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Unlabelled: Respiratory syncytial virus (RSV) is the leading cause of pediatric respiratory disease. RSV has an RNA-dependent RNA polymerase that transcribes and replicates the viral negative-sense RNA genome. The large polymerase subunit (L) has multiple enzymatic activities, having the capability to synthesize RNA and add and methylate a cap on each of the viral mRNAs. Previous studies (H. Xiong et al., Bioorg Med Chem Lett, 23:6789-6793, 2013, http://dx.doi.org/10.1016/j.bmcl.2013.10.018; C. L. Tiong-Yip et al., Antimicrob Agents Chemother, 58:3867-3873, 2014, http://dx.doi.org/10.1128/AAC.02540-14) had identified a small-molecule inhibitor, AZ-27, that targets the L protein. In this study, we examined the effect of AZ-27 on different aspects of RSV polymerase activity. AZ-27 was found to inhibit equally both mRNA transcription and genome replication in cell-based minigenome assays, indicating that it inhibits a step common to both of these RNA synthesis processes. Analysis in an in vitro transcription run-on assay, containing RSV nucleocapsids, showed that AZ-27 inhibits synthesis of transcripts from the 3' end of the genome to a greater extent than those from the 5' end, indicating that it inhibits transcription initiation. Consistent with this finding, experiments that assayed polymerase activity on the promoter showed that AZ-27 inhibited transcription and replication initiation. The RSV polymerase also can utilize the promoter sequence to perform a back-priming reaction. Interestingly, addition of AZ-27 had no effect on the addition of up to three nucleotides by back-priming but inhibited further extension of the back-primed RNA. These data provide new information regarding the mechanism of inhibition by AZ-27. They also suggest that the RSV polymerase adopts different conformations to perform its different activities at the promoter., Importance: Currently, there are no effective antiviral drugs to treat RSV infection. The RSV polymerase is an attractive target for drug development, but this large enzymatic complex is poorly characterized, hampering drug development efforts. AZ-27 is a small-molecule inhibitor previously shown to target the RSV large polymerase subunit (C. L. Tiong-Yip et al., Antimicrob Agents Chemother, 58:3867-3873, 2014, http://dx.doi.org/10.1128/AAC.02540-14), but its inhibitory mechanism was unknown. Understanding this would be valuable both for characterizing the polymerase and for further development of inhibitors. Here, we show that AZ-27 inhibits an early stage in mRNA transcription, as well as genome replication, by inhibiting initiation of RNA synthesis from the promoter. However, the compound does not inhibit back priming, another RNA synthesis activity of the RSV polymerase. These findings provide insight into the different activities of the RSV polymerase and will aid further development of antiviral agents against RSV., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

As obligate parasites of cellular processes, viruses must take over cellular macromolecular machinery. It is also becoming clear that viruses routinely control intracellular signaling pathways through the direct or indirect control of kinases and phosphatases. This control of cellular phosphoproteins is important to promote a variety of viral processes, from control of entry to nuclear function to the stimulation of viral protein synthesis. This review focuses on the takeover of the cellular phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway by a variety of retroviruses, DNA viruses, and RNA viruses, highlighting the functions ascribed to virus activation of PI3K and Akt activity. This review also describes the role that the PI3K/Akt pathway plays in the host response, noting that it that can trigger anti- as well as proviral functions., (Copyright © 2012 Elsevier Inc. All rights reserved.)

We describe a convenient system for analyzing bunyavirus transcription using a recombinant RNA template derived from the plasmid pBUNSCAT, which comprises a negative-sense reporter gene (chloramphenicol acetyltransferase or CAT) flanked by the exact 5' and 3' untranslated regions of the Bunyamwera virus (BUN) S RNA segment. When cells which expressed bunyavirus proteins (either by recombinant vaccinia viruses or by the vaccinia virus-T7 system) were transfected with BUNSCAT RNA, CAT activity could be measured, indicating transcription of the negative-sense reporter RNA into mRNA. The system permits investigation of both the protein and RNA sequence requirements for transcription. Extensions of 2 bases at the 5' end or 11 or 35 bases at the 3' end of BUNSCAT RNA allowed transcription but a lower level than the wild-type template. Deletion of the 5 nucleotides at the 3' end of BUNSCAT RNA reduced CAT activity by > 99%. Investigation of the viral protein requirements of the system showed that only the bunyavirus L and N proteins were needed for CAT activity. The BUN L protein was also able to transcribe the reporter RNA in concert with the N proteins of closely related bunyaviruses such as Batai, Cache Valley, Maguari, Main Drain, and Northway, but only inefficiently with those of Kairi, Guaroa, or Lumbo viruses. When BUN L proteins containing specific mutations were expressed CAT activity was only observed using those mutated L proteins previously reported to be active in a nucleocapsid transfection assay (H. Jin and R. M. Elliott, 1992, J. Gen. Virol. 73, 2235-2244). These results illustrate the utility of this system for a detailed genetic analysis of the factors involved in bunyavirus transcription.