Guanosine inhibits hepatitis C virus replication and increases indel frequencies, associated with altered intracellular nucleotide pools

In the course of experiments aimed at deciphering the inhibition mechanism of mycophenolic acid and ribavirin in hepatitis C virus (HCV) infection, we observed an inhibitory effect of the nucleoside guanosine (Gua). Here, we report that Gua and not the other standard nucleosides inhibits HCV replication in human hepatoma cells. Gua did not directly inhibit the in vitro polymerase activity of NS5B, but it modified the intracellular levels of nucleoside di- and tri-phosphate (NDPs and NTPs), leading to deficient HCV RNA replication and reduction of infectious progeny virus production. Changes in the concentrations of NTP or NDP modified NS5B RNA polymerase activity in vitro, in particular de novo RNA synthesis and template switching. Furthermore, the Gua-mediated changes were associated with a significant increase in the number of indels in viral RNA, which may account for the reduction of the specific infectivity of the viral progeny, suggesting the presence of defective genomes. Thus, a proper NTP:NDP balance appears to be critical to ensure HCV polymerase fidelity and minimal production of defective genomes. Author summary Ribonucleoside metabolism is essential for replication of RNA viruses. In this article we describe the antiviral activity of the natural ribonucleoside guanosine (Gua). We demonstrate that hepatitis C virus (HCV) replication is inhibited in the presence of increasing concentrations of this ribonucleoside and that this inhibition does not occur as a consequence of a direct inhibition of HCV polymerase. Cells exposed to increasing concentrations of Gua show imbalances in the intracellular concentrations of nucleoside-diphosphates and triphosphates and as the virus is passaged in these cells, it accumulates mutations that reduce its infectivity and decimate its normal spreading capacity.

There are several ways to approach the control of RNA viral diseases. Inhibition of HCV functions by direct-acting antiviral agents (DAAs) has yielded sustained virological responses of about 98% [2,3]. Thus, HCV infection may be targeted for eradication by the combined use of different DAAs directed to viral proteins. However, access to this treatment is not affordable in countries with high prevalence rates, and an effective prophylactic vaccine is not available, making global HCV eradication difficult. Consequently, treatment with a combination of pegylated interferon-alpha (PEG-IFNα) plus ribavirin (Rib) is still in use in several countries with high prevalence rates of HCV infection [4].
Rib displays several mechanisms of antiviral activity [5], a major one being the inhibition of inosine-5'-monophosphate (IMP) dehydrogenase (IMPDH), which converts IMP to xanthosine monophosphate (XMP) and thus is involved in the de novo biosynthesis of GTP [6]. Rib also exerts its antiviral activity through lethal mutagenesis [7][8][9][10]. In the course of our experiments on the effect of mycophenolic acid and Rib on HCV clonal population HCV p0 [11] we observed that the presence of guanosine (Gua) during viral replication produced a decrease of up to 100 times in infectious progeny production. Although there are Gua derivatives that have antiviral properties, including Rib itself, natural Gua has never been identified as having antiviral activity [5]. The objective of the present study was to quantify the inhibitory role of Gua on HCV, its specificity, and its mechanism of action. We show that i) Gua inhibits infectious HCV progeny production but does not inhibit directly the HCV polymerase; ii) Gua alters the intracellular pools of di-and triphosphate ribonucleosides (NDP and NTP); iii) the

