The authors have declared that no competing interests exist.
Conceived and designed the experiments: HML ML. Performed the experiments: HML HA. Analyzed the data: HML JO ML. Contributed reagents/materials/analysis tools: HML HA KM. Wrote the paper: HML ML.
Hepatitis C virus (HCV) RNA initiates its replication on a detergent-resistant membrane structure derived from the endoplasmic reticulum (ER) in the HCV replicon cells. By performing a pulse-chase study of BrU-labeled HCV RNA, we found that the newly-synthesized HCV RNA traveled along the anterograde-membrane traffic and moved away from the ER. Presumably, the RNA moved to the site of translation or virion assembly in the later steps of viral life cycle. In this study, we further addressed how HCV RNA translation was regulated by HCV RNA trafficking. When the movement of HCV RNA from the site of RNA synthesis to the Golgi complex was blocked by nocodazole, an inhibitor of ER-Golgi transport, HCV protein translation was surprisingly enhanced, suggesting that the translation of viral proteins occurred near the site of RNA synthesis. We also found that the translation of HCV proteins was dependent on active RNA synthesis: inhibition of viral RNA synthesis by an NS5B inhibitor resulted in decreased HCV viral protein synthesis even when the total amount of intracellular HCV RNA remained unchanged. Furthermore, the translation activity of the replication-defective HCV replicons or viral RNA with an NS5B mutation was greatly reduced as compared to that of the corresponding wildtype RNA. By performing live cell labeling of newly synthesized HCV RNA and proteins, we further showed that the newly synthesized HCV proteins colocalized with the newly synthesized viral RNA, suggesting that HCV RNA replication and protein translation take place at or near the same site. Our findings together indicate that the translation of HCV RNA is coupled to RNA replication and that the both processes may occur at the same subcellular membrane compartments, which we term the replicasome.
Hepatitis C virus (HCV) is a positive-sense RNA virus that is estimated to chronically infect as many as 3% of the world's population. As a member of Flaviviridae, HCV is an enveloped virus with a single, positive-stranded RNA around 9.6 kb in length
Using the HCV subgenomic replicon system as well as infectious virus system, many host factors have been identified to be involved in HCV RNA replication, including the human homologue of the 33-kDa vesicle-associated membrane protein-associated protein (hVAP-33)
The balance between viral RNA transcription and translation is critical for the replication of positive-stranded RNA viruses, since the same RNA is used both for translation and as the template for negative-strand RNA synthesis. Transcription of poliovirus has been reported to be dependent on the translational activity of the viral RNA
HCV replicon cells were labeled with BrUTP (A) or 3H-Uridine (B) in the presence of actinomycin D and chased for up to 180 minutes. (A) Immunofluorescence staining with anti-BrdU and other organelle antibodies shows the colocalization of BrU-labeled HCV RNA with ER initially (30 min) and then with Golgi (180 min). (B) Fractionation of ER and Golgi by sucrose gradient. Fraction numbers and their gradient positions are noted at the bottom. 3H-Uridine-labeled RNA in the ER (fraction 4) and the Golgi (fraction 6–8) fractions were collected, and the radioactivity of 3H-Uridine-labeled RNA was counted. Immunoblotting of ER and Golgi makers demonstrates the separation of ER and Golgi by sucrose gradient fractionation.
In this study, we observed that HCV RNA exit from the site of RNA synthesis to the Golgi complex, a process that can be blocked by nocodazole, an inhibitor of the ER-Golgi transport pathway. Surprisingly, HCV protein translation was enhanced when HCV RNA movement was blocked, suggesting that the translation of viral proteins occurred near the site of RNA synthesis. We also found that the translation of HCV proteins was dependent on active RNA synthesis: inhibition of RNA synthesis resulted in decreased HCV viral protein synthesis before there was significant decrease in the total amount of HCV RNA, and that the replication-defective HCV RNA could not be translated efficiently
Huh7 or Huh7.5 cells were grown at 37°C in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and nonessential amino acids. Huh7 cells were obtained from Dr. Sato's lab
Huh-N1b cells was pre-treated with 20 µM nocodazole for 4 hours and then labeled with BrUTP or with 35S-Methionine. (A) The BrU-labeled RNA remained colocalized with calnexin (an ER amrker) even after 180 min. (B–D) Proteins were immunoprecipitated with (B) HCV patient serum, (C) Goat anti-NS3 antibody, or (D) Rabbit anti-NPT antibody. The immunoprecipitated products were detected by autoradiography. The nocodazole-pretreated Huh-N1b cells showed about 50% increase in NS3 and NPT translated from replicon RNA, whereas NPT translation in the Huh-Neo control cells was not affected by the nocodazole treatment.
