Phenylalanines at positions 88 and 159 of Ebolavirus envelope glycoprotein differentially impact envelope function

The envelope glycoprotein (GP) of Ebolavirus (EBOV) mediates viral entry into host cells. Through mutagenesis, we and other groups reported that two phenylalanines at positions 88 and 159 of GP are critical for viral entry. However, it remains elusive which steps of viral entry are impaired by F88 or F159 mutations and how. In this study, we further characterized these two phenylalanines through mutagenesis and examined the impact on GP expression, function, and structure. Our data suggest that F159 plays an indirect role in viral entry by maintaining EBOV GP's overall structure. In contrast, we did not detect any evidence for conformational differences in GP with F88 mutations. The data suggest that F88 influences viral entry during a step after cathepsin processing, presumably impacting viral fusion.     

Characterization of Marburg virus glycoprotein in viral entry

One major determinant of host tropism for filoviruses is viral glycoprotein (GP), which is involved in receptor binding and viral entry. Compared to Ebola GP (EGP), Marburg GP (MGP) is less well characterized in viral entry. In this study, using a human immunodeficiency virus-based pseudotyped virus as a surrogate system, we have characterized the role of MGP in viral entry. We have shown that like EGP, the mucin-like region of MGP (289-501) is not essential for virus entry. We have developed a viral entry interference assay for filoviruses, and using this assay, we have demonstrated that transfection of EGP or MGP in target cells can interfere with EGP/HIV and MGP/HIV pseudotyped virus entry in a dose-dependent manner. These results are consistent with the notion that Ebola and Marburg viruses use the same or a related host molecule(s) for viral entry. Substitutions of the non-conserved residues in MGP1 did not impair MGP-mediated viral entry. Unlike that of EGP1, individual substitutions of many conserved residues of MGP1 exerted severe defects in MGP expression, incorporation to HIV virions, and thus its ability to mediate viral entry. These results indicate that MGP is more sensitive to substitutions of the conserved residues, suggesting that MGP may fold differently from EGP.     

Characterization of Zaire ebolavirus glycoprotein-specific monoclonal antibodies

Zaire ebolavirus (ZEBOV) can be transmitted by human-to-human contact and causes acute haemorrhagic fever with case fatality rates up to 90%. There are no effective therapeutic or prophylactic treatments available. The sole transmembrane glycoprotein (GP) is the key target for developing neutralizing antibodies. In this study, recombinant VSVΔG/ZEBOVGP was used to generate monoclonal antibodies (MAbs) against the ZEBOV GP. A total of 8 MAbs were produced using traditional hybridoma cell fusion technology, and then characterized by ELISA using ZEBOV VLPs, Western blotting, an immunofluorescence assay, and immunoprecipitation. All 8 MAbs worked in IFA and IP, suggesting that they are all conformational MAbs, however six of them recognized linearized epitopes by WB. ELISA results demonstrated that one MAb bound to a secreted GP (sGP 1-295aa); three bind to a part of the mucin domain (333-458aa); three MAbs recognized epitopes on the C-terminal domain of GP1 (296-501aa); and one bound to full length GP (VLPs/GP1,2 ΔTm). Using a mouse model these MAbs were evaluated for their therapeutic capacity during a lethal infection. All 8 MAb improved survival rates by 33%-100% against a high dose lethal challenge with mouse-adapted ZEBOV. This work has important implications for further development of vaccines and immunotherapies for ZEBOV infection.     

