The Phosphatidylserine and Phosphatidylethanolamine Receptor CD300a Binds Dengue Virus and Enhances Infection.

UNLABELLED: Dengue virus (DENV) is the etiological agent of the major human arboviral disease. We previously demonstrated that the TIM and TAM families of phosphatidylserine (PtdSer) receptors involved in the phagocytosis of apoptotic cells mediate DENV entry into target cells. We show here that human CD300a, a recently identified phospholipid receptor, also binds directly DENV particles and enhances viral entry. CD300a facilitates infection of the four DENV serotypes, as well as of other mosquito-borne viruses such as West Nile virus and Chikungunya virus. CD300a acts as an attachment factor that enhances DENV internalization through clathrin-mediated endocytosis. CD300a recognizes predominantly phosphatidylethanolamine (PtdEth) and to a lesser extent PtdSer associated with viral particles. Mutation of residues in the IgV domain critical for phospholipid binding abrogate CD300a-mediated enhancement of DENV infection. Finally, we show that CD300a is expressed at the surface of primary macrophages and anti-CD300a polyclonal antibodies partially inhibited DENV infection of these cells. Overall, these data indicate that CD300a is a novel DENV binding receptor that recognizes PtdEth and PtdSer present on virions and enhance infection. IMPORTANCE: Dengue disease, caused by dengue virus (DENV), has emerged as the most important mosquito-borne viral disease of humans and is a major global health concern. The molecular bases of DENV-host cell interactions during virus entry are poorly understood, hampering the discovery of new targets for antiviral intervention. We recently discovered that the TIM and TAM proteins, two receptor families involved in the phosphatidylserine (PtdSer)-dependent phagocytic removal of apoptotic cells, interact with DENV particles-associated PtdSer through a mechanism that mimics the recognition of apoptotic cells and mediate DENV infection. In this study, we show that CD300a, a novel identified phospholipid receptor, mediates DENV infection. CD300a-dependent DENV infection relies on the direct recognition of phosphatidylethanolamine and to a lesser extent PtdSer associated with viral particles. This study provides novel insights into the mechanisms that mediate DENV entry and reinforce the concept that DENV uses an apoptotic mimicry strategy for viral entry.

Nitrosopumilus maritimus gen. nov., sp. nov., Nitrosopumilus cobalaminigenes sp. nov., Nitrosopumilus oxyclinae sp. nov., and Nitrosopumilus ureiphilus sp. nov., four marine ammonia-oxidizing archaea of the phylum Thaumarchaeota.

Four mesophilic, neutrophilic, and aerobic marine ammonia-oxidizing archaea, designated strains SCM1T, HCA1T, HCE1T and PS0T, were isolated from a tropical marine fish tank, dimly lit deep coastal waters, the lower euphotic zone of coastal waters, and near-surface sediment in the Puget Sound estuary, respectively. Cells are straight or slightly curved small rods, 0.15-0.26 µm in diameter and 0.50-1.59 µm in length. Motility was not observed, although strain PS0T possesses genes associated with archaeal flagella and chemotaxis, suggesting it may be motile under some conditions. Cell membranes consist of glycerol dibiphytanyl glycerol tetraether (GDGT) lipids, with crenarchaeol as the major component. Strain SCM1T displays a single surface layer (S-layer) with p6 symmetry, distinct from the p3-S-layer reported for the soil ammonia-oxidizing archaeon Nitrososphaera viennensis EN76T. Respiratory quinones consist of fully saturated and monounsaturated menaquinones with 6 isoprenoid units in the side chain. Cells obtain energy from ammonia oxidation and use carbon dioxide as carbon source; addition of an α-keto acid (α-ketoglutaric acid) was necessary to sustain growth of strains HCA1T, HCE1T, and PS0T. Strain PS0T uses urea as a source of ammonia for energy production and growth. All strains synthesize vitamin B1 (thiamine), B2 (riboflavin), B6 (pyridoxine), and B12 (cobalamin). Optimal growth occurs between 25 and 32 °C, between pH 6.8 and 7.3, and between 25 and 37 ‰ salinity. All strains have a low mol% G+C content of 33.0-34.2. Strains are related by 98 % or greater 16S rRNA gene sequence identity, sharing ~85 % 16S rRNA gene sequence identity with Nitrososphaera viennensis EN76T. All four isolates are well separated by phenotypic and genotypic characteristics and are here assigned to distinct species within the genus Nitrosopumilus gen. nov. Isolates SCM1T (=ATCC TSD-97T =NCIMB 15022T), HCA1T (=ATCC TSD-96T), HCE1T (=ATCC TSD-98T), and PS0T (=ATCC TSD-99T) are type strains of the species Nitrosopumilusmaritimus sp. nov., Nitrosopumilus cobalaminigenes sp. nov., Nitrosopumilus oxyclinae sp. nov., and Nitrosopumilus ureiphilus sp. nov., respectively. In addition, we propose the family Nitrosopumilaceae fam. nov. and the order Nitrosopumilales ord. nov. within the class Nitrososphaeria.

