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. 2004 Sep;78(18):10202-5.
doi: 10.1128/JVI.78.18.10202-10205.2004.

Newly synthesized hepatitis C virus replicon RNA is protected from nuclease activity by a protease-sensitive factor(s)

Guang Yang  1 , Daniel C Pevear, Marc S Collett, Srinivas Chunduru, Dorothy C Young, Christopher Benetatos, Robert Jordan
Affiliations

Newly synthesized hepatitis C virus replicon RNA is protected from nuclease activity by a protease-sensitive factor(s)

Guang Yang et al. J Virol. 2004 Sep.

Abstract

Biochemical characterization of hepatitis C virus (HCV) replication using purified, membrane-associated replication complexes is hampered by the presence of endogenous nuclease activity that copurifies with the replication complex. In this study, pulse-chase analyses were used to demonstrate that newly synthesized replicon RNA was protected from nuclease activity by a factor(s) that was sensitive to 0.5% NP-40 or protease treatment. Nuclease susceptibility was not related to disruption of lipid membranes, since NP-40 did not significantly affect the buoyant density of HCV replication complexes or protease susceptibility of HCV NS3 and NS5A proteins. These results suggest that a protease-sensitive factor(s) protects newly synthesized RNA from nuclease degradation.

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Figures

FIG. 1.
FIG. 1.
Stability of IVT replicon RNA and newly synthesized replicon RNA in the presence of membrane fractions. Replication reaction mixtures were supplemented with radiolabeled IVT replicon RNA (A) or 10 μCi of [α-32P]CTP (800 Ci/mmol) (B) to generate de novo-synthesized radiolabeled replicon RNA. The reaction mixtures were incubated for 30 min at 37°C followed by addition of 50 μl of 300 μM unlabeled CTP. 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 to stop the reaction. Distilled water (DW) instead of membrane fractions was added to the reaction mixture in lane 1 (A). For both panels, the RNA products were purified and analyzed by glyoxal gel electrophoresis.
FIG. 2.
FIG. 2.
Stability of newly replicated HCV RNA in the presence of NP-40 or subtilisin treatment. Pulse-chase analysis of radiolabeled replicon RNA from replication reaction mixtures supplemented with membrane fractions from Huh-7 cells (lane 2) or replicon containing cells (lanes 3 to 12) was done. The reaction mixtures were incubated for 30 min at 37°C (pulse) followed by the addition of 50 μl of 300 μM unlabeled CTP (chase) in the presence and absence of 0.5% NP-40 or 0.5, 5, and 50 U of subtilisin/ml. At 10-min intervals (NP-40-treated samples) or at 60 min (subtilisin-treated samples), 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 to stop the reaction. The RNA products were analyzed by glyoxal gel electrophoresis.
FIG. 3.
FIG. 3.
Effects of NP-40 on the subtilisin susceptibilities of HCV NS3, NS5A, and NS5B and the cellular proteins calnexin and calreticulin in purified membrane fractions. Purified membrane fractions (15 μg) were treated with 0.01, 0.1, and 1.0 U of subtilisin/ml for 30 min at 37°C in the presence and absence of 0.5% NP-40. The proteins in the reaction mixtures were resolved by SDS-polyacrylamide gel electrophoresis and transferred to Hybond C+ membranes (Amersham, Inc. Piscataway, N.J.). The membranes were probed with a 1-to-100 dilution of anti-HCV NS3 MAb. The membranes were stripped of primary antibody by incubation in 0.2 N NaOH for 5 min at room temperature and reprobed with a 1-to-100 dilution of anti-HCV NS5A MAb or a 1-to-500 dilution of anticalnexin MAb or anticalreticulin antiserum. The Western blots were developed with an enhanced chemiluminescence detection system (Amersham, Inc.).
FIG. 4.
FIG. 4.
Effects of NP-40 on the integrity of HCV membrane complexes. Membrane fractions were incubated for 15 min at 37°C in the presence and absence of 0.5% NP-40 prior to purification by equilibrium centrifugation. Replicase activity and protein content were measured in purified membrane fractions. The RNA products from the replicase reaction mixtures were resolved by glyoxal gel electrophoresis. (A) Lane IVT, IVT replicon RNA; lane 1, RNA from reaction mixtures supplemented with the 20,000 × g membrane pellet (P20) from Huh-7 cells; lane 2, P20 pellet from replicon containing cells prior to equilibrium sedimentation; lane 3, untreated P20 membrane pellet from replicon containing cells purified by equilibrium centrifugation; lane 4, P20 membrane pellet from replicon containing cells treated with NP-40 prior to equilibrium centrifugation. (B) Western blot analysis of proteins from membranes purified by subcellular fractionation and equilibrium sedimentation. The Western blots were probed with a 1:100 dilution of anti-NS3 MAb and anti-NS5A MAb.
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