Reverse Genetics Demonstrates that Proteolytic Processing of the Ebola Virus Glycoprotein Is Not Essential for Replication in Cell Culture

Ebola virus, a prime example of an emerging pathogen, causes fatal hemorrhagic fever in humans and in nonhuman primates. Identification of major determinants of Ebola virus pathogenicity has been hampered by the lack of effective strategies for experimental mutagenesis. Here we exploit a reverse genetics system that allows the generation of Ebola virus from cloned cDNA to engineer a mutant Ebola virus with an altered furin recognition motif in the glycoprotein (GP). When expressed in cells, the GP of the wild type, but not of the mutant, virus was cleaved into GP1 and GP2. Although posttranslational furin-mediated cleavage of GP was thought to be an essential step in Ebola virus infection, generation of a viable mutant Ebola virus lacking a furin recognition motif in the GP cleavage site demonstrates that GP cleavage is not essential for replication of Ebola virus in cell culture.     

The Ebola Virus Glycoprotein Contributes to but Is Not Sufficient for Virulence In Vivo

Among the Ebola viruses most species cause severe hemorrhagic fever in humans; however, Reston ebolavirus (REBOV) has not been associated with human disease despite numerous documented infections. While the molecular basis for this difference remains unclear, in vitro evidence has suggested a role for the glycoprotein (GP) as a major filovirus pathogenicity factor, but direct evidence for such a role in the context of virus infection has been notably lacking. In order to assess the role of GP in EBOV virulence, we have developed a novel reverse genetics system for REBOV, which we report here. Together with a previously published full-length clone for Zaire ebolavirus (ZEBOV), this provides a unique possibility to directly investigate the role of an entire filovirus protein in pathogenesis. To this end we have generated recombinant ZEBOV (rZEBOV) and REBOV (rREBOV), as well as chimeric viruses in which the glycoproteins from these two virus species have been exchanged (rZEBOV-RGP and rREBOV-ZGP). All of these viruses could be rescued and the chimeras replicated with kinetics similar to their parent virus in tissue culture, indicating that the exchange of GP in these chimeric viruses is well tolerated. However, in a mouse model of infection rZEBOV-RGP demonstrated markedly decreased lethality and prolonged time to death when compared to rZEBOV, confirming that GP does indeed contribute to the full expression of virulence by ZEBOV. In contrast, rREBOV-ZGP did not show any signs of virulence, and was in fact slightly attenuated compared to rREBOV, demonstrating that GP alone is not sufficient to confer a lethal phenotype or exacerbate disease in this model. Thus, while these findings provide direct evidence that GP contributes to filovirus virulence in vivo, they also clearly indicate that other factors are needed for the acquisition of full virulence.     

Induction of Cell-Cell Fusion by Ebola Virus Glycoprotein: Low pH Is Not a Trigger

Ebola virus (EBOV) is a highly pathogenic filovirus that causes hemorrhagic fever in humans and animals. Currently, how EBOV fuses its envelope membrane within an endosomal membrane to cause infection is poorly understood. We successfully measure cell-cell fusion mediated by the EBOV fusion protein, GP, assayed by the transfer of both cytoplasmic and membrane dyes. A small molecule fusion inhibitor, a neutralizing antibody, as well as mutations in EBOV GP known to reduce viral infection, all greatly reduce fusion. By monitoring redistribution of small aqueous dyes between cells and by electrical capacitance measurements, we discovered that EBOV GP-mediated fusion pores do not readily enlarge—a marked difference from the behavior of other viral fusion proteins. EBOV GP must be cleaved by late endosome-resident cathepsins B or L in order to become fusion-competent. Cleavage of cell surface-expressed GP appears to occur in endosomes, as evidenced by the fusion block imposed by cathepsin inhibitors, agents that raise endosomal pH, or an inhibitor of anterograde trafficking. Treating effector cells with a recombinant soluble cathepsin B or thermolysin, which cleaves GP into an active form, increases the extent of fusion, suggesting that a fraction of surface-expressed GP is not cleaved. Whereas the rate of fusion is increased by a brief exposure to acidic pH, fusion does occur at neutral pH. Importantly, the extent of fusion is independent of external pH in experiments in which cathepsin activity is blocked and EBOV GP is cleaved by thermolysin. These results imply that low pH promotes fusion through the well-known pH-dependent activity of cathepsins; fusion induced by cleaved EBOV GP is a process that is fundamentally independent of pH. The cell-cell fusion system has revealed some previously unappreciated features of EBOV entry, which could not be readily elucidated in the context of endosomal entry.     

