Identification of Vaccinia Virus Inhibitors and Cellular Functions Necessary for Efficient Viral Replication by Screening Bioactives and FDA-Approved Drugs

Abstract

Four decades after the eradication of smallpox, poxviruses continue to threaten the health of humans and other animals. Vaccinia virus (VACV) was used as the vaccine that successfully eradicated smallpox and is a prototypic member of the poxvirus family. Many cellular pathways play critical roles in productive poxvirus replication. These pathways provide opportunities to expand the arsenal of poxvirus antiviral development by targeting the cellular functions required for efficient poxvirus replication. In this study, we developed and optimized a secreted Gaussia luciferase-based, simplified assay procedure suitable for high throughput screening. Using this procedure, we screened a customized compound library that contained over 3200 bioactives and FDA (Food and Drug Administration)-approved chemicals, most having known cellular targets, for their inhibitory effects on VACV replication. We identified over 140 compounds that suppressed VACV replication. Many of these hits target cellular pathways previously reported to be required for efficient VACV replication, validating the effectiveness of our screening. Importantly, we also identified hits that target cellular functions with previously unknown roles in the VACV replication cycle. Among those in the latter category, we verified the antiviral role of several compounds targeting the janus kinase/signal transducer and activator of transcription-3 (JAK/STAT3) signaling pathway by showing that STAT3 inhibitors reduced VACV replication. Our findings identify pathways that are candidates for use in the prevention and treatment of poxvirus infections and additionally provide a foundation to investigate diverse cellular pathways for their roles in poxvirus replications.

Viral growth factor- and STAT3 signaling-dependent elevation of the TCA cycle intermediate levels during vaccinia virus infection

“…Our results that STAT3 inhibition reduces VACV replication is somehow discrepant to a previous report that inhibition of STAT3 enhanced the replication of ACAM2000, a VACV strain currently used as a vaccine in mice, and keratinocytes [86]. We have independently confirmed the suppression effects on VACV replication using multiple inhibitors and multiple cell types [57]. We do not fully understand the discrepancy, although it could possibly be explained by different cell types or virus strains used in these studies.…”

“…Notably, stattic, an inhibitor of STAT3 activation [56], significantly reduced the increase in citrate level in VACV-infected cells but not in uninfected cells (Fig 6C). Chemical inhibition of the STAT3 pathway by stattic resulted in a 177-fold reduction [57]. Stattic treatment did not affect HFF viability at the same concentration ( S10B Fig), suggesting that STAT3 signaling is also required for VACV-induced citrate level increase.…”

Inhibition of MEK-ERK pathway enhances oncolytic vaccinia virus replication in doxorubicin-resistant ovarian cancer

“… 54 STAT3 inhibitor has been identified to suppress vaccinia virus replication by screening compound library. 55 These results suggest that STAT3 activation is responsible for the increased OVV replication induced by MEK inhibition.…”

Rapid and quantitative evaluation of vaccinia virus‐induced host shutoff using newly generated cell lines stably expressing secreted Gaussia luciferase

“…We have previously shown that Gaussia luciferase secretion is not affected by VACV infection of HeLa cells using a transfected plasmid 43 . We measured the Gaussia luciferase activities in media and cell lysates, respectively, to rule out the possibility that Gaussia luciferase was retained in the A549‐EF1α‐Gluc cells due to VACV infection.…”

“…We have previously shown that Gaussia luciferase secretion is not affected by VACV infection of HeLa cells using a transfected plasmid. 43 We measured the Gaussia luciferase activities in media and cell lysates, respectively, to rule out the possibility that Gaussia luciferase was retained in the A549-EF1α-Gluc cells due to VACV infection. The results indicated that (i) the vast majority (~99%) of Gaussia luciferase activities were in the media, and (ii) in both the media and the cell lysates, VACV infection led to significantly reduced Gaussia luciferase activities (Figure 2D).…”