Filoviruses Require Endosomal Cysteine Proteases for Entry but Exhibit Distinct Protease Preferences

Abstract

, we found that virus-specific differences in the requirement for cathepsin B are correlated with sequence polymorphisms at residues 47 in GP1 and 584 in GP2. We applied these findings to the analysis of additional ebolavirus isolates and correctly predicted that the newly identified ebolavirus species Bundibugyo, containing D47 and I584, is cathepsin B dependent and that ebolavirus Zaire-1995, the single known isolate of ebolavirus Zaire that lacks D47, is not. We also obtained evidence for virusspecific differences in the role of cathepsin L, including cooperation with cathepsin B. These studies strongly suggest that the use of endosomal cysteine proteases as host factors for entry is a general property of members of the family Filoviridae. E bdaviruses and the closely related marburgvirus comprise the family Filoviridae (6,8,9,16). Several lines of recent investigation have elucidated key steps in the pathway for ebolavirus entry into cells. Ebolavirus particles attach to cells through the binding of their glycoprotein (GP) to cell surface receptors or lectins, such as TIM-1 and DC-SIGN, expressed on the plasma membrane (1,22,27,29,37). Membrane-bound particles are taken up into cells by a macropinocytosis-like mechanism and transported to late endosomes/lysosomes (LE/LY) (20,30,31,34), which contain essential entry host factors. We previously showed that cleavage of ebolavirus Zaire-Mayinga (EBOV-May) GP by endosomal cysteine proteases is required for infection (7). More recent work has revealed a second host factor in LE/LY that is broadly required by filoviruses: Niemann-Pick C1 (NPC1) (5, 10), a multipass transmembrane protein that resides in the limiting membrane (44). According to a recently proposed model, virus GP is cleaved by endosomal cysteine proteases and binds to NPC1 (10).Several studies have examined the role of protease cleavage in more detail for EBOV-May. They show that cathepsin L functions in concert with cathepsin B to cleave the GP1 subunit of virus GP (7,35). Structural and functional studies reveal that proteases remove the heavily glycosylated carboxyl-terminal domain of GP1 to expose a more conserved domain that is closely associated with GP2 (12,19,25) and that is proposed to contain the binding site for the filovirus receptor (4,13,24,28). Further, we recently showed that cleaved, but not uncleaved, GP1 binds to purified LE/LY membranes in an NPC1-dependent manner and coimmunoprecipitates with NPC1 (10). We identified small molecules that target NPC1, inhibit infection, and block the binding of cleaved GP1 to NPC1-containing membranes (10), strongly suggesting that the conserved N-terminal domain of GP1 is a ligand for NPC1. Taken together, these previous findings suggest a model in which proteolytic cleavage of GP to remove the carboxylterminal domain of GP1 and expose its N-terminal domain may be functionally analogous to the role of CD4 binding to HIV gp120 to displace highly variable loops and create the coreceptorbinding site (18). Our recent studies show that NPC1 expression...

Host Cell Factors Involved in Filovirus Infection

“…Of note, α5β1 integrin is required for cathepsins B and L to be catalytically active for GP priming [49], supporting an earlier study showing the importance of integrins for filovirus entry [50]. However, since each filovirus species has a different dependency on cathepsin-processing [51,52] and mouseadapted ebolavirus retains full pathogenicity in mice deficient in either cathepsin B or L [53], other important host proteases are likely involved in proteolytic processing of GPs.…”

Characterization of the Envelope Glycoprotein of a Novel Filovirus, Lloviu Virus

“…FY-dmk at the lowest concentration tested in this study (0.625 M) did not reduce infectivities of VSV⌬G*-Lloviu, VSV⌬G*-Zaire, or VSV⌬G*-Angola, suggesting that cathepsin L is not essential for LLOV infection, similar to the other filoviruses (40,41). It was also shown that cathepsin B and cathepsin L activities are not required for EBOV replication in a mouse model (41).…”

“…However, the requirement of cathepsin L might be controversial, since a high concentration (Ͼ1 M) of FY-dmk was suggested to inhibit not only cathepsin L but also cathepsin B and likely other endosomal cysteine proteases (40). FY-dmk at the lowest concentration tested in this study (0.625 M) did not reduce infectivities of VSV⌬G*-Lloviu, VSV⌬G*-Zaire, or VSV⌬G*-Angola, suggesting that cathepsin L is not essential for LLOV infection, similar to the other filoviruses (40,41).…”

Large-Scale Screening and Identification of Novel Ebola Virus and Marburg Virus Entry Inhibitors

“…Conversely, the fact that the other 19 compounds affected only MARV indicates that specific differences in cell entry may be exploited in studying infection mechanisms unique for each virus. Indeed, this is supported by work showing that each virus has different dependencies on cellular proteases for activation of the GP for membrane fusion (22,23) and is potentially the reason that none of the broadly active compounds were found to be cathepsin protease inhibitors.…”

“…Filoviral GP binds receptors, and the virus is internalized through macropinocytosis (18,19) and trafficked through endosomes, which is dependent on the calcium channel TPC2 (14,20). During trafficking the virus is exposed to an increasingly acidic environment, in which proteases are activated and cleave the GP (21)(22)(23)(24), allowing interaction with NPC1, a key protein found in endosomes (25,26).…”