Vanesa Madan

Viroporins: structure and biological functions

Viroporins are small, hydrophobic proteins that are encoded by a wide range of clinically relevant animal viruses. When these proteins oligomerize in host cell membranes, they form hydrophilic pores that disrupt a number of physiological properties of the cell. Viroporins are crucial for viral pathogenicity owing to their involvement in several diverse steps of the viral life cycle. Thus, these viral proteins, which include influenza A virus matrix protein 2 (M2), HIV-1 viral protein U (Vpu) and hepatitis C virus p7, represent ideal targets for therapeutic intervention, and several compounds that block their pore-forming activity have been identified. Here, we review recent studies in the field that have advanced our knowledge of the structure and function of this expanding family of viral proteins.

Hepatitis C virus RNA replication and assembly: living on the fat of the land.

Hepatitis C virus (HCV) is a major global health burden accounting for around 170 million chronic infections worldwide. Although highly potent direct-acting antiviral drugs to treat chronic hepatitis C have been approved recently, owing to their high costs and limited availability and a large number of undiagnosed infections, the burden of disease is expected to rise in the next few years. In addition, HCV is an excellent paradigm for understanding the tight link between a pathogen and host cell pathways, most notably lipid metabolism. HCV extensively remodels intracellular membranes to establish its cytoplasmic replication factory and also usurps components of the intercellular lipid transport system for production of infectious virus particles. Here, we review the molecular mechanisms of viral replicase function, cellular pathways employed during HCV replication factory biogenesis, and viral, as well as cellular, determinants of progeny virus production.

Cyclosporine A inhibits hepatitis C virus nonstructural protein 2 through cyclophilin A

Numerous anti‐hepatitis C virus (HCV) drugs targeting either the viral nonstructural 3 (NS3) protease or NS5B polymerase are currently in clinical testing. However, rapid resistance development is a major problem and optimal therapy will clearly require a combination of multiple mechanisms of action. Cyclosporine A (CsA) and its nonimmunosuppressant derivatives are among the more promising drugs under development. Based on work with subgenomic HCV replicons it has been thought that they act as NS5B‐inhibitors. In this study we show that CsA inhibits replication of full‐length HCV Japanese Fulminant Hepatitis (JFH1) genomes about 10‐fold more efficiently than subgenomic replicons. This effect is dependent on the presence of NS2 in the viral polyprotein and mediated through cellular cyclophilin A. NS2 is either an additional target for CsA‐dependent inhibition or modulates the antiviral activity against NS3 to NS5B proteins. CsA is thus the first anti‐HCV drug shown to act through NS2. Conclusion: CsA inhibits replication of JFH1 full‐length genomes much more efficiently than subgenomic replicons by targeting cleavage at the NS2/NS3 junction and possibly other nonreplication lifecycle steps. (HEPATOLOGY 2009.)

A Major Determinant of Cyclophilin Dependence and Cyclosporine Susceptibility of Hepatitis C Virus Identified by a Genetic Approach

Since the advent of genome-wide small interfering RNA screening, large numbers of cellular cofactors important for viral infection have been discovered at a rapid pace, but the viral targets and the mechanism of action for many of these cofactors remain undefined. One such cofactor is cyclophilin A (CyPA), upon which hepatitis C virus (HCV) replication critically depends. Here we report a new genetic selection scheme that identified a major viral determinant of HCVs dependence on CyPA and susceptibility to cyclosporine A. We selected mutant viruses that were able to infect CyPA-knockdown cells which were refractory to infection by wild-type HCV produced in cell culture. Five independent selections revealed related mutations in a single dipeptide motif (D316 and Y317) located in a proline-rich region of NS5A domain II, which has been implicated in CyPA binding. Engineering the mutations into wild-type HCV fully recapitulated the CyPA-independent and CsA-resistant phenotype and four putative proline substrates of CyPA were mapped to the vicinity of the DY motif. Circular dichroism analysis of wild-type and mutant NS5A peptides indicated that the D316E/Y317N mutations (DEYN) induced a conformational change at a major CyPA-binding site. Furthermore, nuclear magnetic resonance experiments suggested that NS5A with DEYN mutations adopts a more extended, functional conformation in the putative CyPA substrate site in domain II. Finally, the importance of this major CsA-sensitivity determinant was confirmed in additional genotypes (GT) other than GT 2a. This study describes a new genetic approach to identifying viral targets of cellular cofactors and identifies a major regulator of HCVs susceptibility to CsA and its derivatives that are currently in clinical trials.

Inhibition of HCV replication by cyclophilin antagonists is linked to replication fitness and occurs by inhibition of membranous web formation.

