Limits...
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

Motility of SRV9 in the untreated, nocodazole-treated Vero cells.(A,B) Confocal images of SRV9 (red) in live cells labeled with DiO dye (green) under untreated and nocodazole-treated conditions, respectively. (C, D) Representative trajectories corresponding to the untreated and nocodazole-treated images, respectively. Their instantaneous speeds as a function of time are shown in E and F. The green arrow and the yellow arrow in (C) indicate relatively high transport speed and low transport speed in a limited region, respectively. Scale bar: 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4493577&req=5

f6: Motility of SRV9 in the untreated, nocodazole-treated Vero cells.(A,B) Confocal images of SRV9 (red) in live cells labeled with DiO dye (green) under untreated and nocodazole-treated conditions, respectively. (C, D) Representative trajectories corresponding to the untreated and nocodazole-treated images, respectively. Their instantaneous speeds as a function of time are shown in E and F. The green arrow and the yellow arrow in (C) indicate relatively high transport speed and low transport speed in a limited region, respectively. Scale bar: 10 μm.

Mentions: Figure 6A and Movie S6 in Supplementary Information represent typical examples of SRV9 motility in Vero cells. The Matlab software allowed us to analyze the trajectory of single-virus particle inside the cell, with the ability to distinguish the virions inside or on the cell from outside the cell. The analytical results were displayed in Fig. 6C, showing various types of movements. Most of viruses particles were able to achieve relatively high transport speeds (marked by the green arrow in Fig. 6C), despite some particles showed comparatively low speed motion (marked by the yellow arrow in Fig. 6C). The SRV9 particles were able to achieve speeds nearly up to 0.2 μm s−1 (blue trajectory velocity profile in Fig. 6E). Some of these relatively rapid moving particles displayed highly directed movements. It is noted that the slowly moving particles were localized to vesicular structures nonspecifically stained by the lipophilic dye DiO. However, the relatively rapid moving particles didn’t colocalized with the vesicular structures, which means that these viruses were possibly fused with specific endocytic vesicles that need to be labeled by specific makers or were free inside the cytosol. It is worth noting that the actual speeds are virtually much higher than the speeds calculated from the two-dimensional projection. The reason accounting for the deviation is that the virus particles moved inside the cell typically in all three dimensions, but only a two- dimensional projection of three-dimensional motion was measured.


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)

Motility of SRV9 in the untreated, nocodazole-treated Vero cells.(A,B) Confocal images of SRV9 (red) in live cells labeled with DiO dye (green) under untreated and nocodazole-treated conditions, respectively. (C, D) Representative trajectories corresponding to the untreated and nocodazole-treated images, respectively. Their instantaneous speeds as a function of time are shown in E and F. The green arrow and the yellow arrow in (C) indicate relatively high transport speed and low transport speed in a limited region, respectively. Scale bar: 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: Motility of SRV9 in the untreated, nocodazole-treated Vero cells.(A,B) Confocal images of SRV9 (red) in live cells labeled with DiO dye (green) under untreated and nocodazole-treated conditions, respectively. (C, D) Representative trajectories corresponding to the untreated and nocodazole-treated images, respectively. Their instantaneous speeds as a function of time are shown in E and F. The green arrow and the yellow arrow in (C) indicate relatively high transport speed and low transport speed in a limited region, respectively. Scale bar: 10 μm.
Mentions: Figure 6A and Movie S6 in Supplementary Information represent typical examples of SRV9 motility in Vero cells. The Matlab software allowed us to analyze the trajectory of single-virus particle inside the cell, with the ability to distinguish the virions inside or on the cell from outside the cell. The analytical results were displayed in Fig. 6C, showing various types of movements. Most of viruses particles were able to achieve relatively high transport speeds (marked by the green arrow in Fig. 6C), despite some particles showed comparatively low speed motion (marked by the yellow arrow in Fig. 6C). The SRV9 particles were able to achieve speeds nearly up to 0.2 μm s−1 (blue trajectory velocity profile in Fig. 6E). Some of these relatively rapid moving particles displayed highly directed movements. It is noted that the slowly moving particles were localized to vesicular structures nonspecifically stained by the lipophilic dye DiO. However, the relatively rapid moving particles didn’t colocalized with the vesicular structures, which means that these viruses were possibly fused with specific endocytic vesicles that need to be labeled by specific makers or were free inside the cytosol. It is worth noting that the actual speeds are virtually much higher than the speeds calculated from the two-dimensional projection. The reason accounting for the deviation is that the virus particles moved inside the cell typically in all three dimensions, but only a two- dimensional projection of three-dimensional motion was measured.

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