Rabies virus infection of cultured rat sensory neurons.

The axonal transport of rabies virus (challenge virus strain of fixed virus) was studied in differentiated rat embryonic dorsal root ganglion cells. In addition, we observed the attachment of rabies virus to neuronal extensions and virus production by infected neurons. A compartmentalized cell culture system was used, allowing infection and manipulation of neuronal extensions without exposing the neural soma to the virus. The cultures consisted of 60% large neuronal cells whose extensions exhibited neurofilament structures. Rabies virus demonstrated high binding affinity to unmyelinated neurites, as suggested by assays of virus adsorption and immunofluorescence studies. The rate of axoplasmic transport of virus was 12 to 24 mm/day, including the time required for internalization of the virus into neurites. The virus transport could be blocked by cytochalasin B, vinblastine, and colchicine, none of which negatively affected the production of virus in cells once the infection was established. It was concluded that, for the retrograde transfer of rabies virus by neurites from the periphery to the neuronal soma, the integrity of tubulin- and actin-containing structures is essential. The rat sensory neurons were characterized as permissive, moderately susceptible, but low producers of rabies virus. These neurons were capable of harboring rabies virus for long periods of time and able to release virus into the culture medium without showing any morphological alterations. The involvement of sensory neurons in rabies virus pathogenesis, both in viral transport and as a site for persistent viral infection, is discussed.     

Hepatitis C virus entry into the hepatocyte

Hepatitis C virus (HCV) is a small enveloped virus with a positive stranded RNA genome belonging to the Flaviviridae family. The virion has the unique ability of forming a complex with lipoproteins, which is known as the lipoviroparticle. Lipoprotein components as well as the envelope proteins, E1 and E2, play a key role in virus entry into the hepatocyte. HCV entry is a complex multistep process involving sequential interactions with several cell surface proteins. The virus relies on glycosaminoglycans and possibly the low-density lipoprotein receptors to attach to cells. Furthermore, four specific entry factors are involved in the following steps which lead to virus internalization and fusion in early endosomes. These molecules are the scavenger receptor SRB1, tetraspanin CD81 and two tight junction proteins, Claudin-1 and Occludin. Although they are essential to HCV entry, the precise role of these molecules is not completely understood. Finally, hepatocytes are highly polarized cells and which likely affects the entry process. Our current knowledge on HCV entry is summarized in this review.     

Mechanics of enveloped virus entry into host cells

Enveloped viruses such as HIV-1 enter their hosts by first establishing a contact region at the cell surface, which is stabilized by the formation of receptor-ligand complexes. We show that the favorable contact energy stemming from the formation of the receptor complexes in the interaction zone is sufficient to drive the engulfment of the virus by the cell. Using a continuum model, we show that the equilibrium engulfment depth and the force driving the engulfment are functions of the virus size and the complex formation energy. Resistance to engulfment is dominated by the elastic deformation of the cytoskeleton.     

On resistance to virus entry into host cells

In this article, we adopt a continuum model from Sun and Wirtz (2006. Biophys. J. 90:L10-L12) to show that, for the enveloped virus entry into host cells, the binding energy of the receptor-ligand complex can drive the engulfment of the viral particle to overcome the resistance alternatively dominated by the membrane deformation and cytoskeleton deformation at a different engulfing stage. This is contrary to the conclusions by Sun and Wirtz that the cytoskeleton deformation is always dominant. This discrepancy occurs because the energy of membrane deformation in their article is incorrect. Such an unfortunate small error has led to a severe underestimation of the contribution from membrane deformation to the total energy of the system, which then led them to improperly conclude that the cytoskeleton deformation plays the dominant role in the virus entry into host cell. By using the correct energy expression, our conclusion is justified by energy comparisons under a large range of virus sizes and Young's moduli of cytoskeleton. We even find that a critical radius of virus exists, beyond which the resistance to the virus engulfment becomes dominated by the membrane deformation during the whole stage, contrary to the point of view of Sun and Wirtz.     

Monitoring virus entry into living cells using DiD-labeled dengue virus particles

A variety of approaches can be applied to investigate the multiple steps and interactions that occur during virus entry into the host cell. Single-virus tracking is a powerful real-time imaging technique that offers the possibility to monitor virus-cell binding, internalization, intracellular trafficking behavior, and the moment of membrane fusion of single virus particles in living cells. Here we describe the development and applications of a single-virus tracking assay based on the use of DiD-labeled dengue virus (DENV) in BS-C-1 cells. In addition – and using the same experimental setup – we present a binding and fusion assay that can be used to obtain a rapid insight into the relative extent of virus binding to the cell surface and membrane fusion. Details of virus labeling and characterization, microscopy setup, protocols, data analysis, and hints for troubleshooting are described throughout the paper.     

Characterization of dengue virus entry into HepG2 cells

Background

Despite infections by the dengue virus being a significant problem in tropical and sub-tropical countries, the mechanism by which the dengue virus enters into mammalian cells remains poorly described.

Methods

A combination of biochemical inhibition, dominant negative transfection of Eps15 and siRNA mediated gene silencing was used to explore the entry mechanism of dengue into HepG2 cells.

