Regulation of hepatitis C virus replication via threonine phosphorylation of the NS5A protein

The hepatitis C virus non-structural 5A (NS5A) protein is highly phosphorylated and plays roles in both virus genome replication and assembly of infectious virus particles. NS5A comprises three domains separated by low complexity sequences (LCS). Mass spectrometry analysis of NS5A revealed the existence of a singly phosphorylated tryptic peptide corresponding to the end of LCS I and the beginning of domain II that contained a number of potential phosphorylatable residues (serines and threonines). Here we use a mutagenic approach to investigate the potential role of three of these threonine residues. Phosphomimetic mutations of two of these (T242E and T244E) resulted in significant reductions in virus genome replication and the production of infectious virus, suggesting that the phosphorylation of these residues negatively regulated virus RNA synthesis. Mutation of T245 had no effect, however when T245E was combined with the other two phosphomimetic mutations (TripleE) the inhibitory effect on replication was less pronounced. Effects of the mutations on the ratio of basally/hyperphosphorylated NS5A, together with the apparent molecular weight of the basally phosphorylated species were also observed. Lastly, two of the mutations (T245A and TripleE) resulted in a perinuclear restricted localization of NS5A. These data add further complexity to NS5A phosphorylation and suggest that this analysis be extended outwith the serine-rich cluster within LCS I

The Hepatitis C Virus Nonstructural Protein 2 (NS2): An Up-and-Coming Antiviral Drug Target

Infection with Hepatitis C Virus (HCV) continues to be a major global health problem. To overcome the limitations of current therapies using interferon-α in combination with ribavirin, there is a need to develop drugs that specifically block viral proteins. Highly efficient protease and polymerase inhibitors are currently undergoing clinical testing and will become available in the next few years. However, with resistance mutations emerging quickly, additional enzymatic activities or functions of HCV have to be targeted by novel compounds. One candidate molecule is the nonstructural protein 2 (NS2), which contains a proteolytic activity that is essential for viral RNA replication. In addition, NS2 is crucial for the assembly of progeny virions and modulates various cellular processes that interfere with viral replication. This review describes the functions of NS2 in the life cycle of HCV and highlights potential antiviral strategies involving NS2

Indirect interactions of membrane-adsorbed cylinders

Biological and biomimetic membranes often contain aggregates of embedded or adsorbed macromolecules. In this article, the indirect interactions of cylindrical objects adhering to a planar membrane are considered theoretically. The adhesion of the cylinders causes a local perturbation of the equilibrium membrane shape, which leads to membrane-mediated interactions. For a planar membrane under lateral tension, the interaction is repulsive for a pair of cylinders adhering to the same side of the membrane, and attractive for cylinders adhering at opposite membrane sides. For a membrane in an external harmonic potential, the interaction of adsorbed cylinders is always attractive and increases if forces perpendicular to the membrane act on the cylinders.Comment: 9 pages, 8 figures; typos correcte

Population balances in case of crossing characteristic curves: Application to T-cells immune response

The progression of a cell population where each individual is characterized by the value of an internal variable varying with time (e.g. size, weight, and protein concentration) is typically modeled by a Population Balance Equation, a first order linear hyperbolic partial differential equation. The characteristics described by internal variables usually vary monotonically with the passage of time. A particular difficulty appears when the characteristic curves exhibit different slopes from each other and therefore cross each other at certain times. In particular such crossing phenomenon occurs during T-cells immune response when the concentrations of protein expressions depend upon each other and also when some global protein (e.g. Interleukin signals) is also involved which is shared by all T-cells. At these crossing points, the linear advection equation is not possible by using the classical way of hyperbolic conservation laws. Therefore, a new Transport Method is introduced in this article which allowed us to find the population density function for such processes. The newly developed Transport method (TM) is shown to work in the case of crossing and to provide a smooth solution at the crossing points in contrast to the classical PDF techniques.Comment: 18 pages, 10 figure

