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.     

The Glycoproteins of All Filovirus Species Use the Same Host Factors for Entry into Bat and Human Cells but Entry Efficiency Is Species Dependent

Ebola and marburgviruses, members of the family Filoviridae, can cause severe hemorrhagic fever in humans. The ongoing Ebola virus (EBOV) disease epidemic in Western Africa claimed more than 11,300 lives and was associated with secondary cases outside Africa, demonstrating that filoviruses pose a global health threat. Bats constitute an important natural reservoir of filoviruses, including viruses of the recently identified Cuevavirus genus within the Filoviridae family. However, the interactions of filoviruses with bat cells are incompletely understood. Here, we investigated whether filoviruses employ different strategies to enter human and bat cells. For this, we examined host cell entry driven by glycoproteins (GP) from all filovirus species into cell lines of human and fruit bat origin. We show that all GPs were able to mediate entry into human and most fruit bat cell lines with roughly comparable efficiency. In contrast, the efficiency of entry into the cell line EidNi/41 derived from a straw-colored fruit bat varied markedly between the GPs of different filovirus species. Furthermore, inhibition studies demonstrated that filoviruses employ the same host cell factors for entry into human, non-human primate and fruit bat cell lines, including cysteine proteases, two pore channels and NPC1 (Niemann-Pick C1 molecule). Finally, processing of GP by furin and the presence of the mucin-like domain in GP were dispensable for entry into both human and bat cell lines. Collectively, these results show that filoviruses rely on the same host cell factors for entry into human and fruit bat cells, although the efficiency of the usage of these factors might differ between filovirus species.     

Differential Sensitivity of Bat Cells to Infection by Enveloped RNA Viruses: Coronaviruses,Paramyxoviruses, Filoviruses,and Influenza Viruses

Bats (Chiroptera) host major human pathogenic viruses including corona-, paramyxo, rhabdo- and filoviruses. We analyzed six different cell lines from either Yinpterochiroptera (including African flying foxes and a rhinolophid bat) or Yangochiroptera (genera Carollia and Tadarida) for susceptibility to infection by different enveloped RNA viruses. None of the cells were sensitive to infection by transmissible gastroenteritis virus (TGEV), a porcine coronavirus, or to infection mediated by the Spike (S) protein of SARS-coronavirus (SARS-CoV) incorporated into pseudotypes based on vesicular stomatitis virus (VSV). The resistance to infection was overcome if cells were transfected to express the respective cellular receptor, porcine aminopeptidase N for TGEV or angiotensin-converting enzyme 2 for SARS-CoV. VSV pseudotypes containing the S proteins of two bat SARS-related CoV (Bg08 and Rp3) were unable to infect any of the six tested bat cell lines. By contrast, viral pseudotypes containing the surface protein GP of Marburg virus from the family Filoviridae infected all six cell lines though at different efficiency. Notably, all cells were sensitive to infection by two paramyxoviruses (Sendai virus and bovine respiratory syncytial virus) and three influenza viruses from different subtypes. These results indicate that bat cells are more resistant to infection by coronaviruses than to infection by paramyxoviruses, filoviruses and influenza viruses. Furthermore, these results show a receptor-dependent restriction of the infection of bat cells by CoV. The implications for the isolation of coronaviruses from bats are discussed.     

Lentivirus vectors pseudotyped with filoviral envelope glycoproteins transduce airway epithelia from the apical surface independently of folate receptor alpha

The practical application of gene therapy as a treatment for cystic fibrosis is limited by poor gene transfer efficiency with vectors applied to the apical surface of airway epithelia. Recently, folate receptor alpha (FR alpha), a glycosylphosphatidylinositol-linked surface protein, was reported to be a cellular receptor for the filoviruses. We found that polarized human airway epithelia expressed abundant FR alpha on their apical surface. In an attempt to target these apical receptors, we pseudotyped feline immunodeficiency virus (FIV)-based vectors by using envelope glycoproteins (GPs) from the filoviruses Marburg virus and Ebola virus. Importantly, primary cultures of well-differentiated human airway epithelia were transduced when filovirus GP-pseudotyped FIV was applied to the apical surface. Furthermore, by deleting a heavily O-glycosylated extracellular domain of the Ebola GP, we improved the titer of concentrated vector severalfold. To investigate the folate receptor dependence of gene transfer with the filovirus pseudotypes, we compared gene transfer efficiency in immortalized airway epithelium cell lines and primary cultures. By utilizing phosphatidylinositol-specific phospholipase C (PI-PLC) treatment and FR alpha-blocking antibodies, we demonstrated FR alpha-dependent and -independent entry by filovirus glycoprotein-pseudotyped FIV-based vectors in airway epithelia. Of particular interest, entry independent of FR alpha was observed in primary cultures of human airway epithelia. Understanding viral vector binding and entry pathways is fundamental for developing cystic fibrosis gene therapy applications.     

