Supplementary MaterialsSupp Fig S5. is truncated. Using confocal and super-resolution imaging and high content single particle tracking, we investigated DENV binding, DC-SIGN surface transport, endocytosis, as well as cell infectivity. Cdc14A1 DC-SIGN was found colocalized with DENV inside cells suggesting hand-off at the plasma membrane to another receptor did not occur. Moreover, all three DC-SIGN molecules on NIH3T3 cells supported cell infection. These results imply the involvement of a co-receptor because cells expressing the internalization-deficient mutants could still be infected. trafficking, quantitative colocalization, super-resolution imaging, viral receptor, viral entry, C-type lectin receptor, antigen-presenting cells, fluorescence microscopy Graphical Abstract Whether DC-SIGN functions as merely an attachment factor for dengue virus (DENV) or whether DC-SIGN plays further roles beyond attachment has been controversial. ON-01910 (rigosertib) We use mammalian cell culture models, as well as primary dendritic cells, and high resolution, quantitative fluorescence microscopy to track the movements of DC-SIGN and DENV during viral entry. Our results support a model in which DC-SIGN captures DENV and participates, along with a co-receptor, in DENV internalization via clathrin-coated structures and subsequent trafficking to early endosomes. Introduction Dendritic cells (DCs) are professional and potent antigen-presenting cells in the human immune system. They sample pathogens from peripheral tissue, migrate to lymph nodes, and present antigens to activate both memory and na?ve T cells to initiate immune responses 1. However, DCs that are patrolling the peripheral tissues are also often the first targets of infection by ON-01910 (rigosertib) viruses, such as Ebola, HIV and dengue 2C4. The infected DCs transport viruses to lymph nodes and facilitate infection of other cells and systemic spread of the virus. It is estimated that about 400 million people are infected by dengue virus (DENV) each year 5, and some of these cases of dengue infection lead to fatal dengue hemorrhagic fever and dengue shock syndrome. More knowledge about the mechanism in which dengue infects DCs will provide new potential strategies for preventing infection at its earliest stages and aid the numerous ongoing efforts for vaccine development 6. DCs display on their plasma membranes so-called pattern recognition receptors (PRRs) 7. These receptors bind to carbohydrates on pathogens. Dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) is a key PRR. This molecule, which is a single-pass transmembrane type II protein and contains a distal, extracellular lectin domain, is highly expressed on the surface of immature DCs and mediates the uptake of a variety of viral, bacterial, and yeast pathogens, by binding to their surface carbohydrates, for presentation to other immune cells 7. A number of pathogens use DC-SIGN binding to disrupt DC function and circumvent normal immune surveillance 8,9. For example, SARS 8, Ebola 10, dengue 4 and other viruses 11,12 use DC-SIGN as an initial cell attachment factor and/or entry receptor. In the case of dengue infection, it has been controversial whether DC-SIGN is merely an attachment factor for DENV or plays further roles in viral entry 4,13C17. The attachment factor mechanism was proposed after cells expressing internalization motif-deficient DC-SIGN were shown to still be infected by DENV. In this hypothesis, DC-SIGN binds DENV, but ON-01910 (rigosertib) then the virus is handed over to another, as yet unidentified, co-receptor for DENV entry into the cells 15. However, it is also possible that the DENV/DC-SIGN complex does ON-01910 (rigosertib) not dissociate during entry but, rather, acts in concert with a co-receptor. To explore in greater detail the issue of DENV attachment and entry in DC-SIGN.