Table 1.
Effects of nucleosides on cell viability and HCV replication.
CC50, IC50, and therapeutic index (TI, CC50/IC50) values are shown for Adenosine (Ade), Cytidine (Cyt), Guanosine (Gua), and Uridine (Uri) in Huh-7.5 reporter cells.
Fig 1.
Effect of ribonucleosides on HCV replication.
(A) and (B) Effect of guanosine (Gua) on HCV p0 replication. Infectious progeny obtained in the presence of Gua 500 μM (A) and Gua 800 μM (B); four replicas for each condition are shown (red squares). HCV p0 titer in the absence of treatment (black circles) and values for a HCV lethal mutant GNN (black crosses) are also shown (see Methods). Each passage was done using a fixed volume. (C) and (D) Effect of adenosine (Ade, blue squares), cytidine (Cyt, green squares), and uridine (Uri, orange squares) on HCV p0 replication. Infectious progeny obtained in the presence of the corresponding nucleoside at 500 μM (C) and at 800 μM (D). HCV p0 viral titer in the absence of treatment (black circles) and values for a HCV lethal mutant GNN (black crosses) are also shown. (E) HCV p100 viral titer in the absence (black circles) or presence of Gua 500 μM (yellow symbols), and Gua 800 μM (red symbols). Significance (two-way ANOVA test): **p<0.005. Two replicates each one performed in triplicate are shown for each condition in presence of nucleosides. The discontinuous horizontal line marks the limit of detection of virus infectivity. Procedures for serial infections and titration of infectivity are detailed in Materials and Methods.
Fig 2.
Treatment with Gua causes a reduction in the efficiency of early aspects of the infection.
(A) Impact of nucleoside treatment on single cycle trans-complemented HCV particles (HCVtcp) infection efficiency. Huh-7 cells were pre-treated with 800 μM of nucleoside for 20 hours before inoculation with HCVtcp in the presence or absence of the nucleosides. As a positive inhibition control, target cells were treated with the entry inhibitor hydroxyzine (HDH) at the time of infection (5μM). Single cycle infection efficiency was determined by measuring luciferase activity in total cell extracts 48 hours post-inoculation. (B) Huh-7 cells were pre-treated with 800 μM of nucleoside for 20 hours before transfection with in vitro-transcribed subgenomic viral RNA-containing liposomes in the presence or absence of the nucleosides. As a positive inhibition control, target cells were treated with the replication inhibitor 2´-c-methyladenosine (2mAde) (10 μM). Primary translation (5 hours) and RNA replication efficiency (24 and 48 hours) was determined by measuring luciferase activity in total cell extracts at different times post-transfection. Data are shown as average and standard deviation of two experiments performed in triplicate (N = 6). Significance (Student’s T-test): ***p<0.0005; *p<0.05.
Table 2.
Effects of nucleosides on BHK-21 cells viability and VSV, FMDV, and LCMV replication.
CC50, IC50, and therapeutic index (TI, CC50/IC50) values are shown for Adenosine (Ade), Cytidine (Cyt), Guanosine (Gua), and Uridine (Uri) in BHK-21 cells.
Fig 3.
Effect of nucleosides on FMDV, LCMV and VSV replication.
Effect of adenosine (Ade), cytidine (Cyt), uridine (Uri), and guanosine (Gua) on the production of infectious progeny of FMDV (A), LCMV (B) and VSV (C) in BHK-21 cells at an initial m.o.i. of 0.05 TCID50 per cell, in the absence (C-) or presence of 800 μM of the indicated nucleoside. Infectivity was determined at passage 3 in the cell culture supernatant as described in Materials and Methods. (D) Comparative inhibition of VSV and HCV p0 progeny production in Huh-7.5 reporter cells in the presence of 800 μM Gua. The titer shown for HCV is the average (four replicas) of titers determined at passage 3 in the supernatants of the serial infections (corresponding to Fig 1). Procedures for serial infections and titration of infectivity are detailed in Materials and Methods. Significance (Student’s T-test): ** p < 0.005.
Fig 4.
Effect of Gua on NS5BΔ21 RNA polymerase activity.
(A) Recombinant HCV NS5BΔ21 polymerase was added to a reaction containing a 540-nt RNA template [18], the four nucleoside-triphosphates (ATP, CTP, GTP, and UTP) and the indicated concentrations of Gua. Product quantification from three replicates (average ± SEM) and a representative experiment (below) are shown. Polymerase activity is normalized with respect to its maximum activity. The band indicates a new synthesis RNA product of 540 nt length. (B) A representative experiment as in A, but using the 19-nt LE19 RNA as a template. DN, PE, and TS indicate reaction products of de novo synthesis, primer extension, and template switching, respectively [53]. Procedures are detailed in Materials and Methods. Significance (Student’s T-test): ** p < 0.005.
Fig 5.
Effect of Gua on intracellular nucleotides.
Effect of treatment of Huh-7.5 cells with 500 μM and 800 μM of Ade, Uri, Gua, and Cyt on the level of intracellular nucleoside-triphosphates (A) and intracellular nucleoside-diphosphates (B). Quantification from three different runs of two biological replicates (geometric mean ± SEM) are shown. Significance (Student’s T-test): * p < 0.05; ** p < 0.005; *** p < 0.0005.
