Antiviral development is plagued by drug resistance and genetic barriers to resistance are needed. For HIV and hepatitis C virus (HCV), combination therapy has proved life-saving. The targets of direct-acting antivirals for HCV infection are NS3/4A protease, NS5A phosphoprotein and NS5B polymerase. Differential visualization of drug-resistant and -susceptible RNA genomes within cells revealed that resistant variants of NS3/4A protease and NS5A phosphoprotein are cis-dominant, ensuring their direct selection from complex environments. Confocal microscopy revealed that RNA replication complexes are genome-specific, rationalizing the non-interaction of wild-type and variant products. No HCV antivirals yet display the dominance of drug susceptibility shown for capsid proteins of other viruses. However, effective inhibitors of HCV polymerase exact such high fitness costs for drug resistance that stable genome selection is not observed. Barriers to drug resistance vary with target biochemistry and detailed analysis of these barriers should lead to the use of fewer drugs.
- Karla Kirkegaard
- Nicholas van Buuren
- Nicholas van Buuren
- Karla Kirkegaard
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- David M Knipe, Harvard Medical School, United States
© 2018, van Buuren et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
The environmental pathogen Cryptococcus neoformans claims over 180,000 lives each year. Survival of this basidiomycete at host CO2 concentrations has only recently been considered an important virulence trait. Through screening gene knockout libraries constructed in a CO2-tolerant clinical strain, we found mutations leading to CO2 sensitivity are enriched in pathways activated by heat stress, including calcineurin, Ras1-Cdc24, cell wall integrity, and Regulator of Ace2 and Morphogenesis (RAM). Overexpression of Cbk1, the conserved terminal kinase of the RAM pathway, partially restored defects of these mutants at host CO2 or temperature levels. In ascomycetes such as Saccharomyces cerevisiae and Candida albicans, transcription factor Ace2 is an important target of Cbk1, activating genes responsible for cell separation. However, no Ace2 homolog or any downstream component of the RAM pathway has been identified in basidiomycetes. Through in vitro evolution and comparative genomics, we characterized mutations in suppressors of cbk1Δ in C. neoformans that partially rescued defects in CO2 tolerance, thermotolerance, and morphology. One suppressor is the RNA translation repressor Ssd1, which is highly conserved in ascomycetes and basidiomycetes. The other is a novel ribonuclease domain-containing protein, here named PSC1, which is present in basidiomycetes and humans but surprisingly absent in most ascomycetes. Loss of Ssd1 in cbk1Δ partially restored cryptococcal ability to survive and amplify in the inhalation and intravenous murine models of cryptococcosis. Our discoveries highlight the overlapping regulation of CO2 tolerance and thermotolerance, the essential role of the RAM pathway in cryptococcal adaptation to the host condition, and the potential importance of post-transcriptional control of virulence traits in this global pathogen.
Dysregulation of the imprinted H19/IGF2 locus can lead to Silver-Russell syndrome (SRS) in humans. However, the mechanism of how abnormal H19/IGF2 expression contributes to various SRS phenotypes remains unclear, largely due to incomplete understanding of the developmental functions of these two genes. We previously generated a mouse model with humanized H19/IGF2 imprinting control region (hIC1) on the paternal allele that exhibited H19/Igf2 dysregulation together with SRS-like growth restriction and perinatal lethality. Here, we dissect the role of H19 and Igf2 in cardiac and placental development utilizing multiple mouse models with varying levels of H19 and Igf2. We report severe cardiac defects such as ventricular septal defects and thinned myocardium, placental anomalies including thrombosis and vascular malformations, together with growth restriction in mouse embryos that correlated with the extent of H19/Igf2 dysregulation. Transcriptomic analysis using cardiac endothelial cells of these mouse models shows that H19/Igf2 dysregulation disrupts pathways related to extracellular matrix and proliferation of endothelial cells. Our work links the heart and placenta through regulation by H19 and Igf2, demonstrating that accurate dosage of both H19 and Igf2 is critical for normal embryonic development, especially related to the cardiac-placental axis.