Herpes simplex virus (HSV) entry requires the interaction between the envelope glycoprotein D (gD) and a cellular receptor such as nectin-1 (also named herpesvirus entry mediator C [HveC]) or HveA/HVEM. Nectin-1 is a cell adhesion molecule found at adherens junctions associated with the cytoplasmic actin-binding protein afadin. Nectin-1 can act as its own ligand in a homotypic interaction to bridge cells together. We used a cell aggregation assay to map an adhesive functional site on nectin-1 and identify the effects of gD binding and HSV early infection on nectin-1 function. Soluble forms of nectin-1 and anti-nectin-1 monoclonal antibodies were used to map a functional adhesive site within the first immunoglobulin-like domain (V domain) of nectin-1. This domain also contains the gD-binding site, which appeared to overlap the adhesive site. Thus, soluble forms of gD were able to prevent nectin-1-mediated cell aggregation and to disrupt cell clumps in an affinity-dependent manner. HSV also prevented nectin-1-mediated cell aggregation by occupying the receptor. Early in infection, nectin-1 was not downregulated from the cell surface. Rather, detection of nectin-1 changed gradually over a 30-min period of infection, as reflected by a decrease in the CK41 epitope and an increase in the CK35 epitope. The level of detection of virion gD on the cell surface increased within 5 min of infection in a receptor-dependent manner. These observations suggest that cell surface nectin-1 and gD may undergo conformational changes during HSV entry as part of an evolving interaction between the viral envelope and the cell plasma membrane.The interaction between herpes simplex virus (HSV) envelope glycoprotein D (gD) and a specific cellular receptor is required for virus entry into mammalian cells (2, 48). This essential step follows an initial attachment mediated by HSV gC and gB bound to cell surface heparan sulfate proteoglycans (17,18,56). In addition, fusion of the envelope with the cell plasma membrane involves gB and the gH/gL complex (36,50,55).Receptors for gD belong to at least three unrelated families. The herpesvirus entry mediator A (HveA; also called HVEM and TNFRSF14) belongs to the tumor necrosis factor alpha (TNF) receptor family and mediates entry of most HSV-1 and HSV-2 strains (34, 53). Nectin-1 (HveC; also called PRR1 and CD111) (14, 26) and nectin-2 (HveB; also called PRR2 and CD112) (11, 51) are members of the immunoglobulin (Ig) superfamily. Nectin-1 allows entry of all the HSV-1 and HSV-2 strains tested as well as pseudorabies virus and bovine herpesvirus type 1 (9, 14, 32). In contrast, nectin-2 can be used only by HSV-2, some laboratory strains of HSV-1 (rid1 and ANG), and pseudorabies virus (25, 51). The related poliovirus receptor (PVR) CD155 does not function as an HSV receptor, but can be used by pseudorabies virus and bovine herpesvirus type 1 (14). Lastly, a specific type of heparan sulfate modified by D-glucosaminyl 3-O-sulfotransferase 3 can substitute for HveA or nectin-1 and binds to gD to allow e...

The inner ear forms from paired ectodermal primordia that lie to either side of the developing hindbrain. Initially each primordium forms a shallow depression in the ectodermal surface. Invagination to form an otic pit coincides with the formation of several deep folds in the epithelial surface. An initial fold appears parallel to the embryonic axis and at the junction of the rhombencephalon with somitomeric mesoderm. This is followed by formation of cranial and caudal folds perpendicular to the axis and minor folds that are within the pit formed by earlier folding. The central region of the otic primordium remains in close apposition to the lateral surface of the neural tube during the process of fold formation, until the otic pit becomes quite deep. At that time, mesenchymal cells penetrate between the two layers. Experimental analysis of invagination supports the conclusion that otic invagination is controlled differently from that of similar organ primordia, such as the eye and thyroid. Whereas these other primordia can be stimulated to undergo normal morphogenetic shape changes precociously by treatments that presumably activate motile processes in the cytoskeleton, the same conditions have little effect on the otic placode. Similarly, neither inhibitors of calcium transport nor inactivators of calmodulin activity prevent otic pit formation, while these drugs block invagination of other primordia. These results suggest that otic invagination may be caused by changes in the surrounding tissues rather than by an activation of motility within the primordium.

We have developed a highly sensitive and reproducible method to detect the expression of specific genes in small tissue samples, such as a single embryonic somite. The procedure, which utilizes coupled reverse transcription-polymerase chain reaction (RT-PCR), was developed for evaluating the sequence of gene expression occurring in single somites during chick embryonic development. Comparisons of results obtained from using combinations of various RNA isolation methods and reverse transcription methods demonstrate that a protocol using a commercially available RNA isolation reagent (Tri Reagent) followed by optimized PCR, successfully detects low levels of mRNAs.