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1.
Viruses exploit cellular machinery to gain entry and initiate their replication cycle within host cells. The development of methods to visualize virus entry in live cells has provided new insights to the cellular processes involved in virus entry and the intracellular locations where viral payloads are deposited. The use of fluorescently labeled virus and high-resolution microscopy is currently the method of choice to study virus entry in live cells. While fluorescent protein fusions (e.g. viral proteins fused to GFP) have been used, the labeling of viral proteins that contain a small tetracysteine (tc) tag with biarsenical fluorescent compounds (e.g. FlAsH, ReAsH, Lumio-x) offers several advantages over conventional xFP-fusion constructs. This article describes methods for generating fluorescently labeled viruses encoding tc-tagged proteins that are suitable for the study of virus entry in live cells by fluorescence microscopy. Critical parameters required to quantify fluorescence signals from the labeled, tc-tagged proteins in individual virus particles during the entry process and the subsequent fate of the labeled viral proteins after virus uncoating are also described. 相似文献
2.
A. V. Mamontova A. P. Grigoryev A. S. Tsarkova K. A. Lukyanov A. M. Bogdanov 《Russian Journal of Bioorganic Chemistry》2017,43(6):625-633
In modern biotechnological science, there is a need for visualization of objects under study at the levels of cells, organelles, and individual molecules. Prominent among imaging methods are the methods based on the detection of fluorescence from the fluorophores with which objects under study are labeled. Fluorescent proteins (FPs) are very popular as genetically encoded fluorescent labels for lifetime imaging of target structures and processes in living systems. One of the key characteristics of FPs is their photostability, i.e., their resistance to photochemical reactions that quench the fluorescence signal. This review describes the currently known molecular mechanisms underlying photobleaching and the methods used to improve the photostability of fluorescent proteins. 相似文献
3.
Human cytomegalovirus labeled with green fluorescent protein for live analysis of intracellular particle movements 总被引:4,自引:0,他引:4
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Human cytomegalovirus (HCMV) replicates in the nuclei of infected cells. Successful replication therefore depends on particle movements between the cell cortex and nucleus during entry and egress. To visualize HCMV particles in living cells, we have generated a recombinant HCMV expressing enhanced green fluorescent protein (EGFP) fused to the C terminus of the capsid-associated tegument protein pUL32 (pp150). The resulting UL32-EGFP-HCMV was analyzed by immunofluorescence, electron microscopy, immunoblotting, confocal microscopy, and time-lapse microscopy to evaluate the growth properties of this virus and the dynamics of particle movements. UL32-EGFP-HCMV replicated similarly to wild-type virus in fibroblast cultures. Green fluorescent virus particles were released from infected cells. The fluorescence stayed associated with particles during viral entry, and fluorescent progeny particles appeared in the nucleus at 44 h after infection. Surprisingly, strict colocalization of pUL32 and the major capsid protein pUL86 within nuclear inclusions indicated that incorporation of pUL32 into nascent HCMV particles occurred simultaneously with or immediately after assembly of the capsid. A slow transport of nuclear particles towards the nuclear margin was demonstrated. Within the cytoplasm, most particles performed irregular short-distance movements, while a smaller fraction of particles performed centripetal and centrifugal long-distance movements. Although numerous particles accumulated in the cytoplasm, release of particles from infected cells was a rare event, consistent with a release rate of about 1 infectious unit per h per cell in HCMV-infected fibroblasts as calculated from single-step growth curves. UL32-EGFP-HCMV will be useful for further investigations into the entry, maturation, and release of this virus. 相似文献
4.
