Crimean-Congo Hemorrhagic Fever Virus Glycoprotein Research Articles

Establishment of two serological methods for detecting IgG and neutralizing antibodies against Crimean-Congo hemorrhagic fever virus glycoprotein.

The Crimean-Congo hemorrhagic fever virus (CCHFV), the most geographically widespread tick-borne virus, is endemic in Africa, Eastern Europe and Asia, with infection resulting in mortality in up to 30% of cases. Currently, there are no approved vaccines or effective therapies available for CCHF. The CCHFV should only be manipulated in the BSL-4 laboratory, which has severely hampered basic seroprevalence studies. In the present study, two antibody detection methods in the forms of an enzyme-linked immunosorbent assay (ELISA) and a surrogate virus neutralization test (sPVNT) were developed using a recombinant glycoprotein (rGP) and a vesicular stomatitis virus (VSV)-based virus bearing the CCHFV recombinant glycoprotein (rVSV/CCHFV) in a biosafety level 2 (BSL-2) laboratory, respectively. The rGP-based ELISA and rVSV/CCHFV-based sVNT were established by using the anti-CCHFV pre-GC mAb 11E7, known as a broadly cross-reactive, potently neutralizing antibody, and their applications as diagnostic antigens were validated for the specific detection of CCHFV IgG and neutralizing antibodies in experimental animals. In two tests, mAb clone 11E7 (diluted at 1:163840 or 512) still displayed positive binding and neutralization, and the presence of antibodies (IgG and neutralizing) against the rGP and rVSV/CCHFV was also determined in the sera from the experimental animals. Both mAb 11E7 and animal sera showed a high reactivity to both antigens, indicating that bacterially expressed rGP and rVSV/CCHFV have good immunoreactivity. Apart from establishing two serological testing methods, their results also demonstrated an imperfect correlation between IgG and neutralizing antibodies. Within this limited number of samples, the rGP and rVSV/CCHFV could be safe and convenient tools with significant potential for research on specific antibodies and serological samples.

LDLR is an entry receptor for Crimean-Congo hemorrhagic fever virus

Crimean-Congo hemorrhagic fever virus (CCHFV) is the most widespread tick-born zoonotic bunyavirus that causes severe hemorrhagic fever and death in humans. CCHFV enters the cell via clathrin-mediated endocytosis which is dependent on its surface glycoproteins. However, the cellular receptors that are required for CCHFV entry are unknown. Here we show that the low density lipoprotein receptor (LDLR) is an entry receptor for CCHFV. Genetic knockout of LDLR impairs viral infection in various CCHFV-susceptible human, monkey and mouse cells, which is restored upon reconstitution with ectopically-expressed LDLR. Mutagenesis studies indicate that the ligand binding domain (LBD) of LDLR is necessary for CCHFV infection. LDLR binds directly to CCHFV glycoprotein Gc with high affinity, which supports virus attachment and internalization into host cells. Consistently, a soluble sLDLR–Fc fusion protein or anti-LDLR blocking antibodies impair CCHFV infection into various susceptible cells. Furthermore, genetic knockout of LDLR or administration of an LDLR blocking antibody significantly reduces viral loads, pathological effects and death following CCHFV infection in mice. Our findings suggest that LDLR is an entry receptor for CCHFV and pharmacological targeting of LDLR may provide a strategy to prevent and treat Crimean-Congo hemorrhagic fever.