Adhesion-induced phase separation of multiple species of membrane junctions

A theory is presented for the membrane junction separation induced by the adhesion between two biomimetic membranes that contain two different types of anchored junctions (receptor/ligand complexes). The analysis shows that several mechanisms contribute to the membrane junction separation. These mechanisms include (i) the height difference between type-1 and type-2 junctions is the main factor which drives the junction separation, (ii) when type-1 and type-2 junctions have different rigidities against stretch and compression, the ``softer'' junctions are the ``favored'' species, and the aggregation of the softer junction can occur, (iii) the elasticity of the membranes mediates a non-local interaction between the junctions, (iv) the thermally activated shape fluctuations of the membranes also contribute to the junction separation by inducing another non-local interaction between the junctions and renormalizing the binding energy of the junctions. The combined effect of these mechanisms is that when junction separation occurs, the system separates into two domains with different relative and total junction densities.Comment: 23 pages, 6 figure

Binding cooperativity of membrane adhesion receptors

The adhesion of cells is mediated by receptors and ligands anchored in apposing membranes. A central question is how to characterize the binding affinity of these membrane-anchored molecules. For soluble molecules, the binding affinity is typically quantified by the binding equilibrium constant K3D in the linear relation [RL] = K3D [R][L] between the volume concentration [RL] of bound complexes and the volume concentrations [R] and [L] of unbound molecules. For membrane-anchored molecules, it is often assumed by analogy that the area concentration of bound complexes [RL] is proportional to the product [R][L] of the area concentrations for the unbound receptor and ligand molecules. We show here (i) that this analogy is only valid for two planar membranes immobilized on rigid surfaces, and (ii) that the thermal roughness of flexible membranes leads to cooperative binding of receptors and ligands. In the case of flexible membranes, the area concentration [RL] of receptor-ligand bonds is proportional to the square of [R][L] for typical lengths and concentrations of receptors and ligands in cell adhesion zones. The cooperative binding helps to understand why different experimental methods for measuring the binding affinity of membrane-anchored molecules have led to values differing by several orders of magnitude.Comment: 9 pages, 4 figures; to appear in Soft Matte

Differential segregation in a cell-cell contact interface: the dynamics of the immunological synapse

Receptor-ligand couples in the cell-cell contact interface between a T cell and an antigen-presenting cell form distinct geometric patterns and undergo spatial rearrangement within the contact interface. Spatial segregation of the antigen and adhesion receptors occurs within seconds of contact, central aggregation of the antigen receptor then occurring over 1-5 min. This structure, called the immunological synapse, is becoming a paradigm for localized signaling. However, the mechanisms driving its formation, in particular spatial segregation, are currently not understood. With a reaction diffusion model incorporating thermodynamics, elasticity, and reaction kinetics, we examine the hypothesis that differing bond lengths (extracellular domain size) is the driving force behind molecular segregation. We derive two key conditions necessary for segregation: a thermodynamic criterion on the effective bond elasticity and a requirement for the seeding/nucleation of domains. Domains have a minimum length scale and will only spontaneously coalesce/aggregate if the contact area is small or the membrane relaxation distance large. Otherwise, differential attachment of receptors to the cytoskeleton is required for central aggregation. Our analysis indicates that differential bond lengths have a significant effect on synapse dynamics, i.e., there is a significant contribution to the free energy of the interaction, suggesting that segregation by differential bond length is important in cell-cell contact interfaces and the immunological synapse

