Researchers are actively working to develop inhibitors of several stages of the hepatitis C viral (HCV) lifecycle including entry, replication, and assembly 
. A curative antiviral therapy for HCV-infected patients will likely be comprised of a combination of two or more distinct viral inhibitors. An optimal HCV inhibitor combination will prevent the virus from acquiring resistance mutations and lead to eradication of the virus from the patient.
In recent years, significant progress has been made toward understanding HCV entry 
and developing inhibitors of this process 
. HCV entry is initiated by the attachment of viral envelope proteins (E1 and E2) to glycosaminoglycans 
followed by a post-attachment stage which includes specific binding to cellular receptors and subsequent uptake into the cell. The five cellular receptors known to be utilized by HCV are the tetraspanin protein CD81 
, scavenger receptor class B member 1 
, the Niemann-Pick C1-like 1 cholesterol absorption receptor 
, claudin 1 
, and occludin 
. In addition, the tyrosine kinases epidermal growth factor receptor and ephrin receptor A2 are thought to act as HCV entry co-factors by modulating the interaction between CD81 and claudin 1 
. After receptor binding, HCV undergoes clathrin-mediated endocytosis and fusion between the virion envelope and the endosomal membrane 
. Anti-CD81 antibody (Ab) has been used to successfully block HCV binding of the CD81 receptor and viral uptake into the cell 
. In addition, Entry Inhibitor-1 (EI-1) is a small molecule that inhibits HCV genotype 1a and 1b entry during the post-attachment phase, likely during the fusion step 
Though there has been progress in understanding HCV entry and developing entry inhibitors, HCV viral dynamic models predict that entry inhibitors will have a slow and modest antiviral activity as monotherapies in chronically-infected patients 
. These models predict that entry inhibitors would reduce viral load in a monophasic manner reflecting the slow death rate of infected hepatocytes in vivo
2–70 days) and the protection of naïve uninfected cells from HCV infection. In contrast, replication inhibitors are predicted to reduce viral load in a biphasic manner. The initial rapid reduction phase is due to the inhibition of virus production and elimination of plasma virus (t1/2
~3 hours). The second, slower reduction phase results from the elimination of infected hepatocytes 
. However, for many classes of replication inhibitors, monotherapy leads to the rapid emergence of viral resistance mutations 
. Combining two replication inhibitors with different targets or a replication inhibitor with an entry inhibitor would theoretically impact the emergence of resistance by increasing the number of viral mutations required to break through therapy. Because some mutations are less likely to emerge than others 
and because some mutations reduce viral fitness 
, an optimal combination of inhibitors must be investigated experimentally.
Here we sought to determine if HCV entry inhibitors alone can reduce viral levels in persistently-infected Huh7 cultures. Also we sought to determine if HCV entry inhibitors combined with HCV replication inhibitors can provide a greater reduction in viral levels than either monotherapy in persistently-infected cultures. Finally, we wanted to determine if an entry/replication inhibitor combination could prolong reductions in viral levels relative to replication inhibitor monotherapy. To enable these studies, we first demonstrated that persistently-infected Huh7 cell cultures can be established using tissue-culture adapted HCV and used as a model system to monitor extracellular virus levels during antiviral treatment. Using these persistently-infected cell cultures, we observed that entry and replication inhibitor monotherapies fit the model previously proposed for viral load reduction during short-term treatment. Entry inhibitor monotherapy caused a slow, monophasic reduction in viral levels, while replication inhibitor monotherapy caused a rapid, biphasic reduction. This suggests that entry inhibitors will only have a modest impact on serum HCV RNA levels in chronically-infected patients who have minimal viral spreading. However, our results also demonstrated that the combination of an entry plus replication inhibitor can prolong antiviral suppression, likely due to the delay of viral resistance emergence.