Distinct mechanisms of entry by envelope glycoproteins of Marburg and Ebola (Zaire) viruses

Since the Marburg (MBG) and Ebola (EBO) viruses have sequence homology and cause similar diseases, we hypothesized that they associate with target cells by similar mechanisms. Pseudotype viruses prepared with a luciferase-containing human immunodeficiency virus type 1 backbone and packaged by the MBG virus or the Zaire subtype EBO virus glycoproteins (GP) mediated infection of a comparable wide range of mammalian cell types, and both were inhibited by ammonium chloride. In contrast, they exhibited differential sensitivities to treatment of target cells with tunicamycin, endoglycosidase H, or protease (pronase). Therefore, while they exhibit certain functional similarities, the MBG and EBO virus GP interact with target cells by distinct processes.     

C-type lectins do not act as functional receptors for filovirus entry into cells

Cellular C-type lectins have been reported to facilitate filovirus infection by binding to glycans on filovirus glycoprotein (GP). However, it is not clearly known whether interaction between C-type lectins and GP mediates all the steps of virus entry (i.e., attachment, internalization, and membrane fusion). In this study, we generated vesicular stomatitis viruses pseudotyped with mutant GPs that have impaired structures of the putative receptor binding regions and thus reduced ability to infect the monkey kidney cells that are routinely used for virus propagation. We found that infectivities of viruses with the mutant GPs dropped in C-type lectin-expressing cells, parallel with those in the monkey kidney cells, whereas binding activities of these GPs to the C-type lectins were not correlated with the reduced infectivities. These results suggest that C-type lectin-mediated entry of filoviruses requires other cellular molecule(s) that may be involved in virion internalization or membrane fusion.     

The baculovirus GP64 protein mediates highly stable infectivity of a human respiratory syncytial virus lacking its homologous transmembrane glycoproteins

Baculovirus GP64 is a low-pH-dependent membrane fusion protein required for virus entry and cell-to-cell transmission. Recently, GP64 has generated interest for practical applications in mammalian systems. Here we examined the membrane fusion function of GP64 from Autographa californica multiple nucleopolyhedrovirus (AcMNPV) expressed in mammalian cells, as well as its capacity to functionally complement a mammalian virus, human respiratory syncytial virus (HRSV). Both authentic GP64 and GP(64/F), a chimeric protein in which the GP64 cytoplasmic tail domain was replaced with the 12 C-terminal amino acids of the HRSV fusion (F) protein, induced low-pH-dependent cell-cell fusion when expressed transiently in HEp-2 (human) cells. Levels of surface expression and syncytium formation were substantially higher at 33 degrees C than at 37 degrees C. The open reading frames (ORFs) encoding GP64 or GP(64/F), along with two marker ORFs encoding green fluorescent protein (GFP) and beta-glucuronidase (GUS), were used to replace all three homologous transmembrane glycoprotein ORFs (small hydrophobic SH, attachment G, and F) in a cDNA of HRSV. Infectious viruses were recovered that lacked the HRSV SH, G, and F proteins and expressed instead the GP64 or GP(64/F) protein and the two marker proteins GFP and GUS. The properties of these viruses, designated RSDeltaSH,G,F/GP64 or RSDeltaSH,G,F/GP(64/F), respectively, were compared to a previously described HRSV expressing GFP in place of SH but still containing the wild-type HRSV G and F proteins (RSDeltaSH [A. G. Oomens, A. G. Megaw, and G. W. Wertz, J. Virol., 77:3785-3798, 2003]). By immunoelectron microscopy, the GP64 and GP(64/F) proteins were shown to incorporate into HRSV-induced filaments at the cell surface. Antibody neutralization, ammonium chloride inhibition, and replication levels in cell culture showed that both GP64 proteins efficiently mediated infectivity of the respective viruses in a temperature-sensitive, low-pH-dependent manner. Furthermore, RSDeltaSH,G,F/GP64 and RSDeltaSH,G,F/GP(64/F) replicated to higher levels and had significantly higher stability of infectivity than HRSVs containing the homologous HRSV G and F proteins. Thus, GP64 and a GP64/HRSV F chimeric protein were functional and efficiently complemented an unrelated human virus in mammalian cells, producing stable, infectious virus stocks. These results demonstrate the potential of GP64 for both practical applications requiring stable pseudotypes in mammalian systems and for studies of viral glycoprotein requirements in assembly and pathogenesis.     

