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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

Core-NS5A complexes are transported from perinuclear region to early and late endosomes.(A) The HCV-infected cells (at day 10 p.i.) were labeled with antibodies specific for Core protein (red) and NS5A (green) (upper row) or NS4B (green) (lower row). Plasma membrane and nuclei were stained with WGA Alexa Fluor 647 conjugate (blue) and DAPI (gray), respectively. In parallel with panel A, the HCV-infected cells were labeled with antibodies to Core protein (green) and NS5A (red) or NS4B (red) (B, upper rows). Nuclei were stained with DAPI (blue). Colocalization efficiency between Core protein and NS5A or NS4B was analyzed by using Zeiss LSM Zen software (B, lower panel). Error bars represent standard deviations of the mean from 20 cells in two independent experiments. The images were analyzed by using MetaMorph, and the proportion of cells (of 200 counted) in which the NS5A or NS4B colocalized with Core protein at the cell periphery was calculated (C). (D) Effects of nocodazole (Noc) and vinblastine (VBL) on movement of Core-NS5A protein complex to the cell periphery. The HCV-infected cells (at day 10 p.i.) were treated with nocodazole, vinblastine, or DMSO for 3 h. The cells were labeled with antibodies to Core protein (green), NS5A (red). Nuclei were stained with DAPI (blue). Images with differential interference contrast (DIC) and fluorescence images were then merged. An enlarged view of part of image is shown (inset). In parallel with panel A, the HCV-infected cells were labeled with antibodies to Core protein (red) (E, F, G), NS5A (green, E, F, G) (upper rows), NS4B (green, E, F, G) (lower rows), EEA1 (blue) (F) and Lamp-1 (blue) (G). LDs and nuclei were stained with BODYPI 493/503 (blue) (E) and DAPI (gray), respectively. The second and third panels in each row are magnified views, marked with a white box in the panel at the extreme left, of the perinuclear and the peripheral regions of cytoplasm, respectively (A, E, F, G). Colocalization of Core protein with NS5A or NS4B is depicted as yellow (A, B, D, E, F, G). Colocalization of Core-NS5A or -NS4B complexes with LDs (E), early endosomes (F) or late endosomes (G) is depicted as white. Bars, 10 µm. (H) In parallel, the HCV-infected cells was labeled with anti-NS5A or -NS4B Ab. Bound antibodies were detected using anti-mouse or -rabbit secondary antibodies conjugated to 18- or 12-nm gold particles, respectively. Sections were visualized by EM. Arrows, gold-labeled NS5A (18 nm) and NS4B (12 nm) are indicated. MVB contains several intravesicular vesicles (white arrows). An enlarged view of the perinuclear region of lower left image is shown (inset). Arrows, gold-labeled NS5A and NS4B. PM, plasma membrane; MVB, multivesicular bodies; N, nucleus; NM, nuclear membrane; Bars, 500 nm (left panels) and 100 nm (right panels).
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pone-0099022-g005: Core-NS5A complexes are transported from perinuclear region to early and late endosomes.(A) The HCV-infected cells (at day 10 p.i.) were labeled with antibodies specific for Core protein (red) and NS5A (green) (upper row) or NS4B (green) (lower row). Plasma membrane and nuclei were stained with WGA Alexa Fluor 647 conjugate (blue) and DAPI (gray), respectively. In parallel with panel A, the HCV-infected cells were labeled with antibodies to Core protein (green) and NS5A (red) or NS4B (red) (B, upper rows). Nuclei were stained with DAPI (blue). Colocalization efficiency between Core protein and NS5A or NS4B was analyzed by using Zeiss LSM Zen software (B, lower panel). Error bars represent standard deviations of the mean from 20 cells in two independent experiments. The images were analyzed by using MetaMorph, and the proportion of cells (of 200 counted) in which the NS5A or NS4B colocalized with Core protein at the cell periphery was calculated (C). (D) Effects of nocodazole (Noc) and vinblastine (VBL) on movement of Core-NS5A protein complex to the cell periphery. The HCV-infected cells (at day 10 p.i.) were treated with nocodazole, vinblastine, or DMSO for 3 h. The cells were labeled with antibodies to Core protein (green), NS5A (red). Nuclei were stained with DAPI (blue). Images with differential interference contrast (DIC) and fluorescence images were then merged. An enlarged view of part of image is shown (inset). In parallel with panel A, the HCV-infected cells were labeled with antibodies to Core protein (red) (E, F, G), NS5A (green, E, F, G) (upper rows), NS4B (green, E, F, G) (lower rows), EEA1 (blue) (F) and Lamp-1 (blue) (G). LDs and nuclei were stained with BODYPI 493/503 (blue) (E) and DAPI (gray), respectively. The second and third panels in each row are magnified views, marked with a white box in the panel at the extreme left, of the perinuclear and the peripheral regions of cytoplasm, respectively (A, E, F, G). Colocalization of Core protein with NS5A or NS4B is depicted as yellow (A, B, D, E, F, G). Colocalization of Core-NS5A or -NS4B complexes with LDs (E), early endosomes (F) or late endosomes (G) is depicted as white. Bars, 10 µm. (H) In parallel, the HCV-infected cells was labeled with anti-NS5A or -NS4B Ab. Bound antibodies were detected using anti-mouse or -rabbit secondary antibodies conjugated to 18- or 12-nm gold particles, respectively. Sections were visualized by EM. Arrows, gold-labeled NS5A (18 nm) and NS4B (12 nm) are indicated. MVB contains several intravesicular vesicles (white arrows). An enlarged view of the perinuclear region of lower left image is shown (inset). Arrows, gold-labeled NS5A and NS4B. PM, plasma membrane; MVB, multivesicular bodies; N, nucleus; NM, nuclear membrane; Bars, 500 nm (left panels) and 100 nm (right panels).