Interactome profiling of Crimean-Congo hemorrhagic fever virus glycoproteins

Crimean-Congo hemorrhagic fever virus (CCHFV) is a biosafety level-4 pathogen requiring urgent research and development efforts. The glycoproteins of CCHFV, Gn and Gc, are considered to play multiple roles in the viral life cycle by interactions with host cells; however, these interactions remain largely unclear to date. Here, we analyzed the cellular interactomes of CCHFV glycoproteins and identified 45 host proteins as high-confidence Gn/Gc interactors. These host molecules are involved in multiple cellular biological processes potentially associated with the physiological actions of the viral glycoproteins. Then, we elucidated the role of a representative cellular protein, HAX1. HAX1 interacts with Gn by its C-terminus, while its N-terminal region leads to mitochondrial localization. By the strong interaction, HAX1 sequestrates Gn to mitochondria, thus depriving Gn of its normal Golgi localization that is required for functional glycoprotein-mediated progeny virion packaging. Consistently, the inhibitory activity of HAX1 against viral packaging and hence propagation was further elucidated in the contexts of pseudotyped and authentic CCHFV infections in cellular and animal models. Together, the findings provide a systematic CCHFV Gn/Gc-cell protein-protein interaction map, but also unravel a HAX1/mitochondrion-associated host antiviral mechanism, which may facilitate further studies on CCHFV biology and therapeutic approaches.

GP38 as a vaccine target for Crimean-Congo hemorrhagic fever virus

Crimean-Congo Hemorrhagic Fever Virus (CCHFV) is a tick-borne virus that causes severe hemorrhagic disease in humans. There is a great need for effective vaccines and therapeutics against CCHFV for humans, as none are currently internationally approved. Recently, a monoclonal antibody against the GP38 glycoprotein protected mice against lethal CCHFV challenge. To show that GP38 is required and sufficient for protection against CCHFV, we used three inactivated rhabdoviral-based CCHFV-M vaccines, with or without GP38 in the presence or absence of the other CCHFV glycoproteins. All three vaccines elicited strong antibody responses against the respective CCHFV glycoproteins. However, only vaccines containing GP38 showed protection against CCHFV challenge in mice; vaccines without GP38 were not protective. The results of this study establish the need for GP38 in vaccines targeting CCHFV-M and demonstrate the efficacy of a CCHFV vaccine candidate based on an established vector platform.

Structural characterization of protective non-neutralizing antibodies targeting Crimean-Congo hemorrhagic fever virus

Crimean-Congo Hemorrhagic Fever Virus (CCHFV) causes a life-threatening disease with up to a 40% mortality rate. With no approved medical countermeasures, CCHFV is considered a public health priority agent. The non-neutralizing mouse monoclonal antibody (mAb) 13G8 targets CCHFV glycoprotein GP38 and protects mice from lethal CCHFV challenge when administered prophylactically or therapeutically. Here, we reveal the structures of GP38 bound with a human chimeric 13G8 mAb and a newly isolated CC5-17 mAb from a human survivor. These mAbs bind overlapping epitopes with a shifted angle. The broad-spectrum potential of c13G8 and CC5-17 and the practicality of using them against Aigai virus, a closely related nairovirus were examined. Binding studies demonstrate that the presence of non-conserved amino acids in Aigai virus corresponding region prevent CCHFV mAbs from binding Aigai virus GP38. This information, coupled with in vivo efficacy, paves the way for future mAb therapeutics effective against a wide swath of CCHFV strains.

Cryo-EM structure of glycoprotein C from Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a causative agent of serious hemorrhagic diseases in humans with high mortality rates. CCHFV glycoprotein Gc plays critical roles in mediating virus-host membrane fusion and has been studied extensively as an immunogen. However, the molecular mechanisms involved in membrane fusion and Gc-specific antibody-antigen interactions remain unresolved largely because structural information of this glycoprotein is missing. We designed a trimeric protein including most of the ectodomain region of Gc from the prototype CCHFV strain, IbAr10200, which enabled the cryo-electron microscopy structure to be solved at a resolution of 2.8 ​Å. The structure confirms that CCHFV Gc is a class II fusion protein. Unexpectedly, structural comparisons with other solved Gc trimers in the postfusion conformation revealed that CCHFV Gc adopted hybrid architectural features of the fusion loops from hantaviruses and domain III from phenuiviruses, suggesting a complex evolutionary pathway among these bunyaviruses. Antigenic sites on CCHFV Gc that protective neutralizing antibodies target were mapped onto the CCHFV Gc structure, providing valuable information that improved our understanding of potential neutralization mechanisms of various antibodies.

In silico Design of a Crimean-Congo Hemorrhagic Fever Virus Glycoprotein Multi-Epitope Antigen for Vaccine Development.

