In the present study, we undertook a molecular characterization of the processing of arenavirus GPCs by SKI-1/S1P and reached the following conclusions. (i) We confirmed that the GPCs of LCMV and LASV and cellular substrates undergo processing in different intracellular compartments and found a determining role for the RRLL motif in the processing of the LASV GPC in the ER/cis-Golgi compartment. (ii) Our structure-function analysis revealed that the efficient cleavage of arenavirus GPCs, but not cellular substrates, requires efficient SKI-1/S1P autoprocessing. (iii) Membrane anchorage and the cytosolic domain of SKI-I/S1P were dispensable for arenavirus GPC processing. (iv) However, exogenous soluble SKI-1/S1P was unable to process LCMV and LASV GPCs present at the surface of SKI-I/S1P-deficient cells.
In the host cell, SKI-1/S1P is implicated in the proteolytic processing of a defined set of substrates, including the membrane-bound SREBPs and the ATF6 family of transcription factors (27
). The SKI-1/S1P-mediated processing of these cellular substrates is under tight spatiotemporal control, as illustrated by the activation of ATF6 upon ER stress. In the resting cell, ATF6 is retained in the ER by the interaction of its luminal domain with the chaperone GRP78/BiP. The engagement of GRP78/BiP by unfolded proteins upon ER stress liberates ATF6, allowing its translocation to the Golgi compartment, where processing and activation by SKI-1/S1P occur in a tightly controlled manner in the early Golgi compartment (36
). In contrast to cellular substrates whose processing seems to be confined to the Golgi compartment, a significant fraction of the LASV GPC undergoes processing already in the ER (18
). Here we confirmed these findings by showing that the soluble SKI-1/S1P-KDEL variant retained in the ER/cis
-Golgi compartment efficiently processed the LASV GPC. In contrast, the GPC of LCMV, which is structurally closely related to LASV, is processed in a late Golgi compartment (3
) and, accordingly, was poorly processed by the ER-retained SKI-1/S1P-KDEL construct.
Interestingly, the SKI-1/S1P processing site of LCMV, RRLA, differs by only one amino acid at P1 from the one found in LASV GPC (RRLL). The GPC of the bunyavirus Crimean-Congo hemorrhagic fever (CCHF) virus also contains an RRLL SKI-1/S1P recognition site and undergoes cleavage in the ER/cis
-Golgi compartment (41
), suggesting that the RRLL motif allows the processing of substrates early along the secretory pathway, likely in the ER/cis
-Golgi compartment. To address this issue, we used an LCMV GPC variant containing the RRLL motif (LCMV GPC-RRLL) and, conversely, an LASV GPC mutant bearing the LCMV recognition site RRLA (LASV GPC-RRLA). LCMV GPC-RRLL was efficiently processed by SKI-1/S1P-KDEL in the ER/cis
-Golgi compartment. In contrast, a mutation of the RRLL motif in the LASV GPC to RRLA abrogated its early processing. This finding suggests that the RRLL motif is a discriminatory determinant for the selection of the SKI-1/S1P substrates that would be processed in the ER/cis
-Golgi compartment. Altogether, the data at hand indicate that SKI-1/S1P processes cellular and viral substrates in at least three different subcellular compartments: (i) GPCs of LASV, CCHF virus, and likely other arenaviruses in the ER/cis
-Golgi compartment; (ii) cellular substrates in the medial Golgi compartment; and (iii) the LCMV GPC in late Golgi compartments. It is conceivable that the processing of viral GPCs in distinct compartments may prevent a perturbation of the SKI-1/S1P-mediated processing of cellular substrates. Indeed, our recent studies revealed that arenavirus infection results in a transient induction of ATF6 but does not interfere with ATF6-regulated gene expression upon the induction of ER stress (27
). This ability of arenaviruses to utilize a cellular protease without interfering with its function in the host cell may provide a strategy for the virus to limit cellular damage during its nonlytic infection.
