We have shown that primary cultures of HFLC reliably support productive infection with HCVcc. Virus replication was sensitive to inhibition by the polymerase inhibitor 2′CMA (A, A), and to added IFNs (). Levels of virus replication varied significantly between different donor cell preparations and frequently declined in a manner suggestive of active viral clearance (, ). At present, we do not know whether this variability is due to differences in cellular composition or state of differentiation of hepatocytes within these cultures or to genetic differences between tissue donors. Studies to date have not revealed an association with IL28B genotype.39
V protein-transduced HFLC supported significantly enhanced (
10 to 100-fold) levels of HCV infection relative to untransduced or control vector-transduced HFLC. Infection was assessed by measurement of virus-driven luciferase, by assays for infectious HCV and viral RNA, and by direct visualization of HCV-infected hepatocytes (-5
). Similar results were obtained with micropatterned cocultures of adult human hepatocytes11
(Supporting Fig. S2
Time-lapse live cell imaging of HFLC 48-119 hours postinfection demonstrated little or no spread of infection in the absence of V protein expression. In contrast, V protein-transduced HFLC showed numerous HCV infection events (). To our knowledge, this is the first report of visualization of HCV spread in primary cells. During the course of live cell imaging we observed considerable turnover of HCV-infected cells. Cell turnover could indicate a direct cytopathic effect of HCV infection in primary hepatocytes. However, more comprehensive studies will be required to determine the survival time of HCV-infected cells relative to uninfected hepatocytes in these cultures.
V proteins did not measurably affect levels of expression of HCV entry factors in HFLC (), nor did they function to promote virus entry as assessed by experiments using HCV-enveloped pseudoparticles (Supporting Fig. S3
), or measurements of cell-associated HCV RNA 8 hours postinfection (A). Taken together, these results suggest that V proteins exert their effect by mechanisms independent of an effect on virus entry. Consistent with their known role in counteracting innate immunity during PMV infection, we found V protein expression to efficiently antagonize the HCV-inhibitory effects of added IFNs in HFLC ().
Interestingly, induction of the type III IFN, IL-29, that follows acute HCVcc infection of HFLC39
was inhibited in V protein-transduced cultures (). Studies conducted predominantly in hepatoma lines have defined RIG-I as the primary PRR required for IFN induction after HCV infection.5
Because V proteins do not engage RIG-I, but do bind helicases MDA5 and LGP2,20, 22
our results may suggest that recognition of HCV RNA occurs differently in primary HFLC cultures, and that infection of these cells leads to the generation of RNA species that are capable of directly activating the MDA5 pathway. Alternatively, they may reflect V protein antagonism of the PRR up-regulation that has been shown to accompany IFN stimulation.40
Previous work from our laboratory has shown that overexpression of RIG-I or MDA5 is inhibitory for HCV replication in Huh-7 cells.34
Additional studies are required to further define the mechanisms for V protein-mediated enhancement of HCV replication in primary culture.
Chimeric HCV genomes, such as those used in our study, enable the production of well-defined virus stocks for use in infection experiments. In contrast, HCV derived from patient plasma or tissues may be of variable infectious titer, and frequently complexed with virus-neutralizing antibody. Reports of successful infection of primary hepatocytes with such isolates are relatively rare, and the degree of replication achieved has varied widely between different culture systems.6-11
Few have reported sustained production of titratable infectious virus. Many factors may contribute to this variability, including the differentiation state of cultured hepatocytes and differences between HCV genomes in their relative dependence on cellular cofactors required for virus replication and spread.
Our results with HCVcc suggest that the hepatocyte innate response to infection may provide an additional barrier to productive replication in primary culture. Strategies aimed at dampening this response may be key to the further development of robust HCV culture systems based on infection or transfection of viral genomes.