Mechanisms of pattern formation during T cell adhesion

T cells form intriguing patterns during adhesion to antigen-presenting cells. The patterns at the cell-cell contact zone are composed of two types of domains, which either contain short TCR/MHCp receptor-ligand complexes or the longer LFA-1/ICAM-1 complexes. The final pattern consists of a central TCR/MHCp domain surrounded by a ring-shaped LFA-1/ICAM-1 domain, while the characteristic pattern formed at intermediate times is inverted with TCR/MHCp complexes at the periphery of the contact zone and LFA-1/ICAM-1 complexes in the center. In this article, we present a statistical-mechanical model of cell adhesion and propose a novel mechanism for the T cell pattern formation. Our mechanism for the formation of the intermediate inverted pattern is based (i) on the initial nucleation of numerous TCR/MHCp microdomains, and (ii) on the diffusion of free receptors and ligands into the contact zone. Due to this inward diffusion, TCR/MHCp microdomains at the rim of the contact zone grow faster and form an intermediate peripheral ring for sufficiently large TCR/MHCp concentrations. In agreement with experiments, we find that the formation of the final pattern with a central TCR/MHCp domain requires active cytoskeletal transport processes. Without active transport, the intermediate inverted pattern seems to be metastable in our model, which might explain patterns observed during natural killer (NK) cell adhesion. At smaller TCR/MHCp complex concentrations, we observe a different regime of pattern formation with intermediate multifocal TCR/MHCp patterns which resemble experimental patterns found during thymozyte adhesion.Comment: 12 pages, 8 figure

Memory CD8+ T Cells Are Required for Protection from Persistent Hepatitis C Virus Infection

Few hepatitis C virus (HCV) infections resolve spontaneously but those that do appear to afford protective immunity. Second infections are usually shorter in duration and are less likely to persist but mechanisms of virus control in immune individuals have not been identified. In this study we investigated whether memory helper and/or cytotoxic T lymphocytes provide protection in chimpanzees serially reinfected with the virus. Clearance of the first infection took 3–4 mo and coincided with the delayed onset of CD4+ and CD8+ T cell responses. High frequencies of memory T cells targeting multiple HCV proteins were stable over 7 yr of follow-up. Animals were infected for a second time to assess the protective role of memory T cells. In contrast to the prolonged course of the first infection, viremia was terminated within 14 d. Control of this second infection was kinetically linked to rapid acquisition of virus-specific cytolytic activity by liver resident CD8+ T cells and expansion of memory CD4+ and CD8+ T cells in blood. The importance of memory CD8+ T cells in control of HCV infection was confirmed by antibody-mediated depletion of this lymphocyte subset before a third infection. Virus replication was prolonged despite the presence of memory CD4+ T helper cells primed by the two prior infections and was not terminated until HCV-specific CD8+ T cells recovered in the liver. These experiments demonstrate an essential role for memory CD8+ T cells in long-term protection from chronic hepatitis C

T-Cell activation: a queuing theory analysis at low agonist density

We analyze a simple linear triggering model of the T-cell receptor (TCR) within the framework of queuing theory, in which TCRs enter the queue upon full activation and exit by downregulation. We fit our model to four experimentally characterized threshold activation criteria and analyze their specificity and sensitivity: the initial calcium spike, cytotoxicity, immunological synapse formation, and cytokine secretion. Specificity characteristics improve as the time window for detection increases, saturating for time periods on the timescale of downregulation; thus, the calcium spike (30 s) has low specificity but a sensitivity to single-peptide MHC ligands, while the cytokine threshold (1 h) can distinguish ligands with a 30% variation in the complex lifetime. However, a robustness analysis shows that these properties are degraded when the queue parameters are subject to variation—for example, under stochasticity in the ligand number in the cell-cell interface and population variation in the cellular threshold. A time integration of the queue over a period of hours is shown to be able to control parameter noise efficiently for realistic parameter values when integrated over sufficiently long time periods (hours), the discrimination characteristics being determined by the TCR signal cascade kinetics (a kinetic proofreading scheme). Therefore, through a combination of thresholds and signal integration, a T cell can be responsive to low ligand density and specific to agonist quality. We suggest that multiple threshold mechanisms are employed to establish the conditions for efficient signal integration, i.e., coordinate the formation of a stable contact interface