Chemical intervention of influenza virus mRNA nuclear export.

Influenza A viruses are human pathogens with limited therapeutic options. Therefore, it is crucial to devise strategies for the identification of new classes of antiviral medications. The influenza A virus genome is constituted of 8 RNA segments. Two of these viral RNAs are transcribed into mRNAs that are alternatively spliced. The M1 mRNA encodes the M1 protein but is also alternatively spliced to yield the M2 mRNA during infection. M1 to M2 mRNA splicing occurs at nuclear speckles, and M1 and M2 mRNAs are exported to the cytoplasm for translation. M1 and M2 proteins are critical for viral trafficking, assembly, and budding. Here we show that gene knockout of the cellular protein NS1-BP, a constituent of the M mRNA speckle-export pathway and a binding partner of the virulence factor NS1 protein, inhibits M mRNA nuclear export without altering bulk cellular mRNA export, providing an avenue to preferentially target influenza virus. We performed a high-content, image-based chemical screen using single-molecule RNA-FISH to label viral M mRNAs followed by multistep quantitative approaches to assess cellular mRNA and cell toxicity. We identified inhibitors of viral mRNA biogenesis and nuclear export that exhibited no significant activity towards bulk cellular mRNA at non-cytotoxic concentrations. Among the hits is a small molecule that preferentially inhibits nuclear export of a subset of viral and cellular mRNAs without altering bulk cellular mRNA export. These findings underscore specific nuclear export requirements for viral mRNAs and phenocopy down-regulation of the mRNA export factor UAP56. This RNA export inhibitor impaired replication of diverse influenza A virus strains at non-toxic concentrations. Thus, this screening strategy yielded compounds that alone or in combination may serve as leads to new ways of treating influenza virus infection and are novel tools for studying viral RNA trafficking in the nucleus.

A high throughput screen identifies benzoquinoline compounds as inhibitors of Ebola virus replication.

Ebola virus (EBOV) is an enveloped negative-sense, single-stranded RNA virus of the filovirus family that causes severe disease in humans. Approved therapies for EBOV disease are lacking. EBOV RNA synthesis is carried out by a virus-encoded complex with RNA-dependent RNA polymerase activity that is required for viral propagation. This complex and its activities are therefore potential antiviral targets. To identify potential lead inhibitors of EBOV RNA synthesis, a library of small molecule compounds was screened against a previously established assay of EBOV RNA synthesis, the EBOV minigenome assay (MGA), in 384 well microplate format. The screen identified 56 hits that inhibited EBOV MGA activity by more than 70% while exhibiting less than 20% cell cytotoxicity. Inhibitory chemical scaffolds included angelicin derivatives, derivatives of the antiviral compound GSK983 and benzoquinolines. Structure-activity relationship (SAR) studies of the benzoquinoline scaffold produced ∼50 analogs and led to identification of an optimized compound, SW456, with a submicromolar IC50 in the EBOV MGA and antiviral activity against infectious EBOV in cell culture. The compound was also active against a MGA for another deadly filovirus, Marburg virus. It also exhibited antiviral activity towards a negative-sense RNA virus from the rhabdovirus family, vesicular stomatitis virus, and a positive-sense RNA virus, Zika virus. Overall, these data demonstrate the potential of the EBOV MGA to identify anti-EBOV compounds and identifies the benzoquinoline series as a broad-spectrum antiviral lead.