Effect of ribonucleosides on HCV replication
Before studying the possible anti-HCV effect of natural nucleosides we determined their cytotoxicity (CC50) on Huh-7.5 reporter cells. The cytotoxicity of Gua, adenosine (Ade), cytidine (Cyt) or uridine (Uri) was analyzed in semiconfluent cell monolayers by exposing cells to different nucleoside concentrations (from 0 μM to 800 μM). Cell viability (CC50) was monitored after 72 h of treatment (Table 1)  nucleoside, and infectious progeny production was measured as described in Materials and Methods. A decrease in the production of HCV infectious progeny was observed for Gua and Ade, whereas Cyt and Uri did not show any effect (Table 1). These data yield a therapeutic index (TI), defined as CC50/IC50, of 5.9 and ≥ 4.9 for Ade and Gua, respectively (Table 1).
To further explore the effect of ribonucleosides on HCV replication, HCV p0 was subjected to 5 serial passages in Huh-7.5 reporter cells, using an initial m.o.i. of 0.05 TCID50 per cell, both in the absence and in the presence of ribonucleosides at 500 μM and 800 μM (Fig 1). Results show a consistent decrease in progeny infectivity as a result of Gua treatment (Fig 1A and 1B), but a sustained viral replication in the presence of Ade, Cyt or Uri (Fig 1C, D). In the presence of 500 μM Gua, a decrease in infectivity was detected although only one of the four replicates yielded values below the detection limit ( Fig 1A). A sustained drop in HCV infectivity by Gua 800 μM was achieved, which became undetectable between passages 2 and 4 in all replicates ( Fig 1B).
Therefore, Gua was the only nucleoside that showed antiviral effect without cytotoxicity.
Next, we analyzed the effect of treatment with Gua, Ade, Cyt, and Uri in a surrogate single cycle infection model, taking advantage of spread-deficient bona fide HCV virions bearing a luciferase reporter gene (HCVtcp). This system recapitulates early stages of the infection including viral entry, primary translation and genome replication, overall efficiency of which is proportional to reporter gene activity [12]. The results (Fig 2A) show that a selective HCV entry inhibitor, hydroxyzine, strongly interferes with reporter gene accumulation, as previously documented [13] (Figure 2A). Of the four natural nucleosides, only Gua exerted a significant inhibitory role, as shown by reduced luciferase levels in these cells (Figure 2A), and suggesting that an early step of the infection preceding viral assembly is significantly inhibited by Gua. To further dissect the impact of Gua on HCV replication, we analyzed the effect of Gua, Ade, Cyt and Uri treatment at different times in the replication of a dicistronic subgenomic genotype 2a (JFH-1) replicon bearing a luciferase reporter gene [12]. The objective was to analyze if the effect took place at the level of IRES-dependent translation (5 h post-transfection) or during RNA replication (24 and 48 h post-transfection) [13]. The results ( Fig 2B) show that there are no differences among the different treatment points at 5 h post-transfection, which excludes an effect on HCV IRES-dependent RNA translation or any spurious interference with reporter gene expression. However, Gua-treated cells showed a statistically significant 12-and 5-fold reduction in RNA replication at 24 and 48 hours post-transfection respectively ( Figure 2B). A modest (±2-fold) but significant reduction was also observed in Ade-treated cells. The fact that Ade treatment does not interfere with HCVtcp (trans-complemented HCV particles) infection suggests that only Gua affects significantly with HCV infection by interfering with viral RNA replication, downstream of viral entry and primary translation.

Effect of Gua on a high fitness HCV population
The HCV p100 virus [HCV p0 passaged 100 times in Huh-7.5 reporter cells], shows a relative fitness that is 2.2 times higher than that of the HCV p0 parental population [14]. Since viral fitness can influence the response of the virus to antiviral agents [15][16][17], HCV p100 was used to study the response of a high fitness HCV to Gua. For this, HCV p100 was subjected to 5 serial passages in Huh-7. Gua, respectively. However, no decrease of infectivity below the limit of detection was observed throughout the five passages in any of the replicates (Fig 1E). Only the decrease in progeny production in the presence of 800 µM reached statistical significance ( Fig 1E). Thus, the results showed increased resistance of HCV p100 to Gua compared to HCV p0 (compare Fig 1A, 1B, and 1E) as was previously observed with several antiviral drugs [16,17].