Studies of ebola virus glycoprotein-mediated entry and fusion by using pseudotyped human immunodeficiency virus type 1 virions: involvement of cytoskeletal proteins and enhancement by tumor necrosis factor alpha

The Ebola filoviruses are aggressive pathogens that cause severe and often lethal hemorrhagic fever syndromes in humans and nonhuman primates. To date, no effective therapies have been identified. To analyze the entry and fusion properties of Ebola virus, we adapted a human immunodeficiency virus type 1 (HIV-1) virion-based fusion assay by substituting Ebola virus glycoprotein (GP) for the HIV-1 envelope. Fusion was detected by cleavage of the fluorogenic substrate CCF2 by beta-lactamase-Vpr incorporated into virions and released as a result of virion fusion. Entry and fusion induced by the Ebola virus GP occurred with much slower kinetics than with vesicular stomatitis virus G protein (VSV-G) and were blocked by depletion of membrane cholesterol and by inhibition of vesicular acidification with bafilomycin A1. These properties confirmed earlier studies and validated the assay for exploring other properties of Ebola virus GP-mediated entry and fusion. Entry and fusion of Ebola virus GP pseudotypes, but not VSV-G or HIV-1 Env pseudotypes, were impaired in the presence of the microtubule-disrupting agent nocodazole but were enhanced in the presence of the microtubule-stabilizing agent paclitaxel (Taxol). Agents that impaired microfilament function, including cytochalasin B, cytochalasin D, latrunculin A, and jasplakinolide, also inhibited Ebola virus GP-mediated entry and fusion. Together, these findings suggest that both microtubules and microfilaments may play a role in the effective trafficking of vesicles containing Ebola virions from the cell surface to the appropriate acidified vesicular compartment where fusion occurs. In terms of Ebola virus GP-mediated entry and fusion to various target cells, primary macrophages proved highly sensitive, while monocytes from the same donors displayed greatly reduced levels of entry and fusion. We further observed that tumor necrosis factor alpha, which is released by Ebola virus-infected monocytes/macrophages, enhanced Ebola virus GP-mediated entry and fusion to human umbilical vein endothelial cells. Thus, Ebola virus infection of one target cell may induce biological changes that facilitate infection of secondary target cells that play a key role in filovirus pathogenesis. Finally, these studies indicate that pseudotyping in the HIV-1 virion-based fusion assay may be a valuable approach to the study of entry and fusion properties mediated through the envelopes of other viral pathogens.     

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.     

The Tyro3 receptor kinase Axl enhances macropinocytosis of Zaire ebolavirus

Axl, a plasma membrane-associated Tyro3/Axl/Mer (TAM) family member, is necessary for optimal Zaire ebolavirus (ZEBOV) glycoprotein (GP)-dependent entry into some permissive cells but not others. To date, the role of Axl in virion entry is unknown. The focus of this study was to characterize entry pathways that are used for ZEBOV uptake in cells that require Axl for optimal transduction and to define the role of Axl in this process. Through the use of biochemical inhibitors, interfering RNA (RNAi), and dominant negative constructs, we demonstrate that ZEBOV-GP-dependent entry into these cells occurs through multiple uptake pathways, including both clathrin-dependent and caveola/lipid raft-mediated endocytosis. Other dynamin-dependent and -independent pathways such as macropinocytosis that mediate high-molecular-weight dextran uptake also stimulated ZEBOV-GP entry into these cells, and inhibitors that are known to block macropinocytosis inhibited both dextran uptake and ZEBOV infection. These findings provided strong evidence for the importance of this pathway in filovirus entry. Reduction of Axl expression by RNAi treatment resulted in decreased ZEBOV entry via macropinocytosis but had no effect on the clathrin-dependent or caveola/lipid raft-mediated endocytic mechanisms. Our findings demonstrate for the first time that Axl enhances macropinocytosis, thereby increasing productive ZEBOV entry.     

