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1.
Markers for trans-Golgi Membranes and the Intermediate Compartment Localize to Induced Membranes with Distinct Replication Functions in Flavivirus-Infected Cells
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Replication of the flavivirus Kunjin virus is associated with virus-induced membrane structures within the cytoplasm of infected cells; these membranes appear as packets of vesicles associated with the sites of viral RNA synthesis and as convoluted membranes (CM) and paracrystalline arrays (PC) containing the components of the virus-specified protease (E. G. Westaway, J. M. Mackenzie, M. T. Kenney, M. K. Jones, and A. A. Khromykh, J. Virol. 71:6650-6661, 1997). To determine the cellular origins of these membrane structures, we compared the immunolabelling patterns of several cell markers in relation to these sites by immunofluorescence and immunoelectron microscopy. A marker for the trans-Golgi membranes and the trans-Golgi network, 1,4-galactosyltransferase (GalT), was redistributed to large foci in the cytoplasm of Kunjin virus-infected cells, partially coincident with immunofluorescent foci associated with the putative sites of viral RNA synthesis. As determined by immunoelectron microscopy, the induced vesicle packets contained GalT, whereas the CM and PC contained a specific protein marker for the intermediate compartment (ERGIC53). A further indicator of the role of cellular organelles in their biogenesis was the observation that the Golgi apparatus-disrupting agent brefeldin A prevented further development of immunofluorescent foci of induced membranes if added before the end of the latent period but that once formed, these membrane foci were resistant to brefeldin A dispersion. Reticulum membranes emanating from the induced CM and PC were also labelled with the rough endoplasmic reticulum marker anti-protein disulfide isomerase and were obviously redistributed during infection. This is the first report identifying trans-Golgi membranes and the intermediate compartment as the apparent sources of the flavivirus-induced membranes involved in events of replication. 相似文献
2.
Indirect evidence suggests that legumes can adjust rapidly theresistance of their root nodules to O2 diffusion. Here we describeexperiments using O2 specific micro-electrodes and dark fieldmicroscopy to study directly the operation of this diffusionbarrier. The O2 concentration sensed by the electrode decreasedsharply in the region of the inner cortex and was less than1.0 mmol m3 throughout the infected tissue in nodulesof both pea (Pisum sativum) and french bean (Phaseolus vulgaris).In a number of experiments the ambient O2 concentration wasincreased to 40% while the electrode tip was just inside theinner cortex. In 13 out of 21 cases the O2 concentration atthis position either remained low and unchanged or increasedirreversibly to near ambient values. In the remaining casesthe O2 concentration increased after 1 to 2.5 min and then decreasedto its former value. These results are ascribed to an increasein resistance of the barrier in response to increased O2 fluxinto the nodule. It was shown microscopically that air spacesboth at the boundary between the infected zone and the innercortex, and within the infected zone started to disappear 3min after nodules were exposed to high ambient O2 concentrationsand had disappeared completely after 8 min. These spaces werenot changed by exposure of the nodule for 10 min to either N2or air. Key words: Oxygen, root nodules, air spaces 相似文献
3.
Evolution of the 28S ribosomal RNA gene in anurans: regions of variability and their phylogenetic implications 总被引:1,自引:0,他引:1
Fifteen restriction sites were mapped to the 28S ribosomal RNA gene of
individuals representing 54 species of frogs, two species of salamanders, a
caecilian, and a lungfish. Eight of these sites were present in all species
examined, and two were found in all but one species. Alignment of these
conserved restriction sites revealed, among anuran 28S rRNA genes, five
regions of major length variation that correspond to four of 12 previously
identified divergent domains of this gene. One of the divergent domains
(DD8) consists of two regions of length variation separated by a short
segment that is conserved at least throughout tetrapods. Most of the
insertions, deletions, and restriction-site variations identified in the
28S gene will require sequence-level analysis for a detailed reconstruction
of their history. However, an insertion in DD9 that is coextensive with
frogs in the suborder Neobatrachia, a BstEII site that is limited to
representatives of two leptodactylid subfamilies, and a deletion in DD10
that is found only in three ranoid genera are probably synapomorphies.
相似文献
4.
5.
