Immortalized Human Skin Fibroblast-hTERT

EBOV Infection of Primary and Immortalized Human Skin Fibroblasts

Ebola virus (EBOV) causes severe human disease. During late infection, EBOV virions are on the skin s surface, however, the permissive skin cell types and the route of virus translocation to the epidermal surface are unknown.

This study evaluated EBOV infection in cultured primary and immortalized human skin fibroblasts (NHSF-2) and identified host receptors that mediate virus entry. Primary skin human fibroblasts were infected with EBOV and a dose-dependent increase in virus infection was evident on day 5 (Fig. 1A) and day 7 (Fig. 1B). Gene expression studies in the NHSF-2 cells demonstrated that AXL was more than 100-fold more highly expressed than other known cell surface receptors used by EBOV (Fig. 1D) and treatment of NHSF-2 cells or a primary skin fibroblast population with the AXL inhibitor, bemcentinib, inhibited virus infection (Fig. 1E), indicating that AXL is critical for EBOV entry into fibroblasts. Last, the requirement for the endosomal receptor NPC1 was evaluated in siRNA knockdown and 3.47 inhibition studies. Ruxolitinib enhanced virus infection in the presence of the scrambled siRNA but, as anticipated, decreased rVSV/EBOV GP infection upon NPC1-specific siRNA transfection (Fig. 1, F and G). Treatment of NHSF-2 with NPC1 inhibitor 3.47 also resulted in decreased EBOV GP dependent entry (Fig. 1H). In total, these studies demonstrate that skin fibroblasts support EBOV infection in an AXL-dependent and NPC1-dependent manner.