Uncovering a Genetic Polymorphism Located in Huntingtin Associated Protein 1 in Modulation of Central Pain Sensitization Signaling Pathways.

Fibromyalgia syndrome (FMS) is characterized by widespread pain and increased sensitivity to nociceptive stimulus or tenderness. While familial aggregation could suggest a potential hereditary component in FMS development, isolation of genetic determinants has proven difficult due to the multi-factorial nature and complexity of the syndrome. Central sensitization is thought to be one of the key mechanisms leading to FMS in a subset of patients. Enhanced central pain signaling can be measured using the Nociceptive Flexion Reflex (NFR) or RIII threshold. We performed a genome-wide association study (GWAS) using an array to genotype 258,756 human genetic polymorphisms in 225 FMS patients and 77 healthy volunteers and searched for genetic variants associated with a lowered NFR threshold. We have identified a potential association between a single nucleotide polymorphism resulting in a common non-synonymous coding mutation in the Huntingtin associated protein 1 (HAP1) gene (rs4796604, MAF = 0.5) and the NFR threshold (p = 4.78E-06). The Hap1 protein is involved in trafficking and is particularly enriched in neurons. Our results suggest a possible involvement of the neuronal trafficking protein HAP1 in modulating pain signaling pathways and thus participate in the establishment of the NFR threshold.

A VP1 mutation acquired during an enterovirus 71 disseminated infection confers heparan sulfate binding ability and modulates ex vivo tropism.

Enterovirus 71 (EV71) causes hand, foot and mouth disease, a mild and self-limited illness that is sometimes associated with severe neurological complications. EV71 neurotropic determinants remain ill-defined to date. We previously identified a mutation in the VP1 capsid protein (L97R) that was acquired over the course of a disseminated infection in an immunocompromised host. The mutation was absent in the respiratory tract but was present in the gut (as a mixed population) and in blood and cerebrospinal fluid (as a dominant species). In this study, we demonstrated that this mutation does not alter the dependence of EV71 on the human scavenger receptor class B2 (SCARB2), while it enables the virus to bind to the heparan sulfate (HS) attachment receptor and modifies viral tropism in cell lines and in respiratory, intestinal and neural tissues. Variants with VP197L or VP197R were able to replicate to high levels in intestinal and neural tissues and, to a lesser extent, in respiratory tissues, but their preferred entry site (from the luminal or basal tissue side) differed in respiratory and intestinal tissues and correlated with HS expression levels. These data account for the viral populations sequenced from the patient's respiratory and intestinal samples and suggest that improved dissemination, resulting from an acquired ability to bind HS, rather than specific neurotropism determinants, enabled the virus to reach and infect the central nervous system. Finally, we showed that iota-carrageenan, a highly sulfated polysaccharide, efficiently blocks the replication of HS-dependent variants in cells and 2D neural cultures. Overall, the results of this study emphasize the importance of HS binding in EV71 pathogenesis and open new avenues for the development of antiviral molecules that may prevent this virus's dissemination.

Growth and characterization of different human rhinovirus C types in three-dimensional human airway epithelia reconstituted in vitro.

New molecular diagnostic tools have recently allowed the discovery of human rhinovirus species C (HRV-C) that may be overrepresented in children with lower respiratory tract complications. Unlike HRV-A and HRV-B, HRV-C cannot be propagated in conventional immortalized cell lines and their biological properties have been difficult to study. Recent studies have described the successful amplification of HRV-C15, HRV-C11, and HRV-C41 in sinus mucosal organ cultures and in fully differentiated human airway epithelial cells. Consistent with these studies, we report that a panel of clinical HRV-C specimens including HRV-C2, HRV-C7, HRV-C12, HRV-C15, and HRV-C29 types were all capable of mediating productive infection in reconstituted 3D human primary upper airway epithelial tissues and that the virions enter and exit preferentially through the apical surface. Similar to HRV-A and HRV-B, our data support the acid sensitivity of HRV-C. We observed also that the optimum temperature requirement during HRV-C growth may be type-dependent.

Echovirus 11 infection induces dramatic changes in the actin cytoskeleton of polarized Caco-2 cells.

Binding of echovirus 11 strain 207 (EV11-207) to Caco-2 monolayers results in rapid transfer of the virus to tight junctions prior to uptake. Using a confocal microscopy based-method, this study quantified the spatiotemporal distribution of actin during the time course of infection by EV11-207 in Caco-2 polarized cells. It was found that binding of EV11-207 to the apical surface resulted in rapid rearrangement of the actin cytoskeleton, concomitant with transport of the virus particles to tight junctions. By interfering with the actin network dynamics, the virus remained trapped at the cell surface, leading to abortion of infection. In addition, it was observed that at 4 h post-infection, concomitant with the detection of virus replication, actin filament was depolymerized and degraded. Finally, it was shown that the mechanisms leading to loss of actin were independent of viral genome synthesis, indicating a potential role for the viral protein synthesis seen in late infection. These data confirmed a previous study on the requirement for an intact actin cytoskeleton for EV11-207 to infect cells and reinforce the notion of actin cytoskeleton subversion by picornaviruses during infection in polarized epithelial cells.

Decay-accelerating factor binding determines the entry route of echovirus 11 in polarized epithelial cells.