Pseudorabies Virus Glycoprotein gK Is a Virion Structural Component Involved in Virus Release but Is Not Required for Entry

The pseudorabies virus (PrV) gene homologous to herpes simplex virus type 1 (HSV-1) UL53, which encodes HSV-1 glycoprotein K (gK), has recently been sequenced (J. Baumeister, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 69:5560–5567, 1995). To identify the corresponding protein, a rabbit antiserum was raised against a 40-kDa glutathione S-transferase–gK fusion protein expressed in Escherichia coli. In Western blot analysis, this serum detected a 32-kDa polypeptide in PrV-infected cell lysates as well as a 36-kDa protein in purified virion preparations, demonstrating that PrV gK is a structural component of virions. After treatment of purified virions with endoglycosidase H, a 34-kDa protein was detected, while after incubation with N-glycosidase F, a 32-kDa protein was specifically recognized. This finding indicates that virion gK is modified by N-linked glycans of complex as well as high-mannose type. For functional analysis, the UL53 open reading frame was interrupted after codon 164 by insertion of a gG-lacZ expression cassette into the wild-type PrV genome (PrV-gKβ) or by insertion of the bovine herpesvirus 1 gB gene into a PrV gB⁻ genome (PrV-gK^gB). Infectious mutant virus progeny was obtained only on complementing gK-expressing cells, suggesting that gK has an important function in the replication cycle. After infection of Vero cells with either gK mutant, only single infected cells or small foci of infected cells were visible. In addition, virus yield was reduced approximately 30-fold, and penetration kinetics showed a delay in entry which could be compensated for by phenotypic gK complementation. Interestingly, the plating efficiency of PrV-gKβ was similar to that of wild-type PrV on complementing and noncomplementing cells, pointing to an essential function of gK in virus egress but not entry. Ultrastructurally, virus assembly and morphogenesis of PrV gK mutants in noncomplementing cells were similar to wild-type virus. However, late in infection, numerous nucleocapsids were found directly underneath the plasma membrane in stages typical for the entry process, a phenomenon not observed after wild-type virus infection and also not visible after infection of gK-complementing cells. Thus, we postulate that presence of gK is important to inhibit immediate reinfection.Herpesvirions are complex structures consisting of a nucleoprotein core, capsid, tegument, and envelope. They comprise at least 30 structural proteins (35). Pseudorabies virus (PrV), a member of the Alphaherpesvirinae, is an economically important animal pathogen, causing Aujeszky’s disease in swine. It is also highly pathogenic for most other mammals except higher primates, including humans (28, 45), and a wide range of cultured cells from different species support productive virus replication, reflecting the wide in vivo host range. Envelope glycoproteins play major roles in the early and late interactions between virion and host cell. They are required for virus entry and participate in release of free virions and viral spread by direct cell-to-cell transmission (27, 37). For PrV, 10 glycoproteins, designated gB, gC, gD, gE, gG, gH, gI, gL, gM, and gN, have been characterized (20, 27); these glycoproteins are involved in the attachment of virion to host cell (gC and gD), fusion of viral envelope and cellular cytoplasmic membrane (gB, gD, gH, and gL), spread from infected to noninfected cells (gB, gE, gH, gI, gL, and gM), and egress (gC, gE, and gI) (27, 37). Homologs of these glycoproteins are also present in other alphaherpesviruses (37). The gene coding for a potential 11th PrV glycoprotein, gK, has been described recently (3), but the protein and its function have not been identified.The product of the homologous UL53 open reading frame (ORF) of herpes simplex virus type 1 (HSV-1) is gK (13, 32). gK was detected in nuclear membranes and in membranes of the endoplasmic reticulum but was not observed in the plasma membrane (14). Also, it did not appear to be present in purified virion preparations (15). The latter result was surprising since earlier studies identified several mutations in HSV-1 gK resulting in syncytium-inducing phenotypes (7, 14), which indicates participation of gK in membrane fusion events during HSV-1 infection. Moreover, HSV-1 mutants in gK exhibited a delayed entry into noncomplementing cells, which is difficult to reconcile with absence of gK from virions (31). Mutants deficient for gK expression have been isolated and investigated by different groups (16, 17). Mutant F-gKβ carries a lacZ gene insertion in the HSV-1 strain F gK gene, which interrupts the ORF after codon 112 (16). In mutant ΔgK, derived from HSV-1 KOS, almost all of the UL53 gene was deleted (17). Both mutants formed small plaques on Vero cells, and virus yield was reduced to an extent which varied with the different confluencies of the infected cells, cell types, and mutants used for infection. However, both HSV-1 gK mutants showed a defect in efficient translocation of virions from the cytoplasm to the extracellular space, and only a few enveloped virions were present in the extracellular space after infection of Vero cells (16, 17). The authors therefore suggested that HSV-1 gK plays a role in virion transport during egress.Different routes of final envelopment and egress of alphaherpesvirions are discussed. It has been suggested that HSV-1 nucleocapsids acquire their envelope at the inner nuclear membrane and are transported as enveloped particles through the endoplasmic reticulum to the Golgi stacks, where glycoproteins are modified in situ during transport (5, 6, 19, 39), although other potential egress pathways cannot be excluded (4). In contrast, maturation of varicella-zoster virus and PrV involves primary envelopment at the nuclear membrane, followed by release of nucleocapsids into the cytoplasm and secondary envelopment in the trans-Golgi area (10, 12, 43). Final egress of virions appears to occur via transport vesicles containing one or more virus particles by fusion of vesicle and cell membrane. The possibility of different routes of virion egress is supported by studies of other proteins involved in egress, e.g., the UL20 proteins of HSV-1 and PrV and the PrV UL3.5 protein, which lacks a homolog in the HSV-1 genome (1, 8, 9). In UL20-negative HSV-1, virions accumulated in the perinuclear cisterna of Vero cells (1), while PrV UL20⁻ virions accumulated and were retained in cytoplasmic vesicles (9). PrV UL3.5 is important for budding of nucleocapsids into Golgi-derived vesicles during secondary envelopment (8). Thus, there appear to be profound differences in the egress pathways. Since HSV-1 gK was also implicated in egress, we were interested in identifying the PrV homolog and analyzing its function.     

