Nipah Virus Envelope-Pseudotyped Lentiviruses Efficiently Target ephrinB2-Positive Stem Cell Populations In Vitro and Bypass the Liver Sink When Administered In Vivo

Abstract

Sophisticated retargeting systems for lentiviral vectors have been developed in recent years. Most seek to suppress the viral envelope's natural tropism while modifying the receptor-binding domain such that its tropism is determined by the specificity of the engineered ligand-binding motif. Here we took advantage of the natural tropism of Nipah virus (NiV), whose attachment envelope glycoprotein has picomolar affinity for ephrinB2, a molecule proposed as a molecular marker of "stemness" (present on embryonic, hematopoietic, and neural stem cells) as well as being implicated in tumorigenesis of specific cancers. NiV entry requires both the fusion (F) and attachment (G) glycoproteins. Truncation of the NiV-F cytoplasmic tail (T5F) alone, combined with full-length NiV-G, resulted in optimal titers of NiV-pseudotyped particles (NiVpp) (ϳ10 6 IU/ml), even without ultracentrifugation. To further enhance the infectivity of NiVpp, we engineered a hyperfusogenic NiV-F protein lacking an N-linked glycosylation site (T5F⌬N3). T5F⌬N3/wt G particles exhibited enhanced infectivity on less permissive cell lines and efficiently targeted ephrinB2؉ cells even in a 1,000-fold excess of ephrinB2-negative cells, all without any loss of specificity, as entry was abrogated by soluble ephrinB2. NiVpp also transduced human embryonic, hematopoietic, and neural stem cell populations in an ephrinB2-dependent manner. Finally, intravenous administration of the luciferase reporter NiVpp-T5F⌬N3/G to mice resulted in signals being detected in the spleen and lung but not in the liver. Bypassing the liver sink is a critical barrier for targeted gene therapy. The extraordinary specificity of NiV-G for ephrinB2 holds promise for targeting specific ephrinB2 ؉ populations in vivo or in vitro.

Nipah virus fusion protein: Importance of the cytoplasmic tail for endosomal trafficking and bioactivity

“…Besides the finding that truncation mutants lacking up to 18 of 28 residues less efficiently pseudotyped LV, Khetawat and Broder (2010) demonstrated that F mutants with larger tail deletions no longer containing the Y 525 RSL endocytosis signal (F 24, F 28) supported a 3-10-fold increased LV production. A similar finding was made by Palomares et al (2013) for F 23. Though Witting et al (2013) reported that G tail truncation had a more pronounced effect on LV production as did F tail deletions, they also observed that F proteins with a large tail deletion (F 25) improved the LV titers.…”

“…Glycoprotein G is responsible for binding to ephrin-B2/-B3 on host cells (Bonaparte et al, 2005;Negrete et al, 2005Negrete et al, , 2006 and complexes of G and F mediate fusion between viral and cellular membranes during virus entry and cell-to-cell spread (reviewed in Diederich and Maisner, 2007). Aside of their incorporation into infectious NiV particles, NiV-F and -G can efficiently pseudotype lentiviral vectors thereby allowing specific targeting of ephrin-positive cells (Khetawat and Broder, 2010;Palomares et al, 2013;Witting et al, 2013). http://dx.doi.org/10.1016/j.ejcb.2015.05.005 0171-9335/© 2015 Elsevier GmbH.…”

Nipah and Hendra Virus Glycoproteins Induce Comparable Homologous but Distinct Heterologous Fusion Phenotypes

“…One of our studies showed that chimeras containing the NiV-G head domain and the NDV HN stalk domain could bind ephrinB2 and trigger fusion mediated by NDV F (28). In the present study, to elucidate the protein elements responsible for the fusion phenotypes observed, we created G chimeras in which we exchanged the CT/TM/stalk (residues 1 to 167) or head (residues 168 to 604) domain between HeV and NiV G. Functional properties of the cytoplasmic, stalk, and head domains of G have been described somewhat (54)(55)(56)(57). Our G chimeras revealed that the CT/TM/stalk domain of HeV G is a main determinant of the hypofusogenic NF/HG phenotype and that this may in part be attributed to a stronger binding avidity of the HG CT/TM/stalk domain to NF.…”

Package of NDV-Pseudotyped HIV-Luc Virus and Its Application in the Neutralization Assay for NDV Infection

“…The sensitivity of the pseudovirus-based assay was further confirmed in the detection of neutralizing antibodies (table 1) in the two negative sera as determined with the conventional VN test. These obvious advantages of being a high-throughput, rapid, sensitive, reproducible and less subjective system have encouraged the development of many other pseudotyped lentivirus for the establishment of neutralization assays, such as equine influenza, Nipah virus, Chikungunya Virus, Coxsackievirus A16 and HIV-1 [25,26,45,[59][60][61]. In this novel neutralization assay, there are two methods to titrate NDV-pseudotyped HIV-Luc viruses, one is by measuring the luciferase activity after pseudovirus infection [60], and the other is with the TCID 50 assay.…”