The interaction between echovirus 11 strain 207 (EV11-207) and decay-accelerating factor (DAF or CD55) at the apical surface of polarized Caco-2 cells results in rapid transport of the virus to tight junctions and in its subsequent uptake. A virus mutant (EV11-207R) which differs at 6 amino acids and whose affinity for DAF is apparently significantly lower remains at the apical surface, from where its uptake occurs. Binding of EV11-207 to DAF and its transport to tight junctions result in a loss of function of the junctions. In contrast, the mutant virus EV11-207R is not transferred to tight junctions, nor does it impair the integrity of these junctions. Cholesterol depletion from the apical membrane leads to DAF aggregation and, presumably, internalization and inhibits infection by EV11-207. However, infection by EV11-207R is significantly less sensitive to cholesterol depletion than infection by EV11-207, confirming the DAF requirement for EV11-207, but not EV11-207R, to infect cells. These data strongly indicate that in the case of infection of polarized epithelial cells by echovirus 11, DAF binding appears be a key determinant in the choice of entry pathway, at least in cell culture.

Regulation of the V-ATPase along the endocytic pathway occurs through reversible subunit association and membrane localization.

The lumen of endosomal organelles becomes increasingly acidic when going from the cell surface to lysosomes. Luminal pH thereby regulates important processes such as the release of internalized ligands from their receptor or the activation of lysosomal enzymes. The main player in endosomal acidification is the vacuolar ATPase (V-ATPase), a multi-subunit transmembrane complex that pumps protons from the cytoplasm to the lumen of organelles, or to the outside of the cell. The active V-ATPase is composed of two multi-subunit domains, the transmembrane V(0) and the cytoplasmic V(1). Here we found that the ratio of membrane associated V(1)/Vo varies along the endocytic pathway, the relative abundance of V(1) being higher on late endosomes than on early endosomes, providing an explanation for the higher acidity of late endosomes. We also found that all membrane-bound V-ATPase subunits were associated with detergent resistant membranes (DRM) isolated from late endosomes, raising the possibility that association with lipid-raft like domains also plays a role in regulating the activity of the proton pump. In support of this, we found that treatment of cells with U18666A, a drug that leads to the accumulation of cholesterol in late endosomes, affected acidification of late endosome. Altogether our findings indicate that the activity of the vATPase in the endocytic pathway is regulated both by reversible association/dissociation and the interaction with specific lipid environments.

Late endosomal cholesterol accumulation leads to impaired intra-endosomal trafficking.

BACKGROUND: Pathological accumulation of cholesterol in late endosomes is observed in lysosomal storage diseases such as Niemann-Pick type C. We here analyzed the effects of cholesterol accumulation in NPC cells, or as phenocopied by the drug U18666A, on late endosomes membrane organization and dynamics. METHODOLOGY/PRINCIPAL FINDINGS: Cholesterol accumulation did not lead to an increase in the raft to non-raft membrane ratio as anticipated. Strikingly, we observed a 2-3 fold increase in the size of the compartment. Most importantly, properties and dynamics of late endosomal intralumenal vesicles were altered as revealed by reduced late endosomal vacuolation induced by the mutant pore-forming toxin ASSP, reduced intoxication by the anthrax lethal toxin and inhibition of infection by the Vesicular Stomatitis Virus. CONCLUSIONS/SIGNIFICANCE: These results suggest that back fusion of intralumenal vesicles with the limiting membrane of late endosomes is dramatically perturbed upon cholesterol accumulation.

Diversity of raft-like domains in late endosomes.

BACKGROUND: Late endosomes, the last sorting station in the endocytic pathway before lysosomes, are pleiomorphic organelles composed of tubular elements as well as vesicular regions with a characteristic multivesicular appearance, which play a crucial role in intracellular trafficking. Here, we have investigated whether, in addition to these morphologically distinguishable regions, late endosomal membranes are additionally sub-compartmentalized into membrane microdomains. METHODOLOGY/PRINCIPAL FINDINGS: Using sub-organellar fractionation techniques, both with and without detergents, combined with electron microscopy, we found that both the limiting membrane of the organel and the intraluminal vesicles contain raft-type membrane domains. Interestingly, these differentially localized domains vary in protein composition and physico-chemical properties. CONCLUSIONS/SIGNIFICANCE: In addition to the multivesicular organization, we find that late endosomes contain cholesterol rich microdomains both on their limiting membrane and their intraluminal vesicles that differ in composition and properties. Implications of these findings for late endosomal functions are discussed.

ARF1 regulates Nef-induced CD4 degradation.

BACKGROUND: The HIV Nef protein downregulates CD4 through sequential connection with clathrin-coated pits and the COP1 coatomer, resulting in accelerated endocytosis and lysosomal targeting. RESULTS: Here we report that the small GTPase ARF1 controls the Nef-induced, COP-mediated late-endosomal targeting of CD4. We find that Nef binds ARF1 directly and can recruit the GTPase onto endosomal membranes. Furthermore, a complex comprising Nef, ARF1, and betaCOP can be immunoprecipitated from cells expressing the viral protein. Residues in a C-terminal loop of the viral protein facilitate both these interactions and the targeting of Nef and CD4 to acidic late endosomes, whereas other residues primarily involved in mediating CD4 endocytosis are dispensable for this process. Finally, a dominant-negative ARF1 mutant blocks the migration of the Nef-CD4 complex to lysosomes. CONCLUSIONS: Our results support a model in which ARF1 is the immediate downstream partner of Nef for CD4 lysosomal targeting.