Baculovirus GP64-mediated entry into mammalian cells

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) serves as an efficient viral vector, not only for abundant gene expression in insect cells, but also for gene delivery into mammalian cells. Lentivirus vectors pseudotyped with the baculovirus envelope glycoprotein GP64 have been shown to acquire more potent gene transduction than those with vesicular stomatitis virus (VSV) envelope glycoprotein G. However, there are conflicting hypotheses about the molecular mechanisms of the entry of AcMNPV. Moreover, the mechanisms of the entry of pseudotyped viruses bearing GP64 into mammalian cells are not well characterized. Determination of the entry mechanisms of AcMNPV and the pseudotyped viruses bearing GP64 is important for future development of viral vectors that can deliver genes into mammalian cells with greater efficiency and specificity. In this study, we generated three pseudotyped VSVs, NPVpv, VSVpv, and MLVpv, bearing envelope proteins of AcMNPV, VSV, and murine leukemia virus, respectively. Depletion of membrane cholesterol by treatment with methyl-β-cyclodextrin, which removes cholesterol from cellular membranes, inhibited GP64-mediated internalization in a dose-dependent manner but did not inhibit attachment to the cell surface. Treatment of cells with inhibitors or the expression of dominant-negative mutants for dynamin- and clathrin-mediated endocytosis abrogated the internalization of AcMNPV and NPVpv into mammalian cells, whereas inhibition of caveolin-mediated endocytosis did not. Furthermore, inhibition of macropinocytosis reduced GP64-mediated internalization. These results suggest that cholesterol in the plasma membrane, dynamin- and clathrin-dependent endocytosis, and macropinocytosis play crucial roles in the entry of viruses bearing baculovirus GP64 into mammalian cells.     

Characterization of the receptor-binding domain of Ebola glycoprotein in viral entry

Ebola virus infection causes severe hemorrhagic fever in human and non-human primates with high mortality. Viral entry/infection is initiated by binding of glycoprotein GP protein on Ebola virion to host cells, followed by fusion of virus-cell membrane also mediated by GP. Using an human immunodeficiency virus (HIV)-based pseudotyping system, the roles of 41 Ebola GP1 residues in the receptor-binding domain in viral entry were studied by alanine scanning substitutions. We identified that four residues appear to be involved in protein folding/structure and four residues are important for viral entry. An improved entry interference assay was developed and used to study the role of these residues that are important for viral entry. It was found that R64 and K95 are involved in receptor binding. In contrast, some residues such as I170 are important for viral entry, but do not play a major role in receptor binding as indicated by entry interference assay and/or protein binding data, suggesting that these residues are involved in post-binding steps of viral entry. Furthermore, our results also suggested that Ebola and Marburg viruses share a common cellular molecule for entry.     

Characterization of the Receptor-binding Domain of Ebola Glycoprotein in Viral Entry

Ebola virus infection causes severe hemorrhagic fever in human and non-human primates with high mortality.Viral entry/infection is initiated by binding of glycoprotein GP protein on Ebola virion to host cells,followed by fusion of virus-cell membrane also mediated by GP.Using an human immunodeficiency virus (HIV)-based pseudotyping system,the roles of 41 Ebola GP1 residues in the receptor-binding domain in viral entry were studied by alanine scanning substitutions.We identified that four residues appear to be involved in protein folding/structure and four residues are important for viral entry.An improved entry interference assay was developed and used to study the role of these residues that are important for viral entry.It was found that R64 and K95 are involved in receptor binding.In contrast,some residues such as I170 are important for viral entry,but do not play a major role in receptor binding as indicated by entry interference assay and/or protein binding data,suggesting that these residues are involved in post-binding steps of viral entry.Furthermore,our results also suggested that Ebola and Marburg viruses share a common cellular molecule for entry.     

