An antibody recognizing the apical domain of human transferrin receptor 1 efficiently inhibits the entry of all new world hemorrhagic Fever arenaviruses

Five New World (NW) arenaviruses cause human hemorrhagic fevers. Four of these arenaviruses are known to enter cells by binding human transferrin receptor 1 (hTfR1). Here we show that the fifth arenavirus, Chapare virus, similarly uses hTfR1. We also identify an anti-hTfR1 antibody, ch128.1, which efficiently inhibits entry mediated by the glycoproteins of all five viruses, as well as replication of infectious Junín virus. Our data indicate that all NW hemorrhagic fever arenaviruses utilize a common hTfR1 apical-domain epitope and suggest that therapeutic agents targeting this epitope, including ch128.1 itself, can be broadly effective in treating South American hemorrhagic fevers.     

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.     

Conserved receptor-binding domains of Lake Victoria marburgvirus and Zaire ebolavirus bind a common receptor

The GP(1,2) envelope glycoproteins (GP) of filoviruses (marburg- and ebolaviruses) mediate cell-surface attachment, membrane fusion, and entry into permissive cells. Here we show that a 151-amino acid fragment of the Lake Victoria marburgvirus GP1 subunit bound filovirus-permissive cell lines more efficiently than full-length GP1. An homologous 148-amino acid fragment of the Zaire ebolavirus GP1 subunit similarly bound the same cell lines more efficiently than a series of longer GP1 truncation variants. Neither the marburgvirus GP1 fragment nor that of ebolavirus bound a nonpermissive lymphocyte cell line. Both fragments specifically inhibited replication of infectious Zaire ebolavirus, as well as entry of retroviruses pseudotyped with either Lake Victoria marburgvirus or Zaire ebolavirus GP(1,2). These studies identify the receptor-binding domains of both viruses, indicate that these viruses utilize a common receptor, and suggest that a single small molecule or vaccine can be developed to inhibit infection of all filoviruses.     

Comprehensive analysis of ebola virus GP1 in viral entry

Ebola virus infection is initiated by interactions between the viral glycoprotein GP1 and its cognate receptor(s), but little is known about the structure and function of GP1 in viral entry, partly due to the concern about safety when working with the live Ebola virus and the difficulty of manipulating the RNA genome of Ebola virus. In this study, we have used a human immunodeficiency virus-based pseudotyped virus as a surrogate system to dissect the role of Ebola virus GP1 in viral entry. Analysis of more than 100 deletion and amino acid substitution mutants of GP1 with respect to protein expression, processing, viral incorporation, and viral entry has allowed us to map the region of GP1 responsible for viral entry to the N-terminal 150 residues. Furthermore, six amino acids in this region have been identified as critical residues for early events in Ebola virus entry, and among these, three are clustered and are implicated as part of a potential receptor-binding pocket. In addition, substitutions of some 30 residues in GP1 are shown to adversely affect GP1 expression, processing, and viral incorporation, suggesting that these residues are involved in the proper folding and/or overall conformation of GP. Sequence comparison of the GP1 proteins suggests that the majority of the critical residues for GP folding and viral entry identified in Ebola virus GP1 are conserved in Marburg virus. These results provide information for elucidating the structural and functional roles of the filoviral glycoproteins and for developing potential therapeutics to block viral entry.     

Replicative homeostasis II: Influence of polymerase fidelity on RNA virus quasispecies biology: Implications for immune recognition, viral autoimmunity and other "virus receptor" diseases

Much of the worlds' population is in active or imminent danger from established infectious pathogens, while sporadic and pandemic infections by these and emerging agents threaten everyone. RNA polymerases (RNA_pol) generate enormous genetic and consequent antigenic heterogeneity permitting both viruses and cellular pathogens to evade host defences. Thus, RNA_pol causes more morbidity and premature mortality than any other molecule. The extraordinary genetic heterogeneity defining viral quasispecies results from RNA_pol infidelity causing rapid cumulative genomic RNA mutation a process that, if uncontrolled, would cause catastrophic loss of sequence integrity and inexorable quasispecies extinction. Selective replication and replicative homeostasis, an epicyclical regulatory mechanism dynamically linking RNApol fidelity and processivity with quasispecies phenotypic diversity, modulating polymerase fidelity and, hence, controlling quasispecies behaviour, prevents this happening and also mediates immune escape. Perhaps more importantly, ineluctable generation of broad phenotypic diversity after viral RNA is translated to protein quasispecies suggests a mechanism of disease that specifically targets, and functionally disrupts, the host cell surface molecules – including hormone, lipid, cell signalling or neurotransmitter receptors – that viruses co-opt for cell entry. This mechanism – "Viral Receptor Disease (VRD)" – may explain so-called "viral autoimmunity", some classical autoimmune disorders and other diseases, including type II diabetes mellitus, and some forms of obesity. Viral receptor disease is a unifying hypothesis that may also explain some diseases with well-established, but multi-factorial and apparently unrelated aetiologies – like coronary artery and other vascular diseases – in addition to diseases like schizophrenia that are poorly understood and lack plausible, coherent, pathogenic explanations.     