There is no licensed vaccine available to prevent the severe tick-borne disease Crimean-Congo hemorrhagic fever (CCHF), caused by the CCHF virus (CCHFV). This study sought to show that a combination of computational methods and data from published literature can inform the design of a multi-epitope antigen for CCHFV that has the potential to be immunogenic. Cytotoxic and helper T-cell epitopes were evaluated on the CCHFV GPC using bioinformatic servers, and this data was combined with work from previous studies to identify potentially immunodominant regions of the GPC. Regions of the GPC were selected for generation of a model multi-epitope antigen in silico, and the percent residue identity and similarity of each region was compared across sequences representing the widespread geographical and ecological distribution of CCHFV. Eleven multi-epitope regions were joined together with flexible linkers in silico to generate a model multi-epitope antigen, termed EPIC, which included 812 (75.7%) of all predicted epitopes. EPIC was predicted to be antigenic by two independent bioinformatic servers, suggesting that multi-epitope antigens should be explored further for CCHFV vaccine development. The results presented within this manuscript provide information for potential targets within the CCHFV GPC for guiding future vaccine development.

Computational formulation and immune dynamics of a multi-peptide vaccine candidate against Crimean-Congo hemorrhagic fever virus

The sole objective of this research is to devise an epitope-based vaccine candidate as prophylaxis for the Crimean-Congo hemorrhagic fever virus (CCHFV) using the knowledge of immunoinformatics and structural biology. Importantly, CCHFV outbreaks have increased in several countries resulting in increased mortality up to 40% due to the lack of prospective medication and an efficient vaccine. In this study, we have used several immunoinformatic tools and servers to anticipate potent B-cell and T-cell epitopes from the CCHFV glycoprotein with the highest antigenicity. After a comprehensive evaluation, a vaccine candidate was designed using 6 CD8+, 3 CD4+, and 7 B-cell epitopes with appropriate linkers. To enhance the vaccine's efficiency, we added Mycobacterium tuberculosis lipoprotein LprG (Rv1411c) to the vaccine as an adjuvant. The final construct was composed of a total of 468 amino acid residues. The epitope included in the construct showed 98% worldwide population coverage. Importantly, the construct appeared as antigenic, immunogenic, soluble, and non-allergenic in nature. To explore further, we modelled the three-dimensional (3D) structure of the constructed vaccine. Our chimeric vaccine showed stable and strong interactions for toll-like receptor 2 (TLR2) found on the cell surface. Moreover, the dynamics simulation of immune response showed elevated levels of cellular immune activity and faster clearance of antigen from the body upon repetitive exposure. Finally, the optimized codon (CAI≈1) ensured the marked translation efficiency of the vaccine protein in E. coli strain K12 bacterium followed by the insertion of construct DNA into the cloning vector pET28a (+). We believe that the designed vaccine chimera could be useful in vaccine development to fight CCHFV outbreaks.

Structural Basis of Neutralization by Human Antibodies Targeting Crimean-Congo Hemorrhagic Fever Virus Glycoprotein Gc

Crimean-Congo hemorrhagic fever virus (CCHFV), the only endemic biosafety level 4 pathogen in Europe, is the world’s most widely distributed tick-borne zoonotic virus with a 30% fatality rate in humans, underlining the need for specific therapeutics and vaccines. The CCHFV membrane fusion glycoprotein Gc is the main target of the host neutralizing antibody response. Here we describe the structure of pre-fusion Gc in complex with the antigen-binding fragments (Fabs) corresponding to a therapeutically potent bispecific antibody as well as the structure of unbound Gc in its post-fusion conformation. Our findings suggest that one Fab blocks insertion of the fusion loops into the target membrane, whereas the other blocks formation of the post-fusion trimer. Combined with yeast display of mutagenized Gc, the structures allowed epitope mapping of a large panel of neutralizing antibodies. These data provide the essential molecular underpinnings for developing vaccines and therapeutics against CCHFV.

Structure and Characterization of Crimean-Congo Hemorrhagic Fever Virus GP38.

