Limits...
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.

Show MeSH

Related in: MedlinePlus

Core-NS5A complexes are transported from perinuclear region to the plasma membrane via microtubules.The HCV-infected cells (at day 10 p.i.) were labeled with antibodies to Core protein (red) (A, B), NS5A (blue, A; green, B) (upper rows), NS4B (blue, A; green, B) (lower row) or tubulin (green) (A). Plasma membrane and nuclei were stained with WGA Alexa Fluor 647 conjugate (blue) (B) 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). Colocalization of Core with NS5A or NS4B is depicted as magenta (A) or yellow (B). Colocalization of Core-NS5A or -NS4B protein complexes with microtubules (A) or plasma membrane (B) is depicted as white. The microtubule end (white arrow) is closest to the cell periphery (A). (B) At the right is an enlarged area, marked with a white box, from the merged image. Colocalization of NS5A with Core protein was observed in the membrane curvature (white arrow). PM, plasma membrane; Bars, 10 µm. (C) Quantitation of antibody-labeled signals of viral proteins at the plasma membrane. Images from 20 cells were counted manually using an original magnification of ×630 and followed by a quantitation analysis performed by an observer blinded to the experimental treatment. (D, E) NS5A and Core are localized in clusters or patches on the plasma membrane. In parallel, the HCV-infected cells were labeled with anti-NS5A (D) or anti-Core (E) antibodies. Bound antibodies were detected using anti-mouse secondary antibodies conjugated to 18-nm gold particles. Sections were visualized by EM. Arrows, gold-labeled NS5A (D) or Core protein (E). (F) Immuno-EM of Core-NS5A complex colocalized at the plasma membrane. The HCV-infected cells were co-labeled with antibodies against NS5A (6 nm) and Core (18 nm). Shown is a view of the plasma membrane. Arrowhead, gold-labeled NS5A. Arrow, gold-labeled Core. PM, plasma membrane; Bars, 100 nm (D, F) and 200 nm (E).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4048239&req=5

pone-0099022-g006: Core-NS5A complexes are transported from perinuclear region to the plasma membrane via microtubules.The HCV-infected cells (at day 10 p.i.) were labeled with antibodies to Core protein (red) (A, B), NS5A (blue, A; green, B) (upper rows), NS4B (blue, A; green, B) (lower row) or tubulin (green) (A). Plasma membrane and nuclei were stained with WGA Alexa Fluor 647 conjugate (blue) (B) 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). Colocalization of Core with NS5A or NS4B is depicted as magenta (A) or yellow (B). Colocalization of Core-NS5A or -NS4B protein complexes with microtubules (A) or plasma membrane (B) is depicted as white. The microtubule end (white arrow) is closest to the cell periphery (A). (B) At the right is an enlarged area, marked with a white box, from the merged image. Colocalization of NS5A with Core protein was observed in the membrane curvature (white arrow). PM, plasma membrane; Bars, 10 µm. (C) Quantitation of antibody-labeled signals of viral proteins at the plasma membrane. Images from 20 cells were counted manually using an original magnification of ×630 and followed by a quantitation analysis performed by an observer blinded to the experimental treatment. (D, E) NS5A and Core are localized in clusters or patches on the plasma membrane. In parallel, the HCV-infected cells were labeled with anti-NS5A (D) or anti-Core (E) antibodies. Bound antibodies were detected using anti-mouse secondary antibodies conjugated to 18-nm gold particles. Sections were visualized by EM. Arrows, gold-labeled NS5A (D) or Core protein (E). (F) Immuno-EM of Core-NS5A complex colocalized at the plasma membrane. The HCV-infected cells were co-labeled with antibodies against NS5A (6 nm) and Core (18 nm). Shown is a view of the plasma membrane. Arrowhead, gold-labeled NS5A. Arrow, gold-labeled Core. PM, plasma membrane; Bars, 100 nm (D, F) and 200 nm (E).

Mentions: Furthermore, NS5A and Core protein, but not NS4B, colocalized with the microtubules in the peripheral region of cytoplasm, especially at the microtubule end that is closest to the cell periphery (Fig. 6A). Finally, we characterized the relationship of NS5A-Core complexes with the plasma membrane. HCV-infected cells were co-stained with fluorescent (Alexa 647) WGA, anti-Core, and anti-NS5A or -NS4B Abs. As shown in Fig. 6B, NS5A and Core protein colocalized at the plasma membrane, particularly at the membrane curvature, which has been reported to be essential for virus exit [48]. Quantitative analysis showed that the number of anti-Core and anti-NS5A antibodies-labeled signals at the plasma membrane averaged 63 and 26 per cell, respectively, whereas that for anti-NS4B was less than 1. Interestingly, some signals containing both NS5A and Core (5 per cell on average) were also detected at the plasma membrane, whereas no NS4B-Core signals were found (Fig. 6C). Furthermore, immuno-EM showed that NS5A (Fig. 6D), Core protein (Fig.6E) and the Core-NS5A complex (Fig. 6F) localized mainly to patches or clusters on the plasma membrane. Taken together, these results again suggest that at least some of NS5A-Core complexes (or the assembled virions) are transported from LD through early-to-late endosomes to the plasma membrane via 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)

