Hepatitis C Virus (HCV) is an emerging and challenging virus due to the problematic understanding of its persistence mechanisms. In fact, for many years scientists have been trying to resolve the issue of HCV lymphotropism, as the virus has been detected in peripheral blood mononuclear cells (PBMCs), lymph nodes and brain tissue. HCV isolates detected in these extrahepatic sites have unique mutations at positions 107, 204 and 243, distinct from liver and serum, in the internal ribosomal entry site (IRES) located in the 5’ UTR, one of the most well conserved regions of the HCV genome. In this study, divergent IRES variants were detected in the PBMCs of HCV-infected patients. Differences were found among plasma and PBMC derived 5’ UTRs. Most of the PBMC-derived sequences contained the AAA mutations in the IRES affecting the aforementioned positions, a unique quasispecies population in PBMCs compared with plasma.

The effect of mutations detected in the IRES was predicted based on its secondary structure. The three mutations alone and in combination detected in lymphotropic quasispecies were introduced in the 5’ UTR of HCV isolate JFH-1 and replicon genotype 1b to study the effect of these mutations on HCV replication in different cell lines. The replication capacity of HCV mutants was tested in the Huh7.5, Jurkat, EBV+ B and PBMC cell lines to examine whether such mutations had an effect on the efficiency of the virus to replicate in extrahepatic cell lines. All mutant constructs infected and showed robust replication in Huh7.5 cells. Jurkat cells failed to become infected while EBV+ B lymphocyte cells were shown to support HCV mutant replication. PBMCs failed to become infected with HCV mutants and transfection of such cells with siRNAs against IRF3 and NF-kB did not improve this outcome as they appeared to not have an impact on the innate immune response through failure to inhibit IRF3 and NF-kB gene expression. On the other hand, all cell lines were found to be susceptible to infection with HCV containing plasma. HCV mutants were able to target cells of the immune system but replicated with reduced efficiency compared to hepatocytes. Mutations that were detected in HCV mutants following replication in extrahepatic cells caused changes in the thermodynamic stability of the IRES that resulted in predicted structural changes.