Ultrastructure of Kunjin virus-infected cells: colocalization of NS1 and NS3 with double-stranded RNA, and of NS2B with NS3, in virus-induced membrane structures. 总被引:19,自引:16,他引:3
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E G Westaway J M Mackenzie M T Kenney M K Jones A A Khromykh 《Journal of virology》1997,71(9):6650-6661
The subcellular location of the nonstructural proteins NS1, NS2B, and NS3 in Vero cells infected with the flavivirus Kunjin was investigated using indirect immunofluorescence and cryoimmunoelectron microscopy with monospecific antibodies. Comparisons were also made by dual immunolabelling using antibodies to double-stranded RNA (dsRNA), the putative template in the flavivirus replication complex. At 8 h postinfection, the immunofluorescent patterns showed NS1, NS2B, NS3, and dsRNA located in a perinuclear rim with extensions into the peripheral cytoplasm. By 16 h, at the end of the latent period, all patterns had changed to some discrete perinuclear foci associated with a thick cytoplasmic reticulum. By 24 h, this localization in perinuclear foci was more apparent and some foci were dual labelled with antibodies to dsRNA. In immuno-gold-labelled cryosections of infected cells at 24 h, all antibodies were associated with clusters of induced membrane structures in the perinuclear region. Two important and novel observations were made. First, one set of induced membranes comprised vesicle packets of smooth membranes dual labelled with anti-dsRNA and anti-NS1 or anti-NS3 antibodies. Second, adjacent masses of paracrystalline arrays or of convoluted smooth membranes, which appeared to be structurally related, were strongly labelled only with anti-NS2B and anti-NS3 antibodies. Paired membranes similar in appearance to the rough endoplasmic reticulum were also labelled, but less strongly, with antibodies to the three nonstructural proteins. Other paired membranes adjacent to the structures discussed above enclosed accumulated virus particles but were not labelled with any of the four antibodies. The collection of induced membranes may represent virus factories in which translation, RNA synthesis, and virus assembly occur. 相似文献
6.
D Westaway C A Mirenda D Foster Y Zebarjadian M Scott M Torchia S L Yang H Serban S J DeArmond C Ebeling 《Neuron》1991,7(1):59-68
Prolonged incubation times for experimental scrapie in I/LnJ mice are dictated by a dominant gene linked to the prion protein gene (Prn-p). Transgenic mice were analyzed to discriminate between an effect of the I/LnJ Prn-pb allele and a distinct incubation time locus designated Prn-i. Paradoxically, 4 independent Prn-pb transgenic mouse lines had scrapie incubation times shorter than nontransgenic controls, instead of the anticipated prolonged incubation periods. Aberrant or overexpression of the Prn-pb transgenes may dictate abbreviated incubation times, masking genuine Prn-p/Prn-i congruence; alternatively, a discrete Prn-i gene lies adjacent to Prn-p. 相似文献
7.
Alzheimer’s disease (AD) is a complex multifactorial neurodegenerative disorder believed to be initiated by accumulation of amyloid β (Aβ)-related peptides derived from proteolytic processing of amyloid precursor protein (APP). Research over the past two decades provided a mechanistic link between cholesterol and AD pathogenesis. Genetic polymorphisms in genes regulating the pivotal points in cholesterol metabolism have been suggested to enhance the risk of developing AD. Altered neuronal membrane cholesterol level and/or subcellular distribution have been implicated in aberrant formation, aggregation, toxicity, and degradation of Aβ-related peptides. However, the results are somewhat contradictory and we still do not have a complete understanding on how cholesterol can influence AD pathogenesis. In this review, we summarize our current understanding on the role of cholesterol in regulating the production/function of Aβ-related peptides and also examine the therapeutic potential of regulating cholesterol homeostasis in the treatment of AD pathology. 相似文献
8.
Subgenomic replicons of the flavivirus Kunjin: construction and applications. 总被引:10,自引:7,他引:10
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Several Kunjin virus (KUN) subgenomic replicons containing large deletions in the structural region (C-prM-E) and in the 3' untranslated region (3'UTR) of the genome have been constructed. Replicon RNA deltaME with 1,987 nucleotides deleted (from nucleotide 417 [in codon 108] in the C gene to nucleotide 2403 near the carboxy terminus of the E gene, inclusive) and replicon RNA C20rep with 2,247 nucleotides deleted (from nucleotide 157 [in codon 20] in C to nucleotide 2403) replicated efficiently in electroporated BHK21 cells. A further deletion from C20rep of 53 nucleotides, reducing the coding sequence in core protein to two codons (C2rep RNA), resulted in abolishment of RNA replication. Replicon deltaME/76 with a deletion of 76 nucleotides in the 3'UTR of deltaME RNA (nucleotides 10423 to 10498) replicated efficiently, whereas replicon deltaME/352 with a larger deletion of 352 nucleotides (nucleotides 10423 to 10774), including two conserved sequences RCS3 and CS3, was significantly inhibited in RNA replication. To explore the possibility of using a reporter gene assay to monitor synthesis of the positive strand and the negative strand of KUN RNA, we inserted a chloramphenicol acetyltransferase (CAT) gene into the 3'UTR of deltaME/76 RNA under control of the internal ribosomal entry site (IRES) of encephalomyelocarditis virus RNA in both plus (deltaME/76CAT[+])- and minus (deltaME/76CAT[-])-sense orientations. Although insertion of the IRES-CAT cassette in the plus-sense orientation resulted in a significant (10- to 20-fold) reduction of RNA replication compared to that of the parental deltaME/76 RNA, CAT expression was readily detected in electroporated BHK cells. No CAT expression was detected after electroporation of RNA containing the IRES-CAT cassette inserted in the minus-sense orientation despite its apparently more efficient replication (similar to that of deltaME/76 RNA); this result indicated that KUN negative-strand RNA was probably not released from its template after synthesis. Replacement of the CAT gene in the deltaME/76CAT(+) RNA with the neomycin gene (Neo) enabled selection and recovery of a BHK cell culture in which the majority of cells were continuously expressing the replicon RNA for 41 days (nine passages) without apparent cytopathic effect. The constructed KUN replicons should provide valuable tools to study flavivirus RNA replication as well as providing possible vectors for a long-lasting and noncytopathic RNA virus expression system. 相似文献
9.
