Abstract
Recombinant Sindbis viruses are important tools in neuroscience because they combine rapid and high transgene expression with a capacity to carry large transgenes. Currently, two packaging systems based on the defective helper (DH) RNAs DH(26S)5’SIN and DH-BB(tRNA;TE12) are available for generating recombinant Sindbis virus that is neurotropic (able to infect neurons and potentially other cells). Both systems produce a fraction of viral particles that can propagate beyond the primary infected neuron. When injected into mouse brain, viruses produced using these DH RNAs produce transgene expression at the injection site, but also elsewhere in the brain. Such ectopic labeling caused recombinant Sindbis viruses to be classified as anterograde viruses with limited retrograde spread, and can complicate the interpretation of neuroanatomical and other experiments. Here we describe a new DH RNA, DH-BB(5’SIN;TE12ORF), that can be used to produce virus that is both neurotropic and propagation-incompetent. We show in mice that DH-BB(5’SIN;TE12ORF)-packaged virus eliminates infection of cells outside the injection site. We also provide evidence that ectopically labeled cells observed in previous experiments with recombinant Sindbis virus resulted from secondary infection by propagation-competent virus, rather than from inefficient retrograde spread. Virus produced with our new packaging system retains all the advantages of previous recombinant Sindbis viruses, but minimizes the risks of confounding results with unwanted ectopic labeling. It should therefore be considered in future studies in which a neurotropic, recombinant Sindbis virus is needed.
Highlights
Enveloped, positive strand RNA viruses from the family of the togaviridae are commonly used in neuronscience (Ehrengruber, 2002a,b)
In recombinant Sindbis vectors, the structural protein open reading frames (ORF) is replaced with the gene of interest (Figure 1A bottom; Xiong et al, 1989), allowing insertions of up to 6 kb
Because the region encoding the essential structural proteins has been deleted, a cell infected by only recombinant Sindbis does not produce infectious viral particles
Summary
Positive strand RNA viruses from the family of the togaviridae are commonly used in neuronscience (Ehrengruber, 2002a,b). The DH RNA conventionally used in neuroscience for Sindbis virus production is the interfering particle based DH(26S)5’SIN vector (Geigenmüller-Gnirke et al, 1991; Bredenbeek et al, 1993; Ehrengruber et al, 1999; Malinow et al, 2010) It expresses the TE12 structural proteins, making the resulting virus neurotropic (Lustig et al, 1988; Bredenbeek et al, 1993). It contains the Sindbis packaging signal, and produces virus that contains a fraction of propagation-competent virions (Bredenbeek et al, 1993). Our new DH RNA provides precise spatial control of Sindbis virus-based expression in the mouse brain, and removes ectopic infection as a confounding factor in neuroanatomical and physiological experiments
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