Using WN virus as a model, we recently established three cell-based HTS assays: a full-length reporting virus, packaged VLPs containing replicon RNA, and replicon-harboring cells, each of which contains an Rluc reporter (39
). Here, we have employed these assays to identify and characterize triaryl pyrazoline as a novel inhibitor of flavivirus infection in cell culture. Since the infection assay with full-length reporting virus covers the complete viral life cycle, we chose this system as our primary screening assay (Fig. ). Once a “hit” was identified, the inhibitor was subjected to two other HTS assays (Fig. ) as well as an authentic WN virus infection assay (Fig. ). As summarized in Table , the three HTS assays yielded comparable EC50
values for triaryl pyrazoline. Importantly, these EC50
values are similar to that obtained with the authentic viral infection assay. These results demonstrate that the three HTS systems can be used reliably for anti-WN virus drug discovery.
Comparison of efficacies and cytotoxicities of triaryl pyrazoline in four WN virus assays
We characterized the mode of WN virus inhibition by triaryl pyrazoline. Because the three reporting assays described above encompass multiple but discrete steps of the viral life cycle, they could be used to discriminate among inhibition of viral entry, replication, and virion assembly by any compound. The comparable EC50
values derived from the three reporting assays (Table ) indicate that the compound inhibits WN infection at a step that is shared by all three HTS assays: viral replication. Further analyses using a transient replicon system showed that the compound significantly blocks RNA replication (by ≥95%) without suppression of viral translation (Fig. ). In agreement with the above-described results, time-of-addition experiments showed that a significant suppression in viral titer requires treatment of infected cells with the compound during the first 10 h p.i., after which the inhibitory effects gradually diminished (Fig. ). It should be noted that during BHK-21 cell infection with an Rluc-expressing WN virus or an Rluc-expressing VLP, initial translation occurred during the first 7.5 h p.i., while nascent genomic RNA began to accumulate after 10 h p.i (39
). In combination, the results strongly suggest that triaryl pyrazoline inhibits WN virus through suppression of viral RNA replication.
As with WN virus, the compound inhibited DEN-1 viral infection through suppression of viral replication, as suggested by its antiviral activity in a DEN-1 virus replicon-containing cell line (Fig. ). However, analyses using a transient replicon system of DEN-1 virus consistently showed that besides exerting a significant inhibition of viral RNA replication (by 90%), the compound weakly suppressed viral translation (by 11 to 31%) (Fig. ). These results have raised the question, in the case of DEN-1 virus, of whether the antiviral activity was due to a dual effect of the compound on both viral translation and RNA replication. Alternatively, the observed antiviral activity was caused solely by the compound's effect on translation, which in turn resulted in suppression of viral RNA replication. However, at this point, we could not rule out the possibility that the weak suppression of viral translation of the DEN-1 virus replicon was due to compound-mediated inhibition of FMDV 2A activity. More experiments are required to discriminate among these possibilities.
Besides WN and DEN viruses, triaryl pyrazoline also inhibited other flaviviruses, specifically YF (17D) and SLE viruses. Furthermore, the compound suppressed other plus-strand and minus-strand RNA viruses, as represented by WEE virus, MHV, and VSV. In contrast, the compound did not inhibit HIV-1 and influenza virus (Fig. ). Interestingly, both HIV-1 and influenza virus replicate in the nucleus, whereas the compound-sensitive viruses (flaviviruses, WEE virus, MHV, and VSV) replicate in the cytoplasm. In line with those results, the compound did not inhibit herpes simplex virus (a DNA virus that replicates in the nucleus) (data not shown). These results clearly indicate that the compound inhibits a broad spectrum of RNA viruses with specificity, probably through blocking a target required for replication of the compound-sensitive viruses. It is currently not known whether the compound exerts its functions through direct interaction with a host factor or a viral protein. A number of approaches to define the target of the compound are being explored. Testing the compound in biochemistry assays (RNA-dependent RNA polymerase, protease, helicase, and NTPase) may indicate whether the inhibitor interferes directly with these viral functions. Alternatively, if resistant viruses or replicons could be selected, then sequencing of resistant viruses or replicons may point to the targets of the compound.
It is confirmatory that besides the compound shown in Fig. , one other triaryl pyrazoline analogue from the library was identified as having antiviral activity (data not shown). However, this analogue was less potent, suggesting that different functional groups within this analogue are important for antiviral efficacy. Efforts are being made to generate a triaryl pyrazoline core-based library for improvement of the antiviral potency. At this time, due to the small number of analogues synthesized, we could not conclude any specific structural and functional relationships.
The DEN-1 virus replicon-containing cell lines (Fig. ) and the transient Rluc replicon (Fig. ) described in this study will be useful for HTS of compound libraries for inhibitors of DEN virus and for studying DEN virus replication. The utility of the DEN-1 virus replicon-containing cell line for drug screening was validated in a 96-well format with a known inhibitor (MPA) and the newly identified triaryl pyrazoline. For MPA, the EC50
value (0.3 μM) (Fig. ) derived from the DEN-1 virus replicon cell line was consistent with those derived from the authentic viral titer reduction assay (0.3 to 1.9 μM) (12
). For triaryl pyrazoline, we performed the DEN-1 virus replicon cell line assay and the authentic viral infection assay in Vero cells. The experiments yielded comparable EC50
values of 17 μM (Fig. ) and 23 μM (Fig. ), respectively. The results clearly demonstrate that the DEN-1 virus replicon-containing cell line could be used for HTS of inhibitors of DEN virus. Compared to the traditional viral titer reduction assay, the new system offers superior speed and sensitivity. Establishment of such HTS assays for DEN virus is important, considering the large scale of disease caused by this virus (see Introduction).
It was surprising that the DEN-1 virus replicon containing a direct fusion of the Rluc gene with the N-terminal capsid and the C-terminal envelope (DEN-1 Rluc-Rep) (Fig. ) was replication defective. Replication of the DEN-1 virus replicon requires an FMDV 2A-mediated cleavage between Rluc and the residual envelope fragment (DEN-1 Rluc-2A-Rep) (Fig. ). A similar observation was recently reported for a DEN-2 virus replicon (2
). The requirement for 2A cleavage for DEN-1 virus (this study) and DEN-2 virus (2
) replicons contrasts with our WN virus replicon, whose replication does not require the 2A cleavage between Rluc and its downstream envelope fragment (29
). However, comparison of replication kinetics (as indicated by the Rluc profile) showed a dramatic difference between the WN virus and DEN-1 virus replicons. The first Rluc peak during the initial 10 h p.t. was observed for both WN virus and DEN-1 virus replicons. The second Rluc peak appeared after 24 h p.t. for the WN virus replicon (without 2A cleavage), whereas the second peak appeared immediately after 10 h p.t. for DEN-1 Rluc-2A-Rep (Fig. ). These data indicate that the delay in RNA replication of the WN virus replicon is likely due to the fusion of Rluc to the C-terminal transmembrane domain of the envelope (anchored into the ER membrane), thereby interfering with RNA replication. The data also suggest that replication of DEN-1 virus is more sensitive to such interference than replication of WN virus.
In summary, the identification and characterization of triaryl pyrazoline represent the first step toward the development of this compound for potential antiflavivirus therapy. Its broad spectrum of antiviral activity without detectable cytotoxicity in cell culture warrants future in vivo study. The establishment of luciferase-expressing replicons for DEN-1 virus has made possible HTS of compound libraries for inhibitors of DEN virus.