Nutrient transport suggests an evolutionary basis for charged archaeal surface layer proteins.

Surface layers (S-layers) are two-dimensional, proteinaceous, porous lattices that form the outermost cell envelope component of virtually all archaea and many bacteria. Despite exceptional sequence diversity, S-layer proteins (SLPs) share important characteristics such as their ability to form crystalline sheets punctuated with nano-scale pores, and their propensity for charged amino acids, leading to acidic or basic isoelectric points. However, the precise function of S-layers, or the role of charged SLPs and how they relate to cellular metabolism is unknown. Nano-scale lattices affect the diffusion behavior of low-concentration solutes, even if they are significantly smaller than the pore size. Here, we offer a rationale for charged S-layer proteins in the context of the structural evolution of S-layers. Using the ammonia-oxidizing archaea (AOA) as a model for S-layer geometry, and a 2D electrodiffusion reaction computational framework to simulate diffusion and consumption of the charged solute ammonium (NH4+), we find that the characteristic length scales of nanoporous S-layers elevate the concentration of NH4+ in the pseudo-periplasmic space. Our simulations suggest an evolutionary, mechanistic basis for S-layer charge and shed light on the unique ability of some AOA to oxidize ammonia in environments with nanomolar NH4+ availability, with broad implications for comparisons of ecologically distinct populations.

A multivalent inhibitor of the DC-SIGN dependent uptake of HIV-1 and Dengue virus.

DC-SIGN is a C-type lectin receptor on antigen presenting cells (dendritic cells) which has an important role in some viral infection, notably by HIV and Dengue virus (DV). Multivalent presentation of carbohydrates on dendrimeric scaffolds has been shown to inhibit DC-SIGN binding to HIV envelope glycoprotein gp120, thus blocking viral entry. This approach has interesting potential applications for infection prophylaxis. In an effort to develop high affinity inhibitors of DC-SIGN mediated viral entry, we have synthesized a group of glycodendrimers of different valency that bear different carbohydrates or glycomimetic DC-SIGN ligands and have studied their DC-SIGN binding activity and antiviral properties both in an HIV and a Dengue infection model. Surface Plasmon Resonance (SPR) competition studies have demonstrated that the materials obtained bind efficiently to DC-SIGN with IC50s in the µm range, which depend on the nature of the ligand and on the valency of the scaffold. In particular, a hexavalent presentation of the DC-SIGN selective antagonist 4 displayed high potency, as well as improved accessibility and chemical stability relative to previously reported dendrimers. At low µm concentration the material was shown to block both DC-SIGN mediated uptake of DV by Raji cells and HIV trans-infection of T cells.

The TIM and TAM families of phosphatidylserine receptors mediate dengue virus entry.

Dengue viruses (DVs) are responsible for the most medically relevant arboviral diseases. However, the molecular interactions mediating DV entry are poorly understood. We determined that TIM and TAM proteins, two receptor families that mediate the phosphatidylserine (PtdSer)-dependent phagocytic removal of apoptotic cells, serve as DV entry factors. Cells poorly susceptible to DV are robustly infected after ectopic expression of TIM or TAM receptors. Conversely, DV infection of susceptible cells is inhibited by anti-TIM or anti-TAM antibodies or knockdown of TIM and TAM expression. TIM receptors facilitate DV entry by directly interacting with virion-associated PtdSer. TAM-mediated infection relies on indirect DV recognition, in which the TAM ligand Gas6 acts as a bridging molecule by binding to PtdSer within the virion. This dual mode of virus recognition by TIM and TAM receptors reveals how DVs usurp the apoptotic cell clearance pathway for infectious entry.

Novel roles of hakai in cell proliferation and oncogenesis.

During tumor development, cells acquire multiple phenotypic changes upon misregulation of oncoproteins and tumor suppressor proteins. Hakai was originally identified as an E3 ubiquitin-ligase for the E-cadherin complex that regulates cell-cell contacts. Here, we present evidence that Hakai plays a crucial role in various cellular processes and tumorigenesis. Overexpression of Hakai affects not only cell-cell contacts but also proliferation in both epithelial and fibroblast cells. Furthermore, the knockdown of Hakai significantly suppresses proliferation of transformed epithelial cells. Expression of Hakai is correlated to the proliferation rate in human tissues and is highly up-regulated in human colon and gastric adenocarcinomas. Moreover, we identify PTB-associated splicing factor (PSF), an RNA-binding protein, as a novel Hakai-interacting protein. By using cDNA arrays, we have determined various specific PSF-associated mRNAs encoding proteins that are involved in several cancer-related processes. Hakai affects the ability of PSF to bind these mRNAs, and expression of PSF short hairpin RNA or a dominant-negative PSF mutant significantly suppresses proliferation of Hakai-overexpressing cells. Collectively, these results suggest that Hakai is an important regulator of cell proliferation and that Hakai may be an oncoprotein and a potential molecular target for cancer treatment.