Delayed appearance of pseudotypes between vesicular stomatitis virus influenza virus during mixed infection of MDCK cells.

In intact Madin-Darby canine kidney (MDCK) cell monolayers, vesicular stomatitis virus (VSV) matures only at basolateral membranes beneath tight junctions, whereas influenza virus buds from apical cell surfaces. Early in the growth cycle, the viral glycoproteins are restricted to the membrane domain from which each virus buds. We report here that phenotypic mixing and formation of VSV pseudotypes occurred when influenza virus-infected MDCK cells were superinfected with VSV. Up to 75% of the infectious VSV particles from such experiments were neutralized by antiserum specific for influenza virus, and a smaller proportion (up to 3%) were resistant to neutralization with antiserum specific for VSV. The latter particles, which were neutralized by antiserum to influenza A/WSN virus, are designated as VSV(WSN) pseudotypes. During mixed infections, both wild-type viruses were detected 1 to 2 h before either phenotypically mixed VSV or VSV(WSN) pseudotypes. Coincident with the appearance of cytopathic effects in the monolayer, the yield of pseudotypes rose dramatically. In contrast, in doubly infected BHK-21 cells, which do not show polarity in virus maturation sites and are not connected by tight junctions, VSV(WSN) pseudotypes were detected as soon as VSV titers rose to the minimum levels which allowed detection of pseudotypes, and the proportion observed remained relatively constant at later times. Examination of thin sections of doubly infected MDCK monolayers revealed that polarity in maturation sites was preserved for both viruses until approximately 12 h after inoculation with influenza virus, when disruption of junctional complexes was evident. Even at later periods, the majority of each virus type was associated with its normal membrane domain, suggesting that the sorting mechanisms responsible for directing the glycoproteins of VSV and influenza virus to separate surface domains continue to operate in doubly infected MDCK cells. The time course of VSV(WSN) pseudotype formation and changes in virus maturation sites are compatible with progressive mixing of viral glycoproteins at either intracellular or plasma membranes of doubly infected cells.     

Pseudotyping human immunodeficiency virus type 1 (HIV-1) by the glycoprotein of vesicular stomatitis virus targets HIV-1 entry to an endocytic pathway and suppresses both the requirement for Nef and the sensitivity to cyclosporin A.

Human immunodeficiency virus type 1 (HIV-1) normally enters cells by direct fusion with the plasma membrane. In this report, HIV-1 particles capable of infecting cells through an endocytic pathway are described. Chimeric viruses composed of the HIV-1 core and the envelope glycoprotein of vesicular stomatitis virus (VSV-G) were constructed and are herein termed HIV-1(VSV) pseudotypes. HIV-1(VSV) pseudotypes were 20- to 130-fold more infectious than nonpseudotyped HIV-1. Infection by HIV-1(VSV) pseudotypes was markedly diminished by ammonium chloride and concanamycin A, a selective inhibitor of vacuolar H+ ATPases, demonstrating that these viruses require endosomal acidification to achieve productive infection. HIV-1 is thus capable of performing all of the viral functions necessary for infection when entry is targeted to an endocytic route. Maximal HIV-1 infectivity requires the presence of the viral Nef protein and the cellular protein cyclophilin A (CyPA) during virus assembly. Pseudotyping by VSV-G markedly suppressed the requirement for Nef. HIV-1(VSV) particles were also resistant to inhibition by cyclosporin A; however, the deleterious effect of a gag mutation inhibiting CyPA incorporation was not relieved by VSV-G. These results suggest that Nef acts at a step of the HIV-1 life cycle that is either circumvented or facilitated by targeting virus entry to an endocytic pathway. The findings also support the hypothesis that Nef and CyPA enhance HIV-1 infectivity through independent processes and demonstrate a mechanistic difference between reduction of HIV-1 infectivity by cyclosporin A and gag mutations that decrease HIV-1 incorporation of CyPA.     

Filoviruses Utilize Glycosaminoglycans for Their Attachment to Target Cells

Filoviruses are the cause of severe hemorrhagic fever in human and nonhuman primates. The envelope glycoprotein (GP), responsible for both receptor binding and fusion of the virus envelope with the host cell membrane, has been demonstrated to interact with multiple molecules in order to enhance entry into host cells. Here we have demonstrated that filoviruses utilize glycosaminoglycans, and more specifically heparan sulfate proteoglycans, for their attachment to host cells. This interaction is mediated by GP and does not require the presence of the mucin domain. Both the degree of sulfation and the structure of the carbohydrate backbone play a role in the interaction with filovirus GPs. This new step of filovirus interaction with host cells can potentially be a new target for antiviral strategies. As such, we were able to inhibit filovirus GP-mediated infection using carrageenan, a broad-spectrum microbicide that mimics heparin, and also using the antiviral dendrimeric peptide SB105-A10, which interacts with heparan sulfate, antagonizing the binding of the virus to cells.     

