Viral strategies for studying the brain, including a replication‐restricted self‐amplifying delta‐G vesicular stomatis virus that rapidly expresses transgenes in brain and can generate a multicolor golgi‐like expression

Abstract

Viruses have substantial value as vehicles for transporting transgenes into neurons. Each virus has its own set of attributes for addressing neuroscience-related questions. Here we review some of the advantages and limitations of herpes, pseudorabies, rabies, adeno-associated, lentivirus, and others to study the brain. We then explore a novel recombinant vesicular stomatitis virus (dG-VSV) with the G-gene deleted and transgenes engineered into the first position of the RNA genome, which replicates only in the first brain cell infected, as corroborated with ultrastructural analysis, eliminating spread of virus. Because of its ability to replicate rapidly and to express multiple mRNA copies and additional templates for more copies, reporter gene expression is amplified substantially, over 500-fold in 6 hours, allowing detailed imaging of dendrites, dendritic spines, axons, and axon terminal fields within a few hours to a few days after inoculation. Green fluorescent protein (GFP) expression is first detected within 1 hour of inoculation. The virus generates a Golgilike appearance in all neurons or glia of regions of the brain tested. Whole-cell patch-clamp electrophysiology, calcium digital imaging with fura-2, and time-lapse digital imaging showed that neurons appeared physiologically normal after expressing viral transgenes. The virus has a wide range of species applicability, including mouse, rat, hamster, human, and Drosophila cells. By using dG-VSV, we show efferent projections from the suprachiasmatic nucleus terminating in the periventricular region immediately dorsal to the nucleus. DG-VSVs with genes coding for different color reporters allow multicolor visualization of neurons wherever applied. J. Comp. Neurol. 516:456-481, 2009.

Anterograde or retrograde transsynaptic labeling of CNS neurons with vesicular stomatitis virus vectors

“…We turned to vesicular stomatitis virus (VSV), a negative strand RNA virus that is a member of the Rhabdoviridae family (7). Its use as a gene transfer agent in the CNS had already been established (8), although its native G protein, VSV-G, did not promote specific transsynaptic spread. VSV is lethal to individual cells, and to an animal when injected into the brain, but it is significantly less toxic after natural infections in humans and is being developed as a vaccine vector for humans (9).…”

Peripheral Immunization Blocks Lethal Actions of Vesicular Stomatitis Virus within the Brain

“…(iii) In VSV-CT9-M51, position 51 of the M gene coding for methionine was deleted, and the cytoplasmic tail of VSV-G was reduced from 29 to 9 amino acids. The generation of recombinant VSV strains is described in detail elsewhere (8,17,23,31,37), and they were kindly provided by J. Rose.…”

Viral Mutagenesis as a Means for Generating Novel Proteins

“…Similar to VSV, other RNA viruses with high mutation rates such as Sindbis virus (10,22), measles virus (12,13), or other viruses could be used in place of VSV. For us, VSV makes an attractive choice for random mutagenesis, given its relative safety in the lab, high rate of mutation (7), routine recombinant engineering (6,9,15), the wide spectrum of animal cells that it can infect (11,21,23), and the rapid (1 to 3 h) and robust expression of reporter genes (21). …”

“…One such virus is VSV, a single-stranded, negative-sense RNA virus of the Rhabdoviridae family with a wild-type genome approximately 11.2 kb in length that can be engineered to accept an additional 4.5-kb gene insert (21). Genomic replication of VSV is error prone, with approximately 1 nucleotide mutation per viral genome per replication cycle (7).…”

“…1B) 24 h postinoculation, suggesting the presence of mutant DsRed genes. Recombinant VSV-1ЈDsRed virus (21,24) expressing the DsRed1 gene (Clontech, Palo Alto, CA) in the first gene position was used (from J. Rose and I. Simon, Yale University).…”