Cell-to-cell transmission of HSV1 in human keratinocytes in the absence of the major entry receptor, nectin1.

Herpes simplex virus 1 (HSV1) infects the stratified epithelia of the epidermis, oral or genital mucosa, where the main cell type is the keratinocyte. Here we have used nTERT human keratinocytes to generate a CRISPR-Cas9 knockout (KO) of the primary candidate HSV1 receptor, nectin1, resulting in a cell line that is refractory to HSV1 entry. Nonetheless, a small population of KO cells was able to support infection which was not blocked by a nectin1 antibody and hence was not a consequence of residual nectin1 expression. Strikingly at later times, the population of cells originally resistant to HSV1 infection had also become infected. Appearance of this later population was blocked by inhibition of virus genome replication, or infection with a ΔUL34 virus defective in capsid export to the cytoplasm. Moreover, newly formed GFP-tagged capsids were detected in cells surrounding the initial infected cell, suggesting that virus was spreading following replication in the original susceptible cells. Additional siRNA depletion of the second major HSV1 receptor HVEM, or PTP1B, a cellular factor shown elsewhere to be involved in cell-to-cell transmission, had no effect on virus spread in the absence of nectin1. Neutralizing human serum also failed to block virus transmission in nectin1 KO cells, which was dependent on the receptor binding protein glycoprotein D and the cell-to-cell spread glycoproteins gI and gE, indicating that virus was spreading by direct cell-to-cell transmission. In line with these results, both HSV1 and HSV2 formed plaques on nectin1 KO cells, albeit at a reduced titre, confirming that once the original cell population was infected, the virus could spread into all other cells in the monolayer. We conclude that although nectin1 is required for extracellular entry in to the majority of human keratinocytes, it is dispensable for direct cell-to-cell transmission.

Postnatal nectin-3 knockdown induces structural abnormalities of hippocampal principal neurons and memory deficits in adult mice.

The early postnatal stage is a critical period of hippocampal neurodevelopment and also a period of high vulnerability to adverse life experiences. Recent evidence suggests that nectin-3, a cell adhesion molecule, mediates memory dysfunction and dendritic alterations in the adult hippocampus induced by postnatal stress. But it is unknown whether postnatal nectin-3 reduction alone is sufficient to alter hippocampal structure and function in adulthood. Here, we down regulated hippocampal expression of nectin-3 and its heterophilic adhesion partner nectin-1, respectively, from early postnatal stage by injecting adeno-associated virus (AAV) into the cerebral lateral ventricles of neonatal mice (postnatal day 2). We found that suppression of nectin-3, but not nectin-1, expression from the early postnatal stage impaired hippocampus-dependent novel object recognition and spatial object recognition in adult mice. Moreover, AAV-mediated nectin-3 knockdown significantly reduced dendritic complexity and spine density of pyramidal neurons throughout the hippocampus, whereas nectin-1 knockdown only induced the loss of stubby spines in CA3. Our data provide direct evidence that nectins, especially nectin-3, are necessary for postnatal hippocampal development of memory functions and structural integrity.

Nectin-2α is localized at cholinergic neuron dendrites and regulates synapse formation in the medial habenula.

The medial habenula (MHb) receives afferents from the triangular septum and the medial septal complex, projects efferents to the interpeduncular nucleus (IPN) in the midbrain to regulate dopamine and serotonin levels, and is implicated in stress, depression, memory, and nicotine withdrawal syndrome. We previously showed that the cell adhesion molecule nectin-2α is localized at the boundary between adjacent somata of clustered cholinergic neurons and regulates the voltage-gated A-type K+ channel Kv4.2 localization at membrane specializations in the MHb. This adhesion apparatus, named nectin-2α spots, is not associated with the nectin-binding protein afadin or any classic cadherins and their binding proteins p120-catenin and ß-catenin. We showed here that nectin-2α was additionally localized at cholinergic neuron dendrites in synaptic regions of the MHb. The genetic ablation of nectin-2 reduced the number of synapses in the MHb without affecting their morphology. Nectin-2α was associated with afadin, cadherin-8, p120-catenin, ß-catenin, and αN-catenin, forming puncta adherentia junctions (PAJs). Nectin-2α was observed in the IPN, but not in the triangular septum or the medial septal complex. The genetic ablation of nectin-2 did not affect synapse formation in the IPN. These results indicate that nectin-2α forms two types of adhesion apparatus in the MHb, namely nectin-2α spots at neighboring somata and PAJs at neighboring dendrites, and that dendritic PAJs regulate synapse formation in the MHb.