Determination of Specific Antibody Responses to the Six Species of Ebola and Marburg Viruses by Multiplexed Protein Microarrays

Infectious hemorrhagic fevers caused by the Marburg and Ebola filoviruses result in human mortality rates of up to 90%, and there are no effective vaccines or therapeutics available for clinical use. The highly infectious and lethal nature of these viruses highlights the need for reliable and sensitive diagnostic methods. We assembled a protein microarray displaying nucleoprotein (NP), virion protein 40 (VP40), and glycoprotein (GP) antigens from isolates representing the six species of filoviruses for use as a surveillance and diagnostic platform. Using the microarrays, we examined serum antibody responses of rhesus macaques vaccinated with trivalent (GP, NP, and VP40) virus-like particles (VLP) prior to infection with the Marburg virus (MARV) (i.e., Marburg marburgvirus) or the Zaire virus (ZEBOV) (i.e., Zaire ebolavirus). The microarray-based assay detected a significant increase in antigen-specific IgG resulting from immunization, while a greater level of antibody responses resulted from challenge of the vaccinated animals with ZEBOV or MARV. Further, while antibody cross-reactivities were observed among NPs and VP40s of Ebola viruses, antibody recognition of GPs was very specific. The performance of mucin-like domain fragments of GP (GP mucin) expressed in Escherichia coli was compared to that of GP ectodomains produced in eukaryotic cells. Based on results with ZEBOV and MARV proteins, antibody recognition of GP mucins that were deficient in posttranslational modifications was comparable to that of the eukaryotic cell-expressed GP ectodomains in assay performance. We conclude that the described protein microarray may translate into a sensitive assay for diagnosis and serological surveillance of infections caused by multiple species of filoviruses.     

Alanine substitution of conserved residues in the cytoplasmic tail of herpes simplex virus gB can enhance or abolish cell fusion activity and viral entry

Herpes simplex virus (HSV) glycoprotein B (gB) is one of the four viral glycoproteins required for viral entry and cell fusion and is highly conserved among herpesviruses. Mutants of HSV type 2 gB were generated by substituting conserved residues in the cytoplasmic tail with alanine or by deleting 41 amino acids from the C-terminus. Some of the mutations abolished cell fusion activity and also prevented transport of gB to the cell surface, identifying residues in the gB cytoplasmic tail that are critical for intracellular transport of this glycoprotein. These mutations also prevented production of infectious virus, possibly because the mutant forms of gB were not transported to the site of envelopment. Other mutations, particularly the deletion, significantly enhanced cell fusion activity. These mutations, as well as others described previously, identify regions of the gB cytoplasmic domain that modulate cell fusion activity.     

Interferon regulatory factor-1, interferon-beta,and reovirus-induced myocarditis

Marburg virus, a filovirus, contains only one transmembrane protein (GP) which is responsible for receptor recognition on target cells. GP, a type I membrane protein of approximately 220 kDa, is acylated and highly glycosylated carrying N- and O-linked sugar side chains. GP is transported through the exocytotic pathway toward the plasma membrane where budding of virions takes place. In the trans-Golgi network, GP is proteolytically activated by the prohormone convertase furin into two subunits GP(1) and GP(2). In the present paper, we provide evidence that GP undergoes an additional posttranslational modification; it is phosphorylated at its ectodomain. Phosphorylation takes place at serine residues between amino acid 260 and 273. The respective serines are located in conserved recognition sites for luminal protein kinases (protein kinase CK II and Golgi casein kinase). Consistent with this data, it was found that GP was phosphorylated in the Golgi apparatus of the expressing HeLa cells before cleavage of the molecule. GP is the first example of a viral glycoprotein with a phosphorylated ectodomain.     

Chinese hamster ovary cell lines selected for resistance to ebolavirus glycoprotein mediated infection are defective for NPC1 expression

Ebolavirus causes severe hemorrhagic fever in humans and non-human primates. Entry of ebolavirus is mediated by the viral glycoprotein, GP; however, the required host factors have not been fully elucidated. A screen utilizing a recombinant Vesicular Stomatitis Virus (VSV) encoding Zaire ebolavirus GP identified four Chinese Hamster Ovary (CHO) cell lines resistant to GP-mediated viral entry. Susceptibility to vectors carrying SARS coronavirus S or VSV-G glycoproteins suggests that endocytic and processing pathways utilized by other viruses are intact in these cells. A cathepsin-activated form of the ebolaviral glycoprotein did not overcome the entry restriction, nor did expression of several host factors previously described as important for ebolavirus entry. Conversely, expression of the recently described ebolavirus host entry factor Niemann-Pick Type C1 (NPC1) restored infection. Resistant cells encode distinct mutations in the NPC1 gene, resulting in loss of protein expression. These studies reinforce the importance of NPC1 for ebolavirus entry.     

