Hepatitis C virus infection of a thyroid cell line: implications for pathogenesis of hepatitis C virus and thyroiditis

Abstract

Background: Autoimmune and non-autoimmune thyroiditis frequently occur in persons with hepatitis C virus (HCV) infection. Treatment with interferon alpha (IFNα) is also associated with significant risk for the development of thyroiditis. To explore HCV-thyroid interactions at a cellular level, we evaluated whether a human thyroid cell line (ML1) could be infected productively with HCV in vitro.

Methods and results: ML1 cells showed robust surface expression of the major HCV receptor CD81. Using a highly sensitive, strand-specific reverse transcription polymerase chain reaction assay, positive-sense and negative-sense HCV RNA were detected in ML1 cell lysates at days 3, 7, and 14 postinfection with HCV. HCV core protein was expressed at high levels in ML1 supernatants at days 1, 3, 5, 7, and 14 postinfection. The nonstructural protein NS5A was also detected in ML1 cell lysates by Western blotting. HCV entry into ML1 cells was shown to be dependent on the HCV entry factors CD81 and SR-B1/CLA1, while IFNα inhibited HCV replication in ML1 cells in a dose-dependent manner. Supernatants from HCV-infected ML1 cells were able to infect fresh ML1 cells productively, suggesting that infectious virions could be transferred from infected to naïve thyroid cells in vivo. Additionally, HCV infection of ML1 cells led to increased expression of the pro-inflammatory cytokine IL-8.

Conclusions: For the first time, we have demonstrated that HCV can infect human thyroid cells in vitro. These findings strongly suggest that HCV infection of thyrocytes may play a role in the association between chronic HCV infection and thyroid autoimmunity. Furthermore, the thyroid may serve as an extrahepatic reservoir for HCV viral replication, thus contributing to the persistence of viral infection and to the development of thyroid autoimmunity.

FIG. 1.

Surface expression of CD81 receptor on Huh7.5 (A) or ML1 (B) cells; staining for isotype-matched control antibody is shown in gray.

FIG. 2.

Qualitative reverse transcriptase polymerase chain reaction (PCR) for the detection of positive-sense and negative-sense hepatitis C virus (HCV) RNA at days 3, 7, and 14 postinfection of Huh7.5 (A) or ML1 (B) cells; asterisks denote the 295 bp PCR product.

FIG. 3.

HCV core protein levels at days 1, 3, 5, 7, and 14 postinfection of Huh7.5 (■) or ML1 (□) cells.

FIG. 4.

HCV NS3 protein levels in cell lysates at day 3 postinfection of Huh7.5 (■) or ML1 (□) cells in the presence/absence of anti-CD81 (A) or anti-CLA1 (B) at dilutions of 1:100, 1:500, and 1:2500.

FIG. 5.

HCV core protein levels at day 3 postinfection of Huh7.5 (■) or ML1 (□) cells in the presence/absence of interferon alpha at concentrations of 0.1 ng, 10 ng, and 1000 ng.

FIG. 6.

(A) Qualitative reverse transcriptase PCR for the detection of negative-sense HCV RNA in cell lysates at day 3 postinfection of Huh7.5 or ML1 cells with serum from individuals 1795, 1800, and 1870. The HCVJFH1 cell line is included as a positive control. (B) HCV core protein levels at day 3 postinfection of Huh7.5 (■) or ML1 (□) cells with serum from 1800 in the presence/absence of anti-CD81, anti-CLA1, or isotype control antibodies at dilutions of 1:100. Infection of both cell lines with JFH1 is included as a positive control.

FIG. 7.

ML1 cells were infected with HCV for 4 h, supernatants harvested at day 3 postinfection, and used to infect fresh Huh7.5 (■) or ML1 (□) cells. HCV core protein levels were measured at day 3 postinfection to demonstrate production of infectious virions.

FIG. 8.

Interleukin 8 protein levels in Huh7.5 (■) or ML1 (□) cell culture supernatants—diluted 1:10 with PBS—at day 3 postinfection.