Cathepsin B & L Are Not Required for Ebola Virus Replication

Ebola virus (EBOV), family Filoviridae, emerged in 1976 on the African continent. Since then it caused several outbreaks of viral hemorrhagic fever in humans with case fatality rates up to 90% and remains a serious Public Health concern and biothreat pathogen. The most pathogenic and best-studied species is Zaire ebolavirus (ZEBOV). EBOV encodes one viral surface glycoprotein (GP), which is essential for replication, a determinant of pathogenicity and an important immunogen. GP mediates viral entry through interaction with cellular surface molecules, which results in the uptake of virus particles via macropinocytosis. Later in this pathway endosomal acidification activates the cysteine proteases Cathepsin B and L (CatB, CatL), which have been shown to cleave ZEBOV-GP leading to subsequent exposure of the putative receptor-binding and fusion domain and productive infection. We studied the effect of CatB and CatL on in vitro and in vivo replication of EBOV. Similar to previous findings, our results show an effect of CatB, but not CatL, on ZEBOV entry into cultured cells. Interestingly, cell entry by other EBOV species (Bundibugyo, Côte d''Ivoire, Reston and Sudan ebolavirus) was independent of CatB or CatL as was EBOV replication in general. To investigate whether CatB and CatL have a role in vivo during infection, we utilized the mouse model for ZEBOV. Wild-type (control), catB^−/− and catL^−/− mice were equally susceptible to lethal challenge with mouse-adapted ZEBOV with no difference in virus replication and time to death. In conclusion, our results show that CatB and CatL activity is not required for EBOV replication. Furthermore, EBOV glycoprotein cleavage seems to be mediated by an array of proteases making targeted therapeutic approaches difficult.     

Proteolytic Processing of the Human Immunodeficiency Virus Envelope Glycoprotein Precursor Decreases Conformational Flexibility

The mature envelope glycoprotein (Env) spike on the surface of human immunodeficiency virus type 1 (HIV-1) virions is derived by proteolytic cleavage of a trimeric gp160 glycoprotein precursor. Remarkably, proteolytic processing of the HIV-1 Env precursor results in changes in Env antigenicity that resemble those associated with glutaraldehyde fixation. Apparently, proteolytic processing of the HIV-1 Env precursor decreases conformational flexibility of the Env trimeric complex, differentially affecting the integrity/accessibility of epitopes for neutralizing and nonneutralizing antibodies.     