Antibody-mediated neutralization of Ebola virus can occur by two distinct mechanisms

Human Ebola virus causes severe hemorrhagic fever disease with high mortality and there is no vaccine or treatment. Antibodies in survivors occur early, are sustained, and can delay infection when transferred into nonhuman primates. Monoclonal antibodies (mAbs) from survivors exhibit potent neutralizing activity in vitro and are protective in rodents. To better understand targets and mechanisms of neutralization, we investigated a panel of mAbs shown previously to react with the envelope glycoprotein (GP). While one non-neutralizing mAb recognized a GP epitope in the nonessential mucin-like domain, the rest were specific for GP1, were neutralizing, and could be further distinguished by reactivity with secreted GP. We show that survivor antibodies, human KZ52 and monkey JP3K11, were specific for conformation-dependent epitopes comprising residues in GP1 and GP2 and that neutralization occurred by two distinct mechanisms; KZ52 inhibited cathepsin cleavage of GP whereas JP3K11 recognized the cleaved, fusion-active form of GP.     

Epitopic mapping of linear and conformation-dependent antigenic determinants on GP5 of five U.S. bluetongue viruses.

Two distinct antigenic determinants of the major outer capsid protein, GP5, of five U.S. bluetongue viruses have been identified and mapped using monoclonal and oligoclonal antibodies. One antigenic site, identified by oligoclonal antibody AK-15, was found to be common and conserved in all five U.S. BTV serotypes. This linear epitope was located between amino acid residues 175 and 189 (ALQREAAERSEDEIK). The second determinant identified by monoclonal antibody 34.7 was present in BTV-2, -10, -11, and -17 but absent in BTV-13. The binding of this monoclonal antibody to GP5 could be blocked specifically by one of three short synthetic peptides located among amino acid residues 33-42 (KAAERFAESE), 159-168 (EKILKEEDSK), and 206-215 (EIERDGMQEE), indicating that this antigenic determinant is conformation-dependent. Oligoclonal antibody (AK-15) reacted with denatured GP5 immobilized on nitrocellulose membrane after Western transfer as well as with native GP5 present on the surface of purified BTV virions. Monoclonal antibody (34.7) reacted only with denatured GP5 but not native GP5 using an ELISA assay. However, these two antigenic epitopes alone did not elicit detectable neutralizing antibodies as determined by plaque reduction assay.     

In vivo analysis of the cell cycle dependent association of the bovine papillomavirus E2 protein and ChlR1

Clathrin-mediated endocytosis was previously implicated as one of the cellular pathways involved in filoviral glycoprotein mediated viral entry into target cells. Here we have further dissected the requirements for different components of this pathway in Ebola versus Marburg virus glycoprotein (GP) mediated viral infection. Although a number of these components were involved in both cases; Ebola GP-dependent viral entry specifically required the cargo recognition proteins Eps15 and DAB2 as well as the clathrin adaptor protein AP-2. In contrast, Marburg GP-mediated infection was independent of these three proteins and instead required beta-arrestin 1 (ARRB1). These findings have revealed an unexpected difference between the clathrin pathway requirements for Ebola GP versus Marburg GP pseudovirion infection. Anthrax toxin also uses a clathrin-, and ARRB1-dependent pathway for cellular entry, indicating that the mechanism used by Marburg GP pseudovirions may be more generally important for pathogen entry.     