HCV JFH1 and JFH-GND constructs were obtained from Dr. Wakita’s lab (NIID, Japan)
(A), Mock- or Benzothiadiazine-treated Huh-N1b and Huh-Neo cells were labeled by 35S-Methionine for 4 hours, and followed by immunoprecipitation with anti-NPT or anti-NS3 antibodies or sera from hepatitis C patients. The immunoprecipitates were separated by SDS-PAGE and detected by autoradiography. (B) The intracellular replicon RNA was detected by Northern blotting. (C) Huh7 cells transfected with
Labeling of de novo-synthesized viral RNA, immunofluorescence staining and confocal microscopy were modified from the previously described methods
Huh7.5 cells were infected with HCV JFH-1 strain for 2 days, and then were labeled with Cy5-UTP and BODIPY-FL-Lys-tRNA in the presence of actinomycin D and hippuristanol, which inhibit host RNA and protein synthesis, respectively. The cells were kept in 37°C chamber supplied with CO2 for live cell imaging on a Zeiss LSM 510 laser scanning confocal microscope. Images were taken after 10–40 minutes of chase. Newly-synthesized HCV RNA was the first to be detected (as shown in red) and was in a perinuclear pattern. Newly-translated HCV viral peptides (as shown in green) were detected at later time points, completely co-localized with the sites of RNA synthesis. No significant amount of Cy5-UTP and BODIPY-FL-Lys-tRNA labeling could be detected in naïve Huh7.5 cells (as a negative control) in the presence of actinomycin D and hippuristanol.
To determine the quantity of RNA by real-time PCR, a single-tube reaction was performed by using the TaqMan EZ RT-PCR Core Reagents (Applied Biosystems). Duplicate reactions for RNA standards and the samples were performed in 20-µl volume using 1 µl of HCV RNA, primers from HCV 5′ non-coding region (5′ GA
A proposed model of HCV replication-translation complex “replicasome”. As reported, HCV replication complexes are assembled at ER, and then bud into the ER lumen. Consequently, (A) HCV RNA replication is first initiated in the multi-layered vesicle structure derived from the ER membrane. (B) The newly synthesized HCV RNA is translated around the ER-derived vesicle
Using the ABI Prism 7900 program, standard curves of the assays were obtained automatically by plotting the three hold values against each standard dilution of known concentration (101–106 copies per reaction) of HCV genotype 1b transcript. The same software was used to calculate the coefficients of regression. Values were normalized to that of GAPDH (Applied Biosystems). Each test was done in triplicate and averages were obtained.
The procedure was based on the published method
To visualize the replication of HCV RNA, we performed pulse BrUTP labeling in the actinomycin D-treated Huh-N1b replicon cells; under such conditions, only the viral RNA, which depends on RNA-dependent RNA polymerase, is labeled. After 15-minute labeling, the labeled RNA was chased in non-labeled media for 30 minutes to 3 hours, and the subcellular localization of BrU-labeled RNA was detected with anti-BrdU antibody and co-stained with individual organelle markers for ER (Calnexin), ERGIC (ERGIC53), and Golgi apparatus (GS27), respectively (
The ER-to-Golgi trafficking of the newly synthesized RNA was further confirmed by biochemical analysis. The actinomycin D-treated Huh-N1b cells were labeled with 3H-uridine for 30 minutes and chased for 30 minutes to 3 hours. The labeled cell lysates were separated into ER and Golgi fractions by ultracentrifugation. Immunoblotting studies showed that the ER and the Golgi apparatus were efficiently separated by this procedure (
The ER-to-Golgi apparatus trafficking is known as the anterograde vesicle trafficking pathway, and can be blocked by nocodazole, which depolymerized microtubules and disrupts Golgi apparatus
These results are unexpected, raising a possibility that the newly-synthesized HCV RNA may be used for RNA translation in situ, without being transported away from the site of RNA synthesis. This result brought up an intriguing possibility that HCV RNA replication and translation are coupled and take place in the same replication complex.
To test the idea that the newly synthesized HCV RNA is used for translation in situ, we then investigated whether translation was dependent on active RNA synthesis. We used a specific NS5B polymerase inhibitor, Benzothiadiazine
We first determined the efficiency and specificity of the inhibitor on 3H-uridine incorporation (
We also studied the effects of Benzothiadiazine on the steady-state level of replicon RNA by realtime RT-PCR analysis. The data showed that even after 16 hours of treatment, the total amount of replicon RNA in the cells was not significantly affected (
Having established the specificity of Benzothiadiazine on HCV RNA synthesis, we then labeled the Benzothiadiazine-treated cells with 35S-Met and immunoprecipitated HCV proteins from the cell extracts (
We further used a replication-defective replicon RNA (GND mutation in the NS5B region) to assess if active RNA replication of HCV is required for HCV protein translation. A separate experiment using an
We further performed a similar study using the infectious HCV clone JFH1 and its replication-defective mutant, JFH1-GND, in a time-course study of HCV protein translation. Huh7 cells were transfected with JFH1 or JFH1/GND RNA, labeled with 35S-Met during the 0–8, 8–16, or 16–24 hours post-transfection, and followed by immunoprecipitation. The amounts of these two RNAs at 24 hours post-transfection were almost the same (
We next assessed if the replication and translation of HCV RNA occurred in the same subcellular localizations. Huh7.5 cells were infected with HCV (JFH1); at 2 days after infection, the cells were labeled with Cy5-UTP and BODIPY-FL-lys-tRNA in the presence of hippuristanol and actinomycin D, which blocked eIF4A-dependent translation and host RNA transcription, respectively
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
Although coupling of translation and RNA replication has been reported for many RNA viruses
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 (
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Special thanks are to Specialized Microscopy Core of Doheny Eye Institute at USC, and Microscope Subcore of the USC Center for Liver Disease.