Figure 1

Lentivirus enabled in vitro and in vivo trans-complementation of VSVΔG and transneuronal spread. (A–C) Lentivirus trans-complementation in vitro. (A) VSVΔG was able to infect 293T cells but was unable to spread to neighboring cells, as evident by sparse single-cell infections. (B) In conjunction with lentivirus, VSVΔG was able to both infect and spread, as evident by the presence of large infected plaques. (C) The extent of VSVΔG amplification (as measured by viral titer) is positively correlated with lentivirus titer, expressed in m.o.i. (D) Illustration of viral injection and labeling of the optic nerve. Virus was microinjected into the left eye, which infected the RGC and resulted in fluorescent labeling of the RGC axons (magenta). The layout of the larval CNS is labeled in green, with the olfactory bulb (OB), pallium (P), habenula (H), optic tectum (OT), and cerebellum (Cb) labeled. (E–H)In vivo trans-complementation of VSVΔG by lentivirus. In the absence of lentivirus, VSVΔG infected RGCs and fluorescently labeled the optic nerve, but no spread in the CNS was observed (E). (F,G) When lentivirus and VSVΔG were coinjected, cellular labeling was observed in the CNS (yellow arrowheads), indicating transneuronal spread. (H) Similar patterns of spread was also seen at very high VSVΔG titer (2 × 10⁹ vs. 10⁸ ffu/ml for E–G), suggesting that VSVΔG was able to self-complement. (I–L) Time course of VSVΔG infection and spread with lentivirus trans-complementation, with RFP expression from VSVΔG (magenta) and GFP expression from the vglut2a:GFP transgene (green). Box regions are shown at higher magnifications below (I'–L'). Scale bars are 100 μm. Images in the same row are shown at the same scale.