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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

Association of NS5A and exosomal proteins with the low-density HCV particles.HCV virion populations were analyzed by sucrose density gradient ultracentrifugation. (A) Concentrated culture medium collected from HCV Jc1-infected cells was fractionated using a continuous 10–50% sucrose density gradient. HCV RNA levels were determined in each fraction. (B) Infectivity of each density gradient fraction. An aliquot of each fraction was used to infect naive Huh7.5 cells. Intracellular HCV RNA copy number per µg of total RNA was determined 3 days after infection by qRT-PCR. (C) Western blot analysis of each density gradient fraction, shown in panel A, by using antibodies indicated at the left. (D, E and F) Electron micrograph of the viral spherical structures shown by immunogold labeling. HCV particles were purified from two pooled fractions; the low-density particles (from fractions 7 to 8), and the high-density particles (from fractions 11 to 13), after dialysis and concentration. The virus samples were untreated (D) or treated with 0.01% saponin (E, F), as described in Materials and Methods. Grids were incubated with an untreated or saponin-treated purified HCV and then with antibodies against CD81, E2, or NS5A (D) or with antibodies against Core protein, NS5A or NS4B (E), respectively. Bound antibodies were detected using anti-mouse or -rabbit secondary antibodies conjugated to 12-nm gold particles. (F) Core-NS5A complex localized in the virion core of the low-density particles. Grids were incubated with a saponin-treated purified HCV and then co-labeled with antibodies against NS5A (6 nm) and Core (12 nm). To determine antibody specificity, primary antibodies were replaced with nonspecific normal mouse IgG. Bars, 50 nm. (G) The average diameter of the low- (n = 60) and high-density (n = 90) particles was obtained from the combined analysis of two independent viral preparations and error bars represent standard deviations of the mean. (H) Statistics were performed by counting HCV particles and immunogold labeled HCV particles from the combined analysis of two independent viral preparations. Relative proportion of HCV particles that were labeled with antibodies (n = 60).
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pone-0099022-g007: Association of NS5A and exosomal proteins with the low-density HCV particles.HCV virion populations were analyzed by sucrose density gradient ultracentrifugation. (A) Concentrated culture medium collected from HCV Jc1-infected cells was fractionated using a continuous 10–50% sucrose density gradient. HCV RNA levels were determined in each fraction. (B) Infectivity of each density gradient fraction. An aliquot of each fraction was used to infect naive Huh7.5 cells. Intracellular HCV RNA copy number per µg of total RNA was determined 3 days after infection by qRT-PCR. (C) Western blot analysis of each density gradient fraction, shown in panel A, by using antibodies indicated at the left. (D, E and F) Electron micrograph of the viral spherical structures shown by immunogold labeling. HCV particles were purified from two pooled fractions; the low-density particles (from fractions 7 to 8), and the high-density particles (from fractions 11 to 13), after dialysis and concentration. The virus samples were untreated (D) or treated with 0.01% saponin (E, F), as described in Materials and Methods. Grids were incubated with an untreated or saponin-treated purified HCV and then with antibodies against CD81, E2, or NS5A (D) or with antibodies against Core protein, NS5A or NS4B (E), respectively. Bound antibodies were detected using anti-mouse or -rabbit secondary antibodies conjugated to 12-nm gold particles. (F) Core-NS5A complex localized in the virion core of the low-density particles. Grids were incubated with a saponin-treated purified HCV and then co-labeled with antibodies against NS5A (6 nm) and Core (12 nm). To determine antibody specificity, primary antibodies were replaced with nonspecific normal mouse IgG. Bars, 50 nm. (G) The average diameter of the low- (n = 60) and high-density (n = 90) particles was obtained from the combined analysis of two independent viral preparations and error bars represent standard deviations of the mean. (H) Statistics were performed by counting HCV particles and immunogold labeled HCV particles from the combined analysis of two independent viral preparations. Relative proportion of HCV particles that were labeled with antibodies (n = 60).

Mentions: To confirm that NS5A is released from the plasma membrane as a component of some virion particles, we cultured HCV-infected Huh7.5 cells with dialyzed serum to reduce nonspecific binding of irrelevant proteins to virus particles. The culture supernatant was subject to sucrose gradient sedimentation, and the distribution of the viral RNA and infectivity of HCV were determined. Two peaks, one from fractions 7–8, with density of 1.083 to 1.098 g/ml sucrose (low-density fractions; LDF), and another from fractions 11–13 with density of 1.145 to 1.178 g/ml sucrose (high-density fractions; HDF), were found to contain distinct HCV RNA signals (Fig. 7A). Fraction 12, at 1.162 g/ml sucrose, contains the largest amount of viral RNA, consistent with the previously reported density of free HCV virions [27], [31]. Each fraction was further analyzed for its infectivity on naive Huh7.5 cells. The results showed that both the LDF and HDF from culture supernatant contained infectivity (Fig. 7B), with HDF having approximately 153-fold higher specific infectivity. Thus, both the LDF and HDF are infectious, but LDF contained only 0.65% of the total infectivity; thus, the possibility that the apparent infectivity of LDF may have come from contaminations of HDF cannot be ruled out.


