Showing papers by "Karla Kirkegaard published in 2018"

The exoribonuclease Xrn1 is a post-transcriptional negative regulator of autophagy

Abstract: Macroautophagy/autophagy is a conserved catabolic process that promotes survival during stress. Autophagic dysfunction is associated with pathologies such as cancer and neurodegenerative diseases. Thus, autophagy must be strictly modulated at multiple levels (transcriptional, post-transcriptional, translational and post-translational) to prevent deregulation. Relatively little is known about the post-transcriptional control of autophagy. Here we report that the exoribonuclease Xrn1/XRN1 functions as a negative autophagy factor in the yeast Saccharomyces cerevisiae and in mammalian cells. In yeast, chromosomal deletion of XRN1 enhances autophagy and the frequency of autophagosome formation. Loss of Xrn1 results in the upregulation of autophagy-related (ATG) transcripts under nutrient-replete conditions, and this effect is dependent on the ribonuclease activity of Xrn1. Xrn1 expression is regulated by the yeast transcription factor Ash1 in rich conditions. In mammalian cells, siRNA depletion of XRN1 enhances autophagy and the replication of 2 picornaviruses. This work provides insight into the role of the RNA decay factor Xrn1/XRN1 as a post-transcriptional regulator of autophagy.

Targeting intramolecular proteinase NS2B/3 cleavages for trans-dominant inhibition of dengue virus

Abstract: Many positive-strand RNA viruses translate their genomes as single polyproteins that are processed by host and viral proteinases to generate all viral protein products. Among these is dengue virus, which encodes the serine proteinase NS2B/3 responsible for seven different cleavages in the polyprotein. NS2B/3 has been the subject of many directed screens to find chemical inhibitors, of which the compound ARDP0006 is among the most effective at inhibiting viral growth. We show that at least three cleavages in the dengue polyprotein are exclusively intramolecular. By definition, such a cis-acting defect cannot be rescued in trans. This creates the possibility that a drug-susceptible or inhibited proteinase can be genetically dominant, inhibiting the outgrowth of drug-resistant virus via precursor accumulation. Indeed, an NS3-G459L variant that is incapable of cleavage at the internal NS3 junction dominantly inhibited negative-strand RNA synthesis of wild-type virus present in the same cell. This internal NS3 cleavage site is the junction most inhibited by ARDP0006, making it likely that the accumulation of toxic precursors, not inhibition of proteolytic activity per se, explains the antiviral efficacy of this compound in restraining viral growth. We argue that intramolecularly cleaving proteinases are promising drug targets for viruses that encode polyproteins. The most effective inhibitors will specifically target cleavage sites required for processing precursors that exert trans-dominant inhibition.

Detection and Differentiation of Multiple Viral RNAs Using Branched DNA FISH Coupled to Confocal Microscopy and Flow Cytometry.

Abstract: Due to the exceptionally high mutation rates of RNA-dependent RNA polymerases, infectious RNA viruses generate extensive sequence diversity, leading to some of the lowest barriers to the development of antiviral drug resistance in the microbial world. We have previously discovered that higher barriers to the development of drug resistance can be achieved through dominant suppression of drug-resistant viruses by their drug-susceptible parents. We have explored the existence of dominant drug targets in poliovirus, dengue virus and hepatitis C virus (HCV). The low replication capacity of HCV required the development of novel strategies for identifying cells co-infected with drug-susceptible and drug-resistant strains. To monitor co-infected cell populations, we generated codon-altered versions of the JFH1 strain of HCV. Then, we could differentiate the codon-altered and wild-type strains using a novel type of RNA fluorescent in situ hybridization (FISH) coupled with flow cytometry or confocal microscopy. Both of these techniques can be used in conjunction with standard antibody-protein detection methods. Here, we describe a detailed protocol for both RNA FISH flow cytometry and confocal microscopy.

Transmission genetics of drug-resistant hepatitis C virus

Abstract: Viruses are simple organisms that consist of genetic information and a few types of proteins. They cannot replicate on their own, and instead hijack the molecular machinery of a host cell to produce more of themselves. Inside an infected cell, the genetic information of the virus is replicated and ‘read’ to create viral proteins. These components are then assembled to form a new generation of viruses. During this process, genetic errors may occur that lead to modifications in the viral proteins, and help the virus become resistant to treatment. For instance, a viral protein that used to be targeted by a drug can change slightly and not be recognized anymore. Currently, the most efficient way to fight drug resistance is to use combination therapy, where several drugs are given at the same time. This strategy is successful, for example to treat infections with the hepatitis C virus, but it is also expensive, especially for developing countries. An alternative approach is dominant-drug targeting, which exploits the fact that both drug-resistant and drug-susceptible viruses are ‘born’ in the same cell. There, the susceptible viruses can overwhelm and ‘mask’ the benefits of the resistant ones. For example, proteins from resistant strains, which are no longer detected by a treatment, can bind to proteins from susceptible viruses; drugs will still be able to recognize these resulting viral structures. The proteins that operate in such ways are potential dominant-drug targets. However, resistant and susceptible strains can also cohabit without any contacts if their proteins do not interact with each other. Now, van Buuren et al. screen several viral proteins, including one called NS5A, to test whether a dominant drug target exists for the hepatitis C virus. Only a few molecules of a drug that targets NS5A can stop the virus from growing. In theory, drug-bound NS5A proteins could block their non-drug-bound neighbors, but when these drugs have been used on their own, resistance quickly emerged. Experiments showed that NS5A is not a dominant drug target because the drug-resistant and drug-susceptible proteins do not mix. Unless ‘forced’ in the laboratory, NS5A proteins only bind to the ones produced by the same strain of virus. This explains why resistant viruses quickly take over when NS5A drugs are the sole treatment. However, other hepatitis C proteins, such as the HCV core protein, are known to mix during the assembly of the virus, and thus are likely be dominant drug targets.