No abstract

Ebolavirus (EBOV) entry into cells requires proteolytic disassembly of the viral glycoprotein, GP. This proteolytic processing, unusually extensive for an enveloped virus entry protein, is mediated by cysteine cathepsins, a family of endosomal/lysosomal proteases. Previous work has shown that cleavage of GP by cathepsin B (CatB) is specifically required to generate a critical entry intermediate. The functions of this intermediate are not well understood. We used a forward genetic strategy to investigate this CatB-dependent step. Specifically, we generated a replication-competent recombinant vesicular stomatitis virus bearing EBOV GP as its sole entry glycoprotein and used it to select viral mutants resistant to a CatB inhibitor. We obtained mutations at six amino acid positions in GP that independently confer complete resistance. All of the mutations reside at or near the GP1-GP2 intersubunit interface in the membrane-proximal base of the prefusion GP trimer. This region forms a part of the "clamp" that holds the fusion subunit GP2 in its metastable prefusion conformation. Biochemical studies suggest that most of the mutations confer CatB independence not by altering specific cleavage sites in GP but rather by inducing conformational rearrangements in the prefusion GP trimer that dramatically enhance its susceptibility to proteolysis. The remaining mutants did not show the preceding behavior, indicating the existence of multiple mechanisms for acquiring CatB independence during entry. Altogether, our findings suggest that CatB cleavage is required to facilitate the triggering of viral membrane fusion by destabilizing the prefusion conformation of EBOV GP.Filoviruses are enveloped, filamentous, nonsegmented negative-sense RNA viruses that can cause a deadly hemorrhagic fever with case fatality rates in excess of 90% (see references 4, 20, and 37 for recent reviews). All known filoviruses belong to one of two genera: Ebolavirus (EBOV), consisting of the five species Zaire (ZEBOV), Côte d 'Ivoire, Sudan, Reston, and Bundibugyo (tentative); and Marburgvirus, consisting of the single Lake Victoria species (21, 62).Cell entry by filoviruses is mediated by their envelope glycoprotein, GP (60,68). Mature GP is a trimer of three disulfide-linked GP1-GP2 heterodimers. GP1 and GP2 are generated by endoproteolytic cleavage of the GP0 precursor polypeptide by a furin-like protease during transport to the cell surface (31, 39, 63, 69). The membrane-distal subunit, GP1, mediates viral adhesion to host cells (10,18,38,42,56,59) and regulates the activity of the transmembrane subunit, GP2, which catalyzes fusion of viral and cellular membrane bilayers (30,39,41,64,65). The consequence of membrane fusion is cytoplasmic delivery of the viral nucleocapsid cargo. Lee et al. (39) recently solved the crystal structure of a ZEBOV GP prefusion trimer lacking the heavily glycosylated GP1 mucin domain (Muc) and the GP2 transmembrane domain (see Fig. 5). The three GP1 subunits together form a bowl-like structure encircled by sequences fro...

Ebola virus (EBOV) has been reported to enter cultured cell lines via a dynamin-2-independent macropinocytic pathway or clathrin-mediated endocytosis. The route(s) of productive EBOV internalization into physiologically relevant cell types remain unexplored, and viral-host requirements for this process are incompletely understood. Here, we use electron microscopy and complementary chemical and genetic approaches to demonstrate that the viral glycoprotein, GP, induces macropinocytic uptake of viral particles into cells. GP's highly-glycosylated mucin domain is dispensable for virus-induced macropinocytosis, arguing that interactions between other sequences in GP and the host cell surface are responsible. Unexpectedly, we also found a requirement for the large GTPase dynamin-2, which is proposed to be dispensable for several types of macropinocytosis. Our results provide evidence that EBOV uses an atypical dynamin-dependent macropinocytosis-like entry pathway to enter Vero cells, adherent human peripheral blood-derived monocytes, and a mouse dendritic cell line.