Table 3.
Intracellular NTP/NDP ratio values.
Fig 6.
Effect of guanosine on HCV specific infectivity.
Huh-7.5 reporter cells were infected with HCVp0 at an initial m.o.i. of 0.05 TCID50/cell, in the absence or presence of Gua at the indicated concentrations. HCV GNN infection was used as a negative control. (A) Extracellular viral RNA measured by quantitative RT-PCR in different passages. The populations correspond to those of the experiment described in Fig 1 and the values in each passage are the average of the three replicas; standard deviations are given. (B) Specific infectivities calculated from the infectivity values of the Fig 1A and 1B and the extracellular RNA concentrations indicated in Fig 8A. The horizontal dashed line indicates the limit of detection of viral RNA and specific infectivity. Black, yellow, and red symbols correspond to no drug, Gua 500 μM, and Gua 800 μM, respectively. Values for a HCV lethal mutant GNN (black crosses) are also shown. Details of the procedures are given in Materials and Methods. Significance (Student’s T-test): ** p < 0.005, * p < 0.05.
Fig 7.
Indels found in the mutant spectrum of HCV p0 passaged in the presence of Gua.
The nucleotide sequence of HCV genomic residues 6220 to 6758 was determined for 53 molecular clones derived from the population in absence of Gua, and 132 molecular clones from populations passaged in presence of Gua (data in Table 5). Deduced amino acids (single letter code) are given for residues located at the carboxy-terminal region of NS4B preceding NS5A amino acids. For clarity, only residues around insertions or deletions are shown; three squared points indicate missing amino acids (sequence is that of JFH-1; accession number AB047639). No indels were detected in the population passaged in absence of Gua. (A) Deletions in the population passaged in the presence of 500 μM guanosine. Red boxes indicate nucleotides that were deleted in a component of the mutant spectrum, with the deletion size indicated in the filled boxes. (B) Insertions in the mutant spectrum of the population passaged in the presence of 500 μM Gua are marked with a blue triangle. (C) Deletions found in the HCV populations passaged in the presence of 800 μM Gua. Procedures for HCV genome sequencing are described in Materials and Methods. The name of the indel as detailed in Table 5 is included next to the size.
Table 4.
Mutant spectrum analysis of the hepatitis C virus populations passaged in the absence and presence of guanosine (Gua).
Table 5.
Indels found in the mutant spectra of HCV p0 after 3 passages in the absence and presence of Gua 500 μM and 800 μM.
Fig 8.
Effect of nucleoside-triphosphate concentration on NS5BΔ21 RNA polymerase activity.
(A) Polyacrylamide gel showing the products for de novo (DN), primer extension (PE) and template switching (TS) obtained with HCV NS5BΔ21 at increasing concentrations (100, 500, 800, and 1000 μM) of UTP in the presence of radiolabeled α32P-CTP. ATP and GTP concentrations were maintained at 100 μM and 500 μM, respectively (left panel). Graphic representation of densitometric values obtained from the electropherogram shown in A (red diamonds, yellow triangles, and black squares correspond to de novo (DN), primer extension (PE), and template switching (TS) activities, respectively) (right panel). (B) Corresponds to experiments as in A but for increasing concentrations of ATP, with UTP and GTP maintained at 100 μM and 500 μM, respectively. (C) Corresponds to experiments as in A but for increasing concentrations of GTP, with ATP and UTP both maintained at 100 μM. Activities were normalized to their maximum values. Densitometric data represent the mean of at least three independent experiments. Error bars correspond to standard error of the mean. Horizontal lines indicate statistically significant differences (Student’s T-test) between the activity values that link, using the same color code as the activity type. Details of the activity measurements are given in Materials and Methods. Significance (Student’s T-test): *** p < 0.0005, ** p < 0.005, * p < 0.05.
Fig 9.
Effect of NDPs on NS5BΔ21 RNA polymerase activity.
(A) Polyacrylamide gel showing the products for de novo (DN), primer extension (PE) and template switching (TS) obtained with HCV NS5BΔ21 at increasing concentrations (0, 166, 333, 500, 800, and 1000 μM) of UDP in the presence of ATP and UTP at a final concentration of 100 μM, GTP at 500 μM, and radiolabeled α32P-CTP (left panel). Graphic representation of densitometric values obtained from the electropherogram shown in A (red diamonds, yellow triangles, and black squares correspond to de novo (DN), primer extension (PE), and template switching (TS) activities, respectively) (right panel). (B) Corresponds to experiments as in A but for increasing concentrations of ADP. (C) corresponds to experiments as in A but for increasing concentrations of GDP. Activities were normalized to their maximum values. Densitometric data represent the mean of at least three independent experiments. Error bars correspond to standard error of the mean. Horizontal lines indicate statistically significant differences (Student’s T-test) between activity values, using the same color code as the activity type. Details of the activity measurements are given in Materials and Methods. Significance (Student’s T-test): ** p < 0.005, * p < 0.05.