Breaking Bad:How Viruses Subvert the Cell Cycle

Interactions between the host and viruses during the course of their co-evolution have not only shaped cellular function and the immune system, but also the counter measures employed by viruses. Relatively small genomes and high replication rates allow viruses to accumulate mutations and continuously present the host with new challenges. It is therefore, no surprise that they either escape detection or modulate host physiology, often by redirecting normal cellular pathways to their own advantage. Viruses utilize a diverse array of strategies and molecular targets to subvert host cellular processes, while evading detection. These include cell-cycle regulation, major histocompatibility complex-restricted antigen presentation, intracellular protein transport, apoptosis, cytokine-mediated signaling, and humoral immune responses. Moreover, viruses routinely manipulate the host cell cycle to create a favorable environment for replication, largely by deregulating cell cycle checkpoints. This review focuses on our current understanding of the molecular aspects of cell cycle regulation that are often targeted by viruses. Further study of their interactions should provide fundamental insights into cell cycle regulation and improve our ability to exploit these viruses

Distinct Flippases Translocate Glycerophospholipids and Oligosaccharide Diphosphate Dolichols across the Endoplasmic Reticulum†

Transbilayer movement, or flip-flop, of lipids across the endoplasmic reticulum (ER) is required for membrane biogenesis, protein glycosylation, and GPI anchoring. Specific ER membrane proteins, flippases, are proposed to facilitate lipid flip-flop, but no ER flippase has been biochemically identified. The glycolipid Glc 3Man 9GlcNAc 2-PP-dolichol is the oligosaccharide donor for protein N-glycosylation reactions in the ER lumen. Synthesis of Glc 3Man 9GlcNAc 2-PP-dolichol is initiated on the cytoplasmic side of the ER and completed on the lumenal side, requiring flipping of the intermediate Man 5GlcNAc 2-PP-dolichol (M5-DLO) across the ER. Here we report the reconstitution of M5-DLO flipping in proteoliposomes generated from Triton X-100-extracted Saccharomyces cerevisiae microsomal proteins. Flipping was assayed by using the lectin Concanavalin A to capture M5-DLOs that had been translocated from the inner to the outer leaflet of the vesicles. M5-DLO flipping in the reconstituted system was ATP-independent and trypsin-sensitive and required a membrane protein(s) that sedimented at ∼4 S. Man 7GlcNAc 2-PP-dolichol, a higher-order lipid intermediate, was flipped >10-fold more slowly than M5-DLO at 25°C. Chromatography on Cibacron Blue dye resin enriched M5-DLO flippase activity ∼5-fold and resolved it from both the ER glycerophospholipid flippase activity and the genetically identified flippase candidate Rft1 [Helenius, J., et al. (2002) Nature 415, 447-450]. The latter result indicates that Rft1 is not the M5-DLO flippase. Our data (i) demonstrate that the ER has at least two distinct flippase proteins, each specifically capable of translocating a class of phospholipid, and (ii) provide, for the first time, a biochemical means of identifying the M5-DLO flippase. © 2008 American Chemical Society.link_to_subscribed_fulltex

Viral subversion of selective autophagy is critical for biogenesis of virus replication organelles

Infection by many (+)RNA viruses is accompanied by ER-expansion and membrane remodelling to form viral replication organelles, followed by assembly and secretion of viral progenies. We previously identified that virus-triggered lipophagy was critical for flaviviral assembly, and is driven by the lipid droplet associated protein Ancient ubiquitin protein 1 (Aup1). A ubiquitin conjugating protein Ube2g2 that functions as a co-factor for Aup1 was identified as a host dependency factor in our study. Here we characterized its function: Ube2g2-deficient cells displayed a dramatic reduction in virus production, which could be rescued by reconstituting the wild-type but not the catalytically deficient (C89K) mutant of Ube2g2, suggesting that its enzymatic activity is necessary. Ube2g2 deficiency did not affect entry of virus particles but resulted in a profound loss in formation of replication organelles, and production of infectious progenies. This phenomenon resulted from its dual activity in (i) triggering lipophagy in conjunction with Aup1, and (ii) degradation of ER chaperones such as Herpud1, SEL1L, Hrd1, along with Sec62 to restrict ER-phagy upon Xbp1-IRE1 triggered ER expansion. Our results therefore underscore an exquisite fine-tuning of selective autophagy by flaviviruses that drive host membrane reorganization during infection to enable biogenesis of viral replication organelles

Type I Interferon Imposes a TSG101/ISG15 Checkpoint at the Golgi for Glycoprotein Trafficking during Influenza Virus Infection

SummarySeveral enveloped viruses exploit host pathways, such as the cellular endosomal sorting complex required for transport (ESCRT) machinery, for their assembly and release. The influenza A virus (IAV) matrix protein binds to the ESCRT-I complex, although the involvement of early ESCRT proteins such as Tsg101 in IAV trafficking remain to be established. We find that Tsg101 can facilitate IAV trafficking, but this is effectively restricted by the interferon (IFN)-stimulated protein ISG15. Cytosol from type I IFN-treated cells abolished IAV hemagglutinin (HA) transport to the cell surface in infected semi-intact cells. This inhibition required Tsg101 and could be relieved with deISGylases. Tsg101 is itself ISGylated in IFN-treated cells. Upon infection, intact Tsg101-deficient cells obtained by CRISPR-Cas9 genome editing were defective in the surface display of HA and for infectious virion release. These data support the IFN-induced generation of a Tsg101- and ISG15-dependent checkpoint in the secretory pathway that compromises influenza virus release

OTUB1 Is a Key Regulator of RIG-I-Dependent Immune Signaling and Is Targeted for Proteasomal Degradation by Influenza A NS1.

