Recombinant ZIKV RdRp assays can be useful tools for the screening of both nucleos(t)ide compounds and non-nucleotide metal ion-chelating agents that interfere with ZIKV replication.

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.

Drug resistance mutations (DRMs) have been reported for all currently approved anti-HIV drugs, including the latest integrase strand transfer inhibitors (INSTIs). We previously used the new INSTI dolutegravir (DTG) to select a G118R integrase resistance substitution in tissue culture and also showed that secondary substitutions emerged at positions H51Y and E138K. Now, we have characterized the impact of the G118R substitution, alone or in combination with either H51Y or E138K, on 3= processing and integrase strand transfer activity. The results show that G118R primarily impacted the strand transfer step of integration by diminishing the ability of integrase-long terminal repeat (LTR) complexes to bind target DNA. The addition of H51Y and E138K to G118R partially restored strand transfer activity by modulating the formation of integrase-LTR complexes through increasing LTR DNA affinity and total DNA binding, respectively. This unique mechanism, in which one function of HIV integrase partially compensates for the defect in another function, has not been previously reported. The G118R substitution resulted in low-level resistance to DTG, raltegravir (RAL), and elvitegravir (EVG). The addition of either of H51Y or E138K to G118R did not enhance resistance to DTG, RAL, or EVG. Homology modeling provided insight into the mechanism of resistance conferred by G118R as well as the effects of H51Y or E138K on enzyme activity. The G118R substitution therefore represents a potential avenue for resistance to DTG, similar to that previously described for the R263K substitution. For both pathways, secondary substitutions can lead to either diminished integrase activity and/or increased INSTI susceptibility. The HIV integrase (IN) enzyme catalyzes the insertion of viral DNA into host DNA, a process known as integration (1). In a reaction termed 3= processing, integrase recognizes and cleaves off a dinucleotide GT downstream of a conserved dinucleotide CA signal, located within the last 15 bp of the long terminal repeats (LTR) that flank the viral DNA, and this effectively creates new 3= hydroxyl ends (2). The second step in integration, termed strand transfer, is the integrase-mediated insertion of the processed viral DNA into host DNA by a 5-bp staggered cleavage of target DNA. The exposed 3= hydroxyl groups on the viral insert interact with exposed 5= phosphates on the host DNA. Integration, which occurs primarily in highly expressed genes (3), causes the host machinery to transcribe viral genes and leads to successful propagation of viral particles. Integration is essential for productive infection and the establishment of viral persistence; therefore, integration was an early choice for the development of inhibitory compounds (4).The development of in vitro microtiter plate-based biochemical assays for the measurement of the various biochemical activities of integrase facilitated compound screening and identification of viable integrase inhibitors (5). The first specific integrase inhibitors, identified in 2000 (5), posses...