扎伊尔型埃博拉病毒的实时荧光RT-PCR检测方法研究

目的 建立扎伊尔型埃博拉病毒的核酸检测方法,以期用于埃博拉出血热临床标本的检测.方法 针对扎伊尔型埃博拉病毒核蛋白和糖蛋白基因设计引物和探针,建立单重和双重实时荧光RT-PCR检测方法,利用体外转录病毒RNA和埃博拉病毒系列参考品RNA评价其敏感性,利用马尔堡病毒、健康人、登革热患者和发热伴血小板减少综合征患者血清评价其特异性.结果 所建立的实时荧光RT-PCR检测方法扩增效率在95％～105％,可特异性地检测扎伊尔型埃博拉病毒核蛋白和糖蛋白基因,与马尔堡病毒、登革热和发热伴血小板减少综合征病毒均无交叉反应,体外转录的病毒RNA可检出10～100拷贝/μl.双重检测方法通过细胞培养的扎伊尔型埃博拉病毒RNA验证,可检出100 pfu/ml病毒.结论 本研究建立的检测扎伊尔型埃博拉病毒的实时荧光RT-PCR方法具有良好的特异性和敏感性,可用于埃博拉出血热临床标本的检测.     

Glycosyl-phosphatidylinositol (GPI)-anchored membrane association of the porcine reproductive and respiratory syndrome virus GP4 glycoprotein and its co-localization with CD163 in lipid rafts

The porcine reproductive and respiratory syndrome virus (PRRSV) glycoprotein 4 (GP4) resembles a typical type I membrane protein in its structure but lacks a hydrophilic tail at the C-terminus, suggesting that GP4 may be a lipid-anchored membrane protein. Using the human decay-accelerating factor (DAF; CD55), a known glycosyl-phosphatidylinositol (GPI) lipid-anchored protein, chimeric constructs were made to substitute the GPI-anchor domain of DAF with the putative lipid-anchor domain of GP4, and their membrane association and lipase cleavage were determined in cells. The DAF-GP4 fusion protein was transported to the plasma membrane and was cleaved by phosphatidylinositol-specific phospholipase C (PI-PLC), indicating that the C-terminal domain of GP4 functions as a GPI anchor. Mutational studies for residues adjacent to the GPI modification site and characterization of respective mutant viruses generated from infectious cDNA clones show that the ability of GP4 for membrane association corresponded to virus viability and growth characteristics. The residues T158 (ω − 2, where ω is the GPI moiety at E160), P159 (ω − 1), and M162 (ω + 2) of GP4 were determined to be important for virus replication, with M162 being of particular importance for virus infectivity. The complete removal of the peptide-anchor domain in GP4 resulted in a complete loss of virus infectivity. The depletion of cholesterol from the plasma membrane of cells reduced the virus production, suggesting a role of lipid rafts in PRRSV infection. Remarkably, GP4 was found to co-localize with CD163 in the lipid rafts on the plasma membrane. Since CD163 has been reported as a cellular receptor for PRRSV and GP4 has been shown to interact with this receptor, our data implicates an important role of lipid rafts during entry of the virus.     

Tyrosine kinase receptor Axl enhances entry of Zaire ebolavirus without direct interactions with the viral glycoprotein

In a bioinformatics-based screen for cellular genes that enhance Zaire ebolavirus (ZEBOV) transduction, AXL mRNA expression strongly correlated with ZEBOV infection. A series of cell lines and primary cells were identified that require Axl for optimal ZEBOV entry. Using one of these cell lines, we identified ZEBOV entry events that are Axl-dependent. Interactions between ZEBOV-GP and the Axl ectodomain were not detected in immunoprecipitations and reduction of surface-expressed Axl by RNAi did not alter ZEBOV-GP binding, providing evidence that Axl does not serve as a receptor for the virus. However, RNAi knock down of Axl reduced ZEBOV pseudovirion internalization and α-Axl antisera inhibited pseudovirion fusion with cellular membranes. Consistent with the importance of Axl for ZEBOV transduction, Axl transiently co-localized on the surface of cells with ZEBOV virus particles and was internalized during virion transduction. In total, these findings indicate that endosomal uptake of filoviruses is facilitated by Axl.     

Amino acid residues of the human immunodeficiency virus type I gp120 critical for the binding of rat and human neutralizing antibodies that block the gp120-sCD4 interaction.

