Herpes simplex virus assembly and intracellular trafficking during viral egress
Lee, Grace E.
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Herpes simplex virus (HSV) has evolved to efficiently reorganize the host cell secretory pathway and cytoskeletal elements to achieve virus assembly and exocytosis. The assembly of viral components and the intracellular trafficking of the assembled particles during HSV egress are essential for the spread of disease. However, the mechanisms of those processes are poorly defined at the molecular level.;Tegument residing in the inner surface of the envelope of herpes viruses is critical for the assembly of infectious particles. In this study, tegument assembly was investigated using the tegument protein virion host shutoff (vhs), which is a highly conserved protein among neurotropic herpes viruses. Using an anti-vhs antiserum and Western blotting of cytoplasmic extracts, we found that vhs is predominantly insoluble in HSV-infected cells. Furthermore, a subpopulation of vhs is associated with detergent-insoluble lipid rafts in a cytoplasmic fraction that contains assembling and mature HSV particles. Our data raise the possibility that HSV tegument assembly takes place at a lipid raft region on a cytoplasmic organelle.;The origin and assembly mechanism of the HSV envelope has remained obscure. We previously isolated HSV-bearing organelles from infected cells and showed that they were most likely derived from the trans Golgi network (TGN) or endocytic organelles. Furthermore, several lines of evidence imply that assembly and exocytosis of HSV make use of the microtubule cytoskeleton. Therefore, we hypothesized that HSV particles assemble at the TGN and subsequently traffic along microtubules. We reconstituted HSV transport along microtubules utilizing a green fluorescent protein (GFP)-labeled HSV strain and an in vitro microchamber motility assay with real time fluorescence microscopy. We found that isolated HSV-bearing organelles move along microtubules upon addition of ATP. The movement was bi-directional and severely impaired by a kinesin inhibitor, AMP-PNP. In addition, the motile structures were predominantly immunolabeled with a TGN marker TGN46, but much less with endosomal markers. Taken together, our data suggest that egressing virions bud into the TGN and translocate along the microtubules, mainly mediated by kinesins. The assay system used in this study will facilitate dissection of the molecular details of microtubule-based transport of HSV.
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