Rabies Virus Hijacks and Accelerates the p75NTR Retrograde Axonal Transport Machinery
Figure 4
RABV binds and internalizes with p75NTR in DRG neuron tips.
Co-localization of EGFP-RABV with p75NTR is shown by live TIRF imaging and sub-pixel localization algorithms. (A) RABV-p75 particles shift from the periphery to the center of the growth cone, where they are internalized into the cell. Lower panels zoom in on dashed square, showing co-localized puncta (left) shifting towards the center of the growth cone (middle) until finally internalized (right). (B) Presentation of six separate events of RABV and p75NTR binding and internalization on the surface of the growth cone shown in (A). Colored trajectories denote displacement from point of detection to point of disappearance. (C) RABV and p75NTR are internalized together, illustrated by corresponding plots of puncta intensity over time (normalized to background), calculated for co-localized particles shown in lower panels of (A). Scale bars = 5 µm. (D) Zoom-in on colocalized RABV and p75 spot, taken from panel (A), scale bar = 1 µm. (E) Overlay of 1D-Gaussian fits of p75 and RABV intensity profiles at the x-axis of the image in panel (D). (F) Representative overlay of radial symmetry fits of the x-y intensity profiles of p75 and RABV spots. σ is the standard deviation of each fitting function; distance between the two spot centers is 51.3 nm. (G) Knockdown of p75NTR decreases rabies virus infection for shorts time incubation. DRGs embryonic cells infected with lentiviral vectors (LV) containing 4 different EGFP-tagged shRNA's against p75NTR or LV-EGFP, were transfected with RABV for 30 or 120 minutes. Low levels of infected neurons were found in shRNA-p75-EGFP cells Average RABV infection rates were normalized to LV-EGFP controls (n = 4 experiments, error bars = SEM, *p<0.005, **p<0.0005).