Characterization of the Envelope Glycoprotein of a Novel Filovirus,Lloviu Virus

Lloviu virus (LLOV), a novel filovirus detected in bats, is phylogenetically distinct from viruses in the genera Ebolavirus and Marburgvirus in the family Filoviridae. While filoviruses are known to cause severe hemorrhagic fever in humans and/or nonhuman primates, LLOV is biologically uncharacterized, since infectious LLOV has never been isolated. To examine the properties of LLOV, we characterized its envelope glycoprotein (GP), which likely plays a key role in viral tropism and pathogenicity. We first found that LLOV GP principally has the same primary structure as the other filovirus GPs. Similar to the other filoviruses, virus-like particles (VLPs) produced by transient expression of LLOV GP, matrix protein, and nucleoprotein in 293T cells had densely arrayed GP spikes on a filamentous particle. Mouse antiserum to LLOV VLP was barely cross-reactive to viruses of the other genera, indicating that LLOV is serologically distinct from the other known filoviruses. For functional study of LLOV GP, we utilized a vesicular stomatitis virus (VSV) pseudotype system and found that LLOV GP requires low endosomal pH and cathepsin L, and that human C-type lectins act as attachment factors for LLOV entry into cells. Interestingly, LLOV GP-pseudotyped VSV infected particular bat cell lines more efficiently than viruses bearing other filovirus GPs. These results suggest that LLOV GP mediates cellular entry in a manner similar to that of the other filoviruses while showing preferential tropism for some bat cells.     

Detection of cell-cell fusion mediated by Ebola virus glycoproteins

Ebola viruses (EboV) are enveloped RNA viruses infecting cells by a pH-dependent process mediated by viral glycoproteins (GP) involving endocytosis of virions and their routing into acidic endosomes. As with well-characterized pH-dependent viral entry proteins, in particular influenza virus hemagglutinin, it is thought that EboV GP require activation by low pH in order to mediate fusion of the viral envelope with the membrane of endosomes. However, it has not yet been possible to confirm the direct role of EboV GP in membrane fusion and the requirement for low-pH activation. It was in particular not possible to induce formation of syncytia by exposing cells expressing EboV GP to acidic medium. Here, we have used an assay based on the induction of a beta-galactosidase (lacZ) reporter gene in target cells to detect cytoplasmic exchanges, indicating membrane fusion, with cells expressing EboV GP (Zaire species). Acidic activation of GP-expressing cells was required for efficient fusion with target cells. The direct role of EboV GP in this process is indicated by its inhibition by anti-GP antibodies and by the lack of activity of mutant GP normally expressed at the cell surface but defective for virus entry. Fusion was not observed when target cells underwent acidic treatment, for example, when they were placed in coculture with GP-expressing cells before the activation step. This unexpected feature, possibly related to the nature of the EboV receptor, could explain the impossibility of inducing formation of syncytia among GP-expressing cells.     

Use of a mammalian internal ribosomal entry site element for expression of a foreign protein by a transfectant influenza virus.

The ribonucleoprotein transfection system for influenza virus allowed us to construct two influenza A viruses, GP2/BIP-NA and HGP2/BIP-NA, which contained bicistronic neuraminidase (NA) genes. The mRNAs derived from the bicistronic NA genes have two different open reading frames (ORFs). The first ORF encodes a foreign polypeptide (GP2 or HGP2) containing amino acid sequences derived from the gp41 protein of human immunodeficiency virus type 1. The second ORF encodes the NA protein; its translation is achieved via an internal ribosomal entry site which is derived from the 5' noncoding region of the human immunoglobulin heavy-chain-binding protein mRNA. The GP2 (79 amino acids) and HGP2 (91 amino acids) polypeptides are expressed in cells infected with the corresponding transfectant virus. The HGP2 polypeptide, which contains transmembrane and cytoplasmic domains identical to those of the hemagglutinin (HA) protein of influenza A/WSN/33 virus, is packaged into virus particles. This novel influenza virus system involving an internal ribosomal entry site element may afford a way to express a variety of foreign genes in mammalian cells.     