BACKGROUND & AIMS Replication of hepatitis C virus (HCV) requires host cell factors, such as cyclophilin A (CypA). CypA binds to HCVs nonstructural protein (NS)5A to promote replication of viral RNA. CypA antagonists, such as cyclosporines, are potent inhibitors of HCV replication. NS2 modulates sensitivity of HCV to cyclosporines. We investigated why cyclosporines require NS2 to increase their inhibitory effect and how they block HCV replication. METHODS We determined replication fitness and sensitivity of various HCV replicons, containing or lacking NS2, to cyclosporine and other direct-acting antiviral agents. We also analyzed the effects of cyclosporine on membranous web formation by electron microscopy. RESULTS NS2-5B replicons of genotype 2a (JFH1), but not genotype 1b, had increased sensitivity to cyclosporine. This difference was lost with replication-attenuated NS3-5B JFH1 RNAs, showing that cyclosporine sensitivity is linked to reduced replication fitness of NS2-containing HCV RNAs. Fitness also determined sensitivity to a nucleoside analogue and an NS5A inhibitor, but not to telaprevir. Cyclosporine blocked de novo formation of the membranous web, but had little effect on established membranous replication factories. This block was prevented by cyclosporine resistance mutations in NS5A. CONCLUSIONS Cleavage at the NS2/3 junction is a rate-limiting step in replication of particular HCV isolates and determines their sensitivity to CypA inhibitors. These drugs target de novo formation of the membranous web and RNA replication.

Structural and Functional Properties of the Hepatitis C Virus p7 Viroporin

The high prevalence of hepatitis C virus (HCV) infection in the human population has triggered intensive research efforts that have led to the development of curative antiviral therapy. Moreover, HCV has become a role model to study fundamental principles that govern the replication cycle of a positive strand RNA virus. In fact, for most HCV proteins high-resolution X-ray and NMR (Nuclear Magnetic Resonance)-based structures have been established and profound insights into their biochemical and biological properties have been gained. One example is p7, a small hydrophobic protein that is dispensable for RNA replication, but crucial for the production and release of infectious HCV particles from infected cells. Owing to its ability to insert into membranes and assemble into homo-oligomeric complexes that function as minimalistic ion channels, HCV p7 is a member of the viroporin family. This review compiles the most recent findings related to the structure and dual pore/ion channel activity of p7 of different HCV genotypes. The alternative conformations and topologies proposed for HCV p7 in its monomeric and oligomeric state are described and discussed in detail. We also summarize the different roles p7 might play in the HCV replication cycle and highlight both the ion channel/pore-like function and the additional roles of p7 unrelated to its channel activity. Finally, we discuss possibilities to utilize viroporin inhibitors for antagonizing p7 ion channel/pore-like activity.

Coordination of Hepatitis C Virus Assembly by Distinct Regulatory Regions in Nonstructural Protein 5A

Hepatitis C virus (HCV) nonstructural protein (NS)5A is a RNA-binding protein composed of a N-terminal membrane anchor, a structured domain I (DI) and two intrinsically disordered domains (DII and DIII) interacting with viral and cellular proteins. While DI and DII are essential for RNA replication, DIII is required for assembly. How these processes are orchestrated by NS5A is poorly understood. In this study, we identified a highly conserved basic cluster (BC) at the N-terminus of DIII that is critical for particle assembly. We generated BC mutants and compared them with mutants that are blocked at different stages of the assembly process: a NS5A serine cluster (SC) mutant blocked in NS5A-core interaction and a mutant lacking the envelope glycoproteins (ΔE1E2). We found that BC mutations did not affect core-NS5A interaction, but strongly impaired core–RNA association as well as virus particle envelopment. Moreover, BC mutations impaired RNA-NS5A interaction arguing that the BC might be required for loading of core protein with viral RNA. Interestingly, RNA-core interaction was also reduced with the ΔE1E2 mutant, suggesting that nucleocapsid formation and envelopment are coupled. These findings argue for two NS5A DIII determinants regulating assembly at distinct, but closely linked steps: (i) SC-dependent recruitment of replication complexes to core protein and (ii) BC-dependent RNA genome delivery to core protein, triggering encapsidation that is tightly coupled to particle envelopment. These results provide a striking example how a single viral protein exerts multiple functions to coordinate the steps from RNA replication to the assembly of infectious virus particles.