Production of High-Titer Human Immunodeficiency Virus Type 1 Pseudotyped with Vesicular Stomatitis Virus Glycoprotein

We describe a method for the production of high-titer stocks of human immunodeficiency virus type 1 (HIV-1) pseudotyped with vesicular stomatitis virus envelope glycoprotein (VSV G). VSV G pseudotypes provide several advantages over other retroviral envelope proteins. The VSV G envelope is mechanically stable, enabling ultracentrifugal concentration of virions to high titers, and VSV G has a broad host range, enabling infection of many mammalian and nonmammalian cell types. VSV G pseudotypes of HIV-1 are useful for the study of HIV infection and replication kinetics and for the study of the function of specific viral proteins. We describe applications for the study of HIV-1 using VSV G pseudotypes. Additionally, we describe a method for pseudotyping retroviral vectors with VSV G. The same advantages of VSV G pseudotypes of HIV-1 apply to retroviral vectors; VSV G pseudotyped retroviral vectors may be used to introduce genes of interest into a wide variety of cell lines.     

Pseudotype formation of murine leukemia virus with the G protein of vesicular stomatitis virus.

Mixed infection of a cell by vesicular stomatitis virus (VSV) and retroviruses results in the production of progeny virions bearing the genome of one virus encapsidated by the envelope proteins of the other. The mechanism for the phenomenon of pseudotype formation is not clear, although specific recognition of a viral envelope protein by the nucleocapsid of an unrelated virus is presumably involved. In this study, we used Moloney murine leukemia virus (MoMLV)-based retroviral vectors encoding the gene for neomycin phosphotransferase to investigate the interaction between the VSV G protein and the retroviral nucleocapsid during the formation of MoMLV(VSV) pseudotypes. Our results show that VSV G protein can be incorporated into the virions of retrovirus in the absence of other VSV-encoded proteins or of retroviral envelope protein. Infection of hamster cells by MoMLV(VSV) pseudotypes gave rise to neomycin phosphotransferase-resistant colonies, and addition of anti-VSV serum to the virus preparations completely abolished the infectivity of MoMLV(VSV) pseudotypes. It should be possible to use existing mutants of VSV G protein in the system described here to identify the signals that are important for the formation of MoMLV(VSV) pseudotypes.     

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.     

Cytoplasmic domain requirement for incorporation of a foreign envelope protein into vesicular stomatitis virus.

Incorporation of human immunodeficiency virus type 1 (HIV-1) envelope proteins into vesicular stomatitis virus (VSV) particles was studied in a system that allows expressed envelope proteins to rescue phenotypically a temperature-sensitive mutant of VSV (tsO45). This mutant exhibits defective transport of its own envelope glycoprotein (G) and can be rescued by simultaneous expression of wild-type G protein from cDNA. We report here that a hybrid HIV-1-VSV protein containing the extracellular and transmembrane domains of the HIV-1 envelope protein fused to the cytoplasmic domain of VSV G protein was able to rescue the tsO45 mutant lacking the G protein, while the wild-type HIV-1 envelope protein was not. The VSV(HIV) pseudotypes obtained infected only CD4+ cells and were neutralized specifically by anti-HIV-1 sera. Our results indicate that the cytoplasmic tail of the VSV glycoprotein contains an independent signal capable of directing a foreign protein into VSV particles. The VSV(HIV) pseudotypes generated here were prepared in the absence of HIV-1 and should be useful for identifying molecules that block HIV-1 entry.     

Mechanism of formation of pseudotypes between vesicular stomatitis virus and murine leukemia virus.