Antibody Derived Peptides for Detection of Ebola Virus Glycoprotein

Background

Current Ebola virus (EBOV) detection methods are costly and impractical for epidemic scenarios. Different immune-based assays have been reported for the detection and quantification of Ebola virus (EBOV) proteins. In particular, several monoclonal antibodies (mAbs) have been described that bind the capsid glycoprotein (GP) of EBOV GP. However, the currently available platforms for the design and production of full-length mAbs are cumbersome and costly. The use of antibody fragments, rather than full-length antibodies, might represent a cost-effective alternative for the development of diagnostic and possibly even therapeutic alternatives for EBOV.

Methods/Principal Findings

We report the design and expression of three recombinant anti-GP mAb fragments in Escherichia coli cultures. These fragments contained the heavy and light variable portions of the three well-studied anti-GP full-length mAbs 13C6, 13F6, and KZ52, and are consequently named scFv-13C6, scFv-13F6, and Fab-KZ52, respectively. All three fragments exhibited specific anti-GP binding activity in ELISA experiments comparable to that of full-length anti-GP antibodies (i.e., the same order of magnitude) and they are easily and economically produced in bacterial cultures.

Conclusion/Significance

Antibody fragments might represent a useful, effective, and low cost alternative to full-length antibodies in Ebola related capture and diagnostics applications.     

The Product of the Herpes Simplex Virus Type 1 UL25 Gene Is Required for Encapsidation but Not for Cleavage of Replicated Viral DNA

The herpes simplex virus type 1 (HSV-1) UL25 gene contains a 580-amino-acid open reading frame that codes for an essential protein. Previous studies have shown that the UL25 gene product is a virion component (M. A. Ali et al., Virology 216:278–283, 1996) involved in virus penetration and capsid assembly (C. Addison et al., Virology 138:246–259, 1984). In this study, we describe the isolation of a UL25 mutant (KUL25NS) that was constructed by insertion of an in-frame stop codon in the UL25 open reading frame and propagated on a complementing cell line. Although the mutant was capable of synthesis of viral DNA, it did not form plaques or produce infectious virus in noncomplementing cells. Antibodies specific for the UL25 protein were used to demonstrate that KUL25NS-infected Vero cells did not express the UL25 protein. Western immunoblotting showed that the UL25 protein was associated with purified, wild-type HSV A, B, and C capsids. Transmission electron microscopy indicated that the nucleus of Vero cells infected with KUL25NS contained large numbers of both A and B capsids but no C capsids. Analysis of infected cells by sucrose gradient sedimentation analysis confirmed that the ratio of A to B capsids was elevated in KUL25NS-infected Vero cells. Following restriction enzyme digestion, specific terminal fragments were observed in DNA isolated from KUL25NS-infected Vero cells, indicating that the UL25 gene was not required for cleavage of replicated viral DNA. The latter result was confirmed by pulsed-field gel electrophoresis (PFGE), which showed the presence of genome-size viral DNA in KUL25NS-infected Vero cells. DNase I treatment prior to PFGE demonstrated that monomeric HSV DNA was not packaged in the absence of the UL25 protein. Our results indicate that the product of the UL25 gene is required for packaging but not cleavage of replicated viral DNA.     

Effects of N-Linked Glycan on Lassa Virus Envelope Glycoprotein Cleavage,Infectivity, and Immune Response

