Phosphoinositides (PIs) make up only a small fraction of cellular phospholipids, yet they control almost all aspects of a cell's life and death. These lipids gained tremendous research interest as plasma membrane signaling molecules when discovered in the 1970s and 1980s. Research in the last 15 years has added a wide range of biological processes regulated by PIs, turning these lipids into one of the most universal signaling entities in eukaryotic cells. PIs control organelle biology by regulating vesicular trafficking, but they also modulate lipid distribution and metabolism via their close relationship with lipid transfer proteins. PIs regulate ion channels, pumps, and transporters and control both endocytic and exocytic processes. The nuclear phosphoinositides have grown from being an epiphenomenon to a research area of its own. As expected from such pleiotropic regulators, derangements of phosphoinositide metabolism are responsible for a number of human diseases ranging from rare genetic disorders to the most common ones such as cancer, obesity, and diabetes. Moreover, it is increasingly evident that a number of infectious agents hijack the PI regulatory systems of host cells for their intracellular movements, replication, and assembly. As a result, PI converting enzymes began to be noticed by pharmaceutical companies as potential therapeutic targets. This review is an attempt to give an overview of this enormous research field focusing on major developments in diverse areas of basic science linked to cellular physiology and disease.

Phosphoinositides: Tiny Lipids With Giant Impact on Cell Regulation

More than two decades of intense research has provided a detailed understanding of hepatitis C virus (HCV), which chronically infects 2% of the world's population. This effort has paved the way for the development of antiviral compounds to spare patients from life-threatening liver disease. An exciting new era in HCV therapy dawned with the recent approval of two viral protease inhibitors, used in combination with pegylated interferon-α and ribavirin; however, this is just the beginning. Multiple classes of antivirals with distinct targets promise highly efficient combinations, and interferon-free regimens with short treatment duration and fewer side effects are the future of HCV therapy. Ongoing and future trials will determine the best antiviral combinations and whether the current seemingly rich pipeline is sufficient for successful treatment of all patients in the face of major challenges, such as HCV diversity, viral resistance, the influence of host genetics, advanced liver disease and other co-morbidities.

Understanding the hepatitis C virus life cycle paves the way for highly effective therapies

Hepatitis C virus (HCV) is a highly variable plus-strand RNA virus of the family Flaviviridae . Viral strains are grouped into six epidemiologically relevant genotypes that differ fr

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The HCV Life Cycle: In vitro Tissue Culture Systems and Therapeutic Targets

The availability of the first molecular clone of the hepatitis C virus (HCV) genome allowed the identification and biochemical characterization of two viral enzymes that are targets for antiviral therapy: the protease NS3-4A and the RNA-dependent RNA polymerase NS5B. With the advent of cell culture systems that can recapitulate either the intracellular steps of the viral replication cycle or the complete cycle, additional drug targets have been identified, most notably the phosphoprotein NS5A, but also host cell factors that promote viral replication, such as cyclophilin A. Here, we review insights into the structures of these proteins and the mechanisms by which they contribute to the HCV replication cycle, and discuss how these insights have facilitated the development of new, directly acting antiviral compounds that have started to enter the clinic.

The molecular and structural basis of advanced antiviral therapy for hepatitis C virus infection

Plus-strand RNA virus replication occurs in tight association with cytoplasmic host cell membranes. Both, viral and cellular factors cooperatively generate distinct organelle-like structures, designated viral replication factories. This compartmentalization allows coordination of the different steps of the viral replication cycle, highly efficient genome replication and protection of the viral RNA from cellular defense mechanisms. Electron tomography studies conducted during the last couple of years revealed the three dimensional structure of numerous plus-strand RNA virus replication compartments and highlight morphological analogies between different virus families. Based on the morphology of virus-induced membrane rearrangements, we propose two separate subclasses: the invaginated vesicle/spherule type and the double membrane vesicle type. This review discusses common themes and distinct differences in the architecture of plus-strand RNA virus-induced membrane alterations and summarizes recent progress that has been made in understanding the complex interplay between viral and co-opted cellular factors in biogenesis and maintenance of plus-strand RNA virus replication factories.

Architecture and biogenesis of plus-strand RNA virus replication factories

4-anilino quinazolines have been identified as inhibitors of HCV replication. The target of this class of compounds was proposed to be the viral protein NS5A, although unequivocal proof has never been presented. A 4-anilino quinazoline moiety is often found in kinase inhibitors, leading us to formulate the hypothesis that the anti-HCV activity displayed by these compounds might be due to inhibition of a cellular kinase. Type III phosphatidylinositol 4-kinase α (PI4KIIIα) has recently been identified as a host factor for HCV replication. We therefore evaluated AL-9, a compound prototypical of the 4-anilino quinazoline class, on selected phosphatidylinositol kinases. AL-9 inhibited purified PI4KIIIα and, to a lesser extent, PI4KIIIβ. In Huh7.5 cells, PI4KIIIα is responsible for the phosphatidylinositol-4 phosphate (PI4P) pool present in the plasma membrane. Accordingly, we observed a gradual decrease of PI4P in the plasma membrane upon incubation with AL-9, indicating that this agent inhibits PI4KIIIα also in living cells. Conversely, AL-9 did not affect the level of PI4P in the Golgi membrane, suggesting that the PI4KIIIβ isoform was not significantly inhibited under our experimental conditions. Incubation of cells expressing HCV proteins with AL-9 induced abnormally large clusters of NS5A, a phenomenon previously observed upon silencing PI4KIIIα by RNA interference. In light of our findings, we propose that the antiviral effect of 4-anilino quinazoline compounds is mediated by the inhibition of PI4KIIIα and the consequent depletion of PI4P required for the HCV membranous web. In addition, we noted that HCV has a profound effect on cellular PI4P distribution, causing significant enrichment of PI4P in the HCV-membranous web and a concomitant depletion of PI4P in the plasma membrane. This observation implies that HCV – by recruiting PI4KIIIα in the RNA replication complex – hijacks PI4P metabolism, ultimately resulting in a markedly altered subcellular distribution of the PI4KIIIα product.

Reinaldo Alvarez

Papers

Metabolism of Phosphatidylinositol 4-Kinase IIIα-Dependent PI4P Is Subverted by HCV and Is Targeted by a 4-Anilino Quinazoline with Antiviral Activity