsible for increased permissiveness, since this effect could be reproduced either with alpha interferon or with an HCV NS5B inhibitor. Moreover, adaptive mutations present in the replicon genome used during selection do not influence the permissiveness of the resulting enhanced-cell population, suggesting that the mechanisms governing the permissiveness of enhanced cells are independent from viral adaptation. Because the beta-lactamase reporter allows simultaneous quantitation of replicon-harboring cells and reporter activity, it was possible to investigate the relationship between genome replication activity and the frequency with which transfected genomes can establish persistent replication. Our study demonstrates that differences in the replication potential of the viral genome are manifested primarily in the frequency with which persistent replication is established but modestly affect the number of replicons observed per replicon-harboring cell. Replicon copy number was found to vary over a narrow range that may be defined by a minimal number required for persistent maintenance and a maximum that is limited by the availability of essential host factors. 相似文献
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999.
S-Adenosylmethionine (AdoMet) is the methyl donor of numerous methylation reactions. The current model is that an increased concentration of AdoMet stimulates DNA methyltransferase reactions, triggering hypermethylation and protecting the genome against global hypomethylation, a hallmark of cancer. Using an assay of active demethylation in HEK 293 cells, we show that AdoMet inhibits active demethylation and expression of an ectopically methylated CMV-GFP (green fluorescent protein) plasmid in a dose-dependent manner. The inhibition of GFP expression is specific to methylated GFP; AdoMet does not inhibit an identical but unmethylated CMV-GFP plasmid. S-Adenosylhomocysteine (AdoHcy), the product of methyltransferase reactions utilizing AdoMet does not inhibit demethylation or expression of CMV-GFP. In vitro, AdoMet but not AdoHcy inhibits methylated DNA-binding protein 2/DNA demethylase as well as endogenous demethylase activity extracted from HEK 293, suggesting that AdoMet directly inhibits demethylase activity, and that the methyl residue on AdoMet is required for its interaction with demethylase. Taken together, our data support an alternative mechanism of action for AdoMet as an inhibitor of intracellular demethylase activity, which results in hypermethylation of DNA. 相似文献
1000.
Golabek AA Kida E Walus M Wujek P Mehta P Wisniewski KE 《The Journal of biological chemistry》2003,278(9):7135-7145
Human tripeptidyl-peptidase I (TPP I, CLN2 protein) is a lysosomal serine protease that removes tripeptides from the free N termini of small polypeptides and also shows a minor endoprotease activity. Due to various naturally occurring mutations, an inherited deficiency of TPP I activity causes a fatal lysosomal storage disorder, classic late infantile neuronal ceroid lipofuscinosis (CLN2). In the present study, we analyzed biosynthesis, glycosylation, transport, and proteolytic processing of this enzyme in stably transfected Chinese hamster ovary cells as well as maturation of the endocytosed proenzyme in CLN2 lymphoblasts, fibroblasts, and N2a cells. Human TPP I was initially identified as a single precursor polypeptide of approximately 68 kDa, which, within a few hours, was converted to the mature enzyme of approximately 48 kDa. Compounds affecting the pH of intracellular acidic compartments, those interfering with the intracellular vesicular transport as well as inhibition of the fusion between late endosomes and lysosomes by temperature block or 3-methyladenine, hampered the conversion of TPP I proenzyme into the mature form, suggesting that this process takes place in lysosomal compartments. Digestion of immunoprecipitated TPP I proenzyme with both N-glycosidase F and endoglycosidase H as well as treatment of the cells with tunicamycin reduced the molecular mass of TPP I proenzyme by approximately 10 kDa, which indicates that all five potential N-glycosylation sites in TPP I are utilized. Mature TPP I was found to be partially resistant to endo H treatment; thus, some of its N-linked oligosaccharides are of the complex/hybrid type. Analysis of the effect of various classes of protease inhibitors and mutation of the active site Ser(475) on human TPP I maturation in cultured cells demonstrated that although TPP I zymogen is capable of autoactivation in vitro, a serine protease that is sensitive to AEBSF participates in processing of the proenzyme to the mature, active form in vivo. 相似文献