Pseudotypes of vesicular stomatitis virus (VSV) and Moloney murine leukemia virus (MuLV), defined by their resistance to neutralization by anti-VSV antiserum, are released preferentially at early times after infection of MuLV-producing cells with VSV. At later times, after synthesis of MuLV proteins has been inhibited by the VSV infection, neither MuLV virions nor the VSV (MuLV) pseudotypes are made. Infection of MuLV-producing cells with mutants of VSV having temperature-sensitive lesions in either G or M protein does not generate pseudotypes at nonpermissive temperature, indicating that both proteins are needed for pseudotypes to form. Although the pseudotypes resist neutralization by anti-VSV serum, they are inactivated by anti-VSV serum plus complement, and they can be precipitated by rabbit anti-VSV serum plus goat anti-rabbit IgG. These results, coupled with experiments using a temperature-sensitive mutant of VSV G protein grown at partly restrictive temperature, suggest that small numbers of VSV G protein are obligately incorporated into VSV(MuLV) pseudotypes. There appears to be a stringent requirement for recognition of the viral core by homologous envelope components as the nucleating step in the budding process. Only after such a nucleation can the envelope components of the second virus substitute into the membrane of the budding particle.     

Distinct patterns of IFITM-mediated restriction of filoviruses, SARS coronavirus, and influenza A virus

Interferon-inducible transmembrane proteins 1, 2, and 3 (IFITM1, 2, and 3) are recently identified viral restriction factors that inhibit infection mediated by the influenza A virus (IAV) hemagglutinin (HA) protein. Here we show that IFITM proteins restricted infection mediated by the entry glycoproteins (GP(1,2)) of Marburg and Ebola filoviruses (MARV, EBOV). Consistent with these observations, interferon-β specifically restricted filovirus and IAV entry processes. IFITM proteins also inhibited replication of infectious MARV and EBOV. We observed distinct patterns of IFITM-mediated restriction: compared with IAV, the entry processes of MARV and EBOV were less restricted by IFITM3, but more restricted by IFITM1. Moreover, murine Ifitm5 and 6 did not restrict IAV, but efficiently inhibited filovirus entry. We further demonstrate that replication of infectious SARS coronavirus (SARS-CoV) and entry mediated by the SARS-CoV spike (S) protein are restricted by IFITM proteins. The profile of IFITM-mediated restriction of SARS-CoV was more similar to that of filoviruses than to IAV. Trypsin treatment of receptor-associated SARS-CoV pseudovirions, which bypasses their dependence on lysosomal cathepsin L, also bypassed IFITM-mediated restriction. However, IFITM proteins did not reduce cellular cathepsin activity or limit access of virions to acidic intracellular compartments. Our data indicate that IFITM-mediated restriction is localized to a late stage in the endocytic pathway. They further show that IFITM proteins differentially restrict the entry of a broad range of enveloped viruses, and modulate cellular tropism independently of viral receptor expression.     

Ebolavirus delta-peptide immunoadhesins inhibit marburgvirus and ebolavirus cell entry

With the exception of Reston and Lloviu viruses, filoviruses (marburgviruses, ebolaviruses, and “cuevaviruses”) cause severe viral hemorrhagic fevers in humans. Filoviruses use a class I fusion protein, GP_1,2, to bind to an unknown, but shared, cell surface receptor to initiate virus-cell fusion. In addition to GP_1,2, ebolaviruses and cuevaviruses, but not marburgviruses, express two secreted glycoproteins, soluble GP (sGP) and small soluble GP (ssGP). All three glycoproteins have identical N termini that include the receptor-binding region (RBR) but differ in their C termini. We evaluated the effect of the secreted ebolavirus glycoproteins on marburgvirus and ebolavirus cell entry, using Fc-tagged recombinant proteins. Neither sGP-Fc nor ssGP-Fc bound to filovirus-permissive cells or inhibited GP_1,2-mediated cell entry of pseudotyped retroviruses. Surprisingly, several Fc-tagged Δ-peptides, which are small C-terminal cleavage products of sGP secreted by ebolavirus-infected cells, inhibited entry of retroviruses pseudotyped with Marburg virus GP_1,2, as well as Marburg virus and Ebola virus infection in a dose-dependent manner and at low molarity despite absence of sequence similarity to filovirus RBRs. Fc-tagged Δ-peptides from three ebolaviruses (Ebola virus, Sudan virus, and Taï Forest virus) inhibited GP_1,2-mediated entry and infection of viruses comparably to or better than the Fc-tagged RBRs, whereas the Δ-peptide-Fc of an ebolavirus nonpathogenic for humans (Reston virus) and that of an ebolavirus with lower lethality for humans (Bundibugyo virus) had little effect. These data indicate that Δ-peptides are functional components of ebolavirus proteomes. They join cathepsins and integrins as novel modulators of filovirus cell entry, might play important roles in pathogenesis, and could be exploited for the synthesis of powerful new antivirals.     

