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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.

Many viruses interface with the autophagy pathway, a highly conserved process for recycling cellular components. For three viral infections in which autophagy constituents are proviral (poliovirus, dengue, and Zika), we developed a panel of knockouts (KOs) of autophagy-related genes to test which components of the canonical pathway are utilized. We discovered that each virus uses a distinct set of initiation components; however, all three viruses utilize autophagy-related gene 9 (ATG9), a lipid scavenging protein, and LC3 (light-chain 3), which is involved in membrane curvature. These results show that viruses use noncanonical routes for membrane sculpting and LC3 recruitment. By measuring viral RNA abundance, we also found that poliovirus utilizes these autophagy components for intracellular growth, while dengue and Zika virus only use autophagy components for post-RNA replication processes. Comparing how RNA viruses manipulate the autophagy pathway reveals new noncanonical autophagy routes, explains the exacerbation of disease by starvation, and uncovers common targets for antiviral drugs., Author summary Viruses often co-opt host cellular processes to replicate their genomes and spread to other cells. Many of these cellular pathways provide good targets for antiviral drugs, as they are less likely to develop resistance since they are encoded in the host and not the fast-evolving viral genome. The autophagy pathway is an important stress response pathway that allows cells to recycle cellular components for energy conservation by sequestering cytoplasmic molecules and organelles in double-membraned vesicles (DMVs) and by degrading the contents into reusable elements. Many RNA viruses induce this pathway to provide membrane surfaces for replication and as a source of vesicles for maturation and exit from cells. We developed a panel of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) knockout (KO) human cells lacking individual components of the autophagy pathway to assess what aspects of the pathway diverse RNA viruses utilized. We discovered that poliovirus, dengue virus, and Zika virus all use different initiation components of the autophagy pathway but similar downstream components. Additionally, we found that poliovirus uses autophagy components for genome replication, while dengue and Zika viruses use autophagy components for postreplication processes. Ultimately, we uncovered potential drug targets for multiple RNA viruses.

© 2015 Elsevier Inc. Viral replication significantly alters the gene expression landscape of infected cells. Many of these changes are driven by viral manipulation of host transcription or translation machinery. Several mammalian viruses encode factors that broadly dampen gene expression by directly targeting messenger RNA (mRNA). Here, we highlight how these factors promote mRNA degradation to globally regulate both host and viral gene expression. Although these viral factors are not homologous and use distinct mechanisms to target mRNA, many of them display striking parallels in their strategies for executing RNA degradation and invoke key features of cellular RNA quality control pathways. In some cases, there is a lack of selectivity for degradation of host versus viral mRNA, indicating that the purposes of virus-induced mRNA degradation extend beyond redirecting cellular resources towards viral gene expression. In addition, several antiviral pathways use RNA degradation as a viral restriction mechanism, and we will summarize new findings related to how these host-encoded ribonucleases target and destroy viral RNA.

© 2015 Elsevier Inc. Gamma-herpesviruses encode a cytoplasmic mRNA-targeting endonuclease, SOX, that cleaves most cellular mRNAs. Cleaved fragments are subsequently degraded by the cellular 5′-3′ mRNA exonuclease Xrn1, thereby suppressing cellular gene expression and facilitating viral evasion of host defenses. We reveal that mammalian cells respond to this widespread cytoplasmic mRNA decay by altering RNA Polymerase II (RNAPII) transcription in the nucleus. Measuring RNAPII recruitment to promoters and nascent mRNA synthesis revealed that the majority of affected genes are transcriptionally repressed in SOX-expressing cells. The transcriptional feedback does not occur in response to the initial viral endonuclease-induced cleavage, but instead to degradation of the cleaved fragments by cellular exonucleases. In particular, Xrn1 catalytic activity is required for transcriptional repression. Notably, viral mRNA transcription escapes decay-induced repression, and this escape requires Xrn1. Collectively, these results indicate that mRNA decay rates impact transcription and that gamma-herpesviruses use this feedback mechanism to facilitate viral gene expression.

Lytic gammaherpesvirus infection restricts host gene expression by promoting widespread degradation of cytoplasmic mRNA through the activity of the viral endonuclease SOX. Though generally assumed to be selective for cellular transcripts, the extent to which SOX impacts viral mRNA stability has remained unknown. We addressed this issue using the model murine gammaherpesvirus MHV68 and, unexpectedly, found that all stages of viral gene expression are controlled through mRNA degradation. Using both comprehensive RNA expression profiling and half-life studies we reveal that the levels of the majority of viral mRNAs but not noncoding RNAs are tempered by MHV68 SOX (muSOX) activity. The targeting of viral mRNA by muSOX is functionally significant, as it impacts intracellular viral protein abundance and progeny virion composition. In the absence of muSOX-imposed gene expression control the viral particles display increased cell surface binding and entry as well as enhanced immediate early gene expression. These phenotypes culminate in a viral replication defect in multiple cell types as well as in vivo, highlighting the importance of maintaining the appropriate balance of viral RNA during gammaherpesviral infection. This is the first example of a virus that fails to broadly discriminate between cellular and viral transcripts during host shutoff and instead uses the targeting of viral messages to fine-tune overall gene expression., Author Summary Many viruses restrict host gene expression during infection, presumably to provide a competitive expression advantage to viral transcripts. Not surprisingly, viruses that induce this ‘host shutoff’ phenotype therefore generally possess mechanisms to selectively spare viral genes. Gammaherpesviruses promote host shutoff by inducing widespread mRNA degradation, a process initiated by the viral SOX nuclease. However, the effect of SOX on viral mRNA during infection was unknown. Here, we reveal that during infection with the murine gammaherpesvirus MHV68, the majority of viral transcripts of all kinetic classes are broadly down regulated through the activity of the MHV68 SOX protein (muSOX). We further demonstrate that in the absence of muSOX-induced control of viral mRNA abundance, viral protein levels increase, thereby affecting the composition of progeny viral particles. Altered virion composition directly impacts early events such as entry and induction of lytic gene expression in subsequent rounds of replication. Furthermore, decreasing both virus and host gene expression via global mRNA degradation is critical for viral replication in a cell type specific manner both in vitro and in vivo. This is the first example of a eukaryotic virus whose host shutoff mechanism similarly tempers viral gene expression, and highlights the degree to which gammaherpesviral gene expression must be fine tuned to ensure replicative success.