Crimean-Congo hemorrhagic fever virus (CCHFV) is the causative agent of the most widespread tick-borne viral infection in humans. CCHFV encodes a secreted glycoprotein (GP38) of unknown function that is the target of a protective antibody. Here, we present the crystal structure of GP38 at a resolution of 2.5 Å, which revealed a novel fold primarily consisting of a 3-helix bundle and a β-sandwich. Sequence alignment and homology modeling showed distant homology between GP38 and the ectodomain of Gn (a structural glycoprotein in CCHFV), suggestive of a gene duplication event. Analysis of convalescent-phase sera showed high titers of GP38 antibodies indicating immunogenicity in humans during natural CCHFV infection. The only protective antibody for CCHFV in an adult mouse model reported to date, 13G8, bound GP38 with subnanomolar affinity and protected against heterologous CCHFV challenge in a STAT1-knockout mouse model. Our data strongly suggest that GP38 should be evaluated as a vaccine antigen and that its structure provides a foundation to investigate functions of this protein in the viral life cycle.IMPORTANCE Crimean-Congo hemorrhagic fever virus (CCHFV) is a priority pathogen that poses a high risk to public health. Due to the high morbidity and mortality rates associated with CCHFV infection, there is an urgent need to develop medical countermeasures for disease prevention and treatment. CCHFV GP38, a secreted glycoprotein of unknown function unique to the Nairoviridae family, was recently shown to be the target of a protective antibody against CCHFV. Here, we present the crystal structure of GP38, which revealed a novel fold with distant homology to another CCHFV glycoprotein that is suggestive of a gene duplication event. We also demonstrate that antibody 13G8 protects STAT1-knockout mice against heterologous CCHFV challenge using a clinical isolate from regions where CCHFV is endemic. Collectively, these data advance our understanding of GP38 structure and antigenicity and should facilitate future studies investigating its function.

Efficient production of a lentiviral system for displaying Crimean-Congo hemorrhagic fever virus glycoproteins reveals a broad range of cellular susceptibility and neutralization ability.

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne disease with a mortality rate of up to 50% in humans. To avoid safety concerns associated with the use of live virus in virus neutralization assays and to detect human serum neutralizing antibodies, we prepared lentiviral particles containing the CCHF glycoprotein (lenti-CCHFV-GP). Incorporation of the GP into the lentiviral particle was confirmed by electron microscopy and Western blotting. Lenti-CCHFV-GP was found to be able to infect a wide range of cell lines, including BHK-21, HeLa, HepG2, and AsPC-1 cells. In addition, lenti-CCHFV-GP was successfully used as an alternative to CCHFV for the detection of neutralizing antibodies. Sera collected from CCHF survivors neutralized lenti-CCHFV-GP particles in a dose-dependent manner. Our results suggest that the lenti-CCHFV-GP pseudovirus can be used as a safe tool for neutralization assays in low-containment laboratories.

Vesicular Stomatitis Virus-Based Vaccine Protects Mice against Crimean-Congo Hemorrhagic Fever

Crimean-Congo hemorrhagic fever virus (CCHFV), a tick-borne bunyavirus, can cause a life-threatening hemorrhagic syndrome in humans but not in its animal host. The virus is widely distributed throughout southeastern Europe, the Middle East, Africa, and Asia. Disease management has proven difficult and there are no broadly licensed vaccines or therapeutics. Recombinant vesicular stomatitis viruses (rVSV) expressing foreign glycoproteins (GP) have shown promise as experimental vaccines for several viral hemorrhagic fevers. Here, we developed and assessed a replication competent rVSV vector expressing the CCHFV glycoprotein precursor (GPC), which encodes CCHFV structural glycoproteins. This construct drives strong expression of CCHFV-GP, in vitro. Using these vectors, we vaccinated STAT-1 knock-out mice, an animal model for CCHFV. The vector was tolerated and 100% efficacious against challenge from a clinical strain of CCHFV. Anti-CCHFV-GP IgG and neutralizing antibody titers were observed in surviving animals. This study demonstrates that a rVSV expressing only the CCHFV-GP has the potential to serve as a replication competent vaccine platform against CCHF infections.

