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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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Nocodazole affects the movement of viral replication complexes (RCs) to the LDs.(A) The HCV-infected cells (at day 10 p.i.) were transfected with BrUTP in the presence of actinomycin D for 1 h and then treated with either 10 µM nocodazole or 10 µM taxol for 1 h. The cells were co-stained with mouse MAb against bromodeoxyuridine (green) and rabbit polyclonal antibodies against Core (RR8) (red). LDs and nuclei were stained with BODYPI 493/503 (blue) and DAPI (cyan), respectively. Enlarged views of parts of every image are shown (insets). (B) The number of BrUTP-labeled viral RNA was counted manually using an original magnification of ×630 and followed by a quantitation analysis performed by an observer blinded to the experimental treatment. (C) The average distance between the center of the signal emitted by the BrUTP-labeled viral RNA and the nearest edge of LD (HCV RNA-LD distance) were analyzed by using Zeiss LSM Zen software. A total of 20 cells were used for quantitation and calculation of the HCV RNA-LD distance and the number of BrUTP-labeled viral RNA from two independent experiments and error bars represent standard deviations of the mean. N.S., non-significance; *, P<0.01; Noc, nocodazole. Bars, 10 µm.
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pone-0099022-g004: Nocodazole affects the movement of viral replication complexes (RCs) to the LDs.(A) The HCV-infected cells (at day 10 p.i.) were transfected with BrUTP in the presence of actinomycin D for 1 h and then treated with either 10 µM nocodazole or 10 µM taxol for 1 h. The cells were co-stained with mouse MAb against bromodeoxyuridine (green) and rabbit polyclonal antibodies against Core (RR8) (red). LDs and nuclei were stained with BODYPI 493/503 (blue) and DAPI (cyan), respectively. Enlarged views of parts of every image are shown (insets). (B) The number of BrUTP-labeled viral RNA was counted manually using an original magnification of ×630 and followed by a quantitation analysis performed by an observer blinded to the experimental treatment. (C) The average distance between the center of the signal emitted by the BrUTP-labeled viral RNA and the nearest edge of LD (HCV RNA-LD distance) were analyzed by using Zeiss LSM Zen software. A total of 20 cells were used for quantitation and calculation of the HCV RNA-LD distance and the number of BrUTP-labeled viral RNA from two independent experiments and error bars represent standard deviations of the mean. N.S., non-significance; *, P<0.01; Noc, nocodazole. Bars, 10 µm.

Mentions: We further confirmed the mode of transportation of the HCV RC to the LD by immunofluorescent labeling of newly synthesized HCV RNA with BrUTP [43]. In the DMSO control and taxol-treated cells, the number of BrUTP-labeled speckles averaged 30 per cell, and the average distance between speckle and LD was 0.43 µm (Fig. 4). In the presence of nocodazole, the number of speckles was reduced to an average of 11 per cell (Fig. 4B), indicating that the HCV RNA replication is partially suppressed by nocodazole treatments, consistent with the previous reports [8]–[10]. Correspondingly, the average distance between the speckle center and the nearest edge of LD increased from 0.43 µm in the DMSO controls and taxol-treated cells to 1.63 µm in the nocodazole-treated cells (Fig. 4C), suggesting that the HCV RCs are normally delivered to the vicinity of the LDs through microtubule and that this transport is disrupted by nocodazole. Taken together, these data suggest that NS5A or the NS5A-containing replication complexes and Core protein are transported to the LDs through microtubules.


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 affects the movement of viral replication complexes (RCs) to the LDs.(A) The HCV-infected cells (at day 10 p.i.) were transfected with BrUTP in the presence of actinomycin D for 1 h and then treated with either 10 µM nocodazole or 10 µM taxol for 1 h. The cells were co-stained with mouse MAb against bromodeoxyuridine (green) and rabbit polyclonal antibodies against Core (RR8) (red). LDs and nuclei were stained with BODYPI 493/503 (blue) and DAPI (cyan), respectively. Enlarged views of parts of every image are shown (insets). (B) The number of BrUTP-labeled viral RNA was counted manually using an original magnification of ×630 and followed by a quantitation analysis performed by an observer blinded to the experimental treatment. (C) The average distance between the center of the signal emitted by the BrUTP-labeled viral RNA and the nearest edge of LD (HCV RNA-LD distance) were analyzed by using Zeiss LSM Zen software. A total of 20 cells were used for quantitation and calculation of the HCV RNA-LD distance and the number of BrUTP-labeled viral RNA from two independent experiments and error bars represent standard deviations of the mean. N.S., non-significance; *, P<0.01; Noc, nocodazole. Bars, 10 µm.
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Related In: Results  -  Collection

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pone-0099022-g004: Nocodazole affects the movement of viral replication complexes (RCs) to the LDs.(A) The HCV-infected cells (at day 10 p.i.) were transfected with BrUTP in the presence of actinomycin D for 1 h and then treated with either 10 µM nocodazole or 10 µM taxol for 1 h. The cells were co-stained with mouse MAb against bromodeoxyuridine (green) and rabbit polyclonal antibodies against Core (RR8) (red). LDs and nuclei were stained with BODYPI 493/503 (blue) and DAPI (cyan), respectively. Enlarged views of parts of every image are shown (insets). (B) The number of BrUTP-labeled viral RNA was counted manually using an original magnification of ×630 and followed by a quantitation analysis performed by an observer blinded to the experimental treatment. (C) The average distance between the center of the signal emitted by the BrUTP-labeled viral RNA and the nearest edge of LD (HCV RNA-LD distance) were analyzed by using Zeiss LSM Zen software. A total of 20 cells were used for quantitation and calculation of the HCV RNA-LD distance and the number of BrUTP-labeled viral RNA from two independent experiments and error bars represent standard deviations of the mean. N.S., non-significance; *, P<0.01; Noc, nocodazole. Bars, 10 µm.
Mentions: We further confirmed the mode of transportation of the HCV RC to the LD by immunofluorescent labeling of newly synthesized HCV RNA with BrUTP [43]. In the DMSO control and taxol-treated cells, the number of BrUTP-labeled speckles averaged 30 per cell, and the average distance between speckle and LD was 0.43 µm (Fig. 4). In the presence of nocodazole, the number of speckles was reduced to an average of 11 per cell (Fig. 4B), indicating that the HCV RNA replication is partially suppressed by nocodazole treatments, consistent with the previous reports [8]–[10]. Correspondingly, the average distance between the speckle center and the nearest edge of LD increased from 0.43 µm in the DMSO controls and taxol-treated cells to 1.63 µm in the nocodazole-treated cells (Fig. 4C), suggesting that the HCV RCs are normally delivered to the vicinity of the LDs through microtubule and that this transport is disrupted by nocodazole. Taken together, these data suggest that NS5A or the NS5A-containing replication complexes and Core protein are transported to the LDs through microtubules.

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