Ribonucleoside analogue that blocks replication of bovine viral diarrhea and hepatitis C viruses in culture

Abstract

A base-modified nucleoside analogue, beta-D-N(4)-hydroxycytidine (NHC), was found to have antipestivirus and antihepacivirus activities. This compound inhibited the production of cytopathic bovine viral diarrhea virus (BVDV) RNA in a dose-dependant manner with a 90% effective concentration (EC(90)) of 5.4 microM, an observation that was confirmed by virus yield assays (EC(90) = 2 microM). When tested for hepatitis C virus (HCV) replicon RNA reduction in Huh7 cells, NHC had an EC(90) of 5 microM on day 4. The HCV RNA reduction was incubation time and nucleoside concentration dependent. The in vitro antiviral effect of NHC was additive with recombinant alpha interferon-2a and could be prevented by the addition of exogenous cytidine and uridine but not of other natural ribo- or 2'-deoxynucleosides. When HCV RNA replicon cells were cultured in the presence of increasing concentrations of NHC (up to 40 micro M) for up to 45 cell passages, no resistant replicon was selected. Similarly, resistant BVDV could not be selected after 20 passages. NHC was phosphorylated to the triphosphate form in Huh7 cells, but in cell-free HCV NS5B assays, synthetic NHC-triphosphate (NHC-TP) did not inhibit the polymerization reaction. Instead, NHC-TP appeared to serve as a weak alternative substrate for the viral polymerase, thereby changing the mobility of the product in polyacrylamide electrophoresis gels. We speculate that incorporated nucleoside analogues with the capacity of changing the thermodynamics of regulatory secondary structures (with or without introducing mutations) may represent an important class of new antiviral agents for the treatment of RNA virus infections, especially HCV.

FIG. 1.

Chemical structure of NHC.

FIG. 2.

Antiviral effect of NHC on cpBVDV. EC₉₀s mentioned in the text are deduced from the graphs. Error bars for all data points are included but are very often less than the size of the symbol. (A) Dose-dependent viral RNA yield reduction in MDBK cell supernatant. Cell supernatant fluids were harvested 24 h (on exponentially growing cells) or 72 h (on confluent monolayer cells) postinfection, and viral RNA levels were quantified by Q-RT-PCR. The zero value corresponds to no drug added to the medium. ▿, ribavirin (confluent cells); •, ribavirin (exponentially growing cells); ▪, NHC (confluent cells); ○, NHC (exponentially growing cells); ▾, control compound showing no activity. VL, viral load; S.D., standard deviation. (B) Dose-dependent reduction of viral RNA in a yield reduction assay. cpBVDV was recovered from a single-cycle infection assay, and the virus yield was titrated by means of a plaque assay. •, NHC; ▪, NN-DNJ (47).

FIG. 3.

Dynamics of the cellular and HCV RNA levels. Ct, Q-RT-PCR threshold cycle; ΔCt: subtraction of the one PCR threshold cycle from another; S.D., standard deviation. (A) HCV replicon cells were seeded at 10⁴ cells per well in a 24-well plate. Cells were harvested daily and counted, and total RNA was extracted. ♦, ΔCt_{HCV(day x−day 0)}; ▴, number of viable cells per well. (B) Monitoring of the cellular DNA and RNA levels over a 7-day period. Exponentially growing HCV replicon cells were seeded at 10³ cells/well in a 96-well plate and harvested daily, and total RNA was extracted and quantified. ♦, rRNA gene (DNA); ▪, rRNA; ▴, HCV; ×, ΔCt_{(rRNA gene−rRNA)}; •, ΔCt_(HCV−rRNA).

FIG. 4.

Antiviral effect of NHC on HCV RNA levels. (A) Dynamics of the antiviral effect of NHC over a 12-day treatment period are shown. ΔΔCt_{(HCV Ct−rRNA Ct−no-drug Ct)}; ♦, no treatment; ▪, NHC at 10 μM; ▴, NHC at 100 μM. The antiviral activities at day 4 of IFN-α-2a (100 IU/ml [left bar]) and ribavirin (100 μM [right bar]) are given as references. S.D., standard deviation. (B) Cells were treated for 4 days, followed by RNA extraction and Northern blot analysis. The upper gel shows the positive-strand (+) HCV RNA with a hybridization signal at approximately 8 kb. The lower gel shows the control hybridization of GAPDH mRNA at approximately 1.4 kb. Lane 1, parental Huh7 cell line; lane 2, HCV replicon cells, untreated; lane 3, 100 IU of IFN-α-2a/ml; lane 4, 65 μM NHC. (C) Cells were treated for 4 days, followed by protein extraction and Western blot analysis. The upper gel shows the α-NS5B reactivity of proteins transferred to the membrane; the lower gel is a Coomassie-stained gel illustrating equal amounts of protein in each lane (total cellular protein equivalent of 5 × 10⁴ cells per lane was loaded). Lane 1, recombinant HCV NS5B protein; lane 2, parental Huh7 cell line; lane 3, HCV replicon cells, untreated; lane 4, 100 IU of IFN-α-2a/ml; lane 5, 65 μM NHC. (D) HCV replicon cells were incubated with NHC and IFN-α-2a, either alone or in different combinations. After 94 h of incubation, total RNA was extracted and amplified in multiplex conditions. Analysis was performed by using Combostat software (Jasper, Ga.) (5).