The IG20 gene is overexpressed in human tumors and cancer cell lines, and encodes at least four splice variants (SVs) namely, IG20pa, MADD, IG20-SV2 and DENN-SV. Earlier, gain-of-function studies showed that IG20-SVs can exhibit diverse functions and play a critical role in cell proliferation and apoptosis. Expression of exogenous IG20pa or DENN-SV rendered cells either susceptible or resistant to induced apoptosis, respectively, whereas MADD and IG20-SV2 had no apparent effect. In order to understand the contrasting effects of the IG20-SVs in a physiologically more relevant system, we expressed exonspecific small hairpin RNAs (shRNAs) to selectively knockdown specific IG20-SVs. Consistent with an earlier study, knockdown of all IG20-SVs resulted in spontaneous apoptosis of HeLa and PA-1 cells. In addition, we unambiguously demonstrated that knockdown of MADD can render cells susceptible to spontaneous apoptosis but had no discernible effect on cell proliferation, colony size or cell cycle progression. Moreover, expression of MADD alone, and not DENN-SV, in the absence of endogenous IG20-SVs was sufficient to prevent spontaneous apoptosis. Our results show the utility of shRNAs for selective knockdown of particular IG20-SVs and their potential therapeutic value in cancer. Further, they demonstrate that MADD alone is sufficient and necessary for cancer cell survival.

Ebola virus (EBOV) glycoprotein (GP), responsible for mediating host-cell attachment and membrane fusion, contains a heavily glycosylated mucin-like domain hypothesized to shield GP from neutralizing antibodies. To test whether the mucin-like domain inhibits the production and function of anti-GP antibodies, we vaccinated mice with Ebola virus-like particles (VLPs) that express vesicular stomatitis virus G, wild-type EBOV GP (EBGP), EBOV GP without its mucin-like domain (ΔMucGP), or EBOV GP with a Crimean-Congo hemorrhagic fever virus mucin-like domain substituted for the EBOV mucin-like domain (CMsubGP). EBGP-VLP immunized mice elicited significantly higher serum antibody titers toward EBGP or its mutants, as detected by western blot analysis, than did VLP-ΔMucGP. However, EBGP-, ΔMucGP- and CMsubGP-VLP immunized mouse sera contained antibodies that bound to cell surface-expressed GP at similar levels. Furthermore, low but similar neutralizing antibody titers, measured against a vesicular stomatitis virus (VSV) expressing EBGP or ΔMucGP, were present in EBGP, ΔMucGP, and CMsubGP sera, although a slightly higher neutralizing titer (2- to 2.5-fold) was detected in ΔMucGP sera. We conclude that the EBOV GP mucin-like domain can increase relative anti-GP titers, however these titers appear to be directed, at least partly, to denatured GP. Furthermore, removing the mucin-like domain from immunizing VLPs has modest impact on neutralizing antibody titers in serum.

The IG20 (insulinoma-glucagonoma) gene is implicated in cancer cell proliferation, apoptosis, and survival (1-12), as well as neurotransmission (13-15), neurodegeneration (16), and guanine nucleotide exchange (17)(18)(19)(20). This gene is overexpressed in cancer cells and tissues relative to healthy controls and can encode at least four different splice variants (SVs), 3 namely IG20pa, MADD/DENN, IG20-SV2, and DENN-SV (4). The MADD and DENN-SV isoforms are constitutively expressed, whereas expression of IG20pa and IG20-SV2 appears to be regulated.Earlier gain-of-function studies showed that expression of exogenous IG20pa can render cells more susceptible to induced apoptosis, whereas DENN-SV can confer resistance to the above treatments (4, 6 -8). Interestingly, loss-of-function studies using oligodeoxynucleotides showed that knockdown of all IG20-SVs can result in spontaneous apoptosis of cancer cells, but not normal cells, in vitro as well as in vivo (5, 9). These studies demonstrate an indispensable role for IG20 in cancer cell survival but fail to reveal the relative importance of different IG20-SVs.Using shRNAs that specifically target exon 15, which is expressed in all expressed isoforms of IG20 and designated Mid, or exons 13L and 16, which are differentially expressed in IG20-SVs, we were able to selectively knock down either all or select combinations of IG20-SVs in HeLa and PA-1 cells and determine their role in cell survival. Knockdown of MADD but not other splice variants resulted in spontaneous apoptosis of cancer cells; these cells could be rescued only upon re-expression of MADD but not other splice variants. These studies demonstrated that MADD is required and sufficient for the survival of cancer cells (21).In the present study, we have shown that MADD abrogation can render cells more susceptible to TRAIL-induced apoptosis. Further, we demonstrate that MADD can interact with the death receptors (DRs) but not with either FADD (Fas-associated death domain) or caspase-8, and that the enhanced apoptosis results from activation of caspase-8 at the DRs without an apparent increase in the recruitment of death-inducing signaling complex (DISC) components. These results strongly suggest that under physiological conditions MADD can act as a negative regulator of caspase-8 activity in the DISC.

