Source: Massachusetts Institute of Technology
Summary: Researchers have developed a new viral tool that stops replicating once it infects a cell, allowing it to deliver its genetic cargo without harming the cell.
For the past decade, neuroscientists have been using a modified version of the rabies virus (Recombinant rabies viral vectors) to label neurons and trace the connections between them. Although this technique has proven very useful, it has one major drawback: The virus is toxic to cells and can’t be used for studies longer than about two weeks. Researchers at MIT and the Allen Institute for Brain Science have now developed a new version of this virus that stops replicating once it infects a cell, allowing it to deliver its genetic cargo without harming the cell. Using this technique, scientists should be able to study the infected neurons for several months, enabling longer-term studies of neuron functions and connections. Rabies viruses are well-suited for tracing neural connections because they have evolved to spread from neuron to neuron through junctions known as synapses. The study findings were published in the journal Nature Neuroscience.
The research team have used retrograde targeting (the virus can be injected into a cluster of axon terminals and then travel back to the cell bodies of those axons) to identify populations of neurons of the basolateral amygdala that project to either the nucleus accumbens or the central medial amygdala. To create the second-generation version of this viral tool, Wickersham and his colleagues deleted the gene for the polymerase enzyme, which is necessary for transcribing viral genes. Without this gene, the virus becomes less harmful and infected cells can survive much longer. In this new study, they found that neurons were still functioning normally for up to 4 months after infection. The lack of polymerase also greatly reduces the expression of whichever gene the researchers engineer into the virus, so they need to employ a little extra genetic trickery to achieve their desired outcome.
Senior author, Ian Wickersham said, “The second-generation virus enters a cell with its own few copies of the polymerase protein and is able to start transcribing its genes, including the transgene that we put into it. But then because it’s not able to make more copies of the polymerase, it doesn’t have this exponential takeover of the cell, and in practice it seems to be totally nontoxic.”
More Information: Soumya Chatterjee, “Nontoxic, double-deletion-mutant rabies viral vectors for retrograde targeting of projection neurons”, Nature Neuroscience (2018). nature.com/articles/doi:10.1038/s41593-018-0091-7