The filovirus Ebola (EBOV) causes the most severe hemorrhagic fever known. The EBOV RNA-dependent polymerase complex includes a filovirus-specific VP30, which is critical for the transcriptional but not replication activity of EBOV polymerase; to support transcription, VP30 must be in a dephosphorylated form. Here we show that EBOV VP30 is phosphorylated not only at the N-terminal serine clusters identified previously but also at the threonine residues at positions 143 and 146. We also show that host cell protein phosphatase 1 (PP1) controls VP30 dephosphorylation because expression of a PP1-binding peptide cdNIPP1 increased VP30 phosphorylation. Moreover, targeting PP1 mRNA by shRNA resulted in the overexpression of SIPP1, a cytoplasm-shuttling regulatory subunit of PP1, and increased EBOV transcription, suggesting that cytoplasmic accumulation of PP1 induces EBOV transcription. Furthermore, we developed a small molecule compound, 1E7-03, that targeted a non-catalytic site of PP1 and increased VP30 dephosphorylation. The compound inhibited the transcription but increased replication of the viral genome and completely suppressed replication of EBOV in cultured cells. Finally, mutations of Thr¹⁴³ and Thr¹⁴⁶ of VP30 significantly inhibited EBOV transcription and strongly induced VP30 phosphorylation in the N-terminal Ser residues 29–46, suggesting a novel mechanism of regulation of VP30 phosphorylation. Our findings suggest that targeting PP1 with small molecules is a feasible approach to achieve dysregulation of the EBOV polymerase activity. This novel approach may be used for the development of antivirals against EBOV and other filovirus species.

HIV-1 Tat protein recruits host cell factors including CDK9/cyclin T1 to HIV-1 TAR RNA and thereby induces HIV-1 transcription. An interaction with host Ser/Thr protein phosphatase-1 (PP1) is critical for this function of Tat. PP1 binds to a Tat sequence, Q³⁵VCF³⁸, which resembles the PP1-binding “RVxF” motif present on PP1-binding regulatory subunits. We showed that expression of PP1 binding peptide, a central domain of Nuclear Inhibitor of PP1, disrupted the interaction of HIV-1 Tat with PP1 and inhibited HIV-1 transcription and replication. Here, we report small molecule compounds that target the “RVxF”-binding cavity of PP1 to disrupt the interaction of PP1 with Tat and inhibit HIV-1 replication. Using the crystal structure of PP1, we virtually screened 300,000 compounds and identified 262 small molecules that were predicted to bind the “RVxF”-accommodating cavity of PP1. These compounds were then assayed for inhibition of HIV-1 transcription in CEM T cells. One of the compounds, 1H4, inhibited HIV-1 transcription and replication at non-cytotoxic concentrations. 1H4 prevented PP1-mediated dephosphorylation of a substrate peptide containing an RVxF sequence in vitro. 1H4 also disrupted the association of PP1 with Tat in cultured cells without having an effect on the interaction of PP1 with the cellular regulators, NIPP1 and PNUTS, or on the cellular proteome. Finally, 1H4 prevented the translocation of PP1 to the nucleus. Taken together, our study shows that HIV- inhibition can be achieved through using small molecules to target a non-catalytic site of PP1. This proof-of-principle study can serve as a starting point for the development of novel antiviral drugs that target the interface of HIV-1 viral proteins with their host partners.

Background and Purpose: HIV‐1 transcription is activated by the Tat protein which recruits the cyclin‐dependent kinase CDK9/cyclin T1 to TAR RNA. Tat binds to protein phosphatase‐1 (PP1) through the Q³⁵VCF³⁸ sequence and translocates PP1 to the nucleus. PP1 dephosphorylates CDK9 and activates HIV‐1 transcription. We have synthesized a low MW compound 1H4, that targets PP1 and prevents HIV‐1 Tat interaction with PP1 and inhibits HIV‐1 gene transcription. Here, we report our further work with the 1H4‐derived compounds and analysis of their mechanism of action.Experimental Approach: Using the 1H4‐PP1 complex as a model, we iteratively designed and synthesized follow‐up libraries that were analysed for the inhibition of HIV‐1 transcription and toxicity. We also confirmed the mechanism of action of the PP1‐targeting molecules by determining the affinity of binding of these molecules to PP1, by analysing their effects on PP1 activity, disruption of PP1 binding to Tat and shuttling of PP1 to the nucleus.Key Results: We identified a tetrahydroquinoline derivative, compound 7, which disrupted the interaction of Tat with PP1. We further optimized compound 7 and obtained compound 7c, renamed 1E7‐03, which inhibited HIV‐1 with low IC₅₀ (fivefold lower than the previously reported compound, 1H4), showed no cytotoxicity and displayed a plasma half‐life greater than 8 h in mice. 1E7‐03 bound to PP1 in vitro and prevented shuttling of PP1 into the nucleus.Conclusions and Implications: Our study shows that low MW compounds that functionally mimic the PP1‐binding RVxF peptide can inhibit HIV‐1 transcription by deregulating PP1.