Crimean-Congo hemorrhagic fever virus entry into host cells occurs through the multivesicular body and requires ESCRT regulators.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne bunyavirus causing outbreaks of severe disease in humans, with a fatality rate approaching 30%. There are no widely accepted therapeutics available to prevent or treat the disease. CCHFV enters host cells through clathrin-mediated endocytosis and is subsequently transported to an acidified compartment where the fusion of virus envelope with cellular membranes takes place. To better understand the uptake pathway, we sought to identify host factors controlling CCHFV transport through the cell. We demonstrate that after passing through early endosomes in a Rab5-dependent manner, CCHFV is delivered to multivesicular bodies (MVBs). Virus particles localized to MVBs approximately 1 hour after infection and affected the distribution of the organelle within cells. Interestingly, blocking Rab7 activity had no effect on association of the virus with MVBs. Productive virus infection depended on phosphatidylinositol 3-kinase (PI3K) activity, which meditates the formation of functional MVBs. Silencing Tsg101, Vps24, Vps4B, or Alix/Aip1, components of the endosomal sorting complex required for transport (ESCRT) pathway controlling MVB biogenesis, inhibited infection of wild-type virus as well as a novel pseudotyped vesicular stomatitis virus (VSV) bearing CCHFV glycoprotein, supporting a role for the MVB pathway in CCHFV entry. We further demonstrate that blocking transport out of MVBs still allowed virus entry while preventing vesicular acidification, required for membrane fusion, trapped virions in the MVBs. These findings suggest that MVBs are necessary for infection and are the sites of virus-endosome membrane fusion.

MOLECULAR FARMING, AN EFFECTIVE SYSTEM FOR THE PRODUCTION OF IMMUNOGENIC CRIMEAN-CONGO HEMORRHAGIC FEVER VIRUS GLYCOPROTEIN

The main aim of this study was to obtain the Crimean-Congo hemorrhagic fever virus (CCHFV) glycoprotein, through either stable transgenic plants or using a transient expression system, and determine the yield, quality and finally the immunogenicity of the plant-made CCHFV glycoprotein in a mouse model. We designed and synthesized a codon-optimized G1/G2 gene from the G1 and G2 parts of the CCHFV glycoprotein by bioinformatic analysis. The synthetic construct was cloned into a plant expression vector and tobacco plants were both transiently and stably transformed. The transgenic plantlets or tobacco-derived hairy roots confirmed by PCR and Southern blot analyses. The intact 98 kDa G1/G2 glycoprotein was produced by a transient expression system at as much as 3.3 mg/kg fresh weight. The recombinant G1/G2 protein was analyzed in stable lines by G1/G2 ELISA and Western blot. The yield in the transgenic hairy root line was significantly higher than that in transgenic tobacco lines. Finally, the immunogenicity of the plant-made G1/G2 glycoprotein was evaluated by its subcutaneous administration in mice when compared with positive and negative controls. This plant-purified G1/G2 protein produced a high titer of anti-CCHFV glycoprotein IgG antibodies in mice.

Hypomorphic Mutation in the Site-1 Protease Mbtps1 Endows Resistance to Persistent Viral Infection in a Cell-Specific Manner

The prototypic arenavirus lymphocytic choriomeningitis virus (LCMV), which naturally persists in rodents, represents a model for HIV, HBV, and HCV. Cleavage of the viral glycoprotein precursor by membrane-bound transcription factor peptidase, site 1 (Mbtps1 or site-1 protease), is crucial for the life cycle of arenaviruses and therefore represents a potential target for therapy. Recently, we reported a viable hypomorphic allele of Mbtps1 (woodrat) encoding a protease with diminished enzymatic activity. Using the woodrat allele, we examine the role of Mbtps1 during persistent LCMV infection. Surprisingly, Mbtps1 inhibition limits persistent but not acute viral infection and is associated with an organ/cell type-specific decrease in viral titers. Analysis of bone marrow-derived dendritic cells from woodrat mice supports their specific role in resolving persistent viral infection. These results support in vivo targeting of Mbtps1 in the treatment of arenavirus infections and demonstrate a critical role for dendritic cells in persistent viral infections.

