Tylophorine Analogs Allosterically Regulates Heat Shock Cognate Protein 70 And Inhibits Hepatitis C Virus Replication

Tylophorine analogs have been shown to exhibit diverse activities against cancer, inflammation, arthritis, and lupus in vivo. In this study, we demonstrated that two tylophorine analogs, DCB-3503 and rac-cryptopleurine, exhibit potent inhibitory activity against hepatitis C virus (HCV) replication in genotype 1b Con 1 isolate. The inhibition of HCV replication is at least partially mediated through cellular heat shock cognate protein 70 (Hsc70). Hsc70 associates with the HCV replication complex by primarily binding to the poly U/UC motifs in HCV RNA. The interaction of DCB-3503 and rac-cryptopleurine with Hsc70 promotes the ATP hydrolysis activity of Hsc70 in the presence of the 3′ poly U/UC motif of HCV RNA. Regulating the ATPase activity of Hsc70 may be one of the mechanisms by which tylophorine analogs inhibit HCV replication. This study demonstrates the novel anti-HCV activity of tylophorine analogs. Our results also highlight the importance of Hsc70 in HCV replication.

The HCV pFKI389 lucubineo NS3-3' plasmid was a generous gift from Dr. Ralf Bartenschlager at the University of Heidelberg 1 .
The Hsc70 NBD (residues 1-385) plasmid was constructed by inserting a stop codon after position 1155 in the Hsc70 FL pET28a plasmid using the QuikChange® XL sitedirected mutagenesis kit (Stratagene). The D206S mutation in the Hsc70 gene, rHsc70-1, rHsc70-2, and the D318N mutation in the NS5B gene were introduced using the QuikChange® XL site-directed mutagenesis kit. The ER-Hsc70 plasmid was constructed by inserting the ER localization signal peptide (MSFVSLLLVGILFWATEQLTKCEVFQ) into the N-terminus of HA-Hsc70 plasmid. Primers used for mutagenesis are shown in Table S1. Poly A was cloned from synthetic poly A into a pGL4 vector. To generate the HCV pFKI389 lucubineo NS3-3' plasmid without the poly U/UC motif at the 3' NTR, we cloned the plasmid without the poly U/UC motif by adding a Sca I site to the end of the NS5B coding region, and ligated the linear DNA by the new Sca I site and the existing Sca I site after the poly U/UC motif.

Generation of HLN-cure cells
Highly permissive HLN-cure cells were derived by through interferon treatment similar to the derivation of Huh 7.5 cells described previously 3

Determination of anti-HCV activity by luciferase reporter assay
Firefly-luciferase reporter activity was used to monitor the replication of HCV replicons in Huh-luc/neo-ET cells free from G418 5 . Luciferase activity was measured with a luciferase assay kit (Promega) on Tecan FARCyte Luminometer (GE Healthcare) according to the manufacturer's instruction.

RNA isolation and Real-Time PCR
Total RNA was isolated using the High Pure RNA isolation kit (Roche, Mannheim, Germany). cDNA was synthesized from total RNA using random primers according to the protocol of the iScript TM cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA), and expression levels of specific genes were quantified by Real-Time PCR with genespecific primers (Table S5) using the SsoFast TM EvaGreen supermix (Bio-Rad Laboratories). Absolute copy number of HCV RNA was determined with the standard curve generated using the linearized pFK-I389/NS3-3′ plasmid bearing the HCV genome and 18S rRNA was included as a control.

In vitro transcription assay
HCV subgenomic replicon encoding plasmid pFK I389 lucubineo NS 3-3' was linearized by ScaI. Different fragments of the 3' NTR region from HCV RNA were cloned into a TA-PCRII vector (Invitrogen) with a T7 promoter. Primers used for cloning are provided in Table S1. The linearized plasmids were used as the template for in vitro transcription.
Uncapped and capped HCV replicon mRNAs were generated using the MEGAscript kit containing T7 RNA polymerase (Ambion, Austin, TX). The in vitro transcribed mRNAs were purified using the MEGAclear kit (Ambion). The integrity of the in vitro transcribed mRNAs was confirmed using the Agilent 2100 Bioanalyzer (Foster City, CA). The purified mRNAs were used for in vitro translation experiments, or in vitro RNA binding assays, or ATPase hydrolysis assays.

Antibodies
The following antibodies were used: rabbit and mouse IgG International). All antibodies were used at a 1:2,000 dilution for Western blot analysis.

Optiprep iodixanol continuous density gradient
Optiprep iodixanol density media was purchased from Sigma-Aldrich. Continuous density gradients were performed as described 6 . Briefly, 10 7 Huh-luc/neo-ET cells were

Preparation of HCV crude replication complexes (CRCs)
Preparation of CRCs was adapted from a previous protocol with modification 7

In vitro RNA binding assay
For the in vitro RNA binding assay, 100 μM in vitro transcribed biotinylated HCV RNA (RNA sequence was shown in Figure 5B

Characterization of ATP hydrolysis activity of Hsc70
Characterization of the ATP hydrolysis activity of Hsc70 was described previously 10 .  and HA-NS4B with Hsc70 were examined by immunoprecipitation as described in Figure   4A. (B) Hsc70-associated complex was immunoprecipitated with Hsc70 antibody. The levels of surivin and β-actin RNA were examined by real-time PCR as in Figure 5B.