Cellular PCs are synthesized as inactive zymogens that undergo autocatalytic processing and activation. In most PCs, including furin, the cleaved prosegment remains associated with the enzyme and inhibits is activity, thereby generating a latent form of the protease (23
). Among the PCs, SKI-1/S1P seems unusual, as a mutant impaired in primary B/B′ autoprocessing is still functional in the cleavage of cellular substrates, likely due to its ability to autoprocess itself to a small extent at its secondary C site, even in the absence of primary cleavage at the B/B′ site (11
). Our present study indicates that a variant of SKI-1/S1P deficient in B/B′ processing (SKI-1/S1P-RR) cleaved the GPCs of LCMV and LASV inefficiently and was unable to rescue productive virus infection and cell-to-cell spread in SKI-1/S1P-deficient cells. In line with previously reported work, we were able to show that SKI-1/S1P-RR processed the SREBP2 protein normally and was fully functional in the activation of SREBP2 and ATF6 downstream genes (11
). Thus, contrary to cellular substrates, the SKI-1/S1P processing of viral GPCs seems to critically depend on the efficient autocatalytic processing of the inhibitory prosegment. The underlying reasons for this are currently unclear. One possible explanation could be the markedly different affinities of cellular and viral substrates for SKI-1/S1P. In this scenario, residual amounts of activity in SKI-1/S1P-RR may be sufficient to fully process endogenous cellular transcription factors, whereas the higher load of viral GPCs may overwhelm the system, resulting in the accumulation of the unprocessed GPC, with a negative impact on the formation of infectious virus particles. It is also possible that the intracellular SREBPs and ATF6 are much better SKI-1/S1P substrates than the viral GPCs, hence requiring much less SKI-1/S1P activity for the processing of the former. Alternatively, the distinct activity of SKI-1/S1P-RR toward the SREBP2 protein and viral GPCs may be due to as-yet-unknown differences in the molecular recognition of cellular and viral substrates by SKI-1/S1P. Interestingly, SKI-1/S1P-RR was impaired in the processing of the GPCs of LCMV and LASV that undergo processing in distinct subcellular compartments. This finding indicates that the dependence on SKI-1/S1P autoprocessing is not related to cleavage in a particular compartment but may be a common feature of arenaviral GPCs, which may require high levels of active SKI-1/S1P for their efficient cleavage. This suggests that even a partial inhibition of the cellular SKI-1/S1P activity may be enough to block viral infection without significantly affecting its ability to activate its cognate cellular substrates. This is supported by the observation that the liver-specific knockout of SKI-1/S1P in mice revealed that a more than 95% loss of SKI-1/S1P activity was necessary to observe a loss of SREBP processing and, hence, sterol regulation (45
). Thus, SKI-1/S1P inhibitors may be effective agents against arenavirus infection, since for a lowering of lipid levels, a nearly complete inhibition of SKI-1/S1P is required.
Previous studies identified the SKI-1/S1P-mediated processing of arenavirus GPCs as a promising novel target for the development of antiviral therapeutics (20
). Considering the important roles of SKI-I/S1P in cell biology, the development of SKI-I/S1P inhibitors that specifically block the processing of arenaviral GPCs without affecting cellular substrates remains a major unaddressed and challenging issue. Specific inhibitors of SKI-I/S1P B/B′ prodomain processing are expected to predominantly affect virus infection, with lesser effects on the cellular function of SKI-1/S1P. Our finding that SKI-I/S1P B/B′ prodomain processing is specifically required for the enzyme's ability to efficiently process arenavirus GPCs, but not cellular substrates, may thus have revealed an “Achilles' heel” of the virus to be exploited for the development of novel antiviral strategies.
In the host cell, SKI-1/S1P exists in two active forms: a membrane-associated form that localizes predominantly in the Golgi compartment and, to some extent, endosomal compartments, but not at the cell surface (30
), as well as a soluble secreted form that arises from its autocatalytic shedding (11
). To address the role of the membrane anchorage of SKI-1/S1P in arenavirus GPC processing, we expressed a soluble recombinant form comprising the entire ectodomain in SKI-1/S1P-deficient cells. The efficient rescue of GPC processing and productive virus infection by soluble SKI-1/S1P indicated that membrane anchorage was dispensable for GPC processing. To address a possible specific role for soluble SKI-1/S1P in GPC processing, we employed an SKI-1/S1P form deficient in shedding and found that the lack of shedding had no significant effect on GPC processing. In sum, the data suggest that a critical concentration of enzymatically active SKI-1/S1P in the respective compartment of the secretory pathway is a necessary and sufficient condition for GPC processing.
The efficient processing of the LASV GPC and LCMV GPC by soluble SKI-1/S1P raised the question of whether shed SKI-1/S1P was able to process the viral GPCs at the cell surface. Previous studies showed a normal cell surface transport of unprocessed LCMV GPC (3
) as well as the presence of detectable amounts of uncleaved GPC at the surface of infected cells (16
). By performing medium-swapping experiments, we found no evidence for the processing of the LCMV and LASV GPCs by soluble SKI-1/S1P. These data suggest that arenavirus GPC processing by SKI-1/S1P can occur exclusively in intracellular compartments, a finding that is consistent with the absence of membrane-anchored SKI-1/S1P from the cell surface (30
). In sum, our study revealed the first significant differences in the processing of cellular and viral substrates by SKI-1/S1P. Considering the importance of SKI-1/S1P for normal cell function, and its promise as an antiviral drug target, these findings will help the development of novel therapeutic strategies that specifically target the role of SKI-1/S1P in virus infection.