The role of actin dynamics in clathrin-mediated endocytosis in mammalian cells is unclear. In this study, we define the role of actin cytoskeleton in Kaposi''s sarcoma-associated herpesvirus (KSHV) entry and trafficking in endothelial cells using an immunofluorescence-based assay to visualize viral capsids and the associated cellular components. In contrast to infectivity or reporter assays, this method does not rely on the expression of any viral and reporter genes, but instead directly tracks the accumulation of individual viral particles at the nuclear membrane as an indicator of successful viral entry and trafficking in cells. Inhibitors of endosomal acidification reduced both the percentage of nuclei with viral particles and the total number of viral particles docking at the perinuclear region, indicating endocytosis, rather than plasma membrane fusion, as the primary route for KSHV entry into endothelial cells. Accordingly, a viral envelope protein was only detected on internalized KSHV particles at the early but not late stage of infection. Inhibitors of clathrin- but not caveolae/lipid raft-mediated endocytosis blocked KSHV entry, indicating that clathrin-mediated endocytosis is the major route of KSHV entry into endothelial cells. KSHV particles were colocalized not only with markers of early and recycling endosomes, and lysosomes, but also with actin filaments at the early time points of infection. Consistent with these observations, transferrin, which enters cells by clathrin-mediated endocytosis, was found to be associated with actin filaments together with early and recycling endosomes, and to a lesser degree, with late endosomes and lysosomes. KSHV infection induced dynamic actin cytoskeleton rearrangements. Disruption of the actin cytoskeleton and inhibition of regulators of actin nucleation such as Rho GTPases and Arp2/3 complex profoundly blocked KSHV entry and trafficking. Together, these results indicate an important role for actin dynamics in the internalization and endosomal sorting/trafficking of KSHV and clathrin-mediated endocytosis in endothelial cells. 相似文献
5.
Green fluorescent protein-tagged retroviral envelope protein for analysis of virus-cell interactions
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Fluorescent retroviral envelope (Env) proteins were developed for direct visualization of viral particles. By fusing the enhanced green fluorescent protein (eGFP) to the N terminus of the amphotropic 4070A envelope protein, extracellular presentation of eGFP was achieved. Viruses incorporated the modified Env protein and efficiently infected cells. We used the GFP-tagged viruses for staining retrovirus receptor-positive cells, thereby circumventing indirect labeling techniques. By generating cells which conditionally expressed the GFP-tagged Env protein, we could confirm an inverse correlation between retroviral Env expression and infectivity (superinfection). eGFP-tagged virus particles are suitable for monitoring the dynamics of virus-cell interactions. 相似文献
6.
Fluorescently labeled human immunodeficiency virus (HIV) derivatives, combined with the use of advanced fluorescence microscopy techniques, allow the direct visualization of dynamic events and individual steps in the viral life cycle. HIV proteins tagged with fluorescent proteins (FPs) have been successfully used for live-cell imaging analyses of HIV-cell interactions. However, FPs display limitations with respect to their physicochemical properties, and their maturation kinetics. Furthermore, several independent FP-tagged constructs have to be cloned and characterized in order to obtain spectral variations suitable for multi-color imaging setups. In contrast, the so-called SNAP-tag represents a genetically encoded non-fluorescent tag which mediates specific covalent coupling to fluorescent substrate molecules in a self-labeling reaction. Fusion of the SNAP-tag to the protein of interest allows specific labeling of the fusion protein with a variety of synthetic dyes, thereby offering enhanced flexibility for fluorescence imaging approaches.Here we describe the construction and characterization of the HIV derivative HIV(SNAP), which carries the SNAP-tag as an additional domain within the viral structural polyprotein Gag. Introduction of the tag close to the C-terminus of the matrix domain of Gag did not interfere with particle assembly, release or proteolytic virus maturation. The modified virions were infectious and could be propagated in tissue culture, albeit with reduced replication capacity. Insertion of the SNAP domain within Gag allowed specific staining of the viral polyprotein in the context of virus producing cells using a SNAP reactive dye as well as the visualization of individual virions and viral budding sites by stochastic optical reconstruction microscopy. Thus, HIV(SNAP) represents a versatile tool which expands the possibilities for the analysis of HIV-cell interactions using live cell imaging and sub-diffraction fluorescence microscopy. 相似文献
7.
Rational design of a monomeric and photostable far‐red fluorescent protein for fluorescence imaging in vivo
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William Clay Gustafson Rubén Ruiz‐González Luca Signor Fanny Marzocca Franck Borel Matthew P. Klassen Kalpana Makhijani Antoine Royant Yuh‐Nung Jan William A. Weiss Su Guo Xiaokun Shu 《Protein science : a publication of the Protein Society》2016,25(2):308-315
Fluorescent proteins (FPs) are powerful tools for cell and molecular biology. Here based on structural analysis, a blue‐shifted mutant of a recently engineered monomeric infrared fluorescent protein (mIFP) has been rationally designed. This variant, named iBlueberry, bears a single mutation that shifts both excitation and emission spectra by approximately 40 nm. Furthermore, iBlueberry is four times more photostable than mIFP, rendering it more advantageous for imaging protein dynamics. By tagging iBlueberry to centrin, it has been demonstrated that the fusion protein labels the centrosome in the developing zebrafish embryo. Together with GFP‐labeled nucleus and tdTomato‐labeled plasma membrane, time‐lapse imaging to visualize the dynamics of centrosomes in radial glia neural progenitors in the intact zebrafish brain has been demonstrated. It is further shown that iBlueberry can be used together with mIFP in two‐color protein labeling in living cells and in two‐color tumor labeling in mice. 相似文献
8.