We investigated the physiological role of endogenous MAPKactivating death domain-containing protein (MADD), a splice variant of the IG20 gene, that can interact with TNFR1 in tumor necrosis factor-␣ (TNF␣)-induced activation of NF-B, MAPK, ERK1/2, JNK, and p38. Using exon-specific short hairpin RNAs expressing lentiviruses, we knocked down the expression of all IG20 splice variants or MADD, which is overexpressed in cancer cells. Abrogation of MADD expression rendered cells highly susceptible to TNF␣-induced apoptosis in the absence of cycloheximide. It also resulted in a dramatic loss in TNF␣-induced activation of MAPK without any apparent effect on NF-B activation. This observation was substantiated by an accompanying loss in the activation of p90RSK, a key downstream target of MAPK, whereas the NF-B-regulated interleukin 6 levels remained unaffected. Endogenous MADD knockdown, however, did not affect epidermal growth factor-induced MAPK activation thereby demonstrating the specific requirement of MADD for TNF receptor-mediated MAPK activation. Re-expression of short hairpin RNA-resistant MADD in the absence of endogenous IG20 expression rescued the cells from TNF␣-induced apoptosis. The requirement for MADD was highly specific for TNF␣-induced activation of MAPK but not the related JNK and p38 kinases. Loss of MADD expression resulted in reduced Grb2 and Sos1/2 recruitment to the TNFR1 complex and decreased Ras and MEKK1/2 activation. These results demonstrate the essential role of MADD in protecting cancer cells from TNF␣-induced apoptosis by specifically activating MAPKs through Grb2 and Sos1/2 recruitment, and its potential as a novel cancer therapeutic target.Genes in higher organisms generate alternate transcripts that are translated into closely related proteins with different functions. Perturbations in the tightly regulated alternate splicing of key genes in cancers can result in the accumulation of select splice variants of a particular gene or suppression of others. For instance, some cancers are known to preferentially express the more oncogenic and constitutively active RON⌬ (where RON is recepteur d'origine nantais receptor tyrosine kinase) splice variant of RON receptor tyrosine kinase (1). The study of genes that undergo alternative splicing is therefore likely to unravel novel therapeutic targets against cancer (2-4). The IG20 (insulinoma-glucagonoma) is one such gene previously identified in our laboratory (4) that is implicated in cancer cell survival, proliferation, apoptosis, and other regulated functions through alternative splicing (5-20). The IG20 gene encodes at least six different splice variants (SVs) 3 of which the expression of KIAA0358 and IG20-SV4 isoforms is restricted to certain neuronal tissues (17), with KIAA acting as a Rab3a-GEP (20 -22). The other four, namely IG20pa, MADD, IG20-SV2, and DENN-SV, are expressed more ubiquitously (4). Of these, MADD and DENN-SV are constitutively expressed, whereas the IG20pa and IG20-SV2 may or may not be expressed.Among the IG20 isoforms, ...

The IG20 gene undergoes alternative splicing resulting in the differential expression of six putative splice variants. Four of these (IG20pa, MADD, IG20-SV2, and DENN-SV) are expressed in virtually all human tissues. However, investigations examining alternative splicing of the IG20 gene to date have been largely limited to nonneural malignant and nonmalignant cells. In this study, we investigated the expression of alternative splice isoforms of the IG20 gene in human neuroblastoma cells. We found that six IG20 splice variants (IG20-SVs) were expressed in two human neuroblastoma cell lines (SK-N-SH and SH-SY5Y), highlighted by the expression of two unique splice isoforms (i.e., KIAA0358 and IG20-SV4). Similarly, we found enriched expression of these two IG20-SVs in human neural tissues derived from cerebral cortex, hippocampus, and, to a lesser extent, spinal cord. Using gain-of-function studies and siRNA technology, we determined that these ''neural-enriched isoforms'' exerted significant and contrasting effects on vulnerability to apoptosis in neuroblastoma cells. Specifically, expression of KIAA0358 exerted a potent antiapoptotic effect in both the SK-N-SH and SH-SY5Y neuroblastoma cell lines, whereas expression of IG20-SV4 had proapoptotic effects directly related to the activation of caspase-8 in these cells, which have minimal or absent constitutive caspase-8 expression. These data indicate that the pattern of expression of these neural-enriched IG20-SVs regulates the expression and activation of caspase-8 in certain neuroblastoma cells, and that manipulation of IG20-SV expression pattern may represent a potent therapeutic strategy in the therapy of neuroblastoma and perhaps other cancers. [Cancer Res 2008;68(18):7352-61]