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Hepatitis C virus induced a novel apoptosis-like death of pancreatic beta cells through a caspase 3-dependent pathway.

Wang Q, Chen J, Wang Y, Han X, Chen X - PLoS ONE (2012)

Bottom Line: Pancreatic beta cell failure is central to the progression of type 2 diabetes.Our experiments demonstrate that the HCV virion itself is indispensable and has a dose- and time-dependent cytopathic effect on the cells.However, the fact that HCV-infected cells exhibit a dilated, low-density nucleus with intact plasma and nuclear membrane indicates that a novel apoptosis-like death occurs.

View Article: PubMed Central - PubMed

Affiliation: State Key Lab of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.

ABSTRACT
Epidemiological and experimental studies have suggested that Hepatitis C virus (HCV) infection is associated with the development of type 2 diabetes. Pancreatic beta cell failure is central to the progression of type 2 diabetes. Using virus infection system, we investigate the influence of HCV infection on the fate of the insulinoma cell line, MIN6. Our experiments demonstrate that the HCV virion itself is indispensable and has a dose- and time-dependent cytopathic effect on the cells. HCV infection inhibits cell proliferation and induces death of MIN6 cells with apoptotic characteristics, including cell surface exposure of phosphatidylserine, decreased mitochondrial membrane potential, activation of caspase 3 and poly (ADP-ribose) polymerase, and DNA fragmentation in the nucleus. However, the fact that HCV-infected cells exhibit a dilated, low-density nucleus with intact plasma and nuclear membrane indicates that a novel apoptosis-like death occurs. HCV infection also causes endoplasmic reticulum (ER) stress. Further, HCV RNA replication was detected in MIN6 cells, although the infection efficiency is very low and no progeny virus particle generates. Taken together, our data suggest that HCV infection induces death of pancreatic beta cells through an ER stress-involved, caspase 3-dependent, special pathway.

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Hepatitis C virus replicates in MIN6 cells.(A) Kinetics of detection of positive-strand HCV RNA. MIN6 and Huh7.5.1 cells were mock-infected or infected with 1.0 MOI HCV. At different time points, cells were harvested and total RNA was isolated and reverse-transcribed to cDNA. Single-round PCR products (150 bp) were obtained by amplification with POSF/R primers. Actin was measured as an internal control. (B) Detection of the synthetic negative-strand RNA as in (A). The sample from HCV-infected Huh7.5.1 cells was used as the positive control. Negative controls included PCR amplification from non-infected cells (−) and water (H2O). (C–D) MIN6 cells were mock-infected or infected with 1.0 MOI of HCV at 24 hpi. HCV core (red) and NS5A (green) labeled with respective antibody (C) or HCV dsRNA labeled with J2 antibody (green) (D) were examined by immunofluorescence assay. Blue fluorescence represents DAPI-stained nuclei as observed. (E) Immunoprecipitation and blotting of NS5A purified from 96 h-infected MIN6 and Huh7.5.1 cells. Actin from lysis was used as the internal control. All measurements were done in triplicates. Immunoblots are representative of at least three independent experiments.
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pone-0038522-g007: Hepatitis C virus replicates in MIN6 cells.(A) Kinetics of detection of positive-strand HCV RNA. MIN6 and Huh7.5.1 cells were mock-infected or infected with 1.0 MOI HCV. At different time points, cells were harvested and total RNA was isolated and reverse-transcribed to cDNA. Single-round PCR products (150 bp) were obtained by amplification with POSF/R primers. Actin was measured as an internal control. (B) Detection of the synthetic negative-strand RNA as in (A). The sample from HCV-infected Huh7.5.1 cells was used as the positive control. Negative controls included PCR amplification from non-infected cells (−) and water (H2O). (C–D) MIN6 cells were mock-infected or infected with 1.0 MOI of HCV at 24 hpi. HCV core (red) and NS5A (green) labeled with respective antibody (C) or HCV dsRNA labeled with J2 antibody (green) (D) were examined by immunofluorescence assay. Blue fluorescence represents DAPI-stained nuclei as observed. (E) Immunoprecipitation and blotting of NS5A purified from 96 h-infected MIN6 and Huh7.5.1 cells. Actin from lysis was used as the internal control. All measurements were done in triplicates. Immunoblots are representative of at least three independent experiments.

Mentions: It has been reported that HCV is not strictly hepatotropic and the common presence of negative-strand HCV RNA is detected in pancreas from HCV-infected patients with AIDS [22]. As shown in Fig. 7A, starting from 12 hpi, positive-strand HCV RNA was already detectable in the HCV-infected MIN6 cells and increased with prolongation of infection time. Quantitative RT-PCR analysis confirmed the increase of positive-strand RNA as well (data not shown). The presence of HCV negative strand in infected MIN6 cells was also revealed by using a negative-strand-specific nested-PCR assay (Fig. 7B). Immunostaining for both core and NS5A protein (Fig. 7C) and further HCV dsRNA (Fig. 7D) demonstrated an infection of HCV in MIN6 cells by immunofluorescence assay. Moreover the translation products NS5A in MIN6 cells were probed with anti-NS5A after enrichment by using immunoprecipitation (Fig. 7E). These results revealed that HCV could replicate in the insulinoma MIN6 cells with low efficiency, although the detailed HCV-entry mechanism of MIN6 cells remained obscure.


