School of Biological Sciences, University of Auckland, Auckland 1010, New Zealand.
Ferrier Research Institute, Victoria University of Wellington, 69 Gracefield Rd, Lower Hutt 5010, New Zealand.
ACS Infect Dis. 2023 Aug 11;9(8):1658-1673. doi: 10.1021/acsinfecdis.3c00311. Epub 2023 Jul 24.
Millions of people are infected by the dengue and Zika viruses each year, resulting in significant morbidity and mortality. Galidesivir is an adenosine nucleoside analog that can attenuate flavivirus replication in cell-based assays and animal models of infection. Galidesivir is converted to the triphosphorylated form by host kinases and subsequently incorporated into viral RNA by viral RNA polymerases. This has been proposed to lead to the delayed termination of RNA synthesis. Here, we report direct in vitro testing of the effects of Galidesivir triphosphate on dengue-2 and Zika virus polymerase activity. Galidesivir triphosphate was chemically synthesized, and inhibition of RNA synthesis followed using a dinucleotide-primed assay with a homopolymeric poly(U) template. Galidesivir triphosphate was equipotent against dengue-2 and Zika polymerases, with IC values of 42 ± 12 μM and 47 ± 5 μM, respectively, at an ATP concentration of 20 μM. RNA primer extension assays show that the dengue-2 polymerase stalls while attempting to add a Galidesivir nucleotide to the nascent RNA chain, evidenced by the accumulation of RNA products truncated immediately upstream of Galidesivir incorporation sites. Nevertheless, Galidesivir is incorporated at isolated sites with low efficiency, leading to the subsequent synthesis of full-length RNA with no evidence of delayed chain termination. The incorporation of Galidesivir at consecutive sites is strongly disfavored, highlighting the potential for modulation of inhibitory effects of nucleoside analogs by the template sequence. Our results suggest that attenuation of dengue replication by Galidesivir may not derive from the early termination of RNA synthesis following Galidesivir incorporation.
每年有数百万的人感染登革热和寨卡病毒，导致发病率和死亡率显著上升。加地昔韦是一种腺嘌呤核苷类似物，能够在细胞检测和感染动物模型中减弱黄病毒的复制。加地昔韦在宿主激酶的作用下转化为三磷酸化形式，随后由病毒 RNA 聚合酶将其掺入病毒 RNA 中。这被认为会导致 RNA 合成的延迟终止。在这里，我们报告了加地昔韦三磷酸酯对登革热 2 型和寨卡病毒聚合酶活性的直接体外测试结果。我们通过使用二核苷酸启动的寡聚 U 模板进行聚合酶活性检测，对加地昔韦三磷酸酯进行了化学合成和 RNA 合成抑制测试。加地昔韦三磷酸酯对登革热 2 型和寨卡病毒聚合酶具有相同的抑制作用，在 20 μM 的 ATP 浓度下，其对登革热 2 型和寨卡病毒聚合酶的 IC 值分别为 42±12 μM 和 47±5 μM。RNA 引物延伸试验表明，登革热 2 型聚合酶在尝试将加地昔韦核苷酸添加到新生 RNA 链时会停滞，这表现为在加地昔韦掺入部位上游立即截断的 RNA 产物的积累。然而，加地昔韦以低效率掺入到孤立的部位，导致随后合成全长 RNA，没有证据表明存在延迟的链终止。加地昔韦在连续部位的掺入被强烈抑制，这突显了模板序列对核苷类似物抑制作用的潜在调节作用。我们的结果表明，加地昔韦对登革热复制的抑制作用可能不是源于加地昔韦掺入后 RNA 合成的早期终止。
