No abstract

Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. However, methods to quantify cargo proteins loaded into engineered EVs are lacking. Here, we describe a workflow for EV analysis at the single‐vesicle and single‐molecule level to accurately quantify the efficiency of different EV‐sorting proteins in promoting cargo loading into EVs. Expi293F cells were engineered to express EV‐sorting proteins fused to green fluorescent protein (GFP). High levels of GFP loading into secreted EVs was confirmed by Western blotting for specific EV‐sorting domains, but quantitative single‐vesicle analysis by Nanoflow cytometry detected GFP in less than half of the particles analysed, reflecting EV heterogeneity. Anti‐tetraspanin EV immunostaining in ExoView confirmed a heterogeneous GFP distribution in distinct subpopulations of CD63 + , CD81 + , or CD9 + EVs. Loading of GFP into individual vesicles was quantified by Single‐Molecule Localization Microscopy. The combined results demonstrated TSPAN14, CD63 and CD63/CD81 fused to the PDGFRβ transmembrane domain as the most efficient EV‐sorting proteins, accumulating on average 50–170 single GFP molecules per vesicle. In conclusion, we validated a set of complementary techniques suitable for high‐resolution analysis of EV preparations that reliably capture their heterogeneity, and propose highly efficient EV‐sorting proteins to be used in EV engineering applications.

No abstract

We have previously reported the construction of a murine leukemia virus-based replication-competent gammaretrovirus (SL3-AP) capable of utilizing the human G protein-coupled receptor APJ (hAPJ) as its entry receptor and its natural receptor, the murine Xpr1 receptor, with equal affinities. The apelin receptor has previously been shown to function as a coreceptor for HIV-1, and thus, adaptation of the viral vector to this receptor is of significant interest. Here, we report the molecular evolution of the SL3-AP envelope protein when the virus is cultured in cells harboring either the Xpr1 or the hAPJ receptor. Interestingly, the dual receptor affinity is maintained even after 10 passages in these cells. At the same time, the chimeric viral envelope protein evolves in a distinct pattern in the apelin cassette when passaged on D17 cells expressing hAPJ in three separate molecular evolution studies. This pattern reflects selection for reduced ligand-receptor interaction and is compatible with a model in which SL3-AP has evolved not to activate hAPJ receptor internalization. IMPORTANCEFew successful examples of engineered retargeting of a retroviral vector exist. The engineered SL3-AP envelope is capable of utilizing either the murine Xpr1 or the human APJ receptor for entry. In addition, SL3-AP is the first example of an engineered retrovirus retaining its dual tropism after several rounds of passaging on cells expressing only one of its receptors. We demonstrate that the virus evolves toward reduced ligand-receptor affinity, which sheds new light on virus adaptation. We provide indirect evidence that such reduced affinity leads to reduced receptor internalization and propose a novel model in which too rapid receptor internalization may decrease virus entry. F usion of lipid membranes is not a spontaneous event and thus requires a supply of energy through an active fusion mechanism. Retroviruses are enveloped viruses and hence are strictly dependent on their fusion machinery for entry into host cells. In retroviruses, including murine leukemia virus (MLV), the fusion energy is stored in the envelope (Env) protein that is present in the membrane envelope surrounding the viral particle. Env consists of trimers of two subunits, the N-terminal surface (SU) subunit and the C-terminal transmembrane (TM) subunit. SU is responsible for recognizing and binding cellular receptor proteins, which differ among virus types and define their tropism. The SU and TM subunits remain associated through noncovalent interactions or, as for MLV, through a disulfide bridge that is broken as a result of disulfide isomerization following receptor binding (1). Here, the fusion energy stored in TM and suppressed by SU is released by conformational changes allowing the formation of an elongated triple helix in TM. This leads to insertion of the fusion peptide into the membrane of the host cell and hence induces membrane fusion (2), as reviewed in references 3 and 4. Other reports have suggested a need for proteolytic cleavage of SU in e...