Results

Results were consistent with entry via multiple pathways, specifically via clathrin coated pit mediated endocytosis and macropinocytosis, with clathrin mediated endocytosis being the predominant pathway.

Conclusion

We propose that entry of the dengue virus to mammalian cells can occur by multiple pathways, and this opens the possibility of the virus being directed to multiple cellular compartments. This would have significant implications in understanding the interaction of the dengue virus with the host cell machinery.     

pH-Dependent entry of chikungunya virus fusion into mosquito cells

Background

Millions of human infections caused by arthropod-borne pathogens are initiated by the feeding of an infected mosquito on a vertebrate. However, interactions between the viruses and the mosquito vector, which facilitates successful infection and transmission of virus to a subsequent vertebrate host, are still not fully understood.

Finding

Here we describe early chikungunya virus (CHIKV) infectious events in cells derived from one of the most important CHIKV vectors, Aedes albopictus. We demonstrated that CHIKV infection of mosquito cells depended on acidification of the endosome as indicated by significant inhibition following prophylactic treatment with the lysosomotropic drugs chloroquine, ammonium chloride, and monensin, which is consistent with observations in mammalian cells. While all three agents inhibited CHIKV infection in C6/36 cells, ammonium chloride was less toxic to cells than the other agents.

Conclusion

The observation of similar mechanisms for inhibition of CHIKV infection in mosquito and mammalian cell lines suggests that conserved entry pathways are utilized by CHIKV for vertebrate and invertebrate cell types.

     

非包膜病毒进入细胞基质的研究进展

病毒是最简单的生命形态,必须在宿主细胞中才能繁殖。为了增殖,病毒需要将它们的基因组运送到宿主细胞中。而要达到这一目的,病毒必须通过宿主细胞的主要屏障——细胞质膜,不同病毒会根据宿主细胞膜的特点运用不同的进入策略。根据病毒的外表是否具有脂双层膜可将病毒分成两类:包膜病毒和非包膜病毒。包膜病毒外表有一层来源于宿主细胞的脂双层膜,膜上整合了病毒编码的膜融合蛋白。包膜病毒主要通过膜融合蛋白的作用促使病毒的包膜与细胞膜融合,从而介导病毒核衣壳的侵人。对于非包膜病毒来说,由于缺乏包     

Differential responsiveness of cultured suckling and adult rat hepatocytes to growth-promoting factors: entry into S phase and mitosis

The capacity of suckling and adult rat hepatocytes in culture to enter into S phase and mitosis in response to EGF, insulin, and glucagon was measured. Both cell types were isolated in high yield and purity and cultured in the absence of serum under identical conditions. At the time of isolation, suckling rat hepatocytes were all diploid and in the G1 phase of the cell cycle. Adult rat hepatocytes constituted a population of mixed ploidy level, as shown by flow cytometry. Upon stimulation, both suckling and adult rate hepatocytes entered S phase after a minimum lag period of 24 h. For suckling rat hepatocytes EGF was required, but its stimulating action was dependent on insulin and/or glucagon. In contrast, adult rat hepatocytes entered into S phase in response to EGF alone; insulin and glucagon did not significantly potentiate its effect. Under optimal hormonal stimulation for entry into S phase a large proportion of suckling rat hepatocytes underwent mitosis, whereas only a few mitoses were observed in the case of adult rat hepatocytes. Therefore, there is a differential response of suckling and adult rat hepatocytes to growth factors which correlates with ploidy level, and this difference may be associated with the degree of maturation.     

Vaccinia virus entry into cells via a low-pH-dependent endosomal pathway

Previous studies established that vaccinia virus could enter cells by fusion with the plasma membrane at neutral pH. However, low pH triggers fusion of vaccinia virus-infected cells, a hallmark of viruses that enter by the endosomal route. Here, we demonstrate that entry of mature vaccinia virions is accelerated by brief low-pH treatment and severely reduced by inhibitors of endosomal acidification, providing evidence for a predominant low-pH-dependent endosomal pathway. Entry of vaccinia virus cores into the cytoplasm, measured by expression of firefly luciferase, was increased more than 10-fold by exposure to a pH of 4.0 to 5.5. Furthermore, the inhibitors of endosomal acidification bafilomycin A1, concanamycin A, and monensin each lowered virus entry by more than 70%. This reduction was largely overcome by low-pH-induced entry through the plasma membrane, confirming the specificities of the drugs. Entry of vaccinia virus cores with or without brief low-pH treatment was visualized by electron microscopy of thin sections of immunogold-stained cells. Although some virus particles fused with the plasma membrane at neutral pH, 30 times more fusions and a greater number of cytoplasmic cores were seen within minutes after low-pH treatment. Without low-pH exposure, the number of released cores lagged behind the number of virions in vesicles until 30 min posttreatment, when they became approximately equal, perhaps reflecting the time of endosome acidification and virus fusion. The choice of two distinct pathways may contribute to the ability of vaccinia virus to enter a wide range of cells.     

The entry into host cells of Sindbis virus, vesicular stomatitis virus and Sendai virus.

