Screening for novel, stage-specific inhibitors of herpes simplex virus replication

Herpes simplex virus (HSV) is a clinically significant human pathogen. Early stages of the HSV replication cycle, between attachment and gene expression, are poorly understood compared to later steps such as DNA replication. These early events in viral replication include entry into cells, trafficking to the nucleus, uncoating, and expression of viral genes. Small molecule inhibitors have historically played a major role in elucidating the underlying biology of viruses in their uninhibited states. Therefore, we hypothesized that we could learn more about early steps by identifying novel stage-specific inhibitors of HSV replication. We designed a chemical screening approach to identify small molecules that inhibit HSV replication prior to viral DNA replication. We infected Vero cells with ICP8-GFP, a recombinant HSV that expresses green fluorescent protein (GFP) fused to an HSV early protein, ICP8. Expression of ICP8 is among the last events to occur prior to replication of the viral genome, and the GFP reporter would only be expressed if the prior events occurred successfully. Our screen identified ouabain, a cardiac glycoside. Ouabain decreased viral yield by 100-fold without affecting cellular metabolic activity in an overnight assay. We performed kinetic assays and determined that ouabain did not inhibit viral attachment, viral entry, or transcription of viral immediate early mRNA's. Ouabain did inhibit accumulation of viral IE proteins, and labeling of both cellular and viral proteins in a 35-S methionine assay. Protein stability was not decreased in a pulse-chase assay. Collectively, these data indicate that ouabain has a global effect on translation. To better understand the mechanism of ouabain's antiviral activity, we performed a structure activity relationship assay, and determined that the antiviral potencies of other cardiac glycosides correlated with their potencies against the known target of these compounds, the cellular sodium potassium ATPase. We also determined that inhibition was time-dependent and reversible with removal of drug. Treatment with excess potassium chloride partially alleviated the antiviral effect of ouabain, suggesting that ouabain's effect on translation is due to an effect on cellular potassium.