Summary The most recent Ebola virus outbreak in West Africa – unprecedented in the number of cases and fatalities, geographic distribution, and number of nations affected – highlights the need for safe, effective, and readily available antiviral agents for treatment and prevention of acute Ebola virus (EBOV) disease (EVD) or sequelae1. No antiviral therapeutics have yet received regulatory approval or demonstrated clinical efficacy. Here we describe the discovery of a novel anti-EBOV small molecule antiviral, GS-5734, a monophosphoramidate prodrug of an adenosine analog. GS-5734 exhibits antiviral activity against multiple variants of EBOV in cell-based assays. The pharmacologically active nucleoside triphosphate (NTP) is efficiently formed in multiple human cell types incubated with GS-5734 in vitro, and the NTP acts as an alternate substrate and RNA-chain terminator in primer-extension assays utilizing a surrogate respiratory syncytial virus RNA polymerase. Intravenous administration of GS-5734 to nonhuman primates resulted in persistent NTP levels in peripheral blood mononuclear cells (half-life = 14 h) and distribution to sanctuary sites for viral replication including testes, eye, and brain. In a rhesus monkey model of EVD, once daily intravenous administration of 10 mg/kg GS-5734 for 12 days resulted in profound suppression of EBOV replication and protected 100% of EBOV-infected animals against lethal disease, ameliorating clinical disease signs and pathophysiological markers, even when treatments were initiated three days after virus exposure when systemic viral RNA was detected in two of six treated animals. These results provide the first substantive, post-exposure protection by a small-molecule antiviral compound against EBOV in nonhuman primates. The broad-spectrum antiviral activity of GS-5734 in vitro against other pathogenic RNA viruses – including filoviruses, arenaviruses, and coronaviruses – suggests the potential for expanded indications. GS-5734 is amenable to large-scale manufacturing, and clinical studies investigating the drug safety and pharmacokinetics are ongoing.

The large polymerase subunit (L) of non-segmented negative strand RNA viruses transcribes viral mRNAs and replicates the viral genome. Studies with VSV have shown that conserved region V (CRV) of the L protein is part of the capping domain. However, CRV folds over and protrudes into the polymerization domain, suggesting that it might also have a role in RNA synthesis. In this study, the role of respiratory syncytial virus (RSV) CRV was evaluated using single amino acid substitutions and a small molecule inhibitor called BI-D. Effects were analyzed using cell-based minigenome and in vitro biochemical assays. Several amino acid substitutions inhibited production of capped, full-length mRNA and instead resulted in accumulation of short transcripts of approximately 40 nucleotides in length, confirming that RSV CRV has a role in capping. In addition, all six variants tested were either partially or completely defective in RNA replication. This was due to an inability of the polymerase to efficiently elongate the RNA within the promoter region. BI-D also inhibited transcription and replication. In this case, polymerase elongation activity within the promoter region was enhanced, such that the small RNA transcribed from the promoter was not released and instead was elongated past the first gene start signal. This was accompanied by a decrease in mRNA initiation at the first gene start signal and accumulation of aberrant RNAs of varying length. Thus, in addition to its function in mRNA capping, conserved region V modulates the elongation properties of the polymerase to enable productive transcription and replication to occur.

The respiratory syncytial virus (RSV) RNA dependent RNA polymerase (RdRp) initiates two RNA synthesis processes from the viral promoter: genome replication from position 1U and mRNA transcription from position 3C. Here, we examined the mechanism by which a single promoter can direct initiation from two sites. We show that initiation at 1U and 3C occurred independently of each other, and that the same RdRp was capable of precisely selecting the two sites. The RdRp preferred to initiate at 3C, but initiation site selection could be modulated by the relative concentrations of ATP versus GTP. Analysis of template mutations indicated that the RdRp could bind ATP and CTP, or GTP, independently of template nucleotides. The data suggest a model in which innate affinity of the RdRp for particular NTPs, coupled with a repeating element within the promoter, allows precise initiation of replication at 1U or transcription at 3C.

The ribonucleocapsid complex of respiratory syncytial virus (RSV) is responsible for both viral mRNA transcription and viral replication during infection, though little is known about how this dual function is achieved. Here, we report the use of a recombinant RSV virus with a FLAG-tagged large polymerase protein, L, to characterize and localize RSV ribonucleocapsid structures during the early and late stages of viral infection. Through proximity ligation assays and super-resolution microscopy, viral RNA and proteins in the ribonucleocapsid complex were revealed to dynamically rearrange over time, particularly between 6 and 8 hours post infection, suggesting a connection between the ribonucleocapsid structure and its function. The timing of ribonucleocapsid rearrangement corresponded with an increase in RSV genome RNA accumulation, indicating that this rearrangement is likely involved with the onset of RNA replication and secondary transcription. Additionally, early overexpression of RSV M2-2 from in vitro transcribed mRNA was shown to inhibit virus infection by rearranging the ribonucleocapsid complex. Collectively, these results detail a critical understanding into the localization and activity of RSV L and the ribonucleocapsid complex during RSV infection.

Antimicrobial resistance is rapidly expanding, in a large part due to mobile genetic elements. We screened 94 fecal fluoroquinolone-resistant Escherichia coli isolates from Nigeria for six plasmid-mediated quinolone resistance (PMQR) genes. Sixteen isolates harbored at least one of the PMQR genes and four were positive for aac-6-Ib-cr. In one strain, aac-6-Ib-cr was mapped to a 125 Kb self-transmissible IncFII plasmid, pMB2, which also bears blaCTX-M-15, seven other functional resistance genes and multiple resistance pseudogenes. Laboratory strains carrying pMB2 grew faster than isogenic strains lacking the plasmid in both rich and minimal media. We excised a 32 Kb fragment containing transporter genes and several open-reading frames of unknown function. The resulting 93 Kb mini-plasmid conferred slower growth rates and lower fitness than wildtype pMB2. Trans-complementing the deletion with the cloned sitABCD genes confirmed that they accounted for the growth advantage conferred by pMB2 in iron-depleted media. pMB2 is a large plasmid with a flexible resistance region that contains loci that can account for evolutionary success in the absence of antimicrobials. Ancillary functions conferred by resistance plasmids can mediate their retention and transmissibility, worsening the trajectory for antimicrobial resistance and potentially circumventing efforts to contain resistance through restricted use.