Deubiquitylases (DUBs) regulate critical signaling pathways at the intersection of host immunity and viral pathogenesis. Although RIG-I activation is heavily dependent on ubiquitylation, systematic analyses of DUBs that regulate this pathway have not been performed. Using a ubiquitin C-terminal electrophile, we profile DUBs that function during influenza A virus (IAV) infection and isolate OTUB1 as a key regulator of RIG-I-dependent antiviral responses. Upon infection, OTUB1 relocalizes from the nucleus to mitochondrial membranes together with RIG-I, viral PB2, and NS1. Its expression depends on competing effects of interferon stimulation and IAV-triggered degradation. OTUB1 activates RIG-I via a dual mechanism of K48 polyubiquitin hydrolysis and formation of an E2-repressive complex with UBCH5c. We reconstitute this mechanism in a cell-free system comprising [35S]IRF3, purified RIG-I, mitochondrial membranes, and cytosol expressing OTUB1 variants. A range of IAV NS1 proteins trigger proteasomal degradation of OTUB1, antagonizing the RIG-I signaling cascade and antiviral responses

Enzymatic Blockade of the Ubiquitin-Proteasome Pathway

Ubiquitin-dependent processes control much of cellular physiology. We show that expression of a highly active, Epstein-Barr virus-derived deubiquitylating enzyme (EBV-DUB) blocks proteasomal degradation of cytosolic and ER-derived proteins by preemptive removal of ubiquitin from proteasome substrates, a treatment less toxic than the use of proteasome inhibitors. Recognition of misfolded proteins in the ER lumen, their dislocation to the cytosol, and degradation are usually tightly coupled but can be uncoupled by the EBV-DUB: a misfolded glycoprotein that originates in the ER accumulates in association with cytosolic chaperones as a deglycosylated intermediate. Our data underscore the necessity of a DUB activity for completion of the dislocation reaction and provide a new means of inhibition of proteasomal proteolysis with reduced cytotoxicity.National Institutes of Health (U.S.)EMBO (long term Fellowship 2008-379)Boehringer Ingelheim Fond

Perturbation of Intracellular Cholesterol and Fatty Acid Homeostasis During Flavivirus Infections

Cellular lipid homeostasis is maintained through an intricately linked array of anabolic and catabolic pathways. Upon flavivirus infections, these are significantly altered: on the one hand, these viruses can co-opt lipid metabolic pathways to generate ATP to facilitate replication, or to synthesize membrane components to generate replication sites; on the other hand, more recent evidence suggests counter strategies employed by host cells, which actively modulate several of these networks in response to infection, enhancing interferon signaling by doing so, and thus creating an antiviral environment. In this review, we discuss recent data on mechanisms of alteration of lipid metabolic pathways during infection by flaviviruses, with a focus on cholesterol and fatty acid biosynthesis, which can be manipulated by the invading viruses to support replication, but can also be modulated by the host immune system itself, as a means to fight infection

Modulation of Lipid Droplet Metabolism—A Potential Target for Therapeutic Intervention in Flaviviridae Infections

Lipid droplets (LDs) are endoplasmic reticulum (ER)-related dynamic organelles that store and regulate fatty acids and neutral lipids. They play a central role in cellular energy storage, lipid metabolism and cellular homeostasis. It has become evident that viruses have co-evolved in order to exploit host lipid metabolic pathways. This is especially characteristic of the Flaviviridae family, including hepatitis C virus (HCV) and several flaviviruses. Devoid of an appropriate lipid biosynthetic machinery of their own, these single-strand positive-sense RNA viruses can induce dramatic changes in host metabolic pathways to establish a favorable environment for viral multiplication and acquire essential components to facilitate their assembly and traffic. Here we have reviewed the current knowledge on the intracellular life cycle of those from the Flaviviridae family, with particular emphasis on HCV and dengue virus (DENV), and their association with the biosynthesis and metabolism of LDs, with the aim to identify potential antiviral targets for development of novel therapeutic interventions

Cellular Garbage Disposal: Misfolded Proteins in Normal Biology and Human Disease

The endoplasmic reticulum (ER) orchestrates different cellular processes by which proteins are synthesized, correctly folded, modified and ultimately transported to their final destinations. As part of this crucial biosynthetic process, proteins that are not properly folded and consequently detrimental to normal cellular function are constantly generated. A common signature of many neurodegenerative diseases, including Alzheimer's and Parkinson's, is accumulation and deposition of misfolded proteins that arise when the ability of cells to deal with the burden of misfolded proteins is compromised. In this course, we will explore how the ER quality control machinery ensures that only properly assembled proteins exit the ER while distinguishing between nascent proteins en route to their biologically active folded state from those that are terminally misfolded