Properties associated with filoviral-glycoprotein-mediated entry events in permissive cells

To enter cells, the filovirus, ebolavirus (EBOV), must bind to target cells and internalize into an endocytic vesicle. The properties surrounding filoviral entry into permissive cells remain poorly studied. To date, the kinetics associated with filoviral-glycoprotein (GP)-mediated entry have never been investigated past 6 hours. Our initial entry studies with filoviral-GP pseudotyped retrovirions at 37˚C indicated that virions entered permissive cells with a half time (T50) of ~8 hours. We found that 10 to 20% of retroviral based virions bound to cells in over a one hour period at 4˚C suggesting that virion binding was relatively inefficient. Surprisingly, we also observed that less than half of the retroviral based pseudovirions pre-bound to the cell surface were internalized by 7 hours at 37˚C indicating that virion internalization was a slow process. Consistent with slow internalization of retroviral particles, we observed that, while virus entry lost sensitivity to ammonium chloride treatment with time, 50% of the virions remained sensitive to low pH neutralization for at least 7 hours. These slow entry kinetics for filoviruses have not been appreciated thus far, and could have significant implications in the timing and types of treatments that could be administered to filoviral infected individuals.

We also determined the impact of specific carbohydrate linkages on host cell plasma membrane proteins involved in filoviral entry, by using a series of Chinese hamster ovary (CHO) cell lines deficient in one or more enzymes required for N- and O- linked glycosylation. The LdlD CHO cell line that expresses normal surface N-linked glycans but has abbreviated O-linked surface glycans showed a 50% reduction in transduction by both Zaire (ZEBOV) and Lake Victoria MARV-GP pseudotyped particles as compared to the control wild type parental CHO cell line (Pro5). Use of the novel O-linked inhibitor drug 1-68A allowed us to confirm the necessity of O-linked glycans in efficient ZEBOV entry into additional permissive cells types. Interestingly, loss of terminal sialic acids (Lec2 cells) or galactose (Lec8 cells) on both N- and O- linked sugars resulted in a 2-fold enhancement of filoviral GP mediated entry compared to control. However, Lec1 cells that have wild type O-linked glycans but highly abbreviated N-linked glycans had similar levels of transduction to control Pro5 cells. Further studies indicated that binding of ZEBOV pseudovirions to Pro5 and all mutant CHO cells was equal, indicating that a post-binding defect or enhancement in ZEBOV internalization may be occurring. These data identify the importance of host cell O-linked glycosylation during the initial steps of filovirus infection.

While the receptor(s) used by filoviruses for productive binding and entry into cells remains to be identified, several proteins have been shown to enhance filoviral entry into cells. Axl, a plasma membrane associated Tyro3/Axl/Mer (TAM) family member, is necessary for optimal ZEBOV-GP-dependent entry into some permissive cells, but not others. To date, the precise role of Axl in virion entry is unknown. Through the use of biochemical inhibitors, RNAi, and dominant-negative constructs, we set out to characterize entry pathways used for ZEBOV uptake in cells that require Axl for optimal transduction (Axl-dependent cells) and to define the role of Axl in these processes. We demonstrate that ZEBOV-GP-dependent entry into Axl-dependent cells occurs through multiple pathways including both clathrin-dependent and caveolae/lipid raft-mediated endocytosis. Surprisingly, both dynamin-dependent and -independent fluid-phase uptake (FPU) pathways mediated ZEBOV-GP entry into the Axl-dependent cells as well. Reduction of Axl expression by RNAi treatment resulted in abrogation of ZEBOV entry by FPU-dependent pathways, but had no effect on receptor-mediated endocytosis mechanisms. Our findings demonstrate for the first time that Axl enhances FPU, thereby increasing productive ZEBOV entry, and providing insight into the mechanisms surrounding filoviral entry.