Core-NS5A complexes are transported from perinuclear region to the plasma membrane via microtubules.The HCV-infected cells (at day 10 p.i.) were labeled with antibodies to Core protein (red) (A, B), NS5A (blue, A; green, B) (upper rows), NS4B (blue, A; green, B) (lower row) or tubulin (green) (A). Plasma membrane and nuclei were stained with WGA Alexa Fluor 647 conjugate (blue) (B) 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). Colocalization of Core with NS5A or NS4B is depicted as magenta (A) or yellow (B). Colocalization of Core-NS5A or -NS4B protein complexes with microtubules (A) or plasma membrane (B) is depicted as white. The microtubule end (white arrow) is closest to the cell periphery (A). (B) At the right is an enlarged area, marked with a white box, from the merged image. Colocalization of NS5A with Core protein was observed in the membrane curvature (white arrow). PM, plasma membrane; Bars, 10 µm. (C) Quantitation of antibody-labeled signals of viral proteins at the plasma membrane. Images from 20 cells were counted manually using an original magnification of ×630 and followed by a quantitation analysis performed by an observer blinded to the experimental treatment. (D, E) NS5A and Core are localized in clusters or patches on the plasma membrane. In parallel, the HCV-infected cells were labeled with anti-NS5A (D) or anti-Core (E) antibodies. Bound antibodies were detected using anti-mouse secondary antibodies conjugated to 18-nm gold particles. Sections were visualized by EM. Arrows, gold-labeled NS5A (D) or Core protein (E). (F) Immuno-EM of Core-NS5A complex colocalized at the plasma membrane. The HCV-infected cells were co-labeled with antibodies against NS5A (6 nm) and Core (18 nm). Shown is a view of the plasma membrane. Arrowhead, gold-labeled NS5A. Arrow, gold-labeled Core. PM, plasma membrane; Bars, 100 nm (D, F) and 200 nm (E).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0099022-g006: Core-NS5A complexes are transported from perinuclear region to the plasma membrane via microtubules.The HCV-infected cells (at day 10 p.i.) were labeled with antibodies to Core protein (red) (A, B), NS5A (blue, A; green, B) (upper rows), NS4B (blue, A; green, B) (lower row) or tubulin (green) (A). Plasma membrane and nuclei were stained with WGA Alexa Fluor 647 conjugate (blue) (B) 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). Colocalization of Core with NS5A or NS4B is depicted as magenta (A) or yellow (B). Colocalization of Core-NS5A or -NS4B protein complexes with microtubules (A) or plasma membrane (B) is depicted as white. The microtubule end (white arrow) is closest to the cell periphery (A). (B) At the right is an enlarged area, marked with a white box, from the merged image. Colocalization of NS5A with Core protein was observed in the membrane curvature (white arrow). PM, plasma membrane; Bars, 10 µm. (C) Quantitation of antibody-labeled signals of viral proteins at the plasma membrane. Images from 20 cells were counted manually using an original magnification of ×630 and followed by a quantitation analysis performed by an observer blinded to the experimental treatment. (D, E) NS5A and Core are localized in clusters or patches on the plasma membrane. In parallel, the HCV-infected cells were labeled with anti-NS5A (D) or anti-Core (E) antibodies. Bound antibodies were detected using anti-mouse secondary antibodies conjugated to 18-nm gold particles. Sections were visualized by EM. Arrows, gold-labeled NS5A (D) or Core protein (E). (F) Immuno-EM of Core-NS5A complex colocalized at the plasma membrane. The HCV-infected cells were co-labeled with antibodies against NS5A (6 nm) and Core (18 nm). Shown is a view of the plasma membrane. Arrowhead, gold-labeled NS5A. Arrow, gold-labeled Core. PM, plasma membrane; Bars, 100 nm (D, F) and 200 nm (E).
Mentions: Furthermore, NS5A and Core protein, but not NS4B, colocalized with the microtubules in the peripheral region of cytoplasm, especially at the microtubule end that is closest to the cell periphery (Fig. 6A). Finally, we characterized the relationship of NS5A-Core complexes with the plasma membrane. HCV-infected cells were co-stained with fluorescent (Alexa 647) WGA, anti-Core, and anti-NS5A or -NS4B Abs. As shown in Fig. 6B, NS5A and Core protein colocalized at the plasma membrane, particularly at the membrane curvature, which has been reported to be essential for virus exit [48]. Quantitative analysis showed that the number of anti-Core and anti-NS5A antibodies-labeled signals at the plasma membrane averaged 63 and 26 per cell, respectively, whereas that for anti-NS4B was less than 1. Interestingly, some signals containing both NS5A and Core (5 per cell on average) were also detected at the plasma membrane, whereas no NS4B-Core signals were found (Fig. 6C). Furthermore, immuno-EM showed that NS5A (Fig. 6D), Core protein (Fig.6E) and the Core-NS5A complex (Fig. 6F) localized mainly to patches or clusters on the plasma membrane. Taken together, these results again suggest that at least some of NS5A-Core complexes (or the assembled virions) are transported from LD through early-to-late endosomes to the plasma membrane via 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