The glycoproteins (GP) of enveloped viruses facilitate entry into the host cell by interacting with specific cellular receptors. Despite extensive study, a cellular receptor for the deadly filoviruses Ebolavirus and Marburgvirus has yet to be identified and characterized. Here, we show that T-cell Ig and mucin domain 1 (TIM-1) binds to the receptor binding domain of the Zaire Ebola virus (EBOV) glycoprotein, and ectopic TIM-1 expression in poorly permissive cells enhances EBOV infection by 10-to 30-fold. Conversely, reduction of cell-surface expression of TIM-1 by RNAi decreased infection of highly permissive Vero cells. TIM-1 expression within the human body is broader than previously appreciated, with expression on mucosal epithelia from the trachea, cornea, and conjunctiva-tissues believed to be important during in vivo transmission of filoviruses. Recognition that TIM-1 serves as a receptor for filoviruses on these mucosal epithelial surfaces provides a mechanistic understanding of routes of entry into the human body via inhalation of aerosol particles or hand-to-eye contact. ARD5, a monoclonal antibody against the IgV domain of TIM-1, blocked EBOV binding and infection, suggesting that antibodies or small molecules directed against this cellular receptor may provide effective filovirus antivirals. viral entry | viral receptor | virion internalization T he Filoviridae family of viruses is composed of two genera, Ebolavirus and Marburgvirus, which cause hemorrhagic fever in humans and nonhuman primates. Infection with some strains of filoviruses causes fatality in 50-90% of human cases (1). The viral glycoprotein (GP) of Ebolavirus, which consists of surfaceexposed subunit GP1 attached to membrane-bound subunit GP2 by a disulfide bond (2), mediates binding to, penetration of, and fusion with host-cell membranes (3, 4). Pseudovirions bearing Ebolavirus GP transduce a broad range of cells through interactions that require the GP1 receptor-binding domain (RBD) (5-8). Upon internalization into low-pH endosomes, the filovirus GP1 is proteolyzed by cathepsins B and L, leading to GP2-dependent fusion of the viral and host membranes (9-12). Several proteins enhance filovirus entry in host cells, including the C-type lectins L-SIGN, DC-SIGN, and hMGL, as well as RhoB/C, integrin α5β1, folate receptor-α, and the tyrosine kinase receptor Axl (13-26); however, because none of these molecules has been shown to interact with the RBD of the filovirus GP1, it is unlikely that any of these proteins serve as a receptor for this family of viruses. Thus, we used gene correlation analysis to search for additional potential receptors. Here, we identify T-cell Ig and mucin domain 1 (TIM-1), which interacts with Zaire ebolavirus (EBOV) GP and enhances EBOV infection by 10-to 30-fold upon expression, providing strong evidence that TIM-1 serves as a receptor for EBOV. As we found that TIM-1 is expressed on a number of mucosal epithelial surfaces, we propose that TIM-1/ EBOV interactions may serve as a conduit for filovirus entry into ...

Measles (MV) is an aerosol-transmitted virus that affects more than 10 million children each year and accounts for approximately 120,000 deaths1,2. While it was long believed to replicate in the respiratory epithelium before disseminating, it was recently shown to initially infect macrophages and dendritic cells of the airways using the signaling lymphocytic activation molecule (SLAM, CD150) as receptor3-6. These cells then cross the respiratory epithelium and ferry the infection to lymphatic organs where MV replicates vigorously7. How and where the virus crosses back into the airways has remained unknown. Based on functional analyses of surface proteins preferentially expressed on virus-permissive epithelial cell lines, we identified nectin-48 (poliovirus-receptor-like-4) as a candidate host exit receptor. This adherens junction protein of the immunoglobulin superfamily interacts with the viral attachment protein with high affinity through its membrane-distal domain. Nectin-4 sustains MV entry and non-cytopathic lateral spread in well-differentiated primary human airway epithelial sheets infected basolaterally. It is down-regulated in infected epithelial cells, including those of macaque tracheas. While other viruses use receptors to enter hosts or transit through their epithelial barriers, we suggest that MV targets nectin-4 to emerge in the airways. Nectin-4 is a cellular marker of several types of cancer9-11, which has implications for ongoing MV-based clinical trials of oncolysis12.

The current model of measles virus (MV) pathogenesis implies that apical infection of airway epithelial cells precedes systemic spread. An alternative model suggests that primarily infected lymphatic cells carry MV to the basolateral surface of epithelial cells, supporting MV shedding into the airway lumen and contagion. This model predicts that a mutant MV, unable to enter cells through the unidentified epithelial cell receptor (EpR), would remain virulent but not be shed. To test this model, we identified residues of the MV attachment protein sustaining EpR-mediated cell fusion. These nonpolar or uncharged polar residues defined an area located near the binding site of the signaling lymphocytic activation molecule (SLAM), the receptor for MV on lymphatic cells. We then generated an EpR-blind virus maintaining SLAM-dependent cell entry and inoculated rhesus monkeys intranasally. Hosts infected with the selectively EpR-blind MV developed rash and anorexia while averaging slightly lower viremia than hosts infected with wild-type MV but did not shed virus in the airways. The mechanism restricting shedding was characterized using primary well-differentiated human airway epithelial cells. Wild-type MV infected columnar epithelial cells bearing tight junctions only when applied basolaterally, while the EpR-blind virus did not infect these cells. Thus, EpR is probably a basolateral protein, and infection of the airway epithelium is not essential for systemic spread and virulence of MV.