Ethanol inactivation of orthonairoviruses in ixodid ticks

Experimental and Applied Acarology - Ixodid ticks represent vectors and reservoirs for a broad range of zoonotic pathogens. Collected ticks from field studies are therefore usually stored in...     

The cytoplasmic domain of Marburg virus GP modulates early steps of viral infection

Marburg virus infection is mediated by the only viral surface protein, GP, a trimeric type I transmembrane protein. While its ectodomain mediates receptor binding and fusion of viral and cellular membranes and its transmembrane domain is essential for the recruitment of GP into budding particles by the matrix protein VP40, the role of the short cytoplasmic domain has remained enigmatic. Here we show that a missing cytoplasmic domain did not impair trimerization, intracellular transport, or incorporation of GP into infectious Marburg virus-like particles (iVLPs) but altered the glycosylation pattern as well as the recognition of GP by neutralizing antibodies. These results suggest that subtle conformational changes took place in the ectodomain. To investigate the function of the cytoplasmic domain during viral entry, a novel entry assay was established to monitor the uptake of filamentous VLPs by measuring the occurrence of luciferase-labeled viral nucleocapsids in the cytosol of target cells. This quantitative assay showed that the entry process of VLPs incorporating GP missing its cytoplasmic domain (GPΔCD) was impaired. Supporting these results, iVLPs incorporating a mutant GP missing its cytoplasmic domain were significantly less infectious than iVLPs containing wild-type GP. Taken together, the data indicate that the absence of the short cytoplasmic domain of Marburg virus GP may induce conformational changes in the ectodomain which impact the filoviral entry process.     

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.     

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.     

Clathrin- and caveolin-independent entry of human papillomavirus type 16--involvement of tetraspanin-enriched microdomains (TEMs)

Background

Infectious entry of human papillomaviruses into their host cells is an important step in the viral life cycle. For cell binding these viruses use proteoglycans as initial attachment sites. Subsequent transfer to a secondary receptor molecule seems to be involved in virus uptake. Depending on the papillomavirus subtype, it has been reported that entry occurs by clathrin- or caveolin-mediated mechanisms. Regarding human papillomavirus type 16 (HPV16), the primary etiologic agent for development of cervical cancer, clathrin-mediated endocytosis was described as infectious entry pathway.

Methodology/Principal Findings

Using immunofluorescence and infection studies we show in contrast to published data that infectious entry of HPV16 occurs in a clathrin- and caveolin-independent manner. Inhibition of clathrin- and caveolin/raft-dependent endocytic pathways by dominant-negative mutants and siRNA-mediated knockdown, as well as inhibition of dynamin function, did not impair infection. Rather, we provide evidence for involvement of tetraspanin-enriched microdomains (TEMs) in HPV16 endocytosis. Following cell attachment, HPV16 particles colocalized with the tetraspanins CD63 and CD151 on the cell surface. Notably, tetraspanin-specific antibodies and siRNA inhibited HPV16 cell entry and infection, confirming the importance of TEMs for infectious endocytosis of HPV16.

Conclusions/Significance

Tetraspanins fulfill various roles in the life cycle of a number of important viral pathogens, including human immunodeficiency virus (HIV) and hepatitis C virus (HCV). However, their involvement in endocytosis of viral particles has not been proven. Our data indicate TEMs as a novel clathrin- and caveolin-independent invasion route for viral pathogens and especially HPV16.     

