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Real-time Imaging of Rabies Virus Entry into Living Vero cells.

Xu H, Hao X, Wang S, Wang Z, Cai M, Jiang J, Qin Q, Zhang M, Wang H - Sci Rep (2015)

Bottom Line: Firstly, it was found that the actin-enriched filopodia is in favor of virus reaching to the cell body.Then, our real-time imaging results unambiguously uncover the characteristics of viral internalization and cellular transport dynamics.Significantly, the results provide profound insight into development of novel and effective antiviral targets.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin, P.R. China.

ABSTRACT
Understanding the mechanism of rabies virus (RABV) infection is vital for prevention and therapy of virulent rabies. However, the infection mechanism remains largely uncharacterized due to the limited methods and viral models. Herein, we utilized a powerful single-virus tracking technique to dynamically and globally visualize the infection process of the live attenuated rabies vaccine strain-SRV9 in living Vero cells. Firstly, it was found that the actin-enriched filopodia is in favor of virus reaching to the cell body. Furthermore, by carrying out drug perturbation experiments, we confirmed that RABV internalization into Vero cells proceeds via classical dynamin-dependent clathrin-mediated endocytosis with requirement for intact actin, but caveolae-dependent endocytosis is not involved. Then, our real-time imaging results unambiguously uncover the characteristics of viral internalization and cellular transport dynamics. In addition, our results directly and quantitatively reveal that the intracellular motility of internalized RABV particles is largely microtubule-dependent. Collectively, our work is crucial for understanding the initial steps of RABV infection, and elucidating the mechanisms of post-infection. Significantly, the results provide profound insight into development of novel and effective antiviral targets.

No MeSH data available.


Related in: MedlinePlus

SRV9 Entry depends on clathrin-mediated endocytosis rather than caveola-dependent.The Vero cells were labeled by DiO (green). In the control group, the Vero cells were incubated with Cy5-labeled SRV9 for 30 min at 37 °C and washed by PBS immediately before imaging. In treated group, the Vero cells were pretreated by low temperature, as well as several inhibitors including sucrose (200 mM), CPZ (10μg/ml), CB (20 μM), MβCD (10 mM), nystatin (100 μM), and filipin (10 μg/ml) for 30 min prior to SRV9 infection, respectively. More than 100 cells were examined by CLSM in each treatment. (A) Three-dimensional confocal images of Vero cells infected with SRV9 under different treatment conditions. Successive z-stacks spaced by 200 nm were recorded to construct the 3D image. The XY image plane is located about 4 μm above the bottom of the cell. The XZ image plane that used for recording in all experiments is approximately 1;μm thick as indicated by the white dashed lines. Scale bars: 10 μm. (B) Quantitation of viral entry rate. The viral entry rate was quantified as the average fluorescence intensity of virus-infected cell relative to that for control cells. The viral entry rate of control cells was factitiously set as 100%. Error bars represent SEM. (C) SRV9 particles colocalize with clathrin. Vero cells were transfected with pEGFP-LCa prior to incubation with Cy5-labeled SRV9. The arrows indicate an individual SRV9 particle (red) colocalized with clathrin (green). Scale bars are 10 μm.
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f3: SRV9 Entry depends on clathrin-mediated endocytosis rather than caveola-dependent.The Vero cells were labeled by DiO (green). In the control group, the Vero cells were incubated with Cy5-labeled SRV9 for 30 min at 37 °C and washed by PBS immediately before imaging. In treated group, the Vero cells were pretreated by low temperature, as well as several inhibitors including sucrose (200 mM), CPZ (10μg/ml), CB (20 μM), MβCD (10 mM), nystatin (100 μM), and filipin (10 μg/ml) for 30 min prior to SRV9 infection, respectively. More than 100 cells were examined by CLSM in each treatment. (A) Three-dimensional confocal images of Vero cells infected with SRV9 under different treatment conditions. Successive z-stacks spaced by 200 nm were recorded to construct the 3D image. The XY image plane is located about 4 μm above the bottom of the cell. The XZ image plane that used for recording in all experiments is approximately 1;μm thick as indicated by the white dashed lines. Scale bars: 10 μm. (B) Quantitation of viral entry rate. The viral entry rate was quantified as the average fluorescence intensity of virus-infected cell relative to that for control cells. The viral entry rate of control cells was factitiously set as 100%. Error bars represent SEM. (C) SRV9 particles colocalize with clathrin. Vero cells were transfected with pEGFP-LCa prior to incubation with Cy5-labeled SRV9. The arrows indicate an individual SRV9 particle (red) colocalized with clathrin (green). Scale bars are 10 μm.

Mentions: Although the entry mechanism of RABV has been reported, the reconstructed RABV were used in previous studies827. Here, we employed live SRV9 as the surrogate of wild-type RABV to comprehensively evaluate the endocytosis pathway. Several disruption drugs that specifically hinder different entry pathways were utilized. In the inhibition experiments, cells were pretreated with various inhibitors prior to SRV9 infection. The inhibitors were maintained throughout the experiment process. As positive control, the Vero cells are infected by SRV9 without any drug pretreatment. As shown in Fig. 3A, a large number of virions were seen inside the untreated control cell.