Green fluorescent protein-tagged adeno-associated virus particles allow the study of cytosolic and nuclear trafficking
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Lux K Goerlitz N Schlemminger S Perabo L Goldnau D Endell J Leike K Kofler DM Finke S Hallek M Büning H 《Journal of virology》2005,79(18):11776-11787
To allow the direct visualization of viral trafficking, we genetically incorporated enhanced green fluorescent protein (GFP) into the adeno-associated virus (AAV) capsid by replacement of wild-type VP2 by GFP-VP2 fusion proteins. High-titer virus progeny was obtained and used to elucidate the process of nuclear entry. In the absence of adenovirus 5 (Ad5), nuclear translocation of AAV capsids was a slow and inefficient process: at 2 h and 4 h postinfection (p.i.), GFP-VP2-AAV particles were found in the perinuclear area and in nuclear invaginations but not within the nucleus. In Ad5-coinfected cells, isolated GFP-VP2-AAV particles were already detectable in the nucleus at 2 h p.i., suggesting that Ad5 enhanced the nuclear translocation of AAV capsids. The number of cells displaying viral capsids within the nucleus increased slightly over time, independently of helper virus levels, but the majority of the AAV capsids remained in the perinuclear area under all conditions analyzed. In contrast, independently of helper virus and with 10 times less virions per cell already observed at 2 h p.i., viral genomes were visible within the nucleus. Under these conditions and even with prolonged incubation times (up to 11 h p.i.), no intact viral capsids were detectable within the nucleus. In summary, the results show that GFP-tagged AAV particles can be used to study the cellular trafficking and nuclear entry of AAV. Moreover, our findings argue against an efficient nuclear entry mechanism of intact AAV capsids and favor the occurrence of viral uncoating before or during nuclear entry. 相似文献
9.
Fluorescent proteins (FPs), such as green fluorescent protein (GFP) and its variants, are well-developed visible markers for analyzing bioprocesses. Accurate measurement of fluorescence emitted from FPs in whole cells is complicated by the inner filter effect (IFE), which is caused by intracellular light absorption and scattering by cell particles. The IFE causes nonlinearity between fluorescence intensity and fluorophore concentrations in FP-harboring cells and can significantly influence the accuracy of FP-based analysis, especially at high cell densities. A mathematical model based on detection of fluorescence intensity using a fluorescence spectrophotometer was developed to provide a simple correction for the IFE in fluorescence intensity detection in high-density cultures. The parameters of this model were determined in three different FP-harboring bacterial strains to give the “real fluorescence” intensity without the IFE. Using these parameters, accurate analysis of FP-labeled Escherichia coli at high cell density in pure culture and in mixed cultures with fluorescent and nonfluorescent strains was easily and successfully achieved. 相似文献
10.
《Biophysical journal》2022,121(21):4179-4188
Fluorescent proteins (FPs) are a powerful tool to quantitatively monitor gene expression. The dynamics of a promoter and its regulation can be inferred from fluorescence data. The interpretation of fluorescent data, however, is strongly dependent on the maturation of FPs since different proteins mature in distinct ways. We propose a novel approach for analyzing fluorescent reporter data by incorporating maturation dynamics in the reconstruction of promoter activities. Our approach consists of developing and calibrating mechanistic maturation models for distinct FPs. These models are then used alongside a Bayesian approach to estimate promoter activities from fluorescence data. We demonstrate by means of targeted experiments in Escherichia coli that our approach provides robust estimates and that accounting for maturation is, in many cases, essential for the interpretation of gene expression data. 相似文献
11.