A GP64-null baculovirus pseudotyped with vesicular stomatitis virus G protein

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) GP64 protein is an essential virion protein that is involved in both receptor binding and membrane fusion during viral entry. Genetic studies have shown that GP64-null viruses are unable to move from cell to cell and this results from a defect in the assembly and production of budded virions (BV). To further examine requirements for virion budding, we asked whether a GP64-null baculovirus, vAc(64-), could be pseudotyped by introducing a heterologous viral envelope protein (vesicular stomatitis virus G protein [VSV-G]) into its membrane and whether the resulting virus was infectious. To address this question, we generated a stably transfected insect Sf9 cell line (Sf9(VSV-G)) that inducibly expresses the VSV-G protein upon infection with AcMNPV Sf9(VSV-G) and Sf9 cells were infected with vAc(64-), and cells were monitored for infection and for movement of infection from cell to cell. vAc(64-) formed plaques on Sf9(VSV-G) cells but not on Sf9 cells, and plaques formed on Sf9(VSV-G) cells were observed only after prolonged intervals. Passage and amplification of vAc(64-) on Sf9(VSV-G) cells resulted in pseudotyped virus particles that contained the VSV-G protein. Cell-to-cell propagation of vAc(64-) in the G-expressing cells was delayed in comparison to wild-type (wt) AcMNPV, and growth curves showed that pseudotyped vAc(64-) was generated at titers of approximately 10(6) to 10(7) infectious units (IU)/ml, compared with titers of approximately 10(8) IU/ml for wt AcMNPV. Propagation and amplification of pseudotyped vAc(64-) virions in Sf9(VSV-G) cells suggests that the VSV-G protein may either possess the signals necessary for baculovirus BV assembly and budding at the cell surface or may otherwise facilitate production of infectious baculovirus virions. The functional complementation of GP64-null viruses by VSV-G protein was further demonstrated by identification of a vAc(64-)-derived virus that had acquired the G gene through recombination with Sf9(VSV-G) cellular DNA. GP64-null viruses expressing the VSV-G gene were capable of productive infection, replication, and propagation in Sf9 cells.     

Cell entry by human pathogenic arenaviruses

The arenaviruses Lassa virus (LASV) in Africa and Machupo (MACV), Guanarito (GTOV) and Junin viruses (JUNV) in South America cause severe haemorrhagic fevers in humans with fatality rates of 15–35%. The present review focuses on the first steps of infection with human pathogenic arenaviruses, the interaction with their cellular receptor molecules and subsequent entry into the host cell. While similarities exist in genomic organization, structure and clinical disease caused by pathogenic Old World and New World arenaviruses these pathogens use different primary receptors. The Old World arenaviruses employ α-dystroglycan, a cellular receptor for proteins of the extracellular matrix, and the human pathogenic New World arenaviruses use the cellular cargo receptor transferrin receptor 1. While the New World arenavirus JUNV enters cells via clathrin-dependent endocytosis, evidence occurred for clathrin-independent entry of the prototypic Old World arenavirus lymphocytic choriomeningitis virus. Upon internalization, arenaviruses are delivered to the endosome, where pH-dependent membrane fusion is mediated by the envelope glycoprotein (GP). While arenavirus GPs share characteristics with class I fusion GPs of other enveloped viruses, unusual mechanistic features of GP-mediated membrane fusion have recently been discovered for arenaviruses with important implications for viral entry.     

Human macrophage C-type lectin specific for galactose and N-acetylgalactosamine promotes filovirus entry

Filoviruses cause lethal hemorrhagic disease in humans and nonhuman primates. An initial target of filovirus infection is the mononuclear phagocytic cell. Calcium-dependent (C-type) lectins such as dendritic cell- or liver/lymph node-specific ICAM-3 grabbing nonintegrin (DC-SIGN or L-SIGN, respectively), as well as the hepatic asialoglycoprotein receptor, bind to Ebola or Marburg virus glycoprotein (GP) and enhance the infectivity of these viruses in vitro. Here, we demonstrate that a recently identified human macrophage galactose- and N-acetylgalactosamine-specific C-type lectin (hMGL), whose ligand specificity differs from DC-SIGN and L-SIGN, also enhances the infectivity of filoviruses. This enhancement was substantially weaker for the Reston and Marburg viruses than for the highly pathogenic Zaire virus. We also show that the heavily glycosylated, mucin-like domain on the filovirus GP is required for efficient interaction with this lectin. Furthermore, hMGL, like DC-SIGN and L-SIGN, is present on cells known to be major targets of filoviruses (i.e., macrophages and dendritic cells), suggesting a role for these C-type lectins in viral replication in vivo. We propose that filoviruses use different C-type lectins to gain cellular entry, depending on the cell type, and promote efficient viral replication.     

