CD81 is known to mediate viral entry in HCV infection (10
) and was also implicated in the cell-to-cell transmission of HCV infection (45
). Our study showed that significant differences existed in HCV RNA levels after HCV infection among CD81-H, CD81-L1, and CD81-L2 populations and could not be explained simply by the CD81 entry function. This observation prompted us to investigate whether CD81 is required for additional steps in the HCV life cycle, such as RNA replication. Using a variety of techniques and cell lines, we uncovered a novel function of CD81 in the HCV life cycle that is important for HCV RNA replication. CD81 is a tetraspanin family member and is enriched in the lipid rafts of membranous compartments of the cell, where HCV RNA replication is believed to take place (9
). The requirement for CD81 participation by the HCV replication process can be facilitated by the physical proximity of CD81 to the HCV replicating site.
To explain our data and the proposed dual functions of CD81 in the context of HCV infection and replication, we reason that a low threshold amount of CD81 is required for the HCV entry function but that a much higher level of CD81 is necessary for efficient HCV replication subsequent to viral entry. Koutsoudakis et al. (26
) previously reported that about 70,000 CD81 molecules in a cell appear to be the threshold for viral entry. Our data suggest that the three cell lines with very different levels of CD81 allow similar viral entry but appear to support divergent efficiencies of HCV replication that correlate well with CD81 levels. It is interesting that CD81-L2 cells, despite having very low levels of CD81, can still support viral entry although at a somewhat lower level than the higher-level-CD81-expressing cells. The CD81 expression level of the L2 cells is probably just around the “threshold level” for viral entry.
Our data provide evidence for the mutually exclusive use of HCV RNA as templates for either RNA replication or protein synthesis in infected cell cultures. Two lines of evidence support that the use of HCV RNA for RNA replication is subjected to cellular factor control, such as CD81. One line of evidence is the absence of efficient RNA replication after viral protein translation in HCV-infected and RNA-transfected CD81-L1 and -L2 cells, suggesting that RNA replication could not occur efficiently when the CD81 level was low. The other evidence is that a clear pattern for one dominant template function at a time was shown for infected CD81-H cells, suggesting that there is a coordinated process that directs HCV RNA molecules toward RNA replication function. It is likely that cellular factors are involved in directing viral RNA molecules toward two distinct template functions. On the other hand, viral protein synthesis is negatively correlated with CD81 and HCV RNA levels. These data suggest that the template function of HCV RNA is controlled by cellular factors like CD81, which directs HCV RNA toward its replication function instead of protein translation. However, it is not clear how CD81 exerts this function.
CD81 may assist directly in the assembly of the HCV replicase complex, including NS5B, contributing to viral RNA replication. Alternatively, CD81 may be linked indirectly to a cellular pathway that is critical for efficient viral replication. In a recent study, Brazzoli et al. showed that the engagement of CD81 during HCV infection activates the Raf/MEK/extracellular signal-regulated kinase (ERK) signaling cascade and that this pathway affects postentry events of the HCV life cycle, presumably at the replication step (8
). Further experiments are needed to elucidate the molecular mechanism of this novel CD81 function.
CD81 was previously reported to have diverse functions in biological process. For instance, CD81 is implicated in the metastasis of cancer cells (21
). CD81 can influence the adhesion, morphology, activation, proliferation, and differentiation of B, T, and other cells (22
). In parasite infections, hepatocyte CD81 is required for Plasmodium falciparum
and Plasmodium yoelii
sporozoite infectivity (39
). CD81 was also implicated in the modulation of infectivity, enhancement of viral gene expression, and promotion of virus assembly, budding, and cell-to-cell spread in the HIV life cycle (19
). The identification of this novel CD81 function in HCV replication indicates that CD81 plays a more pleiotropic role in the HCV life cycle besides its well-defined role in viral entry. Our data suggest that CD81 has dual functions in HCV infection: a low threshold level of CD81 required for viral entry and a higher level of CD81 necessary for efficient HCV RNA replication. The dependence of HCV replication on CD81 creates an inherent vulnerability for HCV replication. Thus, CD81 functions could be explored for potential therapeutic development because of the multiple roles of CD81 in HCV infection, as was explored in a recent study of the Alb-uPA/SCID mouse model engrafted with human hepatocytes (29