A Proline-Tryptophan Turn in the Intrinsically Disordered Domain 2 of NS5A Protein is Essential for Hepatitis C Virus RNA Replication

Background: The intrinsically disordered domain 2 of NS5A is required for HCV replication. Results: We characterized a short structural motif in the domain 2 of NS5A. Conclusion: This structural motif in NS5A-D2 is essential for RNA replication. Significance: This work provides a molecular basis for further understanding of the function of the intrinsically disordered domain 2 of HCV NS5A protein. Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) and its interaction with the human chaperone cyclophilin A are both targets for highly potent and promising antiviral drugs that are in the late stages of clinical development. Despite its high interest in regards to the development of drugs to counteract the worldwide HCV burden, NS5A is still an enigmatic multifunctional protein poorly characterized at the molecular level. NS5A is required for HCV RNA replication and is involved in viral particle formation and regulation of host pathways. Thus far, no enzymatic activity or precise molecular function has been ascribed to NS5A that is composed of a highly structured domain 1 (D1), as well as two intrinsically disordered domains 2 (D2) and 3 (D3), representing half of the protein. Here, we identify a short structural motif in the disordered NS5A-D2 and report its NMR structure. We show that this structural motif, a minimal Pro314–Trp316 turn, is essential for HCV RNA replication, and its disruption alters the subcellular distribution of NS5A. We demonstrate that this Pro-Trp turn is required for proper interaction with the host cyclophilin A and influences its peptidyl-prolyl cis/trans isomerase activity on residue Pro314 of NS5A-D2. This work provides a molecular basis for further understanding of the function of the intrinsically disordered domain 2 of HCV NS5A protein. In addition, our work highlights how very small structural motifs present in intrinsically disordered proteins can exert a specific function.

Impact of Vesicular Stomatitis Virus M Proteins on Different Cellular Functions.

Three different matrix (M) proteins termed M1, M2 and M3 have been described in cells infected with vesicular stomatitis virus (VSV). Individual expression of VSV M proteins induces an evident cytopathic effect including cell rounding and detachment, in addition to a partial inhibition of cellular protein synthesis, likely mediated by an indirect mechanism. Analogous to viroporins, M1 promotes the budding of new virus particles; however, this process does not produce an increase in plasma membrane permeability. In contrast to M1, M2 and M3 neither interact with the cellular membrane nor promote the budding of double membrane vesicles at the cell surface. Nonetheless, all three species of M protein interfere with the transport of cellular mRNAs from the nucleus to the cytoplasm and also modulate the redistribution of the splicing factor. The present findings indicate that all three VSV M proteins share some activities that interfere with host cell functions.

Glycine-zipper motifs in hepatitis C virus nonstructural protein 4B are required for the establishment of viral replication organelles

ABSTRACT Hepatitis C virus (HCV) RNA replication occurs in tight association with remodeled host cell membranes, presenting as cytoplasmic accumulations of single-, double-, and multimembrane vesicles in infected cells. Formation of these so-called replication organelles is mediated by a complex interplay of host cell factors and viral replicase proteins. Of these, nonstructural protein 4B (NS4B), an integral transmembrane protein, appears to play a key role, but little is known about the molecular mechanisms of how this protein contributes to organelle biogenesis. Using forward and reverse genetics, we identified glycine zipper motifs within transmembrane helices 2 and 3 of NS4B that are critically involved in viral RNA replication. Foerster resonance energy transfer analysis revealed the importance of the glycine zippers in NS4B homo- and heterotypic self-interactions. Additionally, ultrastructural analysis using electron microscopy unraveled a prominent role of glycine zipper residues for the subcellular distribution and the morphology of HCV-induced double-membrane vesicles. Notably, loss-of-function NS4B glycine zipper mutants prominently induced single-membrane vesicles with secondary invaginations that might represent an arrested intermediate state in double-membrane vesicle formation. These findings highlight a so-far-unknown role of glycine residues within the membrane integral core domain for NS4B self-interaction and functional as well as structural integrity of HCV replication organelles. IMPORTANCE Remodeling of the cellular endomembrane system leading to the establishment of replication organelles is a hallmark of positive-strand RNA viruses. In the case of HCV, expression of the nonstructural proteins induces the accumulation of double-membrane vesicles that likely arise from a concerted action of viral and coopted cellular factors. However, the underlying molecular mechanisms are incompletely understood. Here, we identify glycine zipper motifs within HCV NS4B transmembrane segments 2 and 3 that are crucial for the proteins self-interaction. Moreover, glycine residues within NS4B transmembrane helices critically contribute to the biogenesis of functional replication organelles and, thus, efficient viral RNA replication. These results reveal how glycine zipper motifs in NS4B contribute to structural and functional integrity of the HCV replication organelles and, thus, viral RNA replication.