We have characterized the discontinuous epitopes recognized by two rat and three human neutralizing monoclonal antibodies (mAb) by examining the effect of single amino acid changes in conserved residues of gp120 on mAb recognition. A human mAb derived from an infected individual, 448D, and two rat mAbs, 39.13g and 39.3b, respectively, derived by immunization with native recombinant gp120, recognize similar epitopes. Recognition of the envelope glycoproteins by these mAbs was affected by changes in gp120 amino acid residues 88, 113, 117, 257, 368, or 370. The gp120 amino acids 257, 368, and 370 have previously been reported to be important for CD4 binding, which is consistent with the ability of these mAbs to block the gp120-CD4 interaction. Residues 88, 113, and 117 are not thought to be important for CD4 binding, suggesting that the antibody epitopes overlap, but are distinct from, the CD4 binding region. We also found that some alterations in gp120 residues 88, 117, 368, or 421 reduced the ability of polyclonal sera from HIV-1-infected individuals to inhibit the interaction of the mutant gp120 glycoproteins with soluble CD4. Thus, changes in the HIV-1 gp120 glycoprotein that minimally affect the receptor binding may allow escape from neutralizing antibodies directed against the CD4 binding region.     

Modulation of virion incorporation of Ebolavirus glycoprotein: effects on attachment, cellular entry and neutralization

The filoviruses Ebolavirus (EBOV) and Marburgvirus (MARV) cause severe hemorrhagic fever in humans and are potential agents of biological warfare. The envelope glycoprotein (GP) of filoviruses mediates viral entry into cells and is an attractive target for therapeutic intervention and vaccine design. Here, we asked if the efficiency of virion incorporation of EBOV-GP impacts attachment and entry into target cells and modulates susceptibility to neutralizing antibodies. In order to control the level of EBOV-GP expression, we generated cell lines expressing the GPs of the four known EBOV subspecies in an inducible fashion. Regulated expression of GP on the cell surface allowed production of reporter viruses harboring different amounts of GP. A pronounced reduction of virion incorporation of EBOV-GP had relatively little effect on virion infectivity, suggesting that only a few copies of GP might be sufficient for efficient engagement of cellular receptors. In contrast, optimal interactions with cellular attachment factors like the DC-SIGN protein required incorporation of high amounts of GP. Antibody-mediated neutralization of virions bearing high amounts of GP was slightly more efficient than neutralization of virions harboring low amounts of GP, suggesting that the efficiency of GP incorporation into virions might modulate susceptibility to neutralizing antibodies. Finally, regulated expression of GP in permissive 293 cells did not reduce EBOV-GP-driven infection but diminished vesicular stomatitis virus GP (VSV-G) and amphotropic murine leukemia virus (A-MLV) GP mediated entry in a dose-dependent manner. Therefore, intracellular GP does not seem to downmodulate expression of its receptor(s) but might alter expression and/or function of molecules involved in VSV-G and A-MLV-GP-dependent entry. Our results suggest that the efficiency of virion incorporation of GP could impact EBOV attachment to target cells and might modulate control of viral spread by the humoral immune response.     

The C domain in the surface envelope glycoprotein of subgroup C feline leukemia virus is a second receptor-binding domain

The receptor-binding domain (RBD) in the surface (SU) subunit of gammaretrovirus envelope glycoprotein is critical for determining the host receptor specificity of the virus. This domain is separated from the carboxy terminal C domain (Cdom) of SU by a proline-rich region. In this study, we show that the Cdom region in the SU from subgroup C feline leukemia virus (FeLV-C) forms a second receptor-binding domain that is distinct from its RBD, and which can independently bind to its host receptor FLVCR1, in the absence of RBD. Furthermore, our results suggest that residues located in the C2 disulfide-bonded loop in FeLV-C Cdom are critical for SU binding to FLVCR1 and for virus infection. We propose that binding of FeLV-C SU to FLVCR1 involves interaction of two receptor-binding domains (RBD and Cdom) with FLVCR1, and that this mechanism of interaction is conserved for other gammaretroviruses. Our results could have important implications for designing gammaretrovirus vectors that can efficiently infect specific target cells.     

