The mechanisms of replication and translation of HCV RNA have been extensively studied in the past few years. However, the exact subcellular localization of HCV RNA replication and translation is still unclear. Evidence has previously been presented that HCV RNA replication occurs on the detergent-resistant membrane (DRM) possibly derived from the ER 
. In this report, the newly synthesized RNA was shown to be transported by the anterograde vesicle transport pathway. The microtubule-dependent mobility of newly-synthesized HCV RNA or the replication complex has also been described elsewhere 
. Our data in this study further showed that the nocodazole treatment inhibited the transportation of the newly-synthesized RNA from the ER-derived replication complex to Golgi but did not inhibit the initiation of HCV RNA replication, since BrUTP labeling of HCV RNA occurred normally in the presence of nocodazole (). Intuitively, the newly-synthesized HCV RNA is expected to be transported to the site of the cellular translation machinery, similar to the case for cellular mRNAs, which are synthesized in the nucleus and transported to the cytoplasmic translation machinery for protein synthesis. However, we instead found that the movement of the HCV RNA from the ER to Golgi was not required for HCV translation, suggesting that the newly synthesized HCV RNA is used for translation near the site of HCV RNA synthesis before being transported away. Furthermore, we showed that active RNA replication was a prerequisite for efficient HCV translation. This conclusion was demonstrated using four different approaches, including studying the effects of an HCV RNA polymerase inhibitor on HCV protein translation (), comparing the translation efficiencies of wildtype and replication-defective replicons () and those of infectious and non replicating JFH strain of HCV (Fig. S2A
), and also by determining the relative translation efficiencies of the replicating and non-replicating dual luciferase reporter plasmids (). Finally, we showed that the newly synthesized viral proteins almost completely colocalized with the newly synthesized viral RNA, suggesting that the sites of HCV RNA replication and protein translation nearly overlap. This mechanism of coupled RNA replication and translation may explain the previous findings that many cellular proteins, such as PTB 
, La antigen 
and SYNCRIP 
, are involved in both the replication and translation in the HCV life cycle. The close proximity of these two machineries will allow for ready switches between translation and replication.
Although coupling of translation and RNA replication has been reported for many RNA viruses 
_ENREF_37, the HCV case appears to be unique. For example, translation and replication of poliovirus RNA are coupled, but in the sense that RNA transcription is dependent on viral translation in ci
. Insertion of an early termination codon resulted in lower efficiency of poliovirus RNA replication. The translation and replication are regulated by the binding of different cellular or viral proteins to the 5? UTR of poliovirus RNA 
. Also, the microtubule-dependent movement of poliovirus viral RNA is associated with the replication activity of viral RNA 
. While the inactive replication complexes reside at microtubule-organizing center (MTOC), the replicating viral RNA is localized at the perinuclear sites 
. Thus, the RNA movement is required for poliovirus replication, in contrast to the situation with HCV. In HCV, nocodazole did not inhibit viral RNA replication; also, the newly-synthesized HCV RNA failed to exit from ER after the nocodazole treatment and yet, protein translation increased; thus, the cytoskeleton-assisted movement of the newly-synthesized HCV RNA is not required for RNA translation. Thus, in HCV, the observed movement of the viral RNA from the ER-derived to the Golgi-derived membrane appears to be required for other steps of HCV replication, rather than protein translation. Due to the fact that the viral structural proteins are absent in the HCV replicon cells, this RNA movement is likely mediated by viral NS proteins, such as NS5A, which has been reported to target Golgi apparatus 
. In a kinetics study examining the appearance of the newly synthesized HCV RNA and HCV proteins in the HCV (JFH-1)-infected cell, we also found that all of the newly synthesized proteins were at the site of newly synthesized RNA (). Thus, there appears to be a replication complex that carries out both replication and translation. This concept is novel to the known mechanisms of RNA virus translation and transcription.
These findings raised an important issue, namely, how the initial viral translation is carried out, since, as a positive-strand RNA virus, the very initial round of translation from the incoming HCV viral genome has to take place before viral RNA replication can occur. Conceivably, the free viral RNA genome generated by uncoating of the incoming virion in the endosome (or from the transfected viral RNA or replicons) can associate with ribosomes on the rough ER and be translated in an RNA replication-independent manner. Such translation is likely of low efficiency, but is sufficient to support first round of HCV RNA translation. These initial viral protein products and RNAs will then be encased into the membranous replication complex and become part of the replication-translation machinery. The latter process will then become the main mechanism of HCV replication-translation.
In summary, we propose the following pathway for HCV RNA replication and translation (). Previous studies have shown that HCV RNA replication takes place in an ER-derived membranous vesicle 
. The newly-synthesized viral RNA will be translated immediately after being synthesized in or around the vesicle. Benzothiadiazine treatments inhibited HCV RNA transcription and therefore inhibited HCV RNA translation as well (, A–B). After translation has occurred, HCV RNA is then transported via anterograde vesicle trafficking pathway away from the ER to a membrane compartment associated with the Golgi apparatus (). Several other reports suggest that HCV RNA may be transported along with viral proteins (core and NS5A) to lipid droplets, where viral assembly takes place 
; however, the kinetics of this process is unclear. Further studies in dissecting HCV RNA transportation during HCV infection will be valuable for understanding HCV life cycle comprehensively. In conclusion, this replication and translation machinery constitutes the viral “replicasome”, which may reflect the membranous webs observed previously.
The proposed model of coupled replication/translation of HCV RNA.