Nat Commun 9:1167
Nat Commun 9:1167. decrease in infectivity of neurons. Notably, while Nivocasan (GS-9450) addition of soluble nectin-1 during viral infection inhibits infectivity, addition after infection has no effect on infectivity. Ectopic expression of human nectin-1 in a cell line resistant to productive VZV infection confers susceptibility to infection. In summary, we have identified nectin-1 as a neuronal entry mediator of VZV. IMPORTANCE Varicella-zoster virus (VZV) causes chickenpox, gains access to Rabbit Polyclonal to Mnk1 (phospho-Thr385) neurons during primary infection where it resides lifelong, and can later be reactivated. Reactivation Nivocasan (GS-9450) is associated with shingles and postherpetic neuralgia, as well as with severe neurologic complications, including vasculitis and encephalitis. Although the varicella vaccine substantially decreases morbidity and mortality associated with primary infection, the vaccine cannot prevent the development of neuronal latency, and vaccinated populations are still at risk for reactivation. Furthermore, Nivocasan (GS-9450) immunocompromised individuals are at higher risk for VZV reactivation and associated complications. Little is known regarding how VZV enters neurons. Here, we identify nectin-1 as an entry mediator of VZV in human neurons. Identification of nectin-1 as a neuronal VZV entry mediator could lead to improved treatments and preventative measures to reduce VZV related morbidity and mortality. or did not cause significant reduction of targeted protein expression; as a result, we serially transfected siRNAs every 4?days. Open in a separate window FIG 2 Effect of nectin-1 siRNA knockdown on VZV infection of human neurons. (A) RNA fold changes normalized to HRPT1 expression. The error bars indicate the SEM (values refer to two-way analysis of variance with Bonferroni posttest). (B) Effect of siRNA-mediated knockdown against GAPDH or nectin-1 (50?M) at 7 and 10?days by Western blotting with whole-cell lysates. (C) Effect of siRNA-mediated knockdown on cell surface level of nectin-1 by flow cytometry at 7?days posttransfection. (D) Neurons transfected with siRNA against nectin-1 (7?days) were infected with VZV, and the effect of nectin-1 knockdown on VZV infection was analyzed by Western blotting for VZV gE expression at 4?days after infection. (E) Dose-dependent effect of nectin-1 siRNA (0, 10, 25, and 50?M) on VZV infection was analyzed by Western blotting with anti-VZV gE and pORF63 antibodies. At 7?days after initial transfection (i.e., transfection on day 0 and day 4), nectin-1 and GAPDH protein levels were reduced by 61 and 80%, respectively (Fig. 2B). At 10 days after the initial siRNA transfection (i.e., transfection on days 0, 4, and 8), protein expression was still reduced, though it had begun to recover (34% reduction in GAPDH and 53% reduction in nectin-1; Fig. 2B). Live cell immunostaining with antibody against nectin-1, followed by flow cytometry, confirmed that knockdown of nectin-1 resulted in decreased protein levels at the neuronal cell surface (Fig. 2C). To examine whether downregulation of nectin-1 impacted VZV infection, neurons were infected at day 7 after siRNA treatment, a time point when nectin-1 protein expression was observed to be lowest in silenced neurons. Nectin-1 downregulation blocked VZV infection, as evidenced by the absence of gE in the cells (Fig. 2D). Neither expression of housekeeping genes (GAPDH or beta-actin; Fig. 2D and ?andE)E) nor cell viability (104% 2% with 25?M siRNA treatment and 106% 5% for 50?M siRNA treatment when normalized to mock siRNA treatment) were affected, suggesting that the observed decrease in viral infectivity was not explained by cell death in the setting of nectin-1 knockdown. In separate experiments in which we added increasing concentrations of nectin-1 siRNA, decreasing levels of gE and the VZV immediate early ORF63 protein (pORF63) were observed (Fig. 2E), thus demonstrating a dose-dependent effect of nectin-1 knockdown on VZV infection of neurons. Recombinant nectin-1 modulates VZV infectivity in human neurons. Since nectin-1 functions as an entry receptor for several other alphaherpesviruses (25), we assessed its role as an entry mediator for VZV infection in neurons by utilizing soluble recombinant forms of the protein. While such methods have successfully been used to block entry of HSV into cells, the prominent cell-to-cell spread of VZV in cell culture could potentially confound.