Amino acid residues Tyr-67, Asn-72, and Asp-73 of the TVB receptor are important for subgroup E avian sarcoma and leukosis virus interaction

The chicken TVB(S1) protein serves as the cellular receptor for the cytopathic subgroups B and D avian sarcoma and leukosis viruses (ASLVs) as well as for the non-cytopathic subgroup E ASLV. Previous studies had mapped the subgroup B viral interaction determinants to a region that was located between residues 32 and 46 of TVB(S1) [J. Virol. 76 (2002) 5404]. To gain a greater insight into ASLV Env-receptor interactions and the possible role of these interactions in viral cytopathic effects, we employed a homolog-scanning mutagenesis approach to identify amino acid residues important for subgroup E viral receptor function by exchanging amino acid residues between TVB(S1) and its human homolog, DR5. These studies identified residues Tyr-67, Asn-72, and Asp-73 of TVB(S1) as important subgroup E viral interaction determinants. Intriguingly, these three residues are conserved between TVB(S1) and DR5, demonstrating that the human protein contains critical subgroup E viral interaction determinants, but in this context, they cannot support viral entry. These data confirm that the molecular determinants of the TVB receptor required for subgroup E viral entry are completely distinct from those used by subgroup B viruses.     

Molecular characterization of the env gene of two CCR5/CXCR4‐independent human immunodeficiency 2 primary isolates

Human immunodeficiency virus 2 (HIV‐2) infection is characterized by a slower disease progression and lower transmission rates. The molecular features that could be assigned as directly involved in this in vivo phenotype remain essentially unknown, and the importance of HIV‐2 as a model to understand pathogenicity of HIV infection has been frequently underestimated. The early events of the HIV replication cycle involve the interaction between viral envelope glycoproteins and cellular receptors: the CD4 molecule and a chemokine receptor, usually CCR5 or CXCR4. Despite the importance of these two chemokine receptors in human immunodeficiency virus 1 (HIV‐1) entry into cells, we have previously shown that in some HIV‐2 asymptomatic individuals, a viral population exists that is unable to use both CCR5 and CXCR4. The goal of the present study was to investigate whether possible regions in the env gene of these viruses might account for this phenotype. From the molecular characterization of these env genes we could not detect any correlation between V3 loop sequence and viral phenotype. In contrast, it reveals the existence of remarkable differences in the V1/V2 and C5 regions of the surface glycoprotein, including the loss of a putative glycosilation site. Moreover, in the transmembrane glycoprotein some unique sequence signatures could be detected in the central ectodomain and second heptad repeat (HR2). Some of the mutations affect well‐conserved residues, and may affect the conformation and/or the dynamics of envelope glycoproteins complex, including the SU–TM association and the modulation of viral entry function. J. Med. Virol. 81:1869–1881, 2009. © 2009 Wiley‐Liss, Inc.     

Role of the gp120 inner domain beta-sandwich in the interaction between the human immunodeficiency virus envelope glycoprotein subunits

The inner domain of the human immunodeficiency virus (HIV-1) gp120 glycoprotein has been proposed to mediate the noncovalent interaction with the gp41 transmembrane envelope glycoprotein. We used mutagenesis to investigate the functional importance of a conserved beta-sandwich located within the gp120 inner domain. Changes in aliphatic residues lining a hydrophobic groove on the surface of the beta-sandwich decreased the association of the gp120 and gp41 glycoproteins. Other changes in the base of the hydrophobic groove resulted in envelope glycoproteins that were structurally intact and able to bind receptors, but were inefficient in mediating either syncytium formation or virus entry. These results support a model in which the beta-sandwich in the gp120 inner domain contributes to gp120-gp41 contacts, thereby maintaining the integrity of the envelope glycoprotein complex and allowing adjustments in the gp120-gp41 interaction required for membrane fusion.