High-efficiency intracellular magnetic labeling with novel superparamagnetic-Tat peptide conjugates 总被引:29,自引:0,他引:29
A biocompatible, dextran coated superparamagnetic iron oxide particle was derivatized with a peptide sequence from the HIV-tat protein to improve intracellular magnetic labeling of different target cells. The conjugate had a mean particle size of 41 nm and contained an average of 6.7 tat peptides. Derivatized particles were internalized into lymphocytes over 100-fold more efficiently than nonmodified particles, resulting in up to 12.7 x 10(6) particles/cell. Internalized particles localized in cytoplasm and nuclear compartments as demonstrated by fluorescence microscopy and immunohistochemistry. Labeled cells were highly magnetic, were detectable by NMR imaging, and could be retained on magnetic separation columns. The described method has potential applications for in vivo tracking of magnetically labeled cells by MR imaging and for recovering intracellularly labeled cells from organs. 相似文献
12.
Fluorescent particles are used for a diverse number of biochemical assays including intracellular imaging, cellular tracking, as well as detection of a variety of biomolecules. They are typically prepared by postpolymerization conjugations of dyes onto preformed particles. Herein we report the synthesis of aminomethyl-functionalized fluorescent particles via the synthesis and application of polymerizable fluorescein monomers. These monomers allowed high and controllable fluorophore loading into the particles, resulting in enhanced fluorescence properties in comparison with more commonly used carboxyfluorescein conjugated particles. Furthermore, the particles were rapidly taken up by cells with enhanced fluorescence. The herein presented results demonstrate the advantages of dye polymerization in contrast with more conventional conjugation strategies for fluorescent particle generation with applications in the life sciences. 相似文献
13.
Toivola J Gilbert L Michel P White D Vuento M Oker-Blom C 《Comptes rendus biologies》2005,328(12):1052-1056
Analysis of the breakdown products of engineered viral particles can give useful information on the particle structure. We used various methods to breakdown both a recombinant enveloped virus and virus-like particles (VLPs) from two non-enveloped viruses and analysed the resulting subunits by fluorescence correlation spectroscopy (FCS). Analysis of the enveloped baculovirus, Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), displaying the green fluorescent protein (GFP) fused to its envelope protein gp64 was performed in the presence and absence of 5 mM SDS and 25 mM DTT. Without treatment, the viral particle showed a diffusion time of 3.3 ms. In the presence of SDS, fluorescent subunits with diffusion times of 0.2 ms were observed. Additional treatment with DTT caused a drop in the diffusion time to 0.1 ms. Changes in the amplitude of the autocorrelation function suggested a 3-fold increase in fluorescent particle number when viral particles were treated with SDS, and a further 1.5-fold increase with additional treatment with DTT. Thus, the data showed that an average of 4.5 molecules of gp64-GFP was incorporated in the membrane of the modified baculovirus. Further, this suggests that each fluorescent gp64 trimer carries on average 1.5 fluorescent units. Similar experiments were carried out with two non-enveloped fluorescent virus-like particles (fVLPs) that displayed enhanced green fluorescent protein (EGFP). These, fVLPs of canine and human B19 parvoviruses were treated with 6 M urea and 5 mM SDS, respectively. Correspondingly, the original hydrodynamic radii of 17 and 14 nm were reduced to 9 and 5 nm after treatment. Here, the change in the amplitude of the autocorrelation curve suggested a 10-fold increase in particle number when viral particles of CPV were treated with 6 M urea at 50 degrees C for 10 min. For EGFP-B19, there was a decrease in the amplitude, accompanied by a 9-fold increase in the number of fluorescent units with SDS treatment. The results showed that approximately 10 and 9 fluorescent units were associated with the corresponding CPV and B19 VLPs. In summary, we were able to estimate the number of fluorescent subunits in a baculovirus containing a GFP-fusion with its gp64 envelope protein and in two different parvo-VLPs containing EGFP-fused with their VP2 capsid proteins. 相似文献
14.
Briandet R Lacroix-Gueu P Renault M Lecart S Meylheuc T Bidnenko E Steenkeste K Bellon-Fontaine MN Fontaine-Aupart MP 《Applied and environmental microbiology》2008,74(7):2135-2143
In the natural environment, most of the phages that target bacteria are thought to exist in biofilm ecosystems. The purpose of this study was to gain a clearer understanding of the reactivity of these viral particles when they come into contact with bacteria embedded in biofilms. Experimentally, we quantified lactococcal c2 phage diffusion and reaction through model biofilms using in situ fluorescence correlation spectroscopy with two-photon excitation. Correlation curves for fluorescently labeled c2 phage in nonreacting Stenotrophomonas maltophilia biofilms indicated that extracellular polymeric substances did not provide significant resistance to phage penetration and diffusion, even though penetration and diffusion were sometimes restricted because of the noncontractile tail of the viral particle. Fluctuations in the fluorescence intensity of the labeled phage were detected throughout the thickness of biofilms formed by c2-sensitive and c2-resistant strains of Lactococcus lactis but could never be correlated with time, revealing that the phage was immobile. This finding confirmed that recognition binding receptors for the viral particles were present on the resistant bacterial cell wall. Taken together, our results suggest that biofilms may act as "active" phage reservoirs that can entrap and amplify viral particles and protect them from harsh environments. 相似文献
15.
