Summary Host defense to RNA viruses depends on rapid intracellular recognition of viral RNA by two cytoplasmic RNA helicases, RIG-I and MDA5. RNA transfection experiments indicate that RIG-I responds to naked double-stranded (ds) RNAs with a triphosphorylated 5′ (5′ppp) terminus. However, identity of the RIG-I stimulating viral structures in an authentic infection context remains unresolved. We show that incoming viral nucleocapsids containing a 5′ppp dsRNA “panhandle” structure trigger antiviral signaling that commences with RIG-I, is mediated through the adaptor protein MAVS, and terminates with transcription factor IRF-3. Independent of mammalian cofactors or viral polymerase activity, RIG-I bound to viral nucleocapsids, underwent a conformational switch, and homo-oligomerized. Enzymatic probing and super-resolution microscopy suggest that RIG-I interacts with the panhandle structure of the viral nucleocapsids. These results define cytoplasmic entry of nucleocapsids as the proximal RIG-I-sensitive step during infection, and establish viral nucleocapsids with a 5′ppp dsRNA panhandle as a RIG-I activator.

Olfactory receptors, which belong to the family of G-protein-coupled receptors, are found to be ectopically expressed in non-sensory tissues mediating a variety of cellular functions. In this study we detected the olfactory receptor OR51E2 at the transcript and the protein level in human epidermal melanocytes. Stimulation of primary melanocytes with the OR51E2 ligand β-ionone significantly inhibited melanocyte proliferation. Our results further showed that β-ionone stimulates melanogenesis and dendritogenesis. Using RNA silencing and receptor antagonists, we demonstrated that OR51E2 activation elevated cytosolic Ca(2+) and cAMP, which could mediate the observed increase in melanin synthesis. Co-immunocytochemical stainings using a specific OR51E2 antibody revealed subcellular localization of the receptor in early endosomes associated with EEA-1 (early endosome antigen 1). Plasma membrane preparations showed that OR51E2 protein is present at the melanocyte cell surface. Our findings thus suggest that activation of olfactory receptor signaling by external compounds can influence melanocyte homeostasis.

In the female reproductive tract, mammalian sperm undergo a regulated sequence of prefusion changes that "prime" sperm for fertilization. Among the least understood of these complex processes are the molecular mechanisms that underlie sperm guidance by environmental chemical cues. A "hard-wired" Ca 2؉ signaling strategy that orchestrates specific motility patterns according to given functional requirements is an emerging concept for regulation of sperm swimming behavior. The molecular players involved, the spatiotemporal characteristics of such motility-associated Ca 2؉ dynamics, and the relation between a distinct Ca 2؉ signaling pattern and a behavioral sperm phenotype, however, remain largely unclear. Here, we report the functional characterization of two human sperm chemoreceptors. Using complementary molecular, physiological, and behavioral approaches, we comparatively describe sperm Ca 2؉ responses to specific agonists of these novel receptors and bourgeonal, a known sperm chemoattractant. We further show that individual receptor activation induces specific Ca 2؉ signaling patterns with unique spatiotemporal dynamics. These distinct Ca 2؉ dynamics are correlated to a set of stimulus-specific stereotyped behavioral responses that could play vital roles during various stages of prefusion sperm-egg chemical communication.On their journey to locate the oocyte, navigating mammalian sperm depend on both chemical (1) and physical (2, 3) cues to regulate flagellar motion, switch flagellar beat modes, and, thus, direct their movement. How such environmental signals are detected and translated into changes in motility, however, remains largely unknown.Sperm acquire motility upon vaginal deposition (4). This initial swimming behavior is referred to as activated motility and characterized by a relatively low amplitude/high frequency sinusoidal flagellar motion (5). Motile sperm that pass the cervical mucus barrier must locate and enter the oviduct. Only a fraction of sperm present in the uterus (ϳ10% in humans (6)) accomplishes this task. Within the oviductal isthmus, sperm frequently attach to the highly convoluted epithelial surface (3,7,8), forming a sperm reservoir in close proximity to the fertilization site. Upon ovulation, capacitated sperm that adopt a hyperactivated state (i.e. displaying asymmetrical and relatively high amplitude/low frequency flagellar beating) (4), generate sufficient propulsion force to detach from the epithelium, enter the ampulla, and eventually penetrate the cumulus and zona pellucida surrounding the egg (9).Given the small fraction of sperm that reaches the oviduct, a "competitive race" scenario has fallen out of favor in recent years. Instead, effective sperm guidance mechanisms are required to assure a synchronized arrival and encounter of both gametes at the fertilization site (1). Current models propose a complex multistep process of sperm navigation along thermal (2) and chemical (10) gradients. Accordingly, chemical guidance cues are secreted by both the oocyte and the surroundi...

The detection of external cues is fundamental for human spermatozoa to locate the oocyte in the female reproductive tract. This task requires a specific chemoreceptor repertoire that is expressed on the surface of human spermatozoa, which is not fully identified to date. Olfactory receptors (ORs) are candidate molecules and have been attributed to be involved in sperm chemotaxis and chemokinesis, indicating an important role in mammalian spermatozoa. An increasing importance has been suggested for spermatozoal RNA, which led us to investigate the expression of all 387 OR genes. This study provides the first comprehensive analysis of OR transcripts in human spermatozoa of several individuals by RNA-Seq. We detected 91 different transcripts in the spermatozoa samples that could be aligned to annotated OR genes. Using stranded mRNA-Seq, we detected a class of these putative OR transcripts in an antisense orientation, indicating a different function, rather than coding for a functional OR protein. Nevertheless, we were able to detect OR proteins in various compartments of human spermatozoa, indicating distinct functions in human sperm. A panel of various OR ligands induced Ca2+ signals in human spermatozoa, which could be inhibited by mibefradil. This study indicates that a variety of ORs are expressed at the mRNA and protein level in human spermatozoa.

Olfactory receptors (ORs), which belong to the G-protein coupled receptor family, are expressed in various human tissues, including skin. Cells in non-olfactory tissues tend to express more than one individual OR gene, but function and interaction of two or more ORs in the same cell type has only been marginally analysed. Here, we revealed OR2A4/7 and OR51B5 as two new ORs in human skin cells and identified cyclohexyl salicylate and isononyl alcohol as agonists of these receptors. In cultured human keratinocytes, both odorants induce strong Ca signals that are mediated by OR2A4/7 and OR51B5, as demonstrated by the receptor knockdown experiments. Activation of corresponding receptors induces a cAMP-dependent pathway. Localization studies and functional characterization of both receptors revealed several differences. OR2A4/7 is expressed in suprabasal keratinocytes and basal melanocytes of the epidermis and influences cytokinesis, cell proliferation, phosphorylation of AKT and Chk-2 and secretion of IL-1. In contrast, OR51B5 is exclusively expressed in suprabasal keratinocytes, supports cell migration and regeneration of keratinocyte monolayers, influences Hsp27, AMPK1 and p38MAPK phosphorylation and interestingly, IL-6 secretion. These findings underline that different ORs perform diverse functions in cutaneous cells, and thus offering an approach for the modulated treatment of skin diseases and wound repair.