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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

Biological characterization of purified SRV9.(A) Electron micrograph of density gradient-purified SRV9 negatively stained with PTA. (B) Confocal image of Cy5-stained SRV9 particles immobilized on glass coverslips. (C) The fluorescence image of Vero cells that internalized Cy5-labled SRV9 particles. The nucleus region was circled by white dashed line.
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f1: Biological characterization of purified SRV9.(A) Electron micrograph of density gradient-purified SRV9 negatively stained with PTA. (B) Confocal image of Cy5-stained SRV9 particles immobilized on glass coverslips. (C) The fluorescence image of Vero cells that internalized Cy5-labled SRV9 particles. The nucleus region was circled by white dashed line.

Mentions: The SRV9 belongs to living attenuated rabies vaccine strain with good safety and immunogenicity, resulting from the alteration of Arg→Ser in the 333 position of glycoprotein of rabies viruses31. The amino acid mutation does not alter the features of rabies viruses in morphology. The electron microscopic analysis showed that the SRV9 particles are in bullet shape and measured on average 180nm in length and 80nm in width (Fig. 1A). The SRV9 is morphologically consistent with the wild type (WT) RABV. To carry out single particle fluorescence imaging, we covalently labeled the glycoprotein of SRV9 with fluorescent dye molecules Cy5-NHS. The Cy5 is an amino-active dye that spontaneously binds with the glycoproteins, and its surface density was sufficiently high so that its fluorescence allowed the labeled SRV9 to be clearly detected. The confocal laser scanning microscopy (CLSM) was used to image the Cy5-labeled SRV9 particles adsorbed onto glass coverslips. As shown in Fig. 1B, some scattered individual SRV9 particles were observed, which indicates that the purified SRV9 particles were suitable for single particle tracking experiment. Next, we co-incubated the cells with labeled SRV9 particles for 30 min at 37 °C. The confocal image revealed that numerous virus particles had entered the cells and distributed throughout the interior of the cell, including the cell periphery and perinuclear positions (Fig. 1C). The result indicated that the labeled SRV9 still enable to enter the 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)

Biological characterization of purified SRV9.(A) Electron micrograph of density gradient-purified SRV9 negatively stained with PTA. (B) Confocal image of Cy5-stained SRV9 particles immobilized on glass coverslips. (C) The fluorescence image of Vero cells that internalized Cy5-labled SRV9 particles. The nucleus region was circled by white dashed line.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Biological characterization of purified SRV9.(A) Electron micrograph of density gradient-purified SRV9 negatively stained with PTA. (B) Confocal image of Cy5-stained SRV9 particles immobilized on glass coverslips. (C) The fluorescence image of Vero cells that internalized Cy5-labled SRV9 particles. The nucleus region was circled by white dashed line.
Mentions: The SRV9 belongs to living attenuated rabies vaccine strain with good safety and immunogenicity, resulting from the alteration of Arg→Ser in the 333 position of glycoprotein of rabies viruses31. The amino acid mutation does not alter the features of rabies viruses in morphology. The electron microscopic analysis showed that the SRV9 particles are in bullet shape and measured on average 180nm in length and 80nm in width (Fig. 1A). The SRV9 is morphologically consistent with the wild type (WT) RABV. To carry out single particle fluorescence imaging, we covalently labeled the glycoprotein of SRV9 with fluorescent dye molecules Cy5-NHS. The Cy5 is an amino-active dye that spontaneously binds with the glycoproteins, and its surface density was sufficiently high so that its fluorescence allowed the labeled SRV9 to be clearly detected. The confocal laser scanning microscopy (CLSM) was used to image the Cy5-labeled SRV9 particles adsorbed onto glass coverslips. As shown in Fig. 1B, some scattered individual SRV9 particles were observed, which indicates that the purified SRV9 particles were suitable for single particle tracking experiment. Next, we co-incubated the cells with labeled SRV9 particles for 30 min at 37 °C. The confocal image revealed that numerous virus particles had entered the cells and distributed throughout the interior of the cell, including the cell periphery and perinuclear positions (Fig. 1C). The result indicated that the labeled SRV9 still enable to enter the 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