Acidic pH-Induced Conformations and LAMP1 Binding of the Lassa Virus Glycoprotein Spike

Lassa virus is an enveloped, bi-segmented RNA virus and the most prevalent and fatal of all Old World arenaviruses. Virus entry into the host cell is mediated by a tripartite surface spike complex, which is composed of two viral glycoprotein subunits, GP1 and GP2, and the stable signal peptide. Of these, GP1 binds to cellular receptors and GP2 catalyzes fusion between the viral envelope and the host cell membrane during endocytosis. The molecular structure of the spike and conformational rearrangements induced by low pH, prior to fusion, remain poorly understood. Here, we analyzed the three-dimensional ultrastructure of Lassa virus using electron cryotomography. Sub-tomogram averaging yielded a structure of the glycoprotein spike at 14-Å resolution. The spikes are trimeric, cover the virion envelope, and connect to the underlying matrix. Structural changes to the spike, following acidification, support a viral entry mechanism dependent on binding to the lysosome-resident receptor LAMP1 and further dissociation of the membrane-distal GP1 subunits.     

Effects of a zoonotic pathogen,Borrelia burgdorferi,on the behavior of a key reservoir host

Most emerging infectious diseases of humans are transmitted to humans from other animals. The transmission of these “zoonotic” pathogens is affected by the abundance and behavior of their wildlife hosts. However, the effects of infection with zoonotic pathogens on behavior of wildlife hosts, particularly those that might propagate through ecological communities, are not well understood. Borrelia burgdorferi is a bacterium that causes Lyme disease, the most common vector‐borne disease in the USA and Europe. In its North American range, the pathogen is most frequently transmitted among hosts through the bite of infected blacklegged ticks (Ixodes scapularis). Using sham and true vaccines, we experimentally manipulated infection load with this zoonotic pathogen in its most competent wildlife reservoir host, the white‐footed mouse, Peromyscus leucopus, and quantified the effects of infection on mouse foraging behavior, as well as levels of mouse infestation with ticks. Mice treated with the true vaccine had 20% fewer larval blacklegged ticks infesting them compared to mice treated with the sham vaccine, a significant difference. We observed a nonsignificant trend for mice treated with the true vaccine to be more likely to visit experimental foraging trays (20%–30% effect size) and to prey on gypsy moth pupae (5%–20% effect size) compared to mice treated with the sham vaccine. We observed no difference between mice on true‐ versus sham‐vaccinated grids in risk‐averse foraging. Infection with this zoonotic pathogen appears to elicit behavioral changes that might reduce self‐grooming, but other behaviors were affected subtly or not at all. High titers of B. burgdorferi in mice could elicit a self‐reinforcing feedback loop in which reduced grooming increases tick burdens and hence exposure to tick‐borne pathogens.     

Identification of a GP64 subdomain involved in receptor binding by budded virions of the baculovirus Autographica californica multicapsid nucleopolyhedrovirus

Enveloped virus entry into host cells is typically initiated by an interaction between a viral envelope glycoprotein and a host cell receptor. For budded virions of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus, the envelope glycoprotein GP64 is involved in host cell receptor binding, and GP64 is sufficient to mediate low-pH-triggered membrane fusion. To better define the role of GP64 in receptor binding, we generated and characterized a panel of antisera against subdomains of GP64. Eight subdomain-specific antisera were generated, and their reactivities with GP64 proteins and neutralization of virus infectivity and binding were examined. Antibodies directed against the N-terminal region of GP64 (amino acids 21 to 159) showed strong neutralization of infectivity and effectively inhibited binding of (35)S-labeled budded virions to Sf9 cells. In addition, we generated virions displaying truncated GP64 constructs. A construct displaying the N-terminal 274 amino acids (residues 21 to 294) of the ectodomain was sufficient to mediate virion binding. Additional studies of antisera directed against small subdomains revealed that an antiserum against a 40-amino-acid region (residues 121 to 160) neutralized virus infectivity. Site-directed mutagenesis was subsequently used for functional analysis of that region. Recombinant viruses expressing GP64 proteins with single amino acid substitutions within amino acids 120 to 124 and 142 to 148 replicated to high titers, suggesting that those amino acids were not critical for receptor binding or other important GP64 functions. In contrast, GP64 proteins with single amino acid substitutions of residues 153 and 156 were unable to substitute for wild-type GP64 and did not rescue a gp64 knockout virus. Further analysis showed that these substitutions substantially reduced binding of recombinant virus to Sf9 cells. Thus, the amino acid region from positions 21 to 159 was identified as a putative receptor binding domain, and amino acids 153 and 156 appear to be important for receptor binding.     