Hepatitis C virus induced a novel apoptosis-like death of pancreatic beta cells through a caspase 3-dependent pathway.

Wang Q, Chen J, Wang Y, Han X, Chen X - PLoS ONE (2012)

Hepatitis C virus replicates in MIN6 cells.(A) Kinetics of detection of positive-strand HCV RNA. MIN6 and Huh7.5.1 cells were mock-infected or infected with 1.0 MOI HCV. At different time points, cells were harvested and total RNA was isolated and reverse-transcribed to cDNA. Single-round PCR products (150 bp) were obtained by amplification with POSF/R primers. Actin was measured as an internal control. (B) Detection of the synthetic negative-strand RNA as in (A). The sample from HCV-infected Huh7.5.1 cells was used as the positive control. Negative controls included PCR amplification from non-infected cells (−) and water (H2O). (C–D) MIN6 cells were mock-infected or infected with 1.0 MOI of HCV at 24 hpi. HCV core (red) and NS5A (green) labeled with respective antibody (C) or HCV dsRNA labeled with J2 antibody (green) (D) were examined by immunofluorescence assay. Blue fluorescence represents DAPI-stained nuclei as observed. (E) Immunoprecipitation and blotting of NS5A purified from 96 h-infected MIN6 and Huh7.5.1 cells. Actin from lysis was used as the internal control. All measurements were done in triplicates. Immunoblots are representative of at least three independent experiments.
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pone-0038522-g007: Hepatitis C virus replicates in MIN6 cells.(A) Kinetics of detection of positive-strand HCV RNA. MIN6 and Huh7.5.1 cells were mock-infected or infected with 1.0 MOI HCV. At different time points, cells were harvested and total RNA was isolated and reverse-transcribed to cDNA. Single-round PCR products (150 bp) were obtained by amplification with POSF/R primers. Actin was measured as an internal control. (B) Detection of the synthetic negative-strand RNA as in (A). The sample from HCV-infected Huh7.5.1 cells was used as the positive control. Negative controls included PCR amplification from non-infected cells (−) and water (H2O). (C–D) MIN6 cells were mock-infected or infected with 1.0 MOI of HCV at 24 hpi. HCV core (red) and NS5A (green) labeled with respective antibody (C) or HCV dsRNA labeled with J2 antibody (green) (D) were examined by immunofluorescence assay. Blue fluorescence represents DAPI-stained nuclei as observed. (E) Immunoprecipitation and blotting of NS5A purified from 96 h-infected MIN6 and Huh7.5.1 cells. Actin from lysis was used as the internal control. All measurements were done in triplicates. Immunoblots are representative of at least three independent experiments.
Mentions: It has been reported that HCV is not strictly hepatotropic and the common presence of negative-strand HCV RNA is detected in pancreas from HCV-infected patients with AIDS [22]. As shown in Fig. 7A, starting from 12 hpi, positive-strand HCV RNA was already detectable in the HCV-infected MIN6 cells and increased with prolongation of infection time. Quantitative RT-PCR analysis confirmed the increase of positive-strand RNA as well (data not shown). The presence of HCV negative strand in infected MIN6 cells was also revealed by using a negative-strand-specific nested-PCR assay (Fig. 7B). Immunostaining for both core and NS5A protein (Fig. 7C) and further HCV dsRNA (Fig. 7D) demonstrated an infection of HCV in MIN6 cells by immunofluorescence assay. Moreover the translation products NS5A in MIN6 cells were probed with anti-NS5A after enrichment by using immunoprecipitation (Fig. 7E). These results revealed that HCV could replicate in the insulinoma MIN6 cells with low efficiency, although the detailed HCV-entry mechanism of MIN6 cells remained obscure.

Bottom Line: Pancreatic beta cell failure is central to the progression of type 2 diabetes.Our experiments demonstrate that the HCV virion itself is indispensable and has a dose- and time-dependent cytopathic effect on the cells.However, the fact that HCV-infected cells exhibit a dilated, low-density nucleus with intact plasma and nuclear membrane indicates that a novel apoptosis-like death occurs.

View Article: PubMed Central - PubMed

Affiliation: State Key Lab of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.

ABSTRACT
Epidemiological and experimental studies have suggested that Hepatitis C virus (HCV) infection is associated with the development of type 2 diabetes. Pancreatic beta cell failure is central to the progression of type 2 diabetes. Using virus infection system, we investigate the influence of HCV infection on the fate of the insulinoma cell line, MIN6. Our experiments demonstrate that the HCV virion itself is indispensable and has a dose- and time-dependent cytopathic effect on the cells. HCV infection inhibits cell proliferation and induces death of MIN6 cells with apoptotic characteristics, including cell surface exposure of phosphatidylserine, decreased mitochondrial membrane potential, activation of caspase 3 and poly (ADP-ribose) polymerase, and DNA fragmentation in the nucleus. However, the fact that HCV-infected cells exhibit a dilated, low-density nucleus with intact plasma and nuclear membrane indicates that a novel apoptosis-like death occurs. HCV infection also causes endoplasmic reticulum (ER) stress. Further, HCV RNA replication was detected in MIN6 cells, although the infection efficiency is very low and no progeny virus particle generates. Taken together, our data suggest that HCV infection induces death of pancreatic beta cells through an ER stress-involved, caspase 3-dependent, special pathway.

Show MeSH
Related in: MedlinePlus