Effect of Gua on the replication of other RNA viruses
To determine the specificity of the antiviral action exerted by Gua on HCV and to rule out nonspecific effects that could affect any virus, comparative experiments were conducted with foot-and-mouth disease virus (FMDV), lymphocytic choriomeningitis virus (LCMV), and vesicular stomatitis virus (VSV). First, the CC50 values of Gua, Ade, Cyt, and Uri were determined for BHK-21 cells, as described in Materials and Methods. The values obtained (Table 2) indicate no detectable cytotoxicity of Gua, Cyt and Uri, and a CC 50 value of 391 ± 68 μM for Ade. To determine the IC50 values of these nucleosides, BHK-21 cells were infected with FMDV, LCMV, and VSV, at an initial m.o.i. of 0.05 TCDI50 per cell in the presence of increased nucleoside concentrations and the production of infectious progeny was measured.
The values obtained (Table 2) show that all nucleosides lacked inhibitory profile for FMDV. In contrast, purines were inhibitory for VSV, while all nucleosides were inhibitory for LCMV.
However, the IC50 values were very high and the therapeutic indexes (TI) were consequently low ( Table 2).
As an additional control for the specificity of HCV inhibition by Gua, the response of VSV, results show that the only differences found were those of HCV treatment with Gua at 800 μM ( Fig 1A-B). Finally, to rule out that the inhibitory effect of Gua on HCV was solely due to the action of the nucleoside on the human hepatoma cells used in the experiments, we examined the production of VSV viral progeny in Huh-7.5 reporter cells which this virus also productively infects. High viral titers in the presence of 800 μM Gua were obtained for VSV, confirming a lack of antiviral activity of Gua against this virus also in Huh-7.5 reporter cells (Fig 3D).

Effect of guanosine on HCV NS5B activity
To analyze the mechanism by which Gua inhibits HCV replication we tested the effect of increasing Gua concentrations on HCV polymerase activity in vitro. A 570 nt RNA fragment corresponding to the E1/E2 region of the HCV genome [18] was replicated by HCV recombinant NS5B∆21 in the presence of ATP, CTP, GTP, and UTP, and at increasing concentrations of Gua ( Fig 4A). NS5B polymerase activity increased with Gua concentration up to 500 μM. Even at 1 mM Gua, the RNA polymerase activity was similar to that obtained in absence of Gua. Only at very high Gua concentration (10 mM) the RNA polymerase activity showed a significant reduction (Fig 4A). Similar results were obtained using the 19-mer oligonucleotide LE19 ( Fig 4B). Therefore, according to this in vitro RNA synthesis assay, the inhibition of HCV progeny production by Gua cannot be attributed to direct inhibition of the HCV RNA polymerase.

Effect of guanosine on intracellular nucleotide pools
To investigate whether HCV replication inhibition by Gua could be related to alterations in di-  (Fig 5A). In the case of NDPs a significant increase of 1.7-to 4.1-fold was observed in cells treated with 500 μM Gua (Fig 5B). Treatment with 800 μM Gua resulted in an increase of the concentration (2.1-to 5.4-fold) of all NDP's ( Fig 5B). Therefore, the presence of Gua in the culture medium increased the intracellular levels of nucleoside di-and tri-phosphates Table). The lowest nucleotide concentration was obtained for CDP and CTP independently of the treatment with Gua.

Effect of nucleoside di-and triphosphate imbalance on HCV NS5B activity in vitro
To explore if changes in nucleotide concentrations might affect HCV polymerase activity, we  Table) and CTP was chosen as the carrier of the radioisotope. De novo (DN), primer extension (PE) and template switching (TS) polymerase activities were measured in the presence of increasing concentrations of the corresponding triphosphate nucleosides (Fig 6). A high UTP concentration of 1 mM slightly but significantly decreased primer extension activity ( Fig 6A). However, the main effect of NTP concentration was on the de novo RNA synthesis, with a significant decrease at high ATP concentration (Fig 6B), and a significant increase at high GTP concentration ( Fig 6C). The increase in the de novo RNA synthesis was accompanied by an increase of template switching ( Fig 6C).
Since increasing concentrations of Gua also altered the intracellular NDP concentrations (Fig 5), RNA synthesis, with a significant decrease at high ADP and GDP concentrations. Differences in primer extension and template switching did not reach statistical significance (Fig 7).

