Mechanisms of Hepatitis C virus infection of human T lymphocytes

Hepatitis C virus (HCV) induces one of the most prevalent, potentially fatal chronic viral infections worldwide. This virus symptomatically infects approximately 170 million people (2 .5% of the global population) . Although HCV is primarily a hepatotropic virus, accumulated experimental and clinical evidence indicates that HCV can also replicate in cells of the immune system. The ability of HCV to infect human cells is currently interpreted in the context of the interactions identified between the Japanese fulminant hepatilis -1 (JFH-1) strain of HCV or HCV pseudoparticles and human hepatoma cell lines, particularly Huh7.5 clone. The factors determining HCV tympholropism remained unknown. Based on previous observations demonstrating that wild-type HCV can infect and replicate in human T lymphocytes both in vivo and in vitro , we hypotheSized that HCV utilizes certain T cell-specific molecules to enter these cells and, therefore, that HCV uses (at least in part) different strategy to infect immune cells than Huh7 cells or primary hepatocytes. This thesis is comprised of three related studies In the first study, we established an in vitro infection system applying readily available human T cell lines, such as Molt4 and Jurkat T cells, and naturally occurring wild -type HCV obtained from patients with chronic hepatitis C. Employing this system, we confirmed that HCV infects primary T lymphocytes as well as the mentioned T cell lines, as evident by detection of HCV RNA negative (replicative) strand and virus non-structural (NS5A) and structural (core) proteins. However, some T cell lines were not susceptible to HCV infection. We found that cells prone to HCV infection are endowed with CDS, a scavenger receptor that belongs to the cysteine-rich superfamily. We documented that this molecule serves as HCV T cell receptor by multiple supplementing each other approaches In the subsequent study, we evaluated whether other molecules proposed to serve as HCV receptors based on the JFH-1-Huh7 cell studies may contribute to establishment of HCV infection in T lymphocytes. For this purpose, the mRNA and protein expression of the different HCV cand idate receptors , such as C081 (mRNA only), scavenger receptor class B type1 (SR-B1), occludin (OCLN) , claudin-1 (CLoN-1), claudin-4 (CLoN-4) and claudin-6 (CLoN-6) , in addition to COS molecule identified in the first study, were investigated. We examined T cell lines, peripheral blood mononuclear cells (PBMC) and affinity-purified primary human T cells and their C04+ and CD8+ subsets, and compared to hepatoma Huh7.S and HepG2 cell lines and primary human hepatocytes (PHH). We found that while CDS, C081 , and OCLN are likely involved in mediating T cell susceptibility to HCV infection, SR-B1 and CloN-1 are not. This validated the concept that HCV uses different receptors to infect various cell types naturally targeted by this virus. In general, this study narrowed the range of factors utilized by HCV infecting T lymphocytes amongst those identified using HCV preparations and Huh7.5 cells . In the third study, we tested the ability of the recombinant HCV JFH1 T virus , a culture-adapted JFH-1 strain demonstrating significantly higher infectivity towards Huh7.5 cells than the classical JFH-1 strain, to infect T cell lines, human PBMC and primary T cells , in comparison to the plasma-derived wild-type HCV. We also examined the susceptibility of Huh7.S cells to the plasma-derived wildtype HCV. The resul ts revealed that T cell lines, PBMC and primary human T cells were susceptible to the patient-derived wild-type HCV but not to JFHh clone, while Huh7.S cells were only prone to JFHh but not to wild-type virus. This study demonstrated contrasting differences between wild -type HCV and JFH clones in terms of cell tropism and suggested that HCV -T cell infection system can be more representative of the actual (wild-type) virus behaviour than genetically modified and Huh7.S cell culture-adapted viruses. Overall, our findings have provided conclusive evidence that wild-type HCV can infect and replicate not only in primary human T lymphocytes but also in some readily available human T cell lines . We identified CDS, a glycoprotein belonging to the scavenger receptor cysteine-rich superfamily, as a T cell-specific molecule mediating the cell susceptibility to HCV infection. In addition, CD81 was found to act as a co-receptor, while OClN is likely involved in infection of the virus-prone T cell lines but unlikely primary T cells. In general , our research provided the first insight into the molecular mechanism determining HCV ability to infect cells of the immune system.