Matrix fibronectin binds gammaretrovirus and assists in entry: new light on viral infections 总被引:1,自引:0,他引:1
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A major entry route for the gammaretrovirus amphotropic murine leukemia virus (A-MLV) into NIH 3T3 fibroblasts is via caveola-dependent endocytosis. However, during the infection time, few viral particles can be observed intracellularly. Analyzing the dynamics of the A-MLV infection process by using total internal reflection fluorescence microscopy, we show that the majority of viruses are extracellular and bound to the fibronectin matrix. Moreover, the amounts of bound virus and of fibronectin correlated. Using confocal microscopy, nanoparticles targeted to fibronectin by a III1C-fibronectin fragment or anti-fibronectin antibody were detected intracellularly in NIH 3T3 cells; unconjugated nanoparticles neither bound to cells nor were detectable intracellularly. Furthermore, A-MLV colocalized intracellularly with the fibronectin-targeted nanoparticles, suggesting that they were taken up by the same cellular pathway. Both A-MLV entry and fibronectin turnover depend on caveolar endocytosis, and we found that inhibiting viral binding to the extracellular NIH 3T3 fibronectin-matrix dramatically reduced A-MLV infection, indeed, showing an active role of fibronectin in infection. We suggest that binding to the cellular fibronectin matrix provides a new mechanism by which viruses can enter cells. 相似文献
16.
Nina Senutovitch Lawrence Vernetti Robert Boltz Richard DeBiasio Albert Gough D Lansing Taylor 《Experimental biology and medicine (Maywood, N.J.)》2015,240(6):795-808
This mini-review discusses the evolution of fluorescence as a tool to study living cells and tissues in vitro and the present role of fluorescent protein biosensors (FPBs) in microphysiological systems (MPSs). FPBs allow the measurement of temporal and spatial dynamics of targeted cellular events involved in normal and perturbed cellular assay systems and MPSs in real time. FPBs evolved from fluorescent analog cytochemistry (FAC) that permitted the measurement of the dynamics of purified proteins covalently labeled with environmentally insensitive fluorescent dyes and then incorporated into living cells, as well as a large list of diffusible fluorescent probes engineered to measure environmental changes in living cells. In parallel, a wide range of fluorescence microscopy methods were developed to measure the chemical and molecular activities of the labeled cells, including ratio imaging, fluorescence lifetime, total internal reflection, 3D imaging, including super-resolution, as well as high-content screening. FPBs evolved from FAC by combining environmentally sensitive fluorescent dyes with proteins in order to monitor specific physiological events such as post-translational modifications, production of metabolites, changes in various ion concentrations, and the dynamic interaction of proteins with defined macromolecules in time and space within cells. Original FPBs involved the engineering of fluorescent dyes to sense specific activities when covalently attached to particular domains of the targeted protein. The subsequent development of fluorescent proteins (FPs), such as the green fluorescent protein, dramatically accelerated the adoption of studying living cells, since the genetic “labeling” of proteins became a relatively simple method that permitted the analysis of temporal–spatial dynamics of a wide range of proteins. Investigators subsequently engineered the fluorescence properties of the FPs for environmental sensitivity that, when combined with targeted proteins/peptides, created a new generation of FPBs. Examples of FPBs that are useful in MPS are presented, including the design, testing, and application in a liver MPS. 相似文献
17.
