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Nonstructural protein 5A is incorporated into hepatitis C virus low-density particle through interaction with core protein and microtubules during intracellular transport.

Lai CK, Saxena V, Tseng CH, Jeng KS, Kohara M, Lai MM - PLoS ONE (2014)

Bottom Line: Here, we demonstrate that HCV replication complex along with NS5A and Core protein was transported to the lipid droplet (LD) through microtubules, and NS5A-Core complexes were then transported from LD through early-to-late endosomes to the plasma membrane via microtubules.Furthermore, exosomal markers CD63 and CD81 were also detected in the low-density fractions, but not in the high-density fractions.Overall, our results suggest that HCV NS5A is associated with the core of the low-density virus particles which exit the cell through a preexisting endosome/exosome pathway and may contribute to HCV natural infection.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan; Graduate Institute of Toxicology, National Taiwan University, Taipei, Taiwan.

ABSTRACT
Nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) serves dual functions in viral RNA replication and virus assembly. Here, we demonstrate that HCV replication complex along with NS5A and Core protein was transported to the lipid droplet (LD) through microtubules, and NS5A-Core complexes were then transported from LD through early-to-late endosomes to the plasma membrane via microtubules. Further studies by cofractionation analysis and immunoelectron microscopy of the released particles showed that NS5A-Core complexes, but not NS4B, were present in the low-density fractions, but not in the high-density fractions, of the HCV RNA-containing virions and associated with the internal virion core. Furthermore, exosomal markers CD63 and CD81 were also detected in the low-density fractions, but not in the high-density fractions. Overall, our results suggest that HCV NS5A is associated with the core of the low-density virus particles which exit the cell through a preexisting endosome/exosome pathway and may contribute to HCV natural infection.

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Related in: MedlinePlus

Colocalization of NS5A with Core protein and microtubules in lipid droplets.HCV-infected cells (at day 10 p.i.) were co-stained with anti-tubulin (green), -NS5A (red) (A) and/or -Core (red, B; blue, C) antibodies. Lipid droplets (LDs) were stained with BODYPI 493/503 (blue, A and B; brown, C) and nuclei with DAPI (gray). Stained cells were examined by confocal fluorescence microscope. Merging of the images in the green, red, blue, and gray channels generated the pictures in Fig. A and B. Fig. C was generated by merging the images in the green, red, blue, and brown channels. Yellow indicates overlapping localization of the green and red channels, cyan indicates overlapping localization of the green and blue channels, magenta indicates overlapping localization of the red and blue channels, and white indicates overlapping localization of the red, green, and blue channels. The upper third panels in Fig. A and B and the lower third panel in Fig. C are enlarged areas from the merged image. The same enlarged area is defined in terms of two proteins at a time, as indicated, in the adjoining three panels in Fig. A and B, and six panels in Fig. C. Bars, 10 µm.
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pone-0099022-g001: Colocalization of NS5A with Core protein and microtubules in lipid droplets.HCV-infected cells (at day 10 p.i.) were co-stained with anti-tubulin (green), -NS5A (red) (A) and/or -Core (red, B; blue, C) antibodies. Lipid droplets (LDs) were stained with BODYPI 493/503 (blue, A and B; brown, C) and nuclei with DAPI (gray). Stained cells were examined by confocal fluorescence microscope. Merging of the images in the green, red, blue, and gray channels generated the pictures in Fig. A and B. Fig. C was generated by merging the images in the green, red, blue, and brown channels. Yellow indicates overlapping localization of the green and red channels, cyan indicates overlapping localization of the green and blue channels, magenta indicates overlapping localization of the red and blue channels, and white indicates overlapping localization of the red, green, and blue channels. The upper third panels in Fig. A and B and the lower third panel in Fig. C are enlarged areas from the merged image. The same enlarged area is defined in terms of two proteins at a time, as indicated, in the adjoining three panels in Fig. A and B, and six panels in Fig. C. Bars, 10 µm.

Mentions: Our previous studies have shown that microtubule provides the track for the movement of HCV RCs through NS5A-microtubule interaction [7] and that this transport is required for virus release [10]. We propose that microtubules also provide tracks for the transport of NS5A or the NS5A-containing RC and Core protein to reach the LD, where virus assembly occurs. To test this possibility, we first investigated whether Core-containing LDs colocalized with NS5A and associated with microtubules in HCV-infected Huh7.5 cells [at day 10 postinfection (p.i.)]. Immunofluorescence staining revealed that NS5A and Core protein together (Fig. 1C), or either one alone (Fig. 1A and 1B), is colocalized on the surface of LD and is closely associated with tubulins.