Real-time Imaging of Rabies Virus Entry into Living Vero cells.

Xu H, Hao X, Wang S, Wang Z, Cai M, Jiang J, Qin Q, Zhang M, Wang H - Sci Rep (2015)

SRV9 Entry depends on clathrin-mediated endocytosis rather than caveola-dependent.The Vero cells were labeled by DiO (green). In the control group, the Vero cells were incubated with Cy5-labeled SRV9 for 30 min at 37 °C and washed by PBS immediately before imaging. In treated group, the Vero cells were pretreated by low temperature, as well as several inhibitors including sucrose (200 mM), CPZ (10μg/ml), CB (20 μM), MβCD (10 mM), nystatin (100 μM), and filipin (10 μg/ml) for 30 min prior to SRV9 infection, respectively. More than 100 cells were examined by CLSM in each treatment. (A) Three-dimensional confocal images of Vero cells infected with SRV9 under different treatment conditions. Successive z-stacks spaced by 200 nm were recorded to construct the 3D image. The XY image plane is located about 4 μm above the bottom of the cell. The XZ image plane that used for recording in all experiments is approximately 1;μm thick as indicated by the white dashed lines. Scale bars: 10 μm. (B) Quantitation of viral entry rate. The viral entry rate was quantified as the average fluorescence intensity of virus-infected cell relative to that for control cells. The viral entry rate of control cells was factitiously set as 100%. Error bars represent SEM. (C) SRV9 particles colocalize with clathrin. Vero cells were transfected with pEGFP-LCa prior to incubation with Cy5-labeled SRV9. The arrows indicate an individual SRV9 particle (red) colocalized with clathrin (green). Scale bars are 10 μm.
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Related In: Results  -  Collection

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f3: SRV9 Entry depends on clathrin-mediated endocytosis rather than caveola-dependent.The Vero cells were labeled by DiO (green). In the control group, the Vero cells were incubated with Cy5-labeled SRV9 for 30 min at 37 °C and washed by PBS immediately before imaging. In treated group, the Vero cells were pretreated by low temperature, as well as several inhibitors including sucrose (200 mM), CPZ (10μg/ml), CB (20 μM), MβCD (10 mM), nystatin (100 μM), and filipin (10 μg/ml) for 30 min prior to SRV9 infection, respectively. More than 100 cells were examined by CLSM in each treatment. (A) Three-dimensional confocal images of Vero cells infected with SRV9 under different treatment conditions. Successive z-stacks spaced by 200 nm were recorded to construct the 3D image. The XY image plane is located about 4 μm above the bottom of the cell. The XZ image plane that used for recording in all experiments is approximately 1;μm thick as indicated by the white dashed lines. Scale bars: 10 μm. (B) Quantitation of viral entry rate. The viral entry rate was quantified as the average fluorescence intensity of virus-infected cell relative to that for control cells. The viral entry rate of control cells was factitiously set as 100%. Error bars represent SEM. (C) SRV9 particles colocalize with clathrin. Vero cells were transfected with pEGFP-LCa prior to incubation with Cy5-labeled SRV9. The arrows indicate an individual SRV9 particle (red) colocalized with clathrin (green). Scale bars are 10 μm.
Mentions: Although the entry mechanism of RABV has been reported, the reconstructed RABV were used in previous studies827. Here, we employed live SRV9 as the surrogate of wild-type RABV to comprehensively evaluate the endocytosis pathway. Several disruption drugs that specifically hinder different entry pathways were utilized. In the inhibition experiments, cells were pretreated with various inhibitors prior to SRV9 infection. The inhibitors were maintained throughout the experiment process. As positive control, the Vero cells are infected by SRV9 without any drug pretreatment. As shown in Fig. 3A, a large number of virions were seen inside the untreated control cell.

Bottom Line: Firstly, it was found that the actin-enriched filopodia is in favor of virus reaching to the cell body.Then, our real-time imaging results unambiguously uncover the characteristics of viral internalization and cellular transport dynamics.Significantly, the results provide profound insight into development of novel and effective antiviral targets.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin, P.R. China.

ABSTRACT
Understanding the mechanism of rabies virus (RABV) infection is vital for prevention and therapy of virulent rabies. However, the infection mechanism remains largely uncharacterized due to the limited methods and viral models. Herein, we utilized a powerful single-virus tracking technique to dynamically and globally visualize the infection process of the live attenuated rabies vaccine strain-SRV9 in living Vero cells. Firstly, it was found that the actin-enriched filopodia is in favor of virus reaching to the cell body. Furthermore, by carrying out drug perturbation experiments, we confirmed that RABV internalization into Vero cells proceeds via classical dynamin-dependent clathrin-mediated endocytosis with requirement for intact actin, but caveolae-dependent endocytosis is not involved. Then, our real-time imaging results unambiguously uncover the characteristics of viral internalization and cellular transport dynamics. In addition, our results directly and quantitatively reveal that the intracellular motility of internalized RABV particles is largely microtubule-dependent. Collectively, our work is crucial for understanding the initial steps of RABV infection, and elucidating the mechanisms of post-infection. Significantly, the results provide profound insight into development of novel and effective antiviral targets.

No MeSH data available.


Related in: MedlinePlus