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)

Association of NS5A and exosomal proteins with the low-density HCV particles.HCV virion populations were analyzed by sucrose density gradient ultracentrifugation. (A) Concentrated culture medium collected from HCV Jc1-infected cells was fractionated using a continuous 10–50% sucrose density gradient. HCV RNA levels were determined in each fraction. (B) Infectivity of each density gradient fraction. An aliquot of each fraction was used to infect naive Huh7.5 cells. Intracellular HCV RNA copy number per µg of total RNA was determined 3 days after infection by qRT-PCR. (C) Western blot analysis of each density gradient fraction, shown in panel A, by using antibodies indicated at the left. (D, E and F) Electron micrograph of the viral spherical structures shown by immunogold labeling. HCV particles were purified from two pooled fractions; the low-density particles (from fractions 7 to 8), and the high-density particles (from fractions 11 to 13), after dialysis and concentration. The virus samples were untreated (D) or treated with 0.01% saponin (E, F), as described in Materials and Methods. Grids were incubated with an untreated or saponin-treated purified HCV and then with antibodies against CD81, E2, or NS5A (D) or with antibodies against Core protein, NS5A or NS4B (E), respectively. Bound antibodies were detected using anti-mouse or -rabbit secondary antibodies conjugated to 12-nm gold particles. (F) Core-NS5A complex localized in the virion core of the low-density particles. Grids were incubated with a saponin-treated purified HCV and then co-labeled with antibodies against NS5A (6 nm) and Core (12 nm). To determine antibody specificity, primary antibodies were replaced with nonspecific normal mouse IgG. Bars, 50 nm. (G) The average diameter of the low- (n = 60) and high-density (n = 90) particles was obtained from the combined analysis of two independent viral preparations and error bars represent standard deviations of the mean. (H) Statistics were performed by counting HCV particles and immunogold labeled HCV particles from the combined analysis of two independent viral preparations. Relative proportion of HCV particles that were labeled with antibodies (n = 60).
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Related In: Results  -  Collection

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pone-0099022-g007: Association of NS5A and exosomal proteins with the low-density HCV particles.HCV virion populations were analyzed by sucrose density gradient ultracentrifugation. (A) Concentrated culture medium collected from HCV Jc1-infected cells was fractionated using a continuous 10–50% sucrose density gradient. HCV RNA levels were determined in each fraction. (B) Infectivity of each density gradient fraction. An aliquot of each fraction was used to infect naive Huh7.5 cells. Intracellular HCV RNA copy number per µg of total RNA was determined 3 days after infection by qRT-PCR. (C) Western blot analysis of each density gradient fraction, shown in panel A, by using antibodies indicated at the left. (D, E and F) Electron micrograph of the viral spherical structures shown by immunogold labeling. HCV particles were purified from two pooled fractions; the low-density particles (from fractions 7 to 8), and the high-density particles (from fractions 11 to 13), after dialysis and concentration. The virus samples were untreated (D) or treated with 0.01% saponin (E, F), as described in Materials and Methods. Grids were incubated with an untreated or saponin-treated purified HCV and then with antibodies against CD81, E2, or NS5A (D) or with antibodies against Core protein, NS5A or NS4B (E), respectively. Bound antibodies were detected using anti-mouse or -rabbit secondary antibodies conjugated to 12-nm gold particles. (F) Core-NS5A complex localized in the virion core of the low-density particles. Grids were incubated with a saponin-treated purified HCV and then co-labeled with antibodies against NS5A (6 nm) and Core (12 nm). To determine antibody specificity, primary antibodies were replaced with nonspecific normal mouse IgG. Bars, 50 nm. (G) The average diameter of the low- (n = 60) and high-density (n = 90) particles was obtained from the combined analysis of two independent viral preparations and error bars represent standard deviations of the mean. (H) Statistics were performed by counting HCV particles and immunogold labeled HCV particles from the combined analysis of two independent viral preparations. Relative proportion of HCV particles that were labeled with antibodies (n = 60).
Mentions: To confirm that NS5A is released from the plasma membrane as a component of some virion particles, we cultured HCV-infected Huh7.5 cells with dialyzed serum to reduce nonspecific binding of irrelevant proteins to virus particles. The culture supernatant was subject to sucrose gradient sedimentation, and the distribution of the viral RNA and infectivity of HCV were determined. Two peaks, one from fractions 7–8, with density of 1.083 to 1.098 g/ml sucrose (low-density fractions; LDF), and another from fractions 11–13 with density of 1.145 to 1.178 g/ml sucrose (high-density fractions; HDF), were found to contain distinct HCV RNA signals (Fig. 7A). Fraction 12, at 1.162 g/ml sucrose, contains the largest amount of viral RNA, consistent with the previously reported density of free HCV virions [27], [31]. Each fraction was further analyzed for its infectivity on naive Huh7.5 cells. The results showed that both the LDF and HDF from culture supernatant contained infectivity (Fig. 7B), with HDF having approximately 153-fold higher specific infectivity. Thus, both the LDF and HDF are infectious, but LDF contained only 0.65% of the total infectivity; thus, the possibility that the apparent infectivity of LDF may have come from contaminations of HDF cannot be ruled out.

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