The transposon piggyBac is being used increasingly for genetic studies. Here, we describe modified versions of piggyBac transposase that have potentially wide-ranging applications, such as reversible transgenesis and modified targeting of insertions. piggyBac is distinguished by its ability to excise precisely, restoring the donor site to its pretransposon state. This characteristic makes piggyBac useful for reversible transgenesis, a potentially valuable feature when generating induced pluripotent stem cells without permanent alterations to genomic sequence. To avoid further genome modification following piggyBac excision by reintegration, we generated an excision competent/integration defective (Exc + Int − ) transposase. Our findings also suggest the position of a target DNA-transposase interaction. Another goal of genome engineering is to develop reagents that can guide transgenes to preferred genomic regions. Others have shown that piggyBac transposase can be active when fused to a heterologous DNA-binding domain. An Exc + Int − transposase, the intrinsic targeting of which is defective, might also be a useful intermediate in generating a transposase whose integration activity could be rescued and redirected by fusion to a site-specific DNA-binding domain. We show that fusion to two designed zinc finger proteins rescued the Int − phenotype. Successful guided transgene integration into genomic DNA would have broad applications to gene therapy and molecular genetics. Thus, an Exc + Int − transposase is a potentially useful reagent for genome engineering and provides insight into the mechanism of transposase-target DNA interaction.NA "cut-and-paste" transposable elements are important tools for genome engineering, such as insertional mutagenesis and transgenesis. Research with the DNA transposon Sleeping Beauty, a "resurrected" transposon, has pioneered the use of DNA transposons in mammalian cells (1, 2). piggyBac is also a DNA transposon and a promising alternative to Sleeping Beauty. piggyBac, originally isolated from the cabbage looper moth Trichoplusia ni genome (3), has a large cargo size (4), is highly active in many cell types, and mediates long-term expression in mammalian cells in vivo (5-10). piggyBac is also distinguished by its ability to excise precisely (11), thus restoring the donor site to its pretransposon insertion sequence.Because it can excise precisely, piggyBac is especially useful if a transgene is only transiently required. Transient integration and expression of transcription factors are important approaches to generate transgene-free induced pluripotent stem cells (iPSCs) (12, 13) as well as directed differentiation of specific cell types for both research and clinical use. Removal of the transgenes is key for potential therapeutic applications of iPSCs. piggyBac has been used as a vector for reversible integration; however, reintegration of the transposon catalyzed by piggyBac (PB) transposase occurs in 40-50% of cells (14) Int− transposase whose excision frequency is five-to six-f...

Replication defective vectors derived from simple retroviruses or the more complex genomes of lentiviruses continue to offer the advantages of long-term expression, cell and tissue specific tropism, and large packaging capacity for the delivery of therapeutic genes. The occurrence of adverse events caused by insertional mutagenesis in three patients in a gene therapy trial for X-linked SCID emphasizes the potential for problems in translating this approach to the clinic. Several genome-wide studies of retroviral integration are now providing novel insights into the integration site preferences of different vector classes. We review recent developments in vector design, integration, biosafety, and production.

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␣), 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␣ 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␣-blocking antibodies, we demonstrated FR␣-dependent and -independent entry by filovirus glycoprotein-pseudotyped FIV-based vectors in airway epithelia. Of particular interest, entry independent of FR␣ 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.

Bioluminescence tomography (BLT) is a new molecular imaging mode, which is being actively developed to reveal molecular and cellular signatures as labeled by bioluminescent probes in a living small animal. This technology can help diagnose diseases, evaluate therapies, and facilitate drug development with mouse models. In this paper, we describe in vivo mouse experiments with BLT, and propose the reconstruction procedure of bioluminescent sources from optical data measured on the body surface of the mouse using a modality fusion approach. The results show the feasibility of our methodology for localization and quantification of the bioluminescent activities in vivo.

Gene transfer development for treatment or prevention of cystic fibrosis lung disease has been limited by the inability of vectors to efficiently and persistently transduce airway epithelia. Influenza A is an enveloped virus with natural lung tropism; however, pseudotyping feline immunodeficiency virus (FIV)-based lentiviral vector with the hemagglutinin envelope protein proved unsuccessful. Conversely, pseudotyping FIV with the envelope protein from influenza D (Thogoto virus GP75) resulted in titers of 10 6 transducing units (TU)/ml and conferred apical entry into well-differentiated human airway epithelial cells. Baculovirus GP64 envelope glycoproteins share sequence identity with influenza D GP75 envelope glycoproteins. Pseudotyping FIV with GP64 from three species of baculovirus resulted in titers of 10 7 to 10 9 TU/ml. Of note, GP64 from Autographa californica multicapsid nucleopolyhedrovirus resulted in high-titer FIV preparations (ϳ10 9 TU/ml) and conferred apical entry into polarized primary cultures of human airway epithelia. Using a luciferase reporter gene and bioluminescence imaging, we observed persistent gene expression from in vivo gene transfer in the mouse nose with A. californica GP64-pseudotyped FIV (AcGP64-FIV). Longitudinal bioluminescence analysis documented persistent expression in nasal epithelia for ϳ1 year without significant decline. According to histological analysis using a LacZ reporter gene, olfactory and respiratory epithelial cells were transduced. In addition, methylcellulose-formulated AcGP64-FIV transduced mouse nasal epithelia with much greater efficiency than similarly formulated vesicular stomatitis virus glycoprotein-pseudotyped FIV. These data suggest that AcGP64-FIV efficiently transduces and persistently expresses a transgene in nasal epithelia in the absence of agents that disrupt the cellular tight junction integrity.