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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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Effects of Nocodazole on movement of NS5A and Core protein.Huh7.5 cells were first infected with HCV Jc1 virus at a multiplicity of infection of 0.5 for 3 h, and then the cells were treated with either nocodazole (1 to 4 µM) or taxol (1 to 4 µM) at various concentrations for 2 days for the analysis of colocalization of Core protein or NS5A and calnexin. The cells were co-stained with a polyclonal antibody against calnexin (red) to visualize the ER and a MAb against Core protein (green) (A) or NS5A (green) (C). Nuclei were stained with DAPI (blue). Enlarged views of parts of every image are shown (insets). The same images are shown for Core (green) or NS5A (green) and with DAPI (blue) in the lower panels of Fig. A and C, respectively. Colocalization efficiency between Core protein and calnexin (B) and between NS5A and calnexin (D) was analyzed by using Zeiss LSM Zen software. A total of 20 cells were used for calculation of colocalization efficiency from two independent experiments and error bars represent standard deviations of the mean.
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pone-0099022-g003: Effects of Nocodazole on movement of NS5A and Core protein.Huh7.5 cells were first infected with HCV Jc1 virus at a multiplicity of infection of 0.5 for 3 h, and then the cells were treated with either nocodazole (1 to 4 µM) or taxol (1 to 4 µM) at various concentrations for 2 days for the analysis of colocalization of Core protein or NS5A and calnexin. The cells were co-stained with a polyclonal antibody against calnexin (red) to visualize the ER and a MAb against Core protein (green) (A) or NS5A (green) (C). Nuclei were stained with DAPI (blue). Enlarged views of parts of every image are shown (insets). The same images are shown for Core (green) or NS5A (green) and with DAPI (blue) in the lower panels of Fig. A and C, respectively. Colocalization efficiency between Core protein and calnexin (B) and between NS5A and calnexin (D) was analyzed by using Zeiss LSM Zen software. A total of 20 cells were used for calculation of colocalization efficiency from two independent experiments and error bars represent standard deviations of the mean.

Mentions: Under the control conditions [dimethyl sulfoxide (DMSO)], LD colocalized with Core protein throughout the entire cytoplasm, including the perinuclear region; the proportion of LD that colocalized with Core protein was 69%. When the cells were treated with increasing concentrations of nocodazole, a dose-dependent decrease in the colocalization coefficient of LD with Core protein was observed (Fig. 2A upper panels and 2B). The colocalization coefficient of LD with NS5A also decreased correspondingly (Fig. 2C upper panels and 2D). Taxol did not have effects on this coefficient. In order to rule out the possibility that the dose-dependent decrease in the colocalization of LD with Core or NS5A by nocodazole treatments might have been the consequence of a decrease in the amounts of Core and NS5A proteins, a further analysis of Core protein (Fig. 2A and 3A, lower panels) and NS5A (Fig. 2C and 3C, lower panels) labeling in cells was carried out using the MetaMorph Integrated Morphometry Analysis. In nocodazole-treated cells, there was no significant change on the total fluorescence intensities of Core and NS5A relative to control cells (Fig. 2F and 2G). Immunoblot analysis also showed that the levels of NS5A and Core proteins in the cells were not significantly affected by the low concentration (up to 4 µM) of nocodazole or taxol used (Fig. 2H,). This was in contrast to the effects of nocodazole at 10 or more µM used in most of the published studies, which inhibited HCV RNA replication and thereby HCV protein level [8], [10], [42]. These data support the conclusion that the nocodazole treatment at low concentrations affected the colocalization of Core and NS5A with LD (Fig. 2B and Fig. 2D). Taken together, these results indicated that microtubules are involved in the transport of Core protein and NS5A to the LD.


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)

Effects of Nocodazole on movement of NS5A and Core protein.Huh7.5 cells were first infected with HCV Jc1 virus at a multiplicity of infection of 0.5 for 3 h, and then the cells were treated with either nocodazole (1 to 4 µM) or taxol (1 to 4 µM) at various concentrations for 2 days for the analysis of colocalization of Core protein or NS5A and calnexin. The cells were co-stained with a polyclonal antibody against calnexin (red) to visualize the ER and a MAb against Core protein (green) (A) or NS5A (green) (C). Nuclei were stained with DAPI (blue). Enlarged views of parts of every image are shown (insets). The same images are shown for Core (green) or NS5A (green) and with DAPI (blue) in the lower panels of Fig. A and C, respectively. Colocalization efficiency between Core protein and calnexin (B) and between NS5A and calnexin (D) was analyzed by using Zeiss LSM Zen software. A total of 20 cells were used for calculation of colocalization efficiency from two independent experiments and error bars represent standard deviations of the mean.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0099022-g003: Effects of Nocodazole on movement of NS5A and Core protein.Huh7.5 cells were first infected with HCV Jc1 virus at a multiplicity of infection of 0.5 for 3 h, and then the cells were treated with either nocodazole (1 to 4 µM) or taxol (1 to 4 µM) at various concentrations for 2 days for the analysis of colocalization of Core protein or NS5A and calnexin. The cells were co-stained with a polyclonal antibody against calnexin (red) to visualize the ER and a MAb against Core protein (green) (A) or NS5A (green) (C). Nuclei were stained with DAPI (blue). Enlarged views of parts of every image are shown (insets). The same images are shown for Core (green) or NS5A (green) and with DAPI (blue) in the lower panels of Fig. A and C, respectively. Colocalization efficiency between Core protein and calnexin (B) and between NS5A and calnexin (D) was analyzed by using Zeiss LSM Zen software. A total of 20 cells were used for calculation of colocalization efficiency from two independent experiments and error bars represent standard deviations of the mean.
Mentions: Under the control conditions [dimethyl sulfoxide (DMSO)], LD colocalized with Core protein throughout the entire cytoplasm, including the perinuclear region; the proportion of LD that colocalized with Core protein was 69%. When the cells were treated with increasing concentrations of nocodazole, a dose-dependent decrease in the colocalization coefficient of LD with Core protein was observed (Fig. 2A upper panels and 2B). The colocalization coefficient of LD with NS5A also decreased correspondingly (Fig. 2C upper panels and 2D). Taxol did not have effects on this coefficient. In order to rule out the possibility that the dose-dependent decrease in the colocalization of LD with Core or NS5A by nocodazole treatments might have been the consequence of a decrease in the amounts of Core and NS5A proteins, a further analysis of Core protein (Fig. 2A and 3A, lower panels) and NS5A (Fig. 2C and 3C, lower panels) labeling in cells was carried out using the MetaMorph Integrated Morphometry Analysis. In nocodazole-treated cells, there was no significant change on the total fluorescence intensities of Core and NS5A relative to control cells (Fig. 2F and 2G). Immunoblot analysis also showed that the levels of NS5A and Core proteins in the cells were not significantly affected by the low concentration (up to 4 µM) of nocodazole or taxol used (Fig. 2H,). This was in contrast to the effects of nocodazole at 10 or more µM used in most of the published studies, which inhibited HCV RNA replication and thereby HCV protein level [8], [10], [42]. These data support the conclusion that the nocodazole treatment at low concentrations affected the colocalization of Core and NS5A with LD (Fig. 2B and Fig. 2D). Taken together, these results indicated that microtubules are involved in the transport of Core protein and NS5A to the LD.

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