J Virol. 2011 Sep;85(17):8502-13. doi: 10.1128/JVI.02600-10. Epub 2011 Jun 22.
Ebolavirus delta-peptide immunoadhesins inhibit marburgvirus and ebolavirus cell entry.
Radoshitzky SR1, Warfield KL, Chi X, Dong L, Kota K, Bradfute SB, Gearhart JD, Retterer C, Kranzusch PJ, Misasi JN, Hogenbirk MA, Wahl-Jensen V, Volchkov VE, Cunningham JM, Jahrling PB, Aman MJ, Bavari S, Farzan M, Kuhn JH.
Abstract
With
the exception of Reston and Lloviu viruses, filoviruses
(marburgviruses, ebolaviruses, and "cuevaviruses") cause severe viral
hemorrhagic fevers in humans.
Filoviruses use a class I fusion protein, GP(1,2), to bind to an unknown, but shared, cell surface receptor to initiate virus-cell fusion. In addition to GP(1,2), ebolaviruses and cuevaviruses, but not marburgviruses, express two secreted glycoproteins, soluble GP (sGP) and small soluble GP (ssGP).
All three glycoproteins have identical N termini that include the receptor-binding region (RBR) but differ in their C termini.
We evaluated the effect of the secreted ebolavirus glycoproteins on marburgvirus and ebolavirus cell entry, using Fc-tagged recombinant proteins. Neither sGP-Fc nor ssGP-Fc bound to filovirus-permissive cells or inhibited GP(1,2)-mediated cell entry of pseudotyped retroviruses.
Surprisingly, several Fc-tagged Δ-peptides, which are small C-terminal cleavage products of sGP secreted by ebolavirus-infected cells, inhibited entry of retroviruses pseudotyped with Marburg virus GP(1,2), as well as Marburg virus and Ebola virus infection in a dose-dependent manner and at low molarity despite absence of sequence similarity to filovirus RBRs.
Fc-tagged Δ-peptides from three ebolaviruses (Ebola virus, Sudan virus, and Taï Forest virus) inhibited GP(1,2)-mediated entry and infection of viruses comparably to or better than the Fc-tagged RBRs, whereas the Δ-peptide-Fc of an ebolavirus nonpathogenic for humans (Reston virus) and that of an ebolavirus with lower lethality for humans (Bundibugyo virus) had little effect.
These data indicate that Δ-peptides are functional components of ebolavirus proteomes. They join cathepsins and integrins as novel modulators of filovirus cell entry, might play important roles in pathogenesis, and could be exploited for the synthesis of powerful new antivirals.
Sitaatti sivuilta 11-13
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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, 41). 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 (Fig. 1). 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, 32). 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
Filoviruses use a class I fusion protein, GP(1,2), to bind to an unknown, but shared, cell surface receptor to initiate virus-cell fusion. In addition to GP(1,2), ebolaviruses and cuevaviruses, but not marburgviruses, express two secreted glycoproteins, soluble GP (sGP) and small soluble GP (ssGP).
All three glycoproteins have identical N termini that include the receptor-binding region (RBR) but differ in their C termini.
We evaluated the effect of the secreted ebolavirus glycoproteins on marburgvirus and ebolavirus cell entry, using Fc-tagged recombinant proteins. Neither sGP-Fc nor ssGP-Fc bound to filovirus-permissive cells or inhibited GP(1,2)-mediated cell entry of pseudotyped retroviruses.
Surprisingly, several Fc-tagged Δ-peptides, which are small C-terminal cleavage products of sGP secreted by ebolavirus-infected cells, inhibited entry of retroviruses pseudotyped with Marburg virus GP(1,2), as well as Marburg virus and Ebola virus infection in a dose-dependent manner and at low molarity despite absence of sequence similarity to filovirus RBRs.
Fc-tagged Δ-peptides from three ebolaviruses (Ebola virus, Sudan virus, and Taï Forest virus) inhibited GP(1,2)-mediated entry and infection of viruses comparably to or better than the Fc-tagged RBRs, whereas the Δ-peptide-Fc of an ebolavirus nonpathogenic for humans (Reston virus) and that of an ebolavirus with lower lethality for humans (Bundibugyo virus) had little effect.
These data indicate that Δ-peptides are functional components of ebolavirus proteomes. They join cathepsins and integrins as novel modulators of filovirus cell entry, might play important roles in pathogenesis, and could be exploited for the synthesis of powerful new antivirals.
Sitaatti sivuilta 11-13
----
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, 41). 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 (Fig. 1). 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, 32). 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 (Fig. 7).
The two conserved cysteine residues in ebolavirus Δ-peptides (Fig. 8 gives sequence information) and the homodimerization of the Δ-peptide precursor, pre-sGP (3, 11, 53),
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.
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