Detection of Crimean-Congo hemorrhagic fever virus glycoprotein antigens (38.19)

Abstract Crimean-Congo hemorrhagic fever (CCHF) is a viral infection caused by Crimean-Congo hemorrhagic fever virus (CCHFV), a tick-borne virus (Nairovirus) of Bunyaviridae. Viral antigens of CCHFV can be detected by using various immunologic methodologies. We developed two methodologies to detect viral antigens of CCHFV using polyclonal antibodies isolated either from human plasma or from hyperimmunized horse serum. The first methodology is a western blot using human polyclonal antibodies. The second one is an antigen ELISA using horse antibodies. CCHFV isolates from vero-cell cultures were used as a source of the antigens. Additionally, CCHFV isolates obtained from Centers for Disease Control and Prevention (CDC) of USA were used as positive controls. The human polyclonal antibody pool detected several glycoproteins of various sizes ranging from approximately 35 KDa to 80 KDa or higher indicating that humans can develop antibody responses to different epitopes of the viral glycoproteins. Also, the ELISA can be used successfully to detect CCHFV with quite reproducible results. Our results indicated that both antibody sources could be convenient for rapid detection of CCHFV glycoprotein antigens.

Crimean-Congo Hemorrhagic Fever Virus Glycoprotein Processing by the Endoprotease SKI-1/S1P Is Critical for Virus Infectivity

Crimean-Congo hemorrhagic fever virus (CCHFV) causes severe human disease. The CCHFV medium RNA encodes a polyprotein which is proteolytically processed to yield the glycoprotein precursors PreGn and PreGc, followed by structural glycoproteins Gn and Gc. Subtilisin kexin isozyme-1/site-1 protease (SKI-1/S1P) plays a central role in Gn processing. Here we show that CCHFV-infected cells deficient in SKI-1/S1P produce no infectious virus, although PreGn and PreGc accumulated normally in the Golgi apparatus, the site of virus assembly. Only nucleoprotein-containing particles which lacked virus glycoproteins (Gn/Gc or PreGn/PreGc) were secreted. Complementation of SKI-1/S1P-deficient cells with a SKI-1/S1P expression vector restored release of infectious virus (>10(6) PFU/ml), confirming that SKI-1/S1P processing is required for incorporation of viral glycoproteins. SKI-1/S1P may represent a promising antiviral target.

Crimean-congo hemorrhagic fever virus glycoprotein precursor is cleaved by Furin-like and SKI-1 proteases to generate a novel 38-kilodalton glycoprotein.

Crimean-Congo hemorrhagic fever virus (genus Nairovirus, family Bunyaviridae) genome M segment encodes an unusually large (in comparison to members of other genera) polyprotein (1,684 amino acids in length) containing the two major structural glycoproteins, Gn and Gc, that are posttranslationally processed from precursors PreGn and PreGc by SKI-1 and SKI-1-like proteases, respectively. The characteristics of the N-terminal 519 amino acids located upstream of the mature Gn are unknown. A highly conserved furin/proprotein convertase (PC) cleavage site motif (RSKR247) is located between the variable N-terminal region that is predicted to have mucin-like properties and the rest of PreGn. Mutational analysis of the RSKR247 motif and use of a specific furin/PC inhibitor and brefeldin A demonstrate that furin/PC cleavage occurs at the RSKR247 motif of PreGn as the protein transits the trans Golgi network and generates a novel glycoprotein designated GP38. Immunoprecipitation analysis identified two additional proteins, GP85 and GP160, which contain both mucin and GP38 domain regions, and whose generation does not involve furin/PC cleavage. Consistent with glycosylation predictions, heavy O-linked glycosylation and moderate levels of N-glycans were detected in the GP85 and GP160 proteins, both of which contain the mucin domain. GP38, GP85, and GP160 are likely soluble proteins based on the lack of predicted transmembrane domains, their detection in virus-infected cell supernatants, and the apparent absence from virions. Analogy with soluble glycoproteins and mucin-like proteins encoded by other hemorrhagic fever-associated RNA viruses suggests these proteins could play an important role in viral pathogenesis.

