Abstract
We evaluated Sofosbuvir (SOF), the anti-hepatitis C virus prodrug of β-d-2′-deoxy-2′-α-fluoro-2′-β-C-methyluridine-5′-monophosphate, for potential inhibitory activity against DENV replication. Both cell-based and biochemical assays, based on use of purified DENV full-length NS5 enzyme, were studied. Cytopathic effect protection and virus yield reduction assays confirmed that SOF possessed anti-DENV activity in cell culture with a 50% effective concentration (EC50) of 4.9 µM and 1.4 µM respectively. Real-time RT-PCR verified that SOF inhibits generation of viral RNA with an EC50 of 9.9 µM. Purified DENV NS5 incorporated the active triphosphate form (SOF-TP) into nascent RNA, causing chain-termination. Relative to the natural UTP, the incorporation efficiency of SOF-TP was low (discrimination value = 327.5). In a primer extension assay, SOF-TP was active against DENV NS5 wild-type polymerase activity with an IC50 of 14.7 ± 2.5 µM. The S600T substitution in the B Motif of DENV polymerase conferred 4.3-fold resistance to SOF-TP; this was due to decreased incorporation efficiency rather than enhanced excision of the incorporated SOF nucleotide. SOF has antiviral activity against DENV replication. The high discrimination value in favor of UTP in enzyme assays may not necessarily preclude antiviral activity in cells. SOF may be worthy of evaluation against severe DENV infections in humans.
Highlights
Cytopathic effect protection and virus yield reduction assays confirmed that SOF possessed anti-Dengue virus (DENV) activity in cell culture with a 50% effective concentration (EC50) of 4.9 μM and 1.4 μM respectively
DENV-infected cells were treated with increasing concentrations of SOF or control compound and protection from cytopathic effect (CPE) was measured
We have shown that SOF-TP inhibits DENV NS5 polymerase activity with an IC50 value of 14.7 μM in the presence of competing 0.5 μM UTP, which is a little higher than its K1/2 value
Summary
Cytopathic effect protection and virus yield reduction assays confirmed that SOF possessed anti-DENV activity in cell culture with a 50% effective concentration (EC50) of 4.9 μM and 1.4 μM respectively. Purified DENV NS5 incorporated the active triphosphate form (SOF-TP) into nascent RNA, causing chaintermination. SOF has antiviral activity against DENV replication. Dengue fever is caused by the four serotypes of Dengue virus (DENV) and is the most prevalent arthropod-borne viral disease of humans with major impact on public health[1]. A high degree of structural conservation within the NS5POL domain was observed among Flaviviridae family members[9] Among these domains, the 6 conserved motifs A-F which play key roles in RNA, rNTP, and metal-ion binding and catalysis have been well characterized in DENV RdRp11, 12. The amino acids involved at the catalytic site of DENV RdRp are located within motif A (aspartate at position 533, D533) and the catalytic triad GDD at positions 662–664 in motif C. These aspartate residues are involved in the coordination of two divalent metal ions that are essential for nucleotidyl transfer, termed the “two-metal-ion mechanism”[13]
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