Nonstructural protein 5A is incorporated into hepatitis C virus low-density particle through interaction with core protein and microtubules during intracellular transport.

Lai CK, Saxena V, Tseng CH, Jeng KS, Kohara M, Lai MM - PLoS ONE (2014)

Colocalization of NS5A with Core protein and microtubules in lipid droplets.HCV-infected cells (at day 10 p.i.) were co-stained with anti-tubulin (green), -NS5A (red) (A) and/or -Core (red, B; blue, C) antibodies. Lipid droplets (LDs) were stained with BODYPI 493/503 (blue, A and B; brown, C) and nuclei with DAPI (gray). Stained cells were examined by confocal fluorescence microscope. Merging of the images in the green, red, blue, and gray channels generated the pictures in Fig. A and B. Fig. C was generated by merging the images in the green, red, blue, and brown channels. Yellow indicates overlapping localization of the green and red channels, cyan indicates overlapping localization of the green and blue channels, magenta indicates overlapping localization of the red and blue channels, and white indicates overlapping localization of the red, green, and blue channels. The upper third panels in Fig. A and B and the lower third panel in Fig. C are enlarged areas from the merged image. The same enlarged area is defined in terms of two proteins at a time, as indicated, in the adjoining three panels in Fig. A and B, and six panels in Fig. C. Bars, 10 µm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4048239&req=5

pone-0099022-g001: Colocalization of NS5A with Core protein and microtubules in lipid droplets.HCV-infected cells (at day 10 p.i.) were co-stained with anti-tubulin (green), -NS5A (red) (A) and/or -Core (red, B; blue, C) antibodies. Lipid droplets (LDs) were stained with BODYPI 493/503 (blue, A and B; brown, C) and nuclei with DAPI (gray). Stained cells were examined by confocal fluorescence microscope. Merging of the images in the green, red, blue, and gray channels generated the pictures in Fig. A and B. Fig. C was generated by merging the images in the green, red, blue, and brown channels. Yellow indicates overlapping localization of the green and red channels, cyan indicates overlapping localization of the green and blue channels, magenta indicates overlapping localization of the red and blue channels, and white indicates overlapping localization of the red, green, and blue channels. The upper third panels in Fig. A and B and the lower third panel in Fig. C are enlarged areas from the merged image. The same enlarged area is defined in terms of two proteins at a time, as indicated, in the adjoining three panels in Fig. A and B, and six panels in Fig. C. Bars, 10 µm.
Mentions: Our previous studies have shown that microtubule provides the track for the movement of HCV RCs through NS5A-microtubule interaction [7] and that this transport is required for virus release [10]. We propose that microtubules also provide tracks for the transport of NS5A or the NS5A-containing RC and Core protein to reach the LD, where virus assembly occurs. To test this possibility, we first investigated whether Core-containing LDs colocalized with NS5A and associated with microtubules in HCV-infected Huh7.5 cells [at day 10 postinfection (p.i.)]. Immunofluorescence staining revealed that NS5A and Core protein together (Fig. 1C), or either one alone (Fig. 1A and 1B), is colocalized on the surface of LD and is closely associated with tubulins.

Bottom Line: Here, we demonstrate that HCV replication complex along with NS5A and Core protein was transported to the lipid droplet (LD) through microtubules, and NS5A-Core complexes were then transported from LD through early-to-late endosomes to the plasma membrane via microtubules.Furthermore, exosomal markers CD63 and CD81 were also detected in the low-density fractions, but not in the high-density fractions.Overall, our results suggest that HCV NS5A is associated with the core of the low-density virus particles which exit the cell through a preexisting endosome/exosome pathway and may contribute to HCV natural infection.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan; Graduate Institute of Toxicology, National Taiwan University, Taipei, Taiwan.

ABSTRACT
Nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) serves dual functions in viral RNA replication and virus assembly. Here, we demonstrate that HCV replication complex along with NS5A and Core protein was transported to the lipid droplet (LD) through microtubules, and NS5A-Core complexes were then transported from LD through early-to-late endosomes to the plasma membrane via microtubules. Further studies by cofractionation analysis and immunoelectron microscopy of the released particles showed that NS5A-Core complexes, but not NS4B, were present in the low-density fractions, but not in the high-density fractions, of the HCV RNA-containing virions and associated with the internal virion core. Furthermore, exosomal markers CD63 and CD81 were also detected in the low-density fractions, but not in the high-density fractions. Overall, our results suggest that HCV NS5A is associated with the core of the low-density virus particles which exit the cell through a preexisting endosome/exosome pathway and may contribute to HCV natural infection.

Show MeSH
Related in: MedlinePlus