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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

Colocalization of NS5A and Core protein with exosomal proteins at the plasma membrane.(A) The HCV-infected cells (at day 10 p.i.) were co-stained with anti-Core (red) (left panel) or -NS5A (red) (middle panel) or -NS4B (red) (right panel) and anti-CD81 (green). Plasma membrane and nuclei were stained with WGA Alexa Fluor 647 conjugate (blue) and DAPI (gray), respectively. Enlarged views of parts of every image (insets) are shown. PM, plasma membrane; Bars, 10 µm. (B, C) Immuno-EM of plasma membrane co-labeled with antibodies against Core (6 nm) (B) or NS5A (6 nm) (C) and CD81 (18 nm) are shown. Arrowheads, gold-labeled CD81. Arrows, gold-labeled Core and NS5A. Bars, 200 nm and 50 nm (B and C, left and right panels, respectively).
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pone-0099022-g008: Colocalization of NS5A and Core protein with exosomal proteins at the plasma membrane.(A) The HCV-infected cells (at day 10 p.i.) were co-stained with anti-Core (red) (left panel) or -NS5A (red) (middle panel) or -NS4B (red) (right panel) and anti-CD81 (green). Plasma membrane and nuclei were stained with WGA Alexa Fluor 647 conjugate (blue) and DAPI (gray), respectively. Enlarged views of parts of every image (insets) are shown. PM, plasma membrane; Bars, 10 µm. (B, C) Immuno-EM of plasma membrane co-labeled with antibodies against Core (6 nm) (B) or NS5A (6 nm) (C) and CD81 (18 nm) are shown. Arrowheads, gold-labeled CD81. Arrows, gold-labeled Core and NS5A. Bars, 200 nm and 50 nm (B and C, left and right panels, respectively).

Mentions: The above results prompted us to further characterize the localization of the exosomal proteins relative to NS5A and Core protein on the plasma membrane. The results indicated that NS5A or Core protein colocalized with CD81 at the plasma membrane by immunofluorescence staining (Fig. 8A) and immuno-EM (Fig. 8B and 8C), but not NS4B. These results again suggest that some NS5A-containing HCV particles exit the cells via fusion of MVB with the plasma membrane. Taken together, these data suggest that a minor population of HCV virion exits cells via exosomes, as NS5A-containing, low-density particles, but the majority of virions do not contain NS5A and exit cells through an exosome-independent pathway.


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)

Colocalization of NS5A and Core protein with exosomal proteins at the plasma membrane.(A) The HCV-infected cells (at day 10 p.i.) were co-stained with anti-Core (red) (left panel) or -NS5A (red) (middle panel) or -NS4B (red) (right panel) and anti-CD81 (green). Plasma membrane and nuclei were stained with WGA Alexa Fluor 647 conjugate (blue) and DAPI (gray), respectively. Enlarged views of parts of every image (insets) are shown. PM, plasma membrane; Bars, 10 µm. (B, C) Immuno-EM of plasma membrane co-labeled with antibodies against Core (6 nm) (B) or NS5A (6 nm) (C) and CD81 (18 nm) are shown. Arrowheads, gold-labeled CD81. Arrows, gold-labeled Core and NS5A. Bars, 200 nm and 50 nm (B and C, left and right panels, respectively).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0099022-g008: Colocalization of NS5A and Core protein with exosomal proteins at the plasma membrane.(A) The HCV-infected cells (at day 10 p.i.) were co-stained with anti-Core (red) (left panel) or -NS5A (red) (middle panel) or -NS4B (red) (right panel) and anti-CD81 (green). Plasma membrane and nuclei were stained with WGA Alexa Fluor 647 conjugate (blue) and DAPI (gray), respectively. Enlarged views of parts of every image (insets) are shown. PM, plasma membrane; Bars, 10 µm. (B, C) Immuno-EM of plasma membrane co-labeled with antibodies against Core (6 nm) (B) or NS5A (6 nm) (C) and CD81 (18 nm) are shown. Arrowheads, gold-labeled CD81. Arrows, gold-labeled Core and NS5A. Bars, 200 nm and 50 nm (B and C, left and right panels, respectively).
Mentions: The above results prompted us to further characterize the localization of the exosomal proteins relative to NS5A and Core protein on the plasma membrane. The results indicated that NS5A or Core protein colocalized with CD81 at the plasma membrane by immunofluorescence staining (Fig. 8A) and immuno-EM (Fig. 8B and 8C), but not NS4B. These results again suggest that some NS5A-containing HCV particles exit the cells via fusion of MVB with the plasma membrane. Taken together, these data suggest that a minor population of HCV virion exits cells via exosomes, as NS5A-containing, low-density particles, but the majority of virions do not contain NS5A and exit cells through an exosome-independent pathway.

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