The role of the charged residues of the GP2 helical regions in Ebola entry

The glycoprotein (GP) of Ebola is the sole structural protein that forms the spikes on the viral envelope. The GP contains two subunits, GP1 and GP2, linked by a disulfide bond, which are responsible for receptor binding and membrane fusion, respectively. In this study, the full length of GP gene of Ebola Zaire species, 2028 base pairs in length, was synthesized using 38 overlapping oligonucleotides by multiple rounds of polymerase chain reaction (PCR). The synthesized GP gene was shown to be efficiently expressed in mammalian cells. Furthermore, an efficient HIV-based pseudotyping system was developed using the synthetic GP gene, providing a safe approach to dissecting the entry mechanism of Ebola viruses. Using this pseudotyping system and mutational analysis, the role of the charged residues in the GP2 helical regions was examined. It was found that substitutions of the most charged residues in the regions did not adversely affect GP expression, processing, or viral incorporation, however, most of the mutations greatly impaired the ability of GP to mediate efficient viral infection. These results demonstrate that these charged residues of GP2 play an important role in GP-mediated Ebola entry into its host cells. We propose that these charged residues are involved in forming the intermediate conformation(s) of GP in membrane fusion and Ebola entry.     

Protective effect of ginseng polysaccharides on influenza viral infection

Ginseng polysaccharide has been known to have multiple immunomodulatory effects. In this study, we investigated whether Panax ginseng polysaccharide (GP) would have a preventive effect on influenza infection. Administration of mice with GP prior to infection was found to confer a survival benefit against infection with H1N1 (A/PR/8/34) and H3N2 (A/Philippines/82) influenza viruses. Mice infected with the 2009 H1N1 virus suspended in GP solution showed moderately enhanced survival rates and lower levels of lung viral titers and the inflammatory cytokine (IL-6). Daily treatment of vaccinated mice with GP improved their survival against heterosubtypic lethal challenge. This study demonstrates the first evidence that GP can be used as a remedy against influenza viral infection.     

Evidence for distinct mechanisms of small molecule inhibitors of filovirus entry

Many small molecules have been identified as entry inhibitors of filoviruses. However, a lack of understanding of the mechanism of action for these molecules limits further their development as anti-filoviral agents. Here we provide evidence that toremifene and other small molecule entry inhibitors have at least three distinctive mechanisms of action and lay the groundwork for future development of anti-filoviral agents. The three mechanisms identified here include: (1) direct binding to the internal fusion loop region of Ebola virus glycoprotein (GP); (2) the HR2 domain is likely the main binding site for Marburg virus GP inhibitors and a secondary binding site for some EBOV GP inhibitors; (3) lysosome trapping of GP inhibitors increases drug exposure in the lysosome and further improves the viral inhibition. Importantly, small molecules targeting different domains on GP are synergistic in inhibiting EBOV entry suggesting these two mechanisms of action are distinct. Our findings provide important mechanistic insights into filovirus entry and rational drug design for future antiviral development.     

Conspicuous impacts of inconspicuous hosts on the Lyme disease epidemic

Emerging zoonotic pathogens are a constant threat to human health throughout the world. Control strategies to protect public health regularly fail, due in part to the tendency to focus on a single host species assumed to be the primary reservoir for a pathogen. Here, we present evidence that a diverse set of species can play an important role in determining disease risk to humans using Lyme disease as a model. Host-targeted public health strategies to control the Lyme disease epidemic in North America have focused on interrupting Borrelia burgdorferi sensu stricto (ss) transmission between blacklegged ticks and the putative dominant reservoir species, white-footed mice. However, B. burgdorferi ss infects more than a dozen vertebrate species, any of which could transmit the pathogen to feeding ticks and increase the density of infected ticks and Lyme disease risk. Using genetic and ecological data, we demonstrate that mice are neither the primary host for ticks nor the primary reservoir for B. burgdorferi ss, feeding 10% of all ticks and 25% of B. burgdorferi-infected ticks. Inconspicuous shrews feed 35% of all ticks and 55% of infected ticks. Because several important host species influence Lyme disease risk, interventions directed at a multiple host species will be required to control this epidemic.