Mentions: NS5A, together with Core protein, is involved in virus assembly; the assembled virions are presumably transported to the plasma membrane to be released off the cell. Therefore, we next analyzed whether NS5A is involved in the post-assembly processes of viral life cycle. For this purpose, we examined the colocalization of NS5A with Core protein in two regions of the cytoplasm, viz. the perinuclear (region just around the nucleus) and the peripheral (region just underneath the plasma membrane) regions. HCV-infected cells were co-stained with fluorescent (Alexa 647) WGA (which binds glycoproteins on the cell membrane), which serves as a membrane marker, anti-Core, and either anti-NS5A or -NS4B Abs. As shown in Fig. 5A, NS5A and Core protein colocalized both in the perinuclear region and at the cell periphery. In contrast, NS4B colocalized with the Core protein only in the perinuclear region of cytoplasm, but not in the peripheral region. Analysis of a large number of cells indicated that Core protein was colocalized with NS5A throughout the entire cytoplasm, but not with NS4B (Fig. 5B, upper panels). The total fraction of NS5A that colocalized with Core protein was 76%, while the corresponding NS4B was 51% (Fig. 5B, lower panel). Further, the NS5A-Core complex colocalized in the peripheral region in 33% of the cells, whereas NS4B-Core did not colocalize at all in the same region (Fig. 5C). We next examined the possibility that the Core-NS5A complexes are also transported along microtubule to the cell periphery. In the DMSO-treated cells, NS5A colocalized with Core protein in both the perinuclear and peripheral regions (Fig.5D). After treatment with either nocodazole or vinblastine, the Core-NS5A complexes clustered almost exclusively in the perinuclear region (Fig. 5D). These results suggested that microtubules are required for the transport of Core-NS5A complexes to the cell periphery. This observation further suggests that NS5A, but not NS4B, is involved in the post-assembly transport of virus particles.


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)

