We are currently addressing two hypotheses as to the role of Δ-peptides in pathogenesis. First, the peptides could prevent superinfection of producer cells by binding to a cell surface filovirus receptor. Low-level expression of EBOV spike protein enhances, but high-level expression inhibits, cell transduction with MLV/EBOV, and low-level expression did not have an effect on MLV/MARV cell entry, whereas high-level expression inhibited it (28
). These data imply that ebolavirus-infected cells may be prone to superinfection early in infection and that Δ-peptide could counter this susceptibility. Δ-Peptides also could interfere with steps of the virus entry process other than cell surface factor binding, such as glycoprotein processing by cathepsins or the yet still hypothetical protease-requiring factor that acts after cathepsin cleavage (6
). Preliminary experiments suggest, however, that the peptides do not affect the activity of cathepsin B in in vitro
enzymatic assays (data not shown). Second, it is possible that Δ-peptides prevent the association of maturing ebolavirus GP1,2
with a receptor or a coreceptor during synthesis in the ER of an infected cell and thereby prevent trapping of budding progeny virions. Both strategies are, indeed, being used by other viruses. For instance, HIV-1 Nef downregulates the expression of the HIV-1 receptor CD4 to prevent superinfection (1
) and circumvents premature fusion by inhibiting the engagement of CD4 with the spike protein gp160 in the Golgi network (27
). Overexpression of CD4 in HIV-1-infected cells, on the other hand, reduces infectivity via the sequestering of gp160 by CD4 (22
). Furthermore, HIV-1 Vpu mediates endoplasmic reticulum (ER)-associated protein degradation (ERAD) of CD4 to prevent the formation of CD4-gp160 complexes (58
). In the case of influenza viruses, neuraminidase (NA) limits superinfection of the producer cell by cleaving sialic acids, the receptors of these viruses (15
), an activity that is also necessary for the release of progeny viruses (55
). Marburgviruses, which do not express Δ-peptides, may have developed an alternative way to prevent superinfection and/or tethering to the plasma membrane.
The EBOV RBR is present in GP1
(residues 54 to 201), sGP, and ssGP. We therefore hypothesized that EBOV sGP-Fc and ssGP-Fc bind to the surface of filovirus-permissive cells, but not filovirus-resistant cells, and inhibit filovirus entry, thus mimicking previously described properties of GP1
-Fc and its mutants (21
). Unexpectedly, we discovered that the C-terminal cleavage product that is produced during sGP maturation, Δ-peptide, fulfilled these expectations when fused to Fc, whereas sGP-Fc and ssGP-Fc did not (). The latter observation can be explained if one assumes that the unique C termini of sGP and ssGP influence their tertiary and quaternary structures and thus their ability to modulate entry. Indeed, antibodies from survivors of EBOV infection preferentially react with either GP1,2
or sGP (31
). Moreover, GP1,2
assumes a trimeric conformation (23
), whereas sGP assembles as a parallel homodimer using two intermolecular disulfide bonds at the N and C termini of each monomer (3
). ssGP is secreted as a homodimer that is held together by a single intermolecular disulfide bond (33
). The oligomeric state of Δ-peptides remains to be determined. Initial experiments evaluating SUDV Δ-peptide tagged N-terminally with a FLAG tag instead of a C-terminal Fc tag demonstrate that this most likely monomeric variant does not inhibit Marburg virus infection (). The two conserved cysteine residues in ebolavirus Δ-peptides ( gives sequence information) and the homodimerization of the Δ-peptide precursor, pre-sGP (3
), suggest that Δ-peptides are most likely dimers. If that indeed is the case, it is plausible that the inhibitory effect of Δ-peptide is dependent on its dimeric state, and therefore only an Fc Δ-peptide fusion (dimeric due to the Fc tag) acts as an effective inhibitor. We have thus far failed in raising useful antibodies against Δ-peptides using commercial services and also in procuring sufficient amounts of sera of nonhuman primates that were infected with filoviruses but survived long enough to mount an antibody response. Therefore, it remains to be seen in which quantity and which tissues Δ-peptides are produced in a filovirus-infected animal.
Fig. 7. Evaluation of FLAG-tagged Sudan Δ-peptide as an inhibitor of MARV infection. Fc constructs, N-terminally FLAG-tagged SUDV Δ-peptide, or FLAG tag alone was incubated with Vero E6 cells at the indicated concentrations. Cells were then infected (more ...)
Fig. 8. Sequence alignment of ebolavirus Δ-peptides. Highlighted residues are shaded according to the BLOSUM62 score, which takes into account physico-chemical properties and amino acid characteristics, such as charge or hydrophobicity (4). Conservation (more ...)
The observed effects of Fc-tagged ebolavirus Δ-peptides are most intriguing since thus far they were regarded as nonfunctional by-products of sGP maturation (54
) and because the peptides' primary sequences are not similar to the filovirus RBRs or to any other known protein. SUDV and TAFV Δ-Fc inhibited infectious EBOV and MARV infection more efficiently than EBOV Δ-Fc (), suggesting that they could be developed as novel antivirals. Consequently, we have begun to evaluate their efficacy in rodent models of filovirus disease.
