Reconstitution of herpes simplex virus microtubule-dependent trafficking in vitro.
Microtubule-mediated anterograde transport of herpes simplex virus (HSV) from the neuronal cell body to the axon terminal is crucial for the spread and transmission of the virus. It is therefore of central importance to identify the cellular and viral factors responsible for this trafficking event. In previous studies, we isolated HSV-containing cytoplasmic organelles from infected cells and showed that they represent the first and only destination for HSV capsids after they emerge from the nucleus. In the present study, we tested whether these cytoplasmic compartments were capable of microtubule-dependent traffic. Organelles containing green fluorescent protein-labeled HSV capsids were isolated and found to be able to bind rhodamine-labeled microtubules polymerized in vitro. Following the addition of ATP, the HSV-associated organelles trafficked along the microtubules, as visualized by time lapse microscopy in an imaging microchamber. The velocity and processivity of trafficking resembled those seen for neurotropic herpesvirus traffic in living axons. The use of motor-specific inhibitors indicated that traffic was predominantly kinesin mediated, consistent with the reconstitution of anterograde traffic. Immunocytochemical studies revealed that the majority of HSV-containing organelles attached to the microtubules contained the trans-Golgi network marker TGN46. This simple, minimal reconstitution of microtubule-mediated anterograde traffic should facilitate and complement molecular analysis of HSV egress in vivo.
Lee
Grace E
GE
Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
Murray
John W
JW
Wolkoff
Allan W
AW
Wilson
Duncan W
DW
D001692:Biological Transport; D015415:Biomarkers; D002213:Capsid / Q000378:metabolism; D045744:Cell Line, Tumor; D003593:Cytoplasm / Q000378:metabolism; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism; D046529:Microscopy, Electron, Transmission; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D011485:Protein Binding; D018139:Simplexvirus / Q000378:metabolism* / Q000648:ultrastructure; D014771:Virion / Q000378:metabolism
D015415:Biomarkers; D008562:Membrane Glycoproteins; C072237:TGOLN2 protein, human