Nicholas Hahon 《Applied microbiology》1965,13(6):865-871
A quantitative assay for infective variola virus particles was developed which is based on the enumeration of cells containing fluorescent viral antigen after infection of McCoy cell monolayers. The direct fluorescent-antibody technique was employed to stain cells. The efficiency of virus adsorption was markedly enhanced by centrifugation of virus inoculum onto McCoy cell monolayers at 500 x g for 15 min. By this procedure, a proportionality was obtained between the number of fluorescent cells and volume of inoculum. Observations on the sequential development of viral antigen within cells and counts of fluorescent cells showed that the optimal time for enumerating fluorescent cells was after an incubation period of 16 to 20 hr. A linear function existed between virus concentration and cell-infecting units. Fluorescent cells were distributed randomly in infected cover slip cell monolayers. The assay was demonstrated to be highly sensitive, precise, and reproducible. 相似文献
18.
Murine leukemia virus particle assembly quantitated by fluorescence microscopy: role of Gag-Gag interactions and membrane association
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In order to track the assembly of murine leukemia virus (MLV), we used fluorescence microscopy to visualize particles containing Gag molecules fused to fluorescent proteins (FPs). Gag-FP chimeras budded from cells to produce fluorescent spots, which passed through the same pore-size filters and sedimented at the same velocity as authentic MLV. N-terminal myristylation of Gag-FPs was necessary for particle formation unless wild-type Gag was coexpressed. By labeling nonmyristylated Gag with yellow FP and wild-type Gag with cyan FP, we could quantitate the coincorporation of two proteins into single particles. This experiment showed that nonmyristylated Gag was incorporated into mixed particles at approximately 50% the efficiency of wild-type Gag. Mutations that inhibit Gag-Gag interactions (K. Alin and S. P. Goff, Virology 216:418-424, 1996; K. Alin and S. P. Goff, Virology 222:339-351, 1996) were then introduced into the capsid (CA) region of Gag-FPs. The mutations P150L and R119C/P133L inhibited fluorescent particle formation by these Gag-FPs, but Gag-FPs containing these mutations could be efficiently incorporated into particles when coexpressed with wild-type Gag. When these mutations were introduced into nonmyristylated Gag-FPs, no incorporation into particles in the presence of wild-type Gag was detected. These data suggest that two independent mechanisms, CA interactions and membrane association following myristylation, cooperate in MLV Gag assembly and budding. 相似文献
19.
Analysis of hepatitis C virus superinfection exclusion by using novel fluorochrome gene-tagged viral genomes
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Schaller T Appel N Koutsoudakis G Kallis S Lohmann V Pietschmann T Bartenschlager R 《Journal of virology》2007,81(9):4591-4603
Studies of the complete hepatitis C virus (HCV) life cycle have become possible with the development of an infectious cell culture system using the genotype 2a isolate JFH-1. Taking advantage of this system in the present study, we investigated whether HCV infection leads to superinfection exclusion, a state in which HCV-infected cells are resistant to secondary HCV infection. To discriminate between viral genomes, we inserted genes encoding fluorescent proteins in frame into the 3'-terminal NS5A coding region. These genomes replicated to wild-type levels and supported the production of infectious virus particles. Upon simultaneous infection of Huh-7 cells, co-replication of both viral genomes in the same cell was detected. However, when infections were performed sequentially, secondary infection was severely impaired. This superinfection exclusion was neither due to a reduction of cell surface expression of CD81 and scavenger receptor BI, two molecules implicated in HCV entry, nor due to a functional block at the level of virus entry. Instead, superinfection exclusion was mediated primarily by interference at the level of HCV RNA translation and, presumably, also replication. In summary, our results describe the construction and characterization of viable monocistronic HCV reporter genomes allowing detection of viral replication in infected living cells. By using these genomes, we found that HCV induces superinfection exclusion, which is primarily due to interference at a post-entry step. 相似文献
20.
Pederson T 《European journal of histochemistry : EJH》2004,48(1):57-64
For more than 60 years, RNA has been detectable in fixed cells and tissues by relatively specific staining methods. More recently, it has become possible to study RNA in unfixed, live cells. This review article describes how the intracellular dynamics and localization of RNA in vivo can be studied by microinjection of fluorescent RNA into cells- an approach we have termed Fluorescent RNA Cytochemistry. Depending on the particular RNA species under investigation, Fluorescent RNA Cytochemistry can operate as a "stain" to reveal intracellular sites at which a given RNA resides, or as a "tracer" to allow movements of a dynamically translocating RNA to be followed in the living cell. Several examples of Fluorescent RNA Cytochemistry are presented, collectively illustrating the range of applicability this approach offers in the toolbox of gene expression, studied as in vivo cell biology. 相似文献