Most of the seven flavivirus nonstructural proteins (NS1 to NS5) encoded in the distal two-thirds of the RNA positive-sense genome are believed to be essential components of RNA replication complexes. To explore the functional relationships of these components in RNA replication, we used trans-complementation analysis of full-length infectious RNAs of Kunjin (KUN) virus with a range of lethal in-frame deletions in the nonstructural coding region, using as helper a repBHK cell line stably producing functional replication complexes from KUN replicon RNA. Recently we showed that replication of KUN RNAs with large carboxy-terminal deletions including the entire RNA polymerase region in the NS5 gene, representing 34 to 75% of the NS5 coding content, could be complemented after transfection into repBHK cells. In this study we have demonstrated that KUN RNAs with deletions of 84 to 97% of the NS1 gene, or of 13 to 63% of the NS3 gene including the entire helicase region, were also complemented in repBHK cells with variable efficiencies. In contrast, KUN RNAs with deletions in any of the other four nonstructural genes NS2A, NS2B, NS4A, and NS4B were not complemented. We have also demonstrated successful trans complementation of KUN RNAs containing either combined double deletions in the NS1 and NS5 genes or triple deletions in the NS1, NS3, and NS5 genes comprising as much as 38% of the entire nonstructural coding content. Based on these and our previous complementation results, we have generated a map of cis- and trans-acting elements in RNA replication for the nonstructural coding region of the flavivirus genome. These results are discussed in the context of our model on formation and composition of the flavivirus replication complex, and we suggest molecular mechanisms by which functions of some defective components of the replication complex can be complemented by their wild-type counterparts expressed from another (helper) RNA molecule. 相似文献
10.
Tumor infiltrating lymphocytes (TIL), especially T-cells, have both prognostic and therapeutic applications. The presence of CD8+ effector T-cells and the ratio of CD8+ cells to FOXP3+ regulatory T-cells have been used as biomarkers of disease prognosis to predict response to various immunotherapies. Blocking the interaction between inhibitory receptors on T-cells and their ligands with therapeutic antibodies including atezolizumab, nivolumab, pembrolizumab and tremelimumab increases the immune response against cancer cells and has shown significant improvement in clinical benefits and survival in several different tumor types. The improved clinical outcome is presumed to be associated with a higher tumor infiltration; therefore, it is thought that more accurate methods for measuring the amount of TIL could assist prognosis and predict treatment response. We have developed and validated quantitative immunohistochemistry (IHC) assays for CD3, CD8 and FOXP3 for immunophenotyping T-lymphocytes in tumor tissue. Various types of formalin fixed, paraffin embedded (FFPE) tumor tissues were immunolabeled with anti-CD3, anti-CD8 and anti-FOXP3 antibodies using an IHC autostainer. The tumor area of stained tissues, including the invasive margin of the tumor, was scored by a pathologist (visual scoring) and by computer-based quantitative image analysis. Two image analysis scores were obtained for the staining of each biomarker: the percent positive cells in the tumor area and positive cells/mm2 tumor area. Comparison of visual vs. image analysis scoring methods using regression analysis showed high correlation and indicated that quantitative image analysis can be used to score the number of positive cells in IHC stained slides. To demonstrate that the IHC assays produce consistent results in normal daily testing, we evaluated the specificity, sensitivity and reproducibility of the IHC assays using both visual and image analysis scoring methods. We found that CD3, CD8 and FOXP3 IHC assays met the fit-for-purpose analytical acceptance validation criteria and that they can be used to support clinical studies. 相似文献