We have compared the mechanisms of entry into host cells of three enveloped viruses: Sendai virus, vesicular stomatitis virus (VSV) and Sindbis virus. Virus entry by membrane fusion should antigenically modify the surface of a newly infected cell in such a way that it will be killed by anti-viral antibody and complement. On the other hand, virus entry by a mechanism involving uptake by the cell of the whole virion should not make cells sensitive to antibody and complement. As expected, cells newly infected with Sendai virus were readily and completely lysed by anti-Sendai antibody and complement. In marked contrast, however, cells newly infected with either Sindbis virus or VSV were killed by anti-viral antibody and complement only when infected at an extremely high multiplicity of infection, in excess of 1000 plaque-forming units per cell. We favor the following explanation for these results with Sindbis virus and VSV: a very large majority of the Sindbis and VSV virions entered the infected cells by some means other than membrane fusion, presumably engulfment of the whole particle. Efficient entry by way of membrane fusion may therefore not be a general characteristic of enveloped viruses.     

Differential entry of ricin into malignant and normal rat hepatocytes

We have compared the mechanisms of ricin binding to and entry into Zajdela hepatoma cells (ZHC) and normal rat hepatocytes (HyC). Lactose but not mannan was found to inhibit ricin binding to and toxicity on ZHC and HyC. This finding suggests that ricin binding, entry, and toxicity are expressed only through the galactose binding sites on ZHC and HyC. Nevertheless, the characteristics of ricin binding and its entry pathway appeared to be different in several respects in ZHC and HyC. Scatchard analysis of equilibrium data determined over a wide range of 125I-labeled ricin concentrations yielded a curvilinear plot for ZHC, while a straight line was obtained for HyC. These results indicate that only ZHC possess high-affinity receptors for ricin. Analysis of ricin toxicity on ZHC and HyC, in the presence of ammonium chloride or after K+-depletion in both cell types, suggests that the ricin bound to galactose receptors entered through neutral vesicles in ZHC, and through both neutral and acidic vesicles in HyC. The qualitative and quantitative differences found between the process of receptor-mediated endocytosis of ricin in ZHC and HyC might explain the differential sensitivity of the two cell types toward the toxin.     

Human immunodeficiency virus type 1 entry into macrophages mediated by macropinocytosis

Whereas human immunodeficiency virus (HIV) infects various cell types by fusion at the plasma membrane, we observed a different entry route in human primary macrophages, in which macropinocytosis is active. Shortly after exposure of macrophages to HIV-1 and irrespective of viral envelope-receptor interactions, particles were visible in intracellular vesicles, which were identified as macropinosomes. Most virions appeared subsequently degraded. However, fusion leading to capsid release in the cytosol and productive infection could take place inside vesicles when particles were properly enveloped. These observations provide new insights into HIV-1 interactions with a cell target relevant to pathogenesis. They may have implications for the design of soluble inhibitors aimed at interfering with the fusion or entry processes.     

Mechanisms of herpesvirus infection--virus entry into host cells and virus assembly

Herpesvirus entry into host cells occurs by recognition of specific cellular receptor(s) with viral envelope glycoproteins. Nucleocapsids formed in nucleus are released into cytoplasm, and acquire tegument proteins there. Nucleocapsids with tegument proteins bud into intracellular vesicles formed in infected cells, which are thought to be derived from Golgi apparatus, trans-Golgi network or endosomes. However, the precise mechanisms involved in virus final envelopment are poorly understood. Here, I review our current knowledge regarding herpesvirus entry into host cells and virus assembly.     

On the entry of semliki forest virus into BHK-21 cells

The pathway by which semliki forest virus (SFV), a membrane-containing animal virus, enters BHK-21 cells was studied morphologically and biochemically. After attaching to the cell surface, the majority of viruses was rapidly trapped into coated pits, internalized by endocytosis in coated vesicles, and sequestered into intracellular vacuoles and lysosomes. Direct penetration of viruses through the plasma membrane was never observed. To assess the possible involvement of lysosomes in the release of the genome into the cytoplasm, the effect of five lysosomotropic agents, known to increase the lysosomal pH, was tested. All of these agents inhibited SFV infectivity and one, chloroquine (the agent studied in most detail), inhibited a very early step in the infection but had no effect on binding, endocytosis, or intracellular distribution of SFV. Thus, the inhibitory effect was concluded to be either on penetration of the nucelocapsid into the cytoplasm or on uncoating of the viral RNA. Possible mechanisms for the penetration of the genome into the cytoplasm were studied in vitro, using phospholipids-cholesterol liposomes and isolated SFV. When the pH was 6.0 or lower, efficient fusion of the viral membranes and the liposomal membranes occurred, resulting in the transfer of the nucleocapsid into the liposomes. Infection of cells could also be induced by brief low pH treatment of cells with bound SFV under conditions where the normal infection route was blocked. The results suggest that the penetration of the viral genome into the cytosol takes place intracellularly through fusion between the limiting membrane of intracellular vacuoles and the membrane of viruses contained within them. The low pH required for fusion together with the inhibitory effect of lysosomotropic agents implicate lysosomes, or other intracellular vacuoles with sufficiently low pH, as the main sites of penetration.