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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

Nocodazole blocks the transport of NS5A and Core protein to the lipid droplet in a dose-dependent manner.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. The cells were stained with anti-Core (red) (A) or -NS5A (red) (C) antibodies. LDs and nuclei were stained with BODYPI 493/503 (green) and DAPI (blue), respectively. Enlarged views of parts of every image are shown (insets). The same images are shown for Core (red) or NS5A (red) and with DAPI (blue) in the lower panels of Fig. A and C, respectively. Colocalization efficiency between LD and Core protein (B) and between LD and NS5A (D) was analyzed by using Zeiss LSM Zen software. (E) Analysis of cellular proliferation and survival by MTS assay. (F, G) Quantitation of confocal microscopic fluorescent signals of Core and NS5A in cells. The total fluorescence intensities of Core protein and NS5A were measured using MetaMorph Integrated Morphometry Analysis. A total of 20 cells were used for calculation of colocalization efficiency and total fluorescence intensity from two independent experiments and error bars represent standard deviations of the mean. Noc, nocodazole. Bars, 10 µm. (H) In parallel, the cell lysates were collected and then immunoblotted with antibodies against Core and NS5A. Results were quantified by PhosphorImager counting.
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pone-0099022-g002: Nocodazole blocks the transport of NS5A and Core protein to the lipid droplet in a dose-dependent manner.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. The cells were stained with anti-Core (red) (A) or -NS5A (red) (C) antibodies. LDs and nuclei were stained with BODYPI 493/503 (green) and DAPI (blue), respectively. Enlarged views of parts of every image are shown (insets). The same images are shown for Core (red) or NS5A (red) and with DAPI (blue) in the lower panels of Fig. A and C, respectively. Colocalization efficiency between LD and Core protein (B) and between LD and NS5A (D) was analyzed by using Zeiss LSM Zen software. (E) Analysis of cellular proliferation and survival by MTS assay. (F, G) Quantitation of confocal microscopic fluorescent signals of Core and NS5A in cells. The total fluorescence intensities of Core protein and NS5A were measured using MetaMorph Integrated Morphometry Analysis. A total of 20 cells were used for calculation of colocalization efficiency and total fluorescence intensity from two independent experiments and error bars represent standard deviations of the mean. Noc, nocodazole. Bars, 10 µm. (H) In parallel, the cell lysates were collected and then immunoblotted with antibodies against Core and NS5A. Results were quantified by PhosphorImager counting.

Mentions: We used nocodazole (which induces microtubule depolymerization) or taxol (which stabilizes tubulin polymerization) to examine whether intact microtubules are required for the transport of Core protein, NS5A or viral RCs to the LD. Huh7.5 cells were first infected with HCV, and treated with either nocodazole or taxol at 3 hr p.i. for 2 days. The colocalization coefficient of LD with Core protein or NS5A was then calculated. Under these conditions, cell viability was not affected, as revealed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) assay (Fig. 2E).


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)

Nocodazole blocks the transport of NS5A and Core protein to the lipid droplet in a dose-dependent manner.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. The cells were stained with anti-Core (red) (A) or -NS5A (red) (C) antibodies. LDs and nuclei were stained with BODYPI 493/503 (green) and DAPI (blue), respectively. Enlarged views of parts of every image are shown (insets). The same images are shown for Core (red) or NS5A (red) and with DAPI (blue) in the lower panels of Fig. A and C, respectively. Colocalization efficiency between LD and Core protein (B) and between LD and NS5A (D) was analyzed by using Zeiss LSM Zen software. (E) Analysis of cellular proliferation and survival by MTS assay. (F, G) Quantitation of confocal microscopic fluorescent signals of Core and NS5A in cells. The total fluorescence intensities of Core protein and NS5A were measured using MetaMorph Integrated Morphometry Analysis. A total of 20 cells were used for calculation of colocalization efficiency and total fluorescence intensity from two independent experiments and error bars represent standard deviations of the mean. Noc, nocodazole. Bars, 10 µm. (H) In parallel, the cell lysates were collected and then immunoblotted with antibodies against Core and NS5A. Results were quantified by PhosphorImager counting.
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pone-0099022-g002: Nocodazole blocks the transport of NS5A and Core protein to the lipid droplet in a dose-dependent manner.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. The cells were stained with anti-Core (red) (A) or -NS5A (red) (C) antibodies. LDs and nuclei were stained with BODYPI 493/503 (green) and DAPI (blue), respectively. Enlarged views of parts of every image are shown (insets). The same images are shown for Core (red) or NS5A (red) and with DAPI (blue) in the lower panels of Fig. A and C, respectively. Colocalization efficiency between LD and Core protein (B) and between LD and NS5A (D) was analyzed by using Zeiss LSM Zen software. (E) Analysis of cellular proliferation and survival by MTS assay. (F, G) Quantitation of confocal microscopic fluorescent signals of Core and NS5A in cells. The total fluorescence intensities of Core protein and NS5A were measured using MetaMorph Integrated Morphometry Analysis. A total of 20 cells were used for calculation of colocalization efficiency and total fluorescence intensity from two independent experiments and error bars represent standard deviations of the mean. Noc, nocodazole. Bars, 10 µm. (H) In parallel, the cell lysates were collected and then immunoblotted with antibodies against Core and NS5A. Results were quantified by PhosphorImager counting.
Mentions: We used nocodazole (which induces microtubule depolymerization) or taxol (which stabilizes tubulin polymerization) to examine whether intact microtubules are required for the transport of Core protein, NS5A or viral RCs to the LD. Huh7.5 cells were first infected with HCV, and treated with either nocodazole or taxol at 3 hr p.i. for 2 days. The colocalization coefficient of LD with Core protein or NS5A was then calculated. Under these conditions, cell viability was not affected, as revealed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) assay (Fig. 2E).

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