FIG. 5.

Prevention and rebound of the anti-HCV RNA effect of NHC. HCV replicon cells were treated for 4 days with or without simultaneous incubation of natural nucleosides or deoxynucleosides at 50 μM. (A) Plot showing the differences in Ct outcomes in the different settings with 50 μM NHC. ▴, ΔCt_{(rRNA Ct−no-drug Ct)}; •, ΔCt_{(HCV Ct−no-drug Ct)}. No, incubation with NHC but no competitive nucleosides added; C, cytidine; G, guanosine; A, adenosine; U, uridine; C,G,A,U, mix of the four nucleosides each at 25 μM; dU, 2′-deoxyuridine; dC, 2′-deoxycytidine; dA, 2′-deoxyadenosine; dG, 2′-deoxyguanosine, T, thymidine; dC,dG,dA,T, mix of the four deoxynucleosides each at 25 μM. (B) Dose-dependent antiviral prevention by adding natural nucleosides to the culture medium already containing 100 μM NHC. ▪, uridine; ♦, cytidine. (C) Rebound effect of HCV replicon after removal of NHC from culture supernatant. ♦, no-drug control; ▪, 33 μM NHC; •, 100 μM NHC; ▴, 100 IU of IFN-α-2a/ml. S.D., standard deviation.

FIG. 6.

Cell-free HCV NS5B polymerization reactions in the presence of NHC-TP. Synthetic 5′-UTR IRES template was incubated under conditions supporting NS5B enzyme polymerization in the presence of increasing concentrations of NHC-TP. (A) Gel electrophoresis conditions: 7 M urea-5% acrylamide. [α-³²P]UMP-labeled positive-strand RNA template (artificial RNA derived from an SP6 template) used as size marker (lane 1), polymerization reactions without negative-strand RNA template (lane 2), and polymerization reactions including negative-strand RNA template but with a deficient enzyme (site-directed mutagenesis of the active center from G₃₁₇D₃₁₈D₃₁₉ to G₃₁₇A₃₁₈A₃₁₉) (lane 3) are shown. Full-length polymerization products in the absence of NHC-TP (lane 4), in 5 μM NHC-TP (lane 5), in 10 μM NHC-TP (lane 6), in 20 μM NHC-TP (lane 7), in 40 μM NHC-TP (lane 8), and in 40 μM NHC-TP and the absence of CTP (lane 9) are shown. (B) Gel electrophoresis conditions: 7 M urea-45% (vol/vol) formamide-5% acrylamide. [α-³²P]UMP-labeled positive-strand RNA template (artificial RNA derived from an SP6 template) used as size marker is shown (lane 1). Full-length polymerization products in the absence of NHC-TP (lane 2), in 10 μM NHC-TP (lane 3), and in 20 μM NHC-TP (lane 4) are shown.

FIG. 7.

Long-term treatment of HCV replicon cells with NHC. (A) HCV replicon cells were incubated for the indicated passage number in the presence of G418 (500 μg/ml). The cells were passaged at a ratio of 1:3 during the whole incubation period. (B) Results of an antiviral sensitivity assay are shown. Untreated and NHC-pretreated HCV replicon cells were seeded in a 96-well plate at 10³ cells per well in presence or absence of 100 μM NHC and incubated for 96 h. The ratio HCV RNA to rRNA levels were determined by Q-RT-PCR. ▪, ΔCt_{(rRNA Ct−rRNA-untreated Ct)}; ♦, ΔCt_{(HCV Ct−rRNA-untreated Ct)}. S.D., standard deviation. (C) cpBVDV was grown in the presence of increasing concentrations of compound for 20 passages. (D) cpBVDV, harvested at passage 20, was tested in a 24-h virus yield assay. ▪, passage 20 virus; ♦, control virus.

FIG. 8.

Relative mitDNA and mitRNA levels. HepG2 cells were exposed for 7 days to 0.1, 1, or 10 μM concentrations of compound, and the ratio of mitochondrial to nuclear nucleic acids was determined. D-DDC, ribavirin, and 3′-deoxycytidine (3′-dC) were included as controls. (A) Ratio of COXII DNA to β-actin DNA levels; ΔΔCt_{(COXII Ct−β-actin Ct−no-drug control Ct)}. (B) Ratio of COXII to β-actin RNA levels (RT-PCR).

FIG. 9.

NHC i.p. treatment in mice. Mice were injected i.p. with NHC on day 0 to day 5 (ip treatment), and changes in weight were monitored on the indicated days. ♦, 0 mg/kg/day; ▴, 3 mg/kg/day; •, 10 mg/kg/day; ▪, 33 mg/kg/day; ×, 100 mg/kg/day; *, significantly different from control (P < 0.05). For clarity, standard deviations (S.D.) are only shown for the 0-mg/kg/day and 100-mg/kg/day treatment groups.