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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

The process of individual SRV9 entry into Vero cell.(A) Bright-field image of a Vero cell as circled by blue line. (B–H) Selected frames reveal the process that SRV9 particle was internalized into the Vero cell. In this experiment, Vero cells labeled with DiO (green) were infected with Cy5-labeled SRV9 (red). The imaging was performed at 10 min after the infection. The white dashed circles show the site of SRV9 entry into the Vero cell. Scale bar: 10 μm.
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f4: The process of individual SRV9 entry into Vero cell.(A) Bright-field image of a Vero cell as circled by blue line. (B–H) Selected frames reveal the process that SRV9 particle was internalized into the Vero cell. In this experiment, Vero cells labeled with DiO (green) were infected with Cy5-labeled SRV9 (red). The imaging was performed at 10 min after the infection. The white dashed circles show the site of SRV9 entry into the Vero cell. Scale bar: 10 μm.

Mentions: To investigate the infection process of individual SRV9, we observed the internalization and the transport of SRV9 by tracking single virus in live cells using time-lapse confocal laser scanning microscope. The SRV9 and Vero cell membranes were labeled with Cy5-NHS and DiO, respectively. The cellular vesicles were also nonspecifically stained by the lipophilic dye DiO. Firstly, the viruses were added to Vero cells for 10 min at 37 °C, then tracked in real-time. Figure 4A represents the image of Vero cells in bright field. The border of the Vero cell corresponding to that in the fluorescence images is marked by blue line. It is easy to identify the sites of SRV9 entry through visualizing the fusion site of SRV9 and the Vero cell membrane. The time-lapse confocal microscope image showed that the red fluorescences (Cy5-labeled SRV9) were colocalized with the green fluorescences (DiO-labeled cell membrane) as circled by white dashed line in Fig. 4B. The colocalization indicated that the virus was attached to cell membrane. After attachment, the virus continued to interact with the cell membrane for a short time (Fig. 4B,C). The interaction promoted the cell membrane to invaginate to form vesicle, as shown in Fig. 4D, then the virus was wrapped in the vesicle. At this moment, the vesicle was still located at cell membranes. The preexisting vesicle (pointed by red arrow in Fig. 4B) bound to the new-forming vesicle, which provided tensile force to facilitate the vesicles to detach from the membrane. In Fig. 4E, the single virus had already escaped from the vesicle and entered the cell. Afterwards, the virus fused with the preexisting vesicle and moved towards the cytoplasm. The whole internalization process of SRV9 was recorded in Movie S4 (Supplementary Information).


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)

The process of individual SRV9 entry into Vero cell.(A) Bright-field image of a Vero cell as circled by blue line. (B–H) Selected frames reveal the process that SRV9 particle was internalized into the Vero cell. In this experiment, Vero cells labeled with DiO (green) were infected with Cy5-labeled SRV9 (red). The imaging was performed at 10 min after the infection. The white dashed circles show the site of SRV9 entry into the Vero cell. Scale bar: 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493577&req=5

f4: The process of individual SRV9 entry into Vero cell.(A) Bright-field image of a Vero cell as circled by blue line. (B–H) Selected frames reveal the process that SRV9 particle was internalized into the Vero cell. In this experiment, Vero cells labeled with DiO (green) were infected with Cy5-labeled SRV9 (red). The imaging was performed at 10 min after the infection. The white dashed circles show the site of SRV9 entry into the Vero cell. Scale bar: 10 μm.
Mentions: To investigate the infection process of individual SRV9, we observed the internalization and the transport of SRV9 by tracking single virus in live cells using time-lapse confocal laser scanning microscope. The SRV9 and Vero cell membranes were labeled with Cy5-NHS and DiO, respectively. The cellular vesicles were also nonspecifically stained by the lipophilic dye DiO. Firstly, the viruses were added to Vero cells for 10 min at 37 °C, then tracked in real-time. Figure 4A represents the image of Vero cells in bright field. The border of the Vero cell corresponding to that in the fluorescence images is marked by blue line. It is easy to identify the sites of SRV9 entry through visualizing the fusion site of SRV9 and the Vero cell membrane. The time-lapse confocal microscope image showed that the red fluorescences (Cy5-labeled SRV9) were colocalized with the green fluorescences (DiO-labeled cell membrane) as circled by white dashed line in Fig. 4B. The colocalization indicated that the virus was attached to cell membrane. After attachment, the virus continued to interact with the cell membrane for a short time (Fig. 4B,C). The interaction promoted the cell membrane to invaginate to form vesicle, as shown in Fig. 4D, then the virus was wrapped in the vesicle. At this moment, the vesicle was still located at cell membranes. The preexisting vesicle (pointed by red arrow in Fig. 4B) bound to the new-forming vesicle, which provided tensile force to facilitate the vesicles to detach from the membrane. In Fig. 4E, the single virus had already escaped from the vesicle and entered the cell. Afterwards, the virus fused with the preexisting vesicle and moved towards the cytoplasm. The whole internalization process of SRV9 was recorded in Movie S4 (Supplementary Information).

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