Mutational effects of guanosine
To determine if Gua-related nucleotide pool effects were associated with the mutation repertoire exhibited by HCV during replication in Huh-7.5 cells, the mutant spectrum of the genomic region spanning the last 49 nucleotides of the NS4B gene and the first 490 nucleotides of the NS5A gene, was analyzed using molecular cloning and Sanger sequencing. Following three passages in absence or presence of Gua, the maximum mutation frequency resulted in a significant increase in the HCV populations passaged in the presence of Gua (p<0.0001 and p=0.01 for Gua 500 µM, and Gua 800 µM, respectively; χ 2 test) ( Table 3). A hallmark of virus extinction by lethal mutagenesis is the decrease of specific infectivity (the ratio between viral infectivity and the amount of genomic viral RNA) [7]. Extinction by Gua occurred with a 2.8fold to 11.8-fold decrease of specific infectivity in the first two passages in the presence of the compared drug, as quantified by infectivity and viral RNA in samples of the cell culture supernatants (Fig 8), suggesting that an increase in polymerase error rate was involved. The most remarkable change was that replication in the presence of Gua increased significantly the number of indels in the heteropolymeric genomic regions of the mutant spectrum (Table 4). Indels in homopolymeric regions ─consisting of at least three successive identical nucleotides─ were not considered because control experiments revealed that they can be amplification artifacts [19]. No indels were detected in the 53 molecular clones derived from the population passaged in the absence of Gua. In sharp contrast, 10 deletions and 2 insertions were present in the 64 molecular clones retrieved from the population passaged in the presence of 500 µM Gua, and 5 deletions in 68 molecular clones from the population passaged in the presence of 800 µM Gua ( Table 4). The difference in the number of deletions is highly significant for the populations passaged in the presence of 500 µM and 800 µM Gua (p<0.001; test χ2). The size of the deletions ranged from 1 to 46 nucleotides, some were found in a single clone, others in  (Table 4 and Fig 9). Therefore, the anti-HCV effect of Gua, exerted via nucleotide-mediated alterations of polymerase activity, is associated with the generation of multiple deletions during HCV replication.

Discussion
Nucleoside derivatives are the most important family of drugs targeting viral polymerases, but the antiviral capacity of natural nucleosides has not been described [5]. Interestingly, we observed an inhibitory effect of Gua when it was used in experiments to analyze the impact of mycophenolic acid and ribavirin on HCV progeny production [11]. Here, we document inhibition of HCV replication by Gua in single and serial infections of Huh-7.5 cells that led to loss of infectivity without significant toxicity for the host cells. The antiviral action of Gua was also exerted on high fitness HCV, albeit without loss of infectivity after 5 passages in the presence of Gua, in agreement with the drug resistance phenotype displayed by high fitness HCV (Fig 1) [14][15][16][17]. The antiviral effect of Gua was not observed for FMDV in BHK-21 cells or for LCMV and VSV in Huh-7.5 cells (Fig 3).
RNA synthesis by NS5BΔ21 was not significantly affected by Gua concentrations up to 1 mM. Therefore, the inhibition of virus progeny production is unlikely to be the result of direct polymerase inhibition by Gua. This result is consistent with previous work that showed the ability of NS5B to initiate RNA synthesis with this nucleoside [20]. In contrast to Gua, altered intracellular nucleotide concentrations affected the activity of NS5B, in particular an alteration of the de novo RNA synthesis by the GTP/GDP and/or ATP/ADP balances (Fig 7). The NS5B protein has an allosteric binding site of GTP and the balance between NDP and NTP might

Reagents and plasmids.
Nucleosides Ade, Cyt, Gua, and Uri, as well as nucleoside di-and tri-phosphates were purchased from Sigma-Aldrich. Plasmid pNS5BΔ21 encoding the HCV NS5B that lacks the Cterminal 21 hydrophobic amino acids to enhance solubility has been described previously [54].
The resulting expression vector allows the expression of a tagged NS5BΔ21 with six histidine residues at its C terminus to aid in protein purification.

