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Journal of Virology, September 2004, p. 10202-10205, Vol. 78, No. 18
0022-538X/04/$08.00+0 DOI: 10.1128/JVI.78.18.10202-10205.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
ViroPharma Incorporated, Exton, Pennsylvania
Received 4 March 2004/ Accepted 30 April 2004
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ABSTRACT |
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TEXT |
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One of the drawbacks of the cell-free HCV replication system is the presence of endogenous RNase activity that copurifies with the replicase complex (1). This nuclease activity degrades exogenously added RNA, limiting the utility of the cell-free replication system for studying processes like replication initiation by using defined full-length HCV RNA templates. While nuclease activity degrades exogenously added RNA, newly synthesized RNA and endogenous template RNA are protected from degradation by a detergent-sensitive factor(s) (6). In this report, we demonstrate that an additional subtilisin-sensitive factor(s) is also necessary for protection of newly synthesized RNA.
As a first step towards understanding how newly replicated RNA is protected from endogenous nuclease activity, a cell-free HCV replication system was established by using membrane fractions from Huh-7 cells containing an HCV replicon derived from the infectious Con1 clone (genotype 1b) (3). Replicon-containing cells (107) were scraped into ice-cold 1x phosphate-buffered saline and concentrated by centrifugation at 1,000 x g for 10 min at 4°C. The cell pellet was resuspended in hypotonic lysis buffer (HLB) containing 10 mM HEPES (pH 8.0), 10 mM KCl, 1 mM dithiothreitol (DTT), and 1x protease inhibitor cocktail (Sigma-Aldrich, St. Louis, Mo.) and incubated for 10 min on ice. The cells were lysed by Dounce homogenization using 40 strokes of a type A pestle, and the nuclei and insoluble debris were removed by centrifugation at 1,000 x g for 5 min at 4°C. The supernatant fluid was transferred to a new tube, and the heavy membrane fraction was collected by centrifugation at 20,000 x g (P20) for 20 min at 4°C.
The P20 fraction was further purified by equilibrium centrifugation on a discontinuous sucrose gradient. The gradient was prepared by resuspending the P20 fraction in 72% sucrose in HLB (3 ml). The 72% sucrose solution was overlaid with 1 ml of 55% sucrose in HLB followed by 1 ml of 10% sucrose in HLB. In some samples, NP-40 was added to the P20 fraction to a final concentration of 0.5% prior to equilibrium centrifugation. The gradients were centrifuged at 100,000 x g for 14 h at 4°C. The material that partitioned in the 55-to-72% sucrose interface was collected, diluted in HLB, and concentrated by centrifugation at 100,000 x g for 2 h at 4°C. The membrane pellet was resuspended in 112 µl of HLB containing 5% glycerol, 1 mM DTT, and 1,920 U of RNasin (Promega Inc., Madison, Wis.). The resuspended membrane fraction was stored in 20-µl aliquots at –80°C.
Reaction mixtures (final volume, 50 µl) contained 50 mM HEPES-KOH (pH 8.0), 0.5 mM MgCl2, 1.5 mM MnCl2, 5 mM DTT, 4 µg of actinomycin D/ml, 3.2 U of RNasin/µl, 50 mM potassium acetate, 0.5 mM ATP, UTP, and GTP, 0.5 µM CTP, 1 mM creatine phosphate, 3.5 U of creatine phosphokinase/µl, 10 µCi of [
-32P]CTP (800 Ci/mmol), and 2.5 µl of membrane fractions prepared from either replicon-containing cells or parental Huh-7 cells. The reactions were initiated by the addition of nucleotide triphosphates, and the mixtures were incubated at 37°C. At 0, 5, 10, 15, 25, and 35 min, reactions were terminated by addition of 50 µl of a solution containing 10 mM HEPES (pH 8.0), 1% sodium dodecyl sulfate (SDS), and 50 mM EDTA. The RNA products were purified by phenol extraction and Sephadex G-50 spin column chromatography and concentrated by ethanol precipitation. The ethanol precipitate was resuspended in 10 µl of diethyl pyrocarbonate-treated water and denatured by treatment with 1.2 M glyoxal for 20 min at 50°C. The RNA was resolved by agarose gel electrophoresis in 1x glyoxal gel running buffer (Ambion, Austin, Tex.). The gel was dried under vacuum onto Whatman 3MM paper, and RNA products were visualized and quantified by phosphorimager analysis.
Reaction mixtures supplemented with membranes from replicon-containing cells generated radiolabeled RNA products that comigrated with full-length HCV replicon RNA, as well as products that migrated more slowly than full-length replicon RNA (Fig. 1B, lane 1). The amounts of both RNA species increased linearly as a function of time, suggesting that replicase activity was stable for at least 35 min (data not shown). Upon purification, the full-length replicon RNA products were susceptible to S1 nuclease digestion, while the more slowly migrating RNA species were less susceptible to nuclease treatment (data not shown). In vitro-transcribed (IVT) full-length replicon RNA was also susceptible to S1 nuclease digestion (data not shown). These results suggest that the full-length replicon RNA products were single stranded while the more slowly migrating RNA species contained double-stranded regions, making them less susceptible to S1 nuclease digestion. The more slowly migrating RNA species can be resolved into full-length single-strand replicon RNA and lower-molecular-weight species by methylmercury agarose gel electrophoresis, suggesting that the more slowly migrating RNA species detected on glyoxal gels are likely RNA replication intermediates that are not fully denatured by glyoxal treatment (1).
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The presence of RNase was confirmed using a commercially available test kit (RNaseAlert; Ambion) that quantifies RNase activity by measuring cleavage of an RNA substrate tagged with a fluorescent reporter molecule. Using this assay, RNase activity was readily detected in membrane fractions from replicon containing cells (data not shown).