Persistent equine arteritis virus infection in HeLa cells

The horse-adapted virulent Bucyrus (VB) strain of equine arteritis virus (EAV) established persistent infection in high-passage-number human cervix cells (HeLa-H cells; passages 170 to 221) but not in low-passage-number human cervix cells (HeLa-L cells; passages 95 to 115) or in several other cell lines that were evaluated. However, virus recovered from the 80th passage of the persistently infected HeLa-H cells (HeLa-H-EAVP80) readily established persistent infection in HeLa-L cells. Comparative sequence analysis of the entire genomes of the VB and HeLa-H-EAVP80 viruses identified 16 amino acid substitutions, including 4 in the replicase (nsp1, nsp2, nsp7, and nsp9) and 12 in the structural proteins (E, GP2, GP3, GP4, and GP5). Reverse genetic studies clearly showed that substitutions in the structural proteins but not the replicase were responsible for the establishment of persistent infection in HeLa-L cells by the HeLa-H-EAVP80 virus. It was further demonstrated that recombinant viruses with substitutions in the minor structural proteins E and GP2 or GP3 and GP4 were unable to establish persistent infection in HeLa-L cells but that recombinant viruses with combined substitutions in the E (Ser53→Cys and Val55→Ala), GP2 (Leu15→Ser, Trp31→Arg, Val87→Leu, and Ala112→Thr), GP3 (Ser115→Gly and Leu135→Pro), and GP4 (Tyr4→His and Ile109→Phe) proteins or with a single point mutation in the GP5 protein (Pro98→Leu) were able to establish persistent infection in HeLa-L cells. In summary, an in vitro model of EAV persistence in cell culture was established for the first time. This system can provide a valuable model for studying virus-host cell interactions, especially virus-receptor interactions.     

Different mechanisms of cell entry by human-pathogenic Old World and New World arenaviruses

The Old World arenavirus Lassa virus (LASV) is the causative agent of severe viral hemorrhagic fever (VHF) in humans and is the most prevalent human pathogen among arenaviruses. The present study investigated the largely unknown mechanisms of cell entry of LASV, a process know to be mediated solely by the virus envelope glycoprotein (GP). To circumvent biosafety restrictions associated with the use of live LASV, we used reverse genetics to generate a recombinant variant of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) expressing the LASV GP (rLCMV-LASVGP). The rescued rLCMV-LASVGP grew to titers comparable to that of LCMV and showed the receptor binding characteristics of LASV. We used rLCMV-LASVGP to characterize the cellular mechanisms of LASV entry in the context of a productive arenavirus infection. The kinetics of pH-dependent membrane fusion of rLCMV-LASVGP resembled those of the human-pathogenic New World arenavirus Junin virus (JUNV) and other enveloped viruses that use clathrin-mediated endocytosis for entry. However, rLCMV-LASVGP entered cells predominantly via a clathrin-, caveolin-, and dynamin-independent endocytotic pathway similar to the one recently described for LCMV. Productive infection of rLCMV-LASVGP was only mildly affected by a dominant negative mutant of Rab5 and was independent of Rab7, suggesting an unusual mechanism of delivery to endosomes. In addition, rLCMV-LASVGP infection was independent of actin but required intact microtubules. Our data indicate that LASV enters cells via a pathway distinct from the one used by human-pathogenic New World arenaviruses.     

New World arenavirus clade C, but not clade A and B viruses, utilizes alpha-dystroglycan as its major receptor

Alpha-dystroglycan (alpha-DG) has been identified as a major receptor for lymphocytic choriomeningitis virus (LCMV) and Lassa virus, two Old World arenaviruses. The situation with New World arenaviruses is less clear: previous studies demonstrated that Oliveros virus also exhibited high-affinity binding to alpha-DG but that Guanarito virus did not. To extend these initial studies, several additional Old and New World arenaviruses were screened for entry into mouse embryonic stem cells possessing or lacking alpha-DG. In addition, representative viruses were further analyzed for direct binding to alpha-DG by means of a virus overlay protein blot assay technique. These studies indicate that Old World arenaviruses use alpha-DG as a major receptor, whereas, of the New World arenaviruses, only clade C viruses (i.e., Oliveros and Latino viruses) use alpha-DG as a major receptor. New World clade A and B arenaviruses, which include the highly pathogenic Machupo, Guanarito, Junin, and Sabia viruses, appear to use a different receptor or coreceptor for binding. Previous studies with LCMV have suggested the need for a small aliphatic amino acid at LCMV GP1 glycoprotein amino acid position 260 to allow high-affinity binding to alpha-DG. As reported herein, this requirement appears to be broadly applicable to the arenaviruses as determined by more extensive analysis of alpha-DG receptor usage and GP1 sequences of Old and New World arenaviruses. In addition, GP1 amino acid position 259 also appears to be important, since all arenaviruses showing high-affinity alpha-DG binding possess a bulky aromatic amino acid (tyrosine or phenylalanine) at this position.