Glaucoma, the most frequent optic neuropathy, is a leading cause of blindness worldwide. Death of retinal ganglion cells (RGCs) occurs in all forms of glaucoma and accounts for the loss of vision, however the molecular mechanisms that cause RGC loss remain unclear. The pro-apoptotic molecule, Fas ligand, is a transmembrane protein that can be cleaved from the cell surface by metalloproteinases to release a soluble protein with antagonistic activity. Previous studies documented that constitutive ocular expression of FasL maintained immune privilege and prevented neoangeogenesis. We now show that FasL also plays a major role in retinal neurotoxicity. Importantly, in both TNFα triggered RGC death and a spontaneous model of glaucoma, gene-targeted mice that express only full-length FasL exhibit accelerated RGC death. By contrast, FasL-deficiency, or administration of soluble FasL, protected RGCs from cell death. These data identify membrane-bound FasL as a critical effector molecule and potential therapeutic target in glaucoma.

Short interspersed nuclear elements (SINEs) are highly abundant, RNA polymerase III-transcribed noncoding retrotransposons that are silenced in somatic cells but activated during certain stresses including viral infection. How these induced SINE RNAs impact the host-pathogen interaction is unknown. Here we reveal that during murine gammaherpesvirus 68 (MHV68) infection, rapidly induced SINE RNAs activate the antiviral NF-κB signaling pathway through both mitochondrial antiviral-signaling protein (MAVS)-dependent and independent mechanisms. However, SINE RNA-based signaling is hijacked by the virus to enhance viral gene expression and replication. B2 RNA expression stimulates IKKβ-dependent phosphorylation of the major viral lytic cycle transactivator protein RTA, thereby enhancing its activity and increasing progeny virion production. Collectively, these findings suggest that SINE RNAs participate in the innate pathogen response mechanism, but that herpesviruses have evolved to co-opt retrotransposon activation for viral benefit., Author Summary Short interspersed nuclear elements (SINEs) are noncoding mobile genetic elements that are present at ~106 copies per mammalian genome, roughly comprising 10% of mammalian genomic real estate. SINEs are typically transcriptionally silenced, though in some cases viral infection can promote their expression, yet to an unknown functional outcome. Thus, SINE elements represent the largest class of infection-inducible noncoding RNAs that are functionally uncharacterized. Here, we reveal that SINE RNAs play a critical role in the host-pathogen interaction in that they are required for efficient murine gammaherpesvirus 68 (MHV68) replication and gene expression. We demonstrate that SINE RNAs, both exogenously expressed and infection-induced, are robust activators of the IKKβ kinase, a key signaling molecule in the innate immune response. Activation of the IKKβ kinase by SINE RNA is mediated through both MAVS-dependent and independent mechanisms. Moreover, we demonstrate the activation of the IKKβ via SINE RNA is required to drive the phosphorylation of MHV68 RTA, the main viral transcriptional activator, which enhances its transcriptional activating property. Collectively, we reveal the first example of a role for SINE RNAs in the host-pathogen interaction and identify them as a key immune signaling molecule early during infection. Though SINE RNAs activate the innate immune response, MHV68 has co-opted SINE-mediate innate immune activation to enhance the viral lifecycle.

Primary open angle glaucoma (POAG) is a leading cause of blindness worldwide. The molecular signaling involved in the pathogenesis of POAG remains unknown. Here, we report that mice lacking the α1 subunit of the nitric oxide receptor soluble guanylate cyclase represent a novel and translatable animal model of POAG, characterized by thinning of the retinal nerve fiber layer and loss of optic nerve axons in the context of an open iridocorneal angle. The optic neuropathy associated with soluble guanylate cyclase α1-deficiency was accompanied by modestly increased intraocular pressure and retinal vascular dysfunction. Moreover, data from a candidate gene association study suggests that a variant in the locus containing the genes encoding for the α1 and β1 subunits of soluble guanylate cyclase is associated with POAG in patients presenting with initial paracentral vision loss, a disease subtype thought to be associated with vascular dysregulation. These findings provide new insights into the pathogenesis and genetics of POAG and suggest new therapeutic strategies for POAG.

Background Primary open angle glaucoma (POAG) is a progressive eye disease that leads to blindness due to the irreversible loss of retinal ganglion cells and degeneration of the optic nerve (ON). As of yet, there is no cure for glaucoma. Although available therapies delay disease progression, they offer incomplete protection. Elevated intraocular pressure (IOP) is an important risk factor for POAG. However, the exact molecular mechanisms that trigger increased IOP and glaucomatous optic neuropathy remain incompletely understood. While a few spontaneous murine models of glaucoma exist, none are models of POAG, the most prevalent form of glaucoma. Impaired nitric oxide (NO) signaling has been implicated in the development of glaucoma. To further test the hypothesis that impaired NO/cGMP signaling contributes to the pathogenesis of POAG, we tested whether mice lacking the a1 subunit of the NO receptor soluble guanylate cyclase (sGCa1 -/mice) develop POAG.