Interestingly, Δ-Fc of RESTV, the only ebolavirus nonpathogenic for humans (2
), did not inhibit EBOV or MARV infection (). Since RESTV is as virulent for nonhuman primates, such as cynomolgus macaques, as EBOV and MARV, this observation leads to the fascinating hypothesis that Δ-peptides could play important roles in filovirus pathogenesis in different hosts or in filovirus persistence in their reservoirs—now thought to be frugivorous bats (24
). Indeed, recently performed experiments demonstrated that a recombinant guinea pig-adapted EBOV tailored not to produce sGP/Δ-peptide is severely attenuated in guinea pigs compared to parent guinea pig-adapted virus. Furthermore, passaging of wild-type Ebola virus in Vero E6 cells led to the evolution of variants with a modified GP gene editing site (“8U virus”), which produces predominantly GP1,2
and little sGP/Δ-peptide, whereas infection of guinea pigs with 8U virus rapidly resulted in the evolution of “7U viruses,” thereby “correcting” the ratio of GP1,2
to sGP/Δ-peptide to large amounts of the latter. These data suggest that the wild-type 7U virus has a selective advantage in animals for yet unknown reasons and that the production of larger amounts of sGP and thereby Δ-peptide are important factors in infection (52
Computational analyses revealed that the sequences of Δ-peptides are highly conserved among the variants of each individual ebolavirus (). For instance, the Δ-peptide sequences of all known SUDV strains, isolated over 3 decades from humans, are 100% identical. This extent of conservation is also true for human EBOV variants and mouse- and guinea pig-adapted viruses (EBOV-May BALB/c and -8mc-N3/-GPA-P7, respectively), whereas western lowland gorilla isolates (EBOV-GOR) and central chimpanzee isolates (EBOV-CH) show slight variability. RESTV Δ-peptides of viruses isolated from cynomolgus macaques in 1989, 1992, and 1996 are 100% conserved as well. On the other hand, Δ-peptides of the individual ebolaviruses (the recently described BDBV [46
] and EBOV, RESTV, SUDV, and TAFV) vary greatly among each other but share common elements, suggesting that the function of these peptides is important and is conserved across different ebolavirus hosts ().
Recently, RESTV was isolated from domestic pigs from Bulacan (BulaA) and Pangasinan (PangA and PangE) in the Philippines (2
). Surprisingly, the PangA and PangE Δ-Fcs of these isolates differ slightly, and the BulA Δ-Fc differs drastically, from the other known RESTV Δ-peptides (). The BulaA isolate is additionally characterized by a deleterious mutation in the sGP/Δ-peptide furin cleavage site (RARR → RAQR), suggesting that this most divergent RESTV Δ-peptide might not be produced during infection. Interestingly, one of our experiments suggests that RESTV-BulaA Δ-Fc inhibits at least MARV infection (B). This raises the fascinating hypothesis that Reston viruses have evolved not to produce functional Δ-peptides, either by scrambling their sequence (macaque-derived RESTV) or by preventing their secretion (RESTV-BulaA). Unfortunately, we did not have access to sufficient amounts of high-quality stocks of either macaque- or pig-derived RESTV and could therefore not yet evaluate the effect of RESTV and other filovirus Δ-peptides on RESTV infection. Likewise, one experiment (B) indicates that BDBV Δ-Fc may also be either nonfunctional or specific for BDBV as it had no influence on MARV infection. The function of this particular Δ-peptide will be evaluated in greater detail once we gain access to BDBV and TAFV stocks.
Surprisingly, the conserved sequence in the center of the peptides does not seem to be crucial for their function, as the mutation of several of its residues in SUDV Δ-Fc to alanines (ΔW18A-Fc, ΔR21A-Fc, and ΔW26A-Fc) did not impair cell binding or transduction inhibition considerably (C and E). Similarly, introduction of mutations into the N-terminal half of the peptide (ΔT9A-Fc and ΔS14A-Fc) did not decrease the inhibitory effect on GP1,2
-mediated cell entry. In contrast, consecutive truncation of the C terminus of SUDV Δ-Fc(Δ1-39-Fc, Δ1-33-Fc, Δ1-28-Fc, and Δ1-17-Fc) did result in progressive loss of function. In addition, a chimera consisting of the N-terminal half of SUDV Δ-peptide and the C-terminal half of RESTV Δ-peptide (SRΔ-Fc) behaved like full-length RESTV Δ-Fc and inhibited GP1,2
-mediated cell entry only minimally, whereas a chimera consisting of the N-terminal half of RESTV Δ-peptide and the C-terminal half of SUDV Δ-peptide (RSΔ-Fc) mimicked SUDV Δ-Fc and inhibited transduction efficiently. The Δ28-48-Fc mutant, which consists only of SUDV Δ-peptide's C terminus, barely inhibited GP1,2
-mediated cell entry. These results indicate the following: (i) SUDV Δ-peptide possibly requires its N terminus to ensure correct folding, although the N terminus is not involved in attaching to SUDV Δ-peptide's cell-surface binding partner; (ii) the inhibitory function of SUDV Δ-Fc may be mediated by its C terminus; (iii) T9 of SUDV Δ-Fc, which is probably O
-glycosylated, is not important for function; and (iv) residues S14, R21, W18, and W26 (all located within the N-terminal half of the peptide) and C29/C38 (in the C-terminal half) may not play crucial roles in entry inhibition. A closer look at the C termini of ebolavirus Δ-peptides led us to the conclusion that they might exert their inhibitory effects through charged interactions rather than a common structure as no sequence similarity is obvious (). This is not unusual as structural disorder is a hallmark of proteins associated with signaling or regulation (9
) and is found among numerous virus proteins that are encoded by overlapping ORFs, allowing them to adopt rapidly interconverting structural shapes (36
). Clearly, this hypothesis will have to be tested through the creation of additional mutants.
The observations that several Fc-tagged ebolavirus Δ-peptides bind with high efficiency to filovirus-permissive cells but not to filovirus-resistant cells and even more so that they inhibit MARV spike protein-mediated cell entry as well as EBOV and MARV infection clearly suggest that these peptides interfere with a molecule that is engaged during cell entry by both ebolaviruses and marburgviruses.