Cells and viruses.
The origin of Huh 7.5, Huh 7-Lunet, Huh-7.5 reporter cell lines and procedures used for cell growth in Dulbecco's modification of Eagle's medium (DMEM), have been described [11]. Cell lines were incubated at 37°C and 5% CO2. We used the following viruses in the experiments: Trans-encapsidated HCV virions (HCVtcp) were produced by electroporation into packaging cells of a subgenomic, dicistronic HCV replicon bearing a luciferase gene, as previously described [12]. Supernatants of the electroporated cells were titrated to determine the optimal dose rendering detectable luciferase activity at 48 hours post-inoculation. The same subgenomic replicon was used for lipofection experiments, using lipofectamine2000 transfection reagent as previously described [13].

Production of viral progeny and titration of infectivity.
The procedures used to obtain the initial virus HCV p0 and for serial infections of the hepatoma Huh-7.5 reporter cells have been described [14]. Briefly, electroporation of Huh-7 Lunet cells was performed with 10 μg of the transcript of HCVcc (Jc1 or the negative control GNN) (260 volts, 950 μF). Then, electroporated cells were passaged every 3-4 days before cells became confluent; passages were continued until 30 days post-electroporation. Subsequently, the cell culture supernatants were pooled to concentrate the virus 20 times using 10,000 MWCO spin columns (Millipore), and aliquots were stored at −70°C [14]. For titration of HCV infectivity, cell culture supernatants were serially diluted and applied to Huh-7.5 cells. After 3 days postinfection the cell monolayers were washed with PBS, fixed with ice-cold methanol, and stained with anti-NS5A monoclonal antibody 9E10 [14]. reverse transcriptase (Promega), and subsequent PCR amplification was carried out using AccuScript (Agilent Technologies), with specific primers. Primers for the HCV amplification and the sequencing have been described [11,14,17,19]. Agarose gel electrophoresis was used to analyze the amplification products, using HindIII-digested Φ-29 DNA as a molecular mass standard. In parallel, mixtures without template RNA were reverse transcribed and amplified to monitor the absence of cross-contamination by template nucleic acids. Nucleotide sequences of HCV RNA were determined on the two strands of the cDNA copies [11,55]; only mutations detected in the two strands were considered. To analyze the complexity of mutant spectra by molecular cloning and Sanger sequencing, HCV RNA was extracted and subjected to RT-PCR to amplify the NS5A-coding regions, as has been previously described [11]. Amplifications with template preparations diluted 1:10 and 1:100 were performed to ensure that an excess of template in the amplifications was used in the mutant spectrum analysis; the molecular cloning was performed from the undiluted template only when the 1:100-diluted template produced also a DNA band; this procedure avoids complexity biases due to redundant amplifications of the same initial RNA templates [11]. Control analyses to confirm that mutation frequencies were not affected by the basal error rate during amplification have been previously described [57].

Amplified DNA was ligated to the vector pGEM-T (Amersham) and used to transform
Escherichia coli DH5α; individual colonies were taken for PCR amplification and nucleotide sequencing, as previously described [56].

