TMEM16F is a Ca2+ -gated ion channel that is required for Ca2+ -activated phosphatidylserine exposure on the surface of many eukaryotic cells. TMEM16F is widely expressed and has roles in platelet activation during blood clotting, bone formation and T cell activation. By combining microscopy and patch clamp recording we demonstrate that activation of TMEM16F by Ca2+ ionophores in Jurkat T cells triggers large-scale surface membrane expansion in parallel with phospholipid scrambling. With continued ionophore application,TMEM16F-expressing cells then undergo extensive shedding of ectosomes. The T cell co-receptor PD-1 is selectively incorporated into ectosomes. This selectivity depends on its transmembrane sequence. Surprisingly, cells lacking TMEM16F not only fail to expand surface membrane in response to elevated cytoplasmic Ca2+, but instead undergo rapid massive endocytosis with PD-1 internalisation. These results establish a new role for TMEM16F as a regulator of Ca2+ activated membrane trafficking.

Retroviral and lentiviral vectors often use the envelope G protein from the vesicular stomatitis virus Indiana strain (VSVind.G). However, lentivector producer cell lines that stably express VSVind.G have not been reported, presumably because of its cytotoxicity, preventing simple scale-up of vector production. Interestingly, we showed that VSVind.G and other vesiculovirus G from the VSV New Jersey strain (VSVnj), Cocal virus (COCV), and Piry virus (PIRYV) could be constitutively expressed and supported lentivector production for up to 10 weeks. All G-enveloped particles were robust, allowing concentration and freeze-thawing. COCV.G and PIRYV.G were resistant to complement inactivation, and, using chimeras between VSVind.G and COCV.G, the determinant for complement inactivation of VSVind.G was mapped to amino acid residues 136–370. Clonal packaging cell lines using COCV.G could be generated; however, during attempts to establish LV producer cells, vector superinfection was observed following the introduction of a lentivector genome. This could be prevented by culturing the cells with the antiviral drug nevirapine. As an alternative countermeasure, we demonstrated that functional lentivectors could be reconstituted by admixing supernatant from stable cells producing unenveloped virus with supernatant containing envelopes harvested from cells stably expressing VSVind.G, COCV.G, or PIRYV.G.

VSVind.G is currently regarded as the gold-standard envelope glycoprotein to pseudotype lentiviral vectors. However, recently other G proteins derived from vesiculoviruses have been proposed as alternative envelopes. Here, we investigated two commercially available anti-VSVind.G monoclonal antibodies for their ability to cross-react with other vesiculovirus G proteins, identified the epitopes they recognize, and explored their neutralization activity. We have identified 8G5F11, for the first time, as a cross-neutralizing antibody against several vesiculovirus G proteins. Furthermore, we elucidated the two different neutralization mechanisms employed by these two monoclonal antibodies. Understanding how cross-neutralizing antibodies interact with other G proteins may be of interest in the context of host-pathogen interaction and coevolution, as well as providing the opportunity to modify the G proteins and improve G protein-containing medicinal products and vaccine vectors.