Here, we provide evidence for an involvement of a proteasome subunit with HCV translation by demonstrating that inhibition of HCV IRES activity by the ribozyme Rz3′X, originally engineered to target the 3′X sequence of the HCV minus strand, was mediated by targeting the proteasome subunit PSMA7 RNA. Rz3′X was found to induce GCV-resistant colonies in a bicistronic HCV IRES-HSV TK reporter system and reduced HCV IRES-mediated core protein translation, indicating an inhibitory effect of the ribozyme on HCV IRES activity. Using C-SPACE, a ribozyme cleavage-based technique for target gene identification, we discovered human 20S proteasome α-subunit PSMA7 mRNA as a target RNA cleaved by Rz3′X. Additional ribozymes directed against PSMA7 RNA also substantially inhibited HCV IRES activity in a stable HeLa IRES-TK reporter system as well as in an independent transient Huh7 IRES-luciferase reporter assay. Rz3′X and PSMA7-directed ribozymes reduced HCV IRES activity independently of HCV core protein and did not significantly influence EMCV IRES or HRV IRES activities in transiently transfected Huh7 cells. These data indicate that the reduction in core protein associated with expression of active ribozymes in the stably selected GCV-resistant cells was not caused by decreased core protein stability and that the IRES-inhibitory effect is mediated by factors other than the core protein. The data obtained from different reporter systems and in particular the lack of activity on other viral IRESs suggest a preferential effect of the ribozymes on HCV IRES and indicate that the ribozymes do not generally decrease overall translation or protein stability.
Interestingly, we observed that a 30% decrease in PSMA7 RNA was sufficient to induce a 70 to 80% reduction in HCV IRES translation. Similarly, Rz3′X induces a high number of GCV-resistant colonies, while only moderately decreasing PSMA7 RNA and protein expression in the stable selection system presented here. In contrast, VRz4 exactly matching the Rz3′X target site within PSMA7 RNA revealed less GCV-resistant colonies, while a stronger reduction in PSMA7 RNA and protein was noted. Additionally, in the transient luciferase reporter system, VRz4 caused a higher inhibition of IRES activity than Rz3′X. This is in concordance with our previous observations that a biologically relevant change of function was often achieved by a moderate knock-down of target RNA expression by hairpin ribozymes (3
). Our results also suggest that, depending on the target molecule, a more active ribozyme with perfectly matching binding arms can exhibit cellular toxicity in s
table selection systems, whereas this effect is less important for transient assay systems.
The ubiquitin proteasome pathway is a highly conserved intracellular pathway for the degradation of proteins. Many of the short-lived regulatory proteins that govern cell division, growth, activation, signaling, and transcription are substrates degraded by the proteasome (1
). The 26S proteasome is a multisubunit protease complex that catalyzes the final step of intracellular protein degradation. It consists of a cylindrical 20S catalytic chamber, a barrel-shaped structure, shown by electron microscopy to comprise four rings, each containing seven subunits, which is capped on both ends by a 19S regulatory complex. All 14 20S proteasomal subunit sequences may be classified into two groups, α and β, each group showing distinct structural and functional roles (13
). Ubiquinated proteins are recognized and unfolded by the regulatory complex and threaded through the small pores at the ends of the catalytic chamber, where they are degraded by different protease activities. The resulting peptides exit from the cylinder, upon which the ubiquitin chains from the degraded protein are recycled. A variety of proteins are processed via the ubiquitin-proteasome pathway, such as proteins involved in cell proliferation or cell cycle control, transcriptional regulators, cytosolic proteins, membrane proteins, and major histocompatibility complex class I (MHC-I) antigen processing (17
). Viral proteins synthesized in virus-infected cells are (partially) degraded by the proteasome (5
). The resulting peptides are bound to MHC-I molecules and presented on the cell surface to initiate antiviral immune defenses. Recent data suggested an additional antiviral function of the proteasome at the cellular level by degradation of incoming human immunodeficiency virus proteins (20
). In addition, the 20S proteasome associates with a number of viral RNAs which are cleaved by proteasome-associated endonuclease activity (6
) and it is very likely that additional interactions of proteasome subunits with viral RNA or protein exist.
A dose-dependent inhibitory effect on HCV IRES-mediated translation was demonstrated for short-term application of MG132 in transient and stable HCV IRES-dependent luciferase reporter assays prior to the onset of cellular toxicity. However, when we applied MG132 or lactacystin to HeLa 5′tk reporter cells (1, 10, or 20 μM for 6 h) prior to GCV selection, all cells pretreated with the proteasome inhibitors died during selection with GCV (data not shown). The dose-dependent toxic effects observed upon the application of proteasome inhibitors were not observed when Rz3′X or anti-PSMA7 ribozymes were applied to inhibit proteasome function. This suggests distinct mechanisms of ribozyme-mediated inhibition of PSMA7 RNA and peptide inhibition by proteasome inhibitors.
Since the knowledge about the exact functions of the proteasome subunits and the interaction between proteasomes and viral gene products is still limited, additional studies are required to evaluate the effects of ribozyme-mediated cleavage of proteasomal subunits on modulation of proteasome function and viral antigen processing and presentation as well as viral replication in host cells. Does the proteasome influence HCV IRES translation directly or by influencing degradation of specific cellular (or viral) translation factors mediating this effect? Is this reduction in IRES activity specific for HCV IRES and not applicable to other cellular IRESs? Are the effects reproducible for subgenomic replicons (4
) or the potential full-length HCV replication system?
Rz3′X might confer synergistic antiviral effects on HCV by (i) cleavage of the highly conserved antigenomic 3′X sequence of HCV 3′-UTR and (ii) a reduction in IRES activity mediated by inhibition of PSMA7 RNA. Since the HCV 3′-terminal sequence is essential for productive replication in vivo (12
) and significantly enhances translation of HCV RNA from the HCV IRES when supplied in cis
), a ribozyme targeting this crucial HCV sequence might substantially inhibit translation of viral gene products and viral replication. We are currently investigating the antiviral effects of Rz3′X in cell culture systems of HCV replication.
Finally, the development of specific, nontoxic proteasome inhibitors, such as ribozymes, antisense oligonucleotides, and small molecules that inhibit active sites of proteasome subunits, might prove useful for investigating the role of the ubiquitin-proteasome pathway in diverse cellular processes, such as inflammatory events, cellular immune surveillance, tumorigenesis, and chronic infectious diseases.