While exogenously added RNA was rapidly degraded in membrane fractions, newly synthesized RNA products appeared to be resistant to degradation. This observation suggested that newly synthesized RNA was protected from nuclease activity. To test this hypothesis, the stability of newly synthesized RNA was measured by pulse-chase analysis (Fig. 1B). Replication reaction mixtures were incubated for 30 min in the presence of 10 µCi of [-32P]CTP (800 Ci/mmol) (pulse) to generate radiolabeled RNA. The reaction mixtures were then supplemented with 300 µM unlabeled CTP (chase), and at 10-min intervals, a portion of the reaction mixture was removed and added to an equal volume of 10 mM Tris (pH 7.5), 1 mM EDTA, 150 mM NaCl, and 0.5% SDS (stop buffer) to terminate the reaction. The RNA was purified from the reaction mixture and analyzed by glyoxal agarose gel electrophoresis. Newly synthesized full-length replicon RNA was found to be stable for up to 60 min in the presence of membrane fractions (Fig. 1B). These results suggest that newly synthesized full-length replicon RNA is protected from endogenous nuclease activity.
To characterize the nature of the activity that protects full-length HCV replicon RNA from nuclease degradation, the stability of newly synthesized RNA was measured under pulse-chase labeling conditions in the presence and absence of NP-40 or the protease subtilisin. Replicase reaction mixtures were incubated for 30 min in the presence of 10 µCi of [-32P]CTP (800 Ci/mmol) (pulse) to generate radiolabeled RNA. The reaction mixtures were then supplemented with 300 µM unlabeled CTP (chase) in the presence of either 0.5% NP-40 or 0.5, 5, or 50 U of subtilisin/µl. At 10-min intervals, the reactions were terminated by the addition of stop buffer, and the RNA was purified and analyzed by glyoxal agarose gel electrophoresis. The results show that treatment with NP-40 or subtilisin during the chase period rendered newly synthesized full-length replicon RNA fully susceptible to nuclease digestion (Fig. 2). The more slowly migrating RNA species were resistant to degradation under these conditions. These results are consistent with the observation that purified full-length replicon RNA is susceptible to S1 nuclease digestion while the more slowly migrating RNA species is resistant to S1 nuclease digestion (data not shown). This suggests that the more slowly migrating RNA species may be a replication intermediate containing portions of double-stranded RNA that are resistant to nuclease digestion. The ability of NP-40 to destabilize newly synthesized full-length replicon RNA is consistent with the observation that replicon RNA in permeabilized replicon-containing cells was sensitive to nuclease digestion after addition of detergent (6). These results suggest that the replicon RNA is protected by a component that is sensitive to both detergent and protease treatment.
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Membrane fractions from replicon-containing cells were treated with 0, 0.01, 0.1, and 1.0 U of subtilisin/ml in the presence and absence of 0.5% NP-40. The protease susceptibilities of HCV proteins were determined by Western blot analysis using monoclonal antibody (MAb) NCL-HCV-NS3 (Novocastra Laboratories Ltd., Newcastle, United Kingdom) and MAb 1877 (ViroStat Inc., Portland, Maine), which are specific for the HCV NS3 and NS5A proteins, respectively (Fig. 3) NP-40 had little effect on the susceptibility of NS3 to subtilisin digestion. Full-length NS3 was cleaved with increasing concentrations of subtilisin. At the highest subtilisin concentration (1.0 U/ml), a protease-resistant domain of NS3 of 53 kDa was observed. This protease-resistant domain was also detected after subtilisin treatment of purified NS3 protein, suggesting that protease resistance is an intrinsic property of NS3 (data not shown). NP-40 had a slight effect on the susceptibility of NS5A to subtilisin digestion. Approximately 90% of NS5A was degraded by subtilisin in the absence of NP-40, while in the presence of NP-40, NS5A was completely digested (Fig. 3).
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Purified membrane fractions from replicon containing cells were treated with subtilisin at 0, 0.01, 0.1, and 1 U/ml in the presence and absence of 0.5% NP-40, and the protease susceptibilities of calreticulin and calnexin were measured by Western blot analysis using rabbit polyclonal antiserum PA3-900 (Affinity BioReagents, Golden, Colo.), which is specific for calreticulin, and MAb RDI-CALNEabm (Research Diagnostics Inc., Flanders, N.J.), which is specific for calnexin (Fig. 3). NP-40 treatment of membrane fractions did not significantly change the protease susceptibility of calnexin and calreticulin at 0.01 and 0.1 U of subtilisin/ml (Fig. 4). Importantly, calreticulin, which resides entirely in the ER lumen, was fully protected from protease treatment, while a portion of calnexin was protease resistant. The protease-resistant region of calnexin had the same molecular weight as the luminal domain of calnexin, suggesting that protease resistance was likely due to the presence of intact ER membranes. At the highest concentration of subtilisin (1 U/ml), partial protease susceptibility of both calreticulin and the protease resistant domain of calnexin was observed in the presence of NP-40. These results suggest that 0.5% NP-40 had a slight effect on the integrity of ER-derived microsomes; however, detergent treatment did not completely disrupt ER-derived microsomal membranes.
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Taken together, the results presented here suggest that under conditions where viral membrane complexes remain intact, both NP-40 and subtilisin treatment were able to render newly synthesized HCV replicon RNA susceptible to nuclease digestion. These results suggest that newly synthesized HCV replicon RNA is protected by proteins within the replication complex.
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ACKNOWLEDGMENTS |
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This work was supported by ViroPharma Incorporated.
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FOOTNOTES |
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