Lassa virus (LASV) belongs to the Mammarenavirus genus (family Arenaviridae) and causes severe hemorrhagic fever in humans. The glycoprotein complex (GPC) contains eleven N-linked glycans that play essential roles in GPC functionalities such as cleavage, transport, receptor recognition, epitope shielding, and immune response. We used three mutagenesis strategies (asparagine to glutamine, asparagine to alanine, and serine/tyrosine to alanine mutants) to abolish individual glycan chain on GPC and found that all the three strategies led to cleavage inefficiency on the 2nd (N89), 5th (N119), or 8th (N365) glycosylation motif. To evaluate N to Q mutagenesis for further research, it was found that deletion of the 2nd (N89Q) or 8th (N365Q) glycan completely inhibited the transduction efficiency of pseudotyped particles. We further investigated the role of individual glycan on GPC-mediated immune response by DNA immunization of mice. Deletion of the individual 1st (N79Q), 3rd (N99Q), 5th (N119Q), or 6th (N167Q) glycan significantly enhanced the proportion of effector CD4+ cells, whereas deletion of the 1st (N79Q), 2nd (N89Q), 3rd (N99Q), 4th (N109Q), 5th (N119Q), 6th (N167Q), or 9th (N373Q) glycan enhanced the proportion of CD8+ effector T cells. Deletion of specific glycan improves the Th1-type immune response, and abolishment of glycan on GPC generally increases the antibody titer to the glycan-deficient GPC. However, the antibodies from either the mutant or WT GPC-immunized mice show little neutralization effect on wild-type LASV. The glycan residues on GPC provide an immune shield for the virus, and thus represent a target for the design and development of a vaccine.     

Foamy Virus Gag p71-p68 Cleavage Is Required for Template Switch of the Reverse Transcriptase

In contrast to orthoretroviruses, processing of foamy viral p71 Gag is limited to a single cleavage site. Nevertheless, Gag maturation is essential for infectivity, but deletion of p3 results in a modest drop in infectivity. Here, we show that Gag processing of p71 to p68 and p3 is essential for full-length cDNA synthesis, while inactivation of Gag cleavage results in cDNAs containing only the RU5 region; cDNAs encompassing the U3 region were almost undetectable.     

The Myeloid LSECtin Is a DAP12-Coupled Receptor That Is Crucial for Inflammatory Response Induced by Ebola Virus Glycoprotein

Fatal Ebola virus infection is characterized by a systemic inflammatory response similar to septic shock. Ebola glycoprotein (GP) is involved in this process through activating dendritic cells (DCs) and macrophages. However, the mechanism is unclear. Here, we showed that LSECtin (also known as CLEC4G) plays an important role in GP-mediated inflammatory responses in human DCs. Anti-LSECtin mAb engagement induced TNF-α and IL-6 production in DCs, whereas silencing of LSECtin abrogated this effect. Intriguingly, as a pathogen-derived ligand, Ebola GP could trigger TNF-α and IL-6 release by DCs through LSECtin. Mechanistic investigations revealed that LSECtin initiated signaling via association with a 12-kDa DNAX-activating protein (DAP12) and induced Syk activation. Mutation of key tyrosines in the DAP12 immunoreceptor tyrosine-based activation motif abrogated LSECtin-mediated signaling. Furthermore, Syk inhibitors significantly reduced the GP-triggered cytokine production in DCs. Therefore, our results demonstrate that LSECtin is required for the GP-induced inflammatory response, providing new insights into the EBOV-mediated inflammatory response.     

Dimerization of the Pseudomonas aeruginosa Translocator Chaperone PcrH Is Required for Stability,Not Function

Type III secretion systems rely on hydrophobic translocator proteins that form a pore in the host cell membrane to deliver effector proteins into targeted host cells. These translocator proteins are stabilized in the cytoplasm and targeted for export with the help of specific chaperone proteins. In Pseudomonas aeruginosa, the chaperone of the pore-forming translocator proteins is PcrH. Although all translocator chaperones dimerize, the location of the dimerization interface is in dispute. Moreover, it has been reported that interfering with dimerization interferes with chaperone function. However, binding of P. aeruginosa chaperone PcrH to its cognate secretion substrate, PopD, results in dissociation of the PcrH dimer in vitro, arguing that dimerization of PcrH is likely not important for substrate binding or targeting translocators for export. We demonstrate that PcrH dimerization occurs in vivo in P. aeruginosa and used a genetic screen to identify a dimerization mutant of PcrH. The mutant protein is fully functional in that it can both stabilize PopB and PopD in the cytoplasm and promote their export via the type III secretion system. The location of the mutation suggests that the dimerization interface of PcrH mirrors that of the Yersinia homolog SycD and not the dimerization interface that had previously been reported for PcrH based on crystallographic evidence. Finally, we present data that the dimerization mutant of PcrH is less stable than the wild-type protein in P. aeruginosa, suggesting that the function of dimerization is stabilization of PcrH in the absence of its cognate cargo.