NDP and NTP pool analysis.
The procedure used has been previously described [11]. Briefly, Huh-7.5 cells (2×10 6 cells) were washed with PBS and incubated with 900 μl of 0.6 M trichloroacetic acid on ice for 10 min. A precooled mixture of 180 µl of Tri-n-octylamine (Sigma) and 720 µl of Uvasol® (1,1,2trichlorotrifluoroethane, Sigma) was added to the 900 µl extract, vortexed for 10 s, centrifuged 30 s at 12,000  g at 4 °C, and stored at −80 °C prior to analysis. One hundred µl samples were applied to a Partisil 10 SAX analytical column (4.6 mm×250 mm) (Whatman) with a Partisil 10 SAX guard cartridge column (4.6×30 mm) (Capital HPLC) using an Alliance 2695 HPLC system connected to a 2996 photodiode array detector (Waters). NDP and NTP were separated at a eluent flow rate of 0.8 ml/min and detected with ultraviolet light at a wavelength of 254 nm.
The column was pre-equilibrated with 60 ml of 7 mM NH4H2PO4, µl of 20 pmol/µl UTP, CTP, ATP and GTP (Jena Bioscience), were separated prior to sample analysis. The HPLC analysis did not separate rNTPs from dNTPs, or rNDPs from dNDPs.
However, since the absolute concentration of rNTPs and rNDPs is several orders of magnitude greater than that of dNTPs dNDPs, we consider the value obtained as the concentration of rNTPs and rNDPs. Determinations were carried out with two independent biological samples, each one in triplicate for NDPs and NTPs. The amount of each nucleoside in cell extracts was normalized relative to the number of cells.

Quantification of HCV RNA.
Real time quantitative RT-PCR was performed with the Light Cycler RNA Master SYBR Green I kit (Roche), following the manufacturer's instructions, as previously described [14]. The 5′-UTR non-coding region of the HCV genome was amplified using as primers oligonucleotide HCV-5UTR-F2 (5′-TGAGGAACTACTGTCTTCACGCAGAAAG; sense orientation; the 5′ nucleotide corresponds to HCV genomic residue 47), and oligonucleotide HCV-5UTR-R2 (5′-TGCTCATGGTGCACGGTCTACGAG; antisense orientation; the 5′ nucleotide corresponds to HCV genomic residue 347). Quantification was relative to a standard curve obtained with known amounts of HCV RNA, obtained by in vitro transcription of plasmid GNN DNA [55].
Reaction specificity was monitored by determining the denaturation curve of the amplified DNAs. Mixture without template RNA and RNA from mock-infected cells were run in parallel to ascertain absence of contamination with undesired templates.

NS5B∆21 polymerase expression and purification.
NS5B from strain pJ4-HC with a deletion of 21 aa at the C-terminal end (NS5B∆21) was obtained as previously described [54,58]. This truncated protein displays polymerase activities that were not distinguished from those of the full-length enzyme [59]. Briefly, NS5B∆21 was were monitored by SDS-PAGE and Coomassie brilliant blue staining. Protein was quantified by SDS-PAGE gel imaging and protein determination using the Bradford assay.

In vitro RdRP replication assays.
RNA polymerase assays were carried out using two different RNA substrates, the symmetric substrate LE-19 (sequence 5' UGUUAUAAUAAUUGUAUAC 3'), which is capable of primerextension (PE), de novo initiation (DN), and template switching (TS) [54,58], and an RNA fragment encompassing HCV E1/E2 region (570 nt) [18]. Except when indicated otherwise, E1/E2 products were resolved using 1% agarose gel electrophoresis. Agarose gels were dried in an electrophoresis gel dryer (BioRad). LE19 products were resolved using denaturing polyacrylamide (23% PAA, 7 M urea) gel electrophoresis. Gels were exposed to phosphorimager screens and scanned with a Typhoon 9600 phosphorimager (Molecular Dynamics). Sample quantification was performed from parallel experiments. Band volume values were obtained by using the ImageQuant software provided with the apparatus (GE Healthcare).

Statistical analyses.
The statistical significance of differences between mutation frequencies was evaluated by the chi-square test. Statistical comparisons among groups were performed with Student's T-tests.
No indels were detected in the population passaged in absence of Gua.