Members of social networks use their connections to communicate, learn, and influence others, a pattern familiar to neuroscientists who have been grappling with interconnections — of the neural variety — for decades. Early efforts to trace connections between neurons with high-resolution imaging helped build spectacular maps of circuit architecture. Despite these successes, interest has gravitated toward functional neuroanatomy, an ambitious goal that demands development of even more sophisticated tools. In an article published in Neuron, Osakada et al. present a suite of rabies-based viral vectors for labeling, monitoring, and manipulating neural neighborhoods. Rabies virus, which spreads across synapses in the retrograde direction and does not kill infected cells for a fortnight or so, had previously been engineered into a GFP-expressing vector capable of revealing neural connections. In the new work, the methods for producing recombinant viruses have been refined to improve recovery efficiency and titer, and, building upon this more robust preparation system, the repertoire of vector functionality has been substantially expanded. Of the 12 new variants, three swap in other fluorescent proteins in place of GFP, giving the option of simultaneously tracking different viruses. Equally useful is expression of two different genes from a single virus. The authors present a virus capable of expressing a calcium sensor along with the DsRed fluorophore. After stereotaxic injection of this construct into mice and two-photon imaging, DsRed handily pinpointed the infected neurons for characterization of functional responses by the calcium sensor. Notably, this imaging could be performed at depths greater than 500 µm and more than 10 days after infection. To control neural activity, the authors created a recombinant containing a light-activated ion channel protein. Electrophysiological studies confirmed light-induced depolarization in infected cells, suggesting that this construct will be useful in optogenetics applications. In another vector, a receptor protein that responds to a peptide ligand by inducing hyperpolarization showed the feasibility of ligand-inducible silencing of neural activity. The authors rounded out the suite of rabies virus variants with constructs harboring the Tet-On transactivator, tamoxifen-inducible Cre recombinase, or FLP recombinase.
Osakada et al. 2011. New rabies virus variants for monitoring and manipulating activity and gene expression in defined neural circuits. Neuron. 71:617-31.From Artifact to Tool
A trick for maximizing titers of retroviral vectors involves pseudotyping with the envelope glycoprotein of vesicular stomatitis virus (VSV-G). VSV-G has an exceptionally broad host-cell range and tends to form stable virions, making it easy to concentrate vector stocks to high titers. However, users of VSV-G-pseudotyped vectors will be familiar with “pseudotransduction”, the transient appearance of exogenous proteins in target cells. Writing in Molecular Therapy, Mangeot et al consider whether this experimental artifact might be harnessed for protein transduction. Existing protein transfer methods tend to require purifying recombinant protein and coaxing it to enter target cells with the help of cell-penetrating peptides or proteoliposomes. Though virus-like particles have been used to transfer proteins from one set of cells to another, this technique has been limited to immune stimulation. Similarly, a method that repurposes a retroviral structural factor for protein transfer requires specialized engineering of the protein cargo. By contrast, the new method simply requires that the cargo protein be coexpressed with VSV-G. The supernatant is collected, filtered to remove cell debris, ultracentrifuged, and then incubated with target cells. The authors dub the transducing entity a “gesicle”. One application the authors propose for gesicles is to render human cells permissive to ecotropic retroviruses, which typically infect murine cells only. For this application, gesicles that contain the receptor for the murine leukemia virus ecotropic envelope are transferred to human cells. While amphotropic retroviral vectors bearing certain transgenes may not be handled outside of biosafety level 3 containment, ecotropic retroviruses are permissible in level 2 facilities. Thus, by using gesicles to prepare transiently permissive human fibroblasts, obtaining induced pluripotent stem cells may no longer mean donning a biohazard suit. Although most of the work shown in the paper pertains to membrane and cytosolic proteins, gesicles do transduce nuclear proteins, as shown by transfer of Tetregulated transactivators. In this case, best incorporation was seen with variants that are farnesylated, a modification that does not noticeably interfere with transactivation, suggesting the technique could be useful for temporal control of gene expression. Since gesicles cannot exclude undesirable proteins or nucleic acids, they may not be suitable for sensitive applications. Nonetheless, the convenience of gesicles may well convince many to embrace this one-time artifact as a time-saving tool.
Mangeot et al. 2011. Protein Transfer Into Human Cells by VSV-G-induced Nanovesicles. Mol Ther. 19:1656-66.