Cellular Localization and Antigenic Characterization of Crimean-Congo Hemorrhagic Fever Virus Glycoproteins

Crimean-Congo hemorrhagic fever virus (CCHFV), a member of the genus Nairovirus of the family Bunyaviridae, causes severe disease with high rates of mortality in humans. The CCHFV M RNA segment encodes the virus glycoproteins G(N) and G(C). To understand the processing and intracellular localization of the CCHFV glycoproteins as well as their neutralization and protection determinants, we produced and characterized monoclonal antibodies (MAbs) specific for both G(N) and G(C). Using these MAbs, we found that G(N) predominantly colocalized with a Golgi marker when expressed alone or with G(C), while G(C) was transported to the Golgi apparatus only in the presence of G(N). Both proteins remained endo-beta-N-acetylglucosaminidase H sensitive, indicating that the CCHFV glycoproteins are most likely targeted to the cis Golgi apparatus. Golgi targeting information partly resides within the G(N) ectodomain, because a soluble version of G(N) lacking its transmembrane and cytoplasmic domains also localized to the Golgi apparatus. Coexpression of soluble versions of G(N) and G(C) also resulted in localization of soluble G(C) to the Golgi apparatus, indicating that the ectodomains of these proteins are sufficient for the interactions needed for Golgi targeting. Finally, the mucin-like and P35 domains, located at the N terminus of the G(N) precursor protein and removed posttranslationally by endoproteolysis, were required for Golgi targeting of G(N) when it was expressed alone but were dispensable when G(C) was coexpressed. In neutralization assays on SW-13 cells, MAbs to G(C), but not to G(N), prevented CCHFV infection. However, only a subset of G(C) MAbs protected mice in passive-immunization experiments, while some nonneutralizing G(N) MAbs efficiently protected animals from a lethal CCHFV challenge. Thus, neutralization of CCHFV likely depends not only on the properties of the antibody, but on host cell factors as well. In addition, nonneutralizing antibody-dependent mechanisms, such as antibody-dependent cell-mediated cytotoxicity, may be involved in the in vivo protection seen with the MAbs to G(C).

Development of Protein-based Inhibitors of the Proprotein of Convertase SKI-1/S1P: PROCESSING OF SREBP-2, ATF6, AND A VIRAL GLYCOPROTEIN

Processing of membrane-bound transcription factors such as sterol regulatory element-binding proteins (SREBPs) and the ER-stress response factor ATF6, and glycoproteins of some hemorrhagic fever viruses are initiated by the proprotein convertase SKI-1/S1P. So far, no cellular protein-based inhibitor of the hydrophobic-amino acid specific SKI-1 is known. The prosegment of the basic-amino acid specific convertases (e.g. furin and PC5) or alpha(1)-PDX, a variant of alpha(1)-antitrypsin (alpha(1)-AT) exhibiting an RIPR(358) sequence at the reactive site loop, were shown to potently inhibit these secretory proteinases. Accordingly, we tested the SKI-1-inhibitory potential of various point mutants of either the 198 amino acid preprosegment of SKI-1-(1-198) or alpha(1)-AT. Transient transfections data showed that, out of numerous mutants studied, the R134E prosegment mutant or the alpha(1)-AT reactive site loop variants RRVL(358), RRYL(358) and RRIL(358) are the best specific cellular inhibitors of SKI-1. The observed inhibition of the processing of endogenous SREBP-2, exogenous ATF6 and a PDGF-A (RRLL(86)) variant were >55% and reach approximately 80% in stable transfectants. We also show that SKI-1 forms SDS-stable complexes with these alpha(1)-AT variants, but not with wild-type alpha(1)-AT or alpha(1)-PDX. Finally, these inhibitors were also shown to affect the processing and stability of the Crimean-Congo hemorrhagic fever virus glycoprotein.