Ebola virus entry requires the host-programmed recognition of an intracellular receptor

Ebola and Marburg filoviruses cause deadly outbreaks of haemorrhagic fever. Despite considerable efforts, no essential cellular receptors for filovirus entry have been identified. We showed previously that Niemann-Pick C1 (NPC1), a lysosomal cholesterol transporter, is required for filovirus entry. Here, we demonstrate that NPC1 is a critical filovirus receptor. Human NPC1 fulfills a cardinal property of viral receptors: it confers susceptibility to filovirus infection when expressed in non-permissive reptilian cells. The second luminal domain of NPC1 binds directly and specifically to the viral glycoprotein, GP, and a synthetic single-pass membrane protein containing this domain has viral receptor activity. Purified NPC1 binds only to a cleaved form of GP that is generated within cells during entry, and only viruses containing cleaved GP can utilize a receptor retargeted to the cell surface. Our findings support a model in which GP cleavage by endosomal cysteine proteases unmasks the binding site for NPC1, and GP-NPC1 engagement within lysosomes promotes a late step in entry proximal to viral escape into the host cytoplasm. NPC1 is the first known viral receptor that recognizes its ligand within an intracellular compartment and not at the plasma membrane.     

Formation of vesicular stomatitis virus pseudotypes bearing surface proteins of hepatitis B virus

It has been difficult to propagate and titrate hepatitis B virus (HBV) in tissue culture. We examined whether vesicular stomatitis virus (VSV) pseudotypes bearing HBV surface (HBs) proteins infectious for human cell lines could be prepared. For this, expression plasmids for three surface proteins, L, M, and S, of HBV were made. 293T cells were then transfected with these plasmids either individually or in different combinations. 293T cells expressing HBs proteins were infected with VSVdeltaG*-G, a recombinant VSV expressing green fluorescent protein (GFP), to make VSV pseudotypes. Culture supernatants together with cells were harvested and sonicated for a short time. The infectivities of freshly harvested supernatants were determined by quantifying the number of cells expressing GFP after neutralization with anti-VSV serum and mouse monoclonal antibodies (MAbs) against HBs protein. Among 14 cell lines tested for susceptibility to HBV pseudotype samples, HepG2, JHH-7, and 293T cells were judged to be the most susceptible. Namely, the infectious units (IU) of the culture supernatant samples neutralized with anti-VSV in the absence and presence of anti-HBs S MAbs and titrated on HepG2 cells ranged from 1,000 to 4,000 IU/ml and 200 to 400 IU/ml, respectively, suggesting the presence of VSVdeltaG*(HBV) pseudotypes. This infectivity was inhibited by treatment with lactoferrin or dextran sulfate. Pretreatment of the cells with trypsin or tunicamycin inhibited plating of the pseudotype samples. The HBV pseudotypes can be used to analyze early steps of HBV infection, including the entry mechanism of HBV.     

Durability of a Vesicular Stomatitis Virus-Based Marburg Virus Vaccine in Nonhuman Primates

The filoviruses, Marburg virus (MARV) and Ebola virus, causes severe hemorrhagic fever with high mortality in humans and nonhuman primates. A promising filovirus vaccine under development is based on a recombinant vesicular stomatitis virus (rVSV) that expresses individual filovirus glycoproteins (GPs) in place of the VSV glycoprotein (G). These vaccines have shown 100% efficacy against filovirus infection in nonhuman primates when challenge occurs 28–35 days after a single injection immunization. Here, we examined the ability of a rVSV MARV-GP vaccine to provide protection when challenge occurs more than a year after vaccination. Cynomolgus macaques were immunized with rVSV-MARV-GP and challenged with MARV approximately 14 months after vaccination. Immunization resulted in the vaccine cohort of six animals having anti-MARV GP IgG throughout the pre-challenge period. Following MARV challenge none of the vaccinated animals showed any signs of clinical disease or viremia and all were completely protected from MARV infection. Two unvaccinated control animals exhibited signs consistent with MARV infection and both succumbed. Importantly, these data are the first to show 100% protective efficacy against any high dose filovirus challenge beyond 8 weeks after final vaccination. These findings demonstrate the durability of VSV-based filovirus vaccines.