Core-NS5A complexes are transported from perinuclear region to early and late endosomes.(A) The HCV-infected cells (at day 10 p.i.) were labeled with antibodies specific for Core protein (red) and NS5A (green) (upper row) or NS4B (green) (lower row). Plasma membrane and nuclei were stained with WGA Alexa Fluor 647 conjugate (blue) and DAPI (gray), respectively. In parallel with panel A, the HCV-infected cells were labeled with antibodies to Core protein (green) and NS5A (red) or NS4B (red) (B, upper rows). Nuclei were stained with DAPI (blue). Colocalization efficiency between Core protein and NS5A or NS4B was analyzed by using Zeiss LSM Zen software (B, lower panel). Error bars represent standard deviations of the mean from 20 cells in two independent experiments. The images were analyzed by using MetaMorph, and the proportion of cells (of 200 counted) in which the NS5A or NS4B colocalized with Core protein at the cell periphery was calculated (C). (D) Effects of nocodazole (Noc) and vinblastine (VBL) on movement of Core-NS5A protein complex to the cell periphery. The HCV-infected cells (at day 10 p.i.) were treated with nocodazole, vinblastine, or DMSO for 3 h. The cells were labeled with antibodies to Core protein (green), NS5A (red). Nuclei were stained with DAPI (blue). Images with differential interference contrast (DIC) and fluorescence images were then merged. An enlarged view of part of image is shown (inset). In parallel with panel A, the HCV-infected cells were labeled with antibodies to Core protein (red) (E, F, G), NS5A (green, E, F, G) (upper rows), NS4B (green, E, F, G) (lower rows), EEA1 (blue) (F) and Lamp-1 (blue) (G). LDs and nuclei were stained with BODYPI 493/503 (blue) (E) and DAPI (gray), respectively. The second and third panels in each row are magnified views, marked with a white box in the panel at the extreme left, of the perinuclear and the peripheral regions of cytoplasm, respectively (A, E, F, G). Colocalization of Core protein with NS5A or NS4B is depicted as yellow (A, B, D, E, F, G). Colocalization of Core-NS5A or -NS4B complexes with LDs (E), early endosomes (F) or late endosomes (G) is depicted as white. Bars, 10 µm. (H) In parallel, the HCV-infected cells was labeled with anti-NS5A or -NS4B Ab. Bound antibodies were detected using anti-mouse or -rabbit secondary antibodies conjugated to 18- or 12-nm gold particles, respectively. Sections were visualized by EM. Arrows, gold-labeled NS5A (18 nm) and NS4B (12 nm) are indicated. MVB contains several intravesicular vesicles (white arrows). An enlarged view of the perinuclear region of lower left image is shown (inset). Arrows, gold-labeled NS5A and NS4B. PM, plasma membrane; MVB, multivesicular bodies; N, nucleus; NM, nuclear membrane; Bars, 500 nm (left panels) and 100 nm (right panels).
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pone-0099022-g005: Core-NS5A complexes are transported from perinuclear region to early and late endosomes.(A) The HCV-infected cells (at day 10 p.i.) were labeled with antibodies specific for Core protein (red) and NS5A (green) (upper row) or NS4B (green) (lower row). Plasma membrane and nuclei were stained with WGA Alexa Fluor 647 conjugate (blue) and DAPI (gray), respectively. In parallel with panel A, the HCV-infected cells were labeled with antibodies to Core protein (green) and NS5A (red) or NS4B (red) (B, upper rows). Nuclei were stained with DAPI (blue). Colocalization efficiency between Core protein and NS5A or NS4B was analyzed by using Zeiss LSM Zen software (B, lower panel). Error bars represent standard deviations of the mean from 20 cells in two independent experiments. The images were analyzed by using MetaMorph, and the proportion of cells (of 200 counted) in which the NS5A or NS4B colocalized with Core protein at the cell periphery was calculated (C). (D) Effects of nocodazole (Noc) and vinblastine (VBL) on movement of Core-NS5A protein complex to the cell periphery. The HCV-infected cells (at day 10 p.i.) were treated with nocodazole, vinblastine, or DMSO for 3 h. The cells were labeled with antibodies to Core protein (green), NS5A (red). Nuclei were stained with DAPI (blue). Images with differential interference contrast (DIC) and fluorescence images were then merged. An enlarged view of part of image is shown (inset). In parallel with panel A, the HCV-infected cells were labeled with antibodies to Core protein (red) (E, F, G), NS5A (green, E, F, G) (upper rows), NS4B (green, E, F, G) (lower rows), EEA1 (blue) (F) and Lamp-1 (blue) (G). LDs and nuclei were stained with BODYPI 493/503 (blue) (E) and DAPI (gray), respectively. The second and third panels in each row are magnified views, marked with a white box in the panel at the extreme left, of the perinuclear and the peripheral regions of cytoplasm, respectively (A, E, F, G). Colocalization of Core protein with NS5A or NS4B is depicted as yellow (A, B, D, E, F, G). Colocalization of Core-NS5A or -NS4B complexes with LDs (E), early endosomes (F) or late endosomes (G) is depicted as white. Bars, 10 µm. (H) In parallel, the HCV-infected cells was labeled with anti-NS5A or -NS4B Ab. Bound antibodies were detected using anti-mouse or -rabbit secondary antibodies conjugated to 18- or 12-nm gold particles, respectively. Sections were visualized by EM. Arrows, gold-labeled NS5A (18 nm) and NS4B (12 nm) are indicated. MVB contains several intravesicular vesicles (white arrows). An enlarged view of the perinuclear region of lower left image is shown (inset). Arrows, gold-labeled NS5A and NS4B. PM, plasma membrane; MVB, multivesicular bodies; N, nucleus; NM, nuclear membrane; Bars, 500 nm (left panels) and 100 nm (right panels).
Mentions: NS5A, together with Core protein, is involved in virus assembly; the assembled virions are presumably transported to the plasma membrane to be released off the cell. Therefore, we next analyzed whether NS5A is involved in the post-assembly processes of viral life cycle. For this purpose, we examined the colocalization of NS5A with Core protein in two regions of the cytoplasm, viz. the perinuclear (region just around the nucleus) and the peripheral (region just underneath the plasma membrane) regions. HCV-infected cells were co-stained with fluorescent (Alexa 647) WGA (which binds glycoproteins on the cell membrane), which serves as a membrane marker, anti-Core, and either anti-NS5A or -NS4B Abs. As shown in Fig. 5A, NS5A and Core protein colocalized both in the perinuclear region and at the cell periphery. In contrast, NS4B colocalized with the Core protein only in the perinuclear region of cytoplasm, but not in the peripheral region. Analysis of a large number of cells indicated that Core protein was colocalized with NS5A throughout the entire cytoplasm, but not with NS4B (Fig. 5B, upper panels). The total fraction of NS5A that colocalized with Core protein was 76%, while the corresponding NS4B was 51% (Fig. 5B, lower panel). Further, the NS5A-Core complex colocalized in the peripheral region in 33% of the cells, whereas NS4B-Core did not colocalize at all in the same region (Fig. 5C). We next examined the possibility that the Core-NS5A complexes are also transported along microtubule to the cell periphery. In the DMSO-treated cells, NS5A colocalized with Core protein in both the perinuclear and peripheral regions (Fig.5D). After treatment with either nocodazole or vinblastine, the Core-NS5A complexes clustered almost exclusively in the perinuclear region (Fig. 5D). These results suggested that microtubules are required for the transport of Core-NS5A complexes to the cell periphery. This observation further suggests that NS5A, but not NS4B, is involved in the post-assembly transport of virus particles.

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