Recent studies have demonstrated that human tau can be secreted by neurons and non-neuronal cells, an event linked to the propagation of tau pathology in the brain. In the present study, we confirmed that under physiological conditions, one tau-positive band was detected in the culture medium with an anti-tau antibody recognizing total tau and the Tau-1 antibody directed against unphosphorylated tau. We then examined whether tau secretion was modified upon insults. Tau secretion was increased by starvation [Earle's Balanced Salt Solution (EBSS)], inhibition of lysosomal function (leupeptin) and when both of these conditions were superimposed, this combined treatment having the most important effects on tau secretion. Interestingly, the pattern of tau secretion was distinct from that of control neurons when neurons were treated either with EBSS alone or EBSS + leupeptin. In these conditions, three tau-positive bands were detected in the culture medium. Two of these three bands were immunoreactive to Tau-1 antibody revealing that at least two tau species were released upon these treatments. Collectively, our results indicate that insults such as nutrient deprivation and lysosomal dysfunction observed in neurodegenerative diseases could result in an increase of tau secretion and propagation of tau pathology in the brain.

† The first two authors equally contributed to this paper.

Herpes simplex virus type 1 particles are multilayered structures with a DNA genome surrounded by a capsid, tegument, and envelope. While the protein content of mature virions is known, the sequence of addition of the tegument and the intracellular compartments where this occurs are intensely debated. To probe this process during the initial stages of egress, we used two approaches: an in vitro nuclear egress assay, which reconstitutes the exit of nuclear capsids to the cytoplasm, and a classical nuclear capsid sedimentation assay. As anticipated, in vitro cytoplasmic capsids did not harbor U L 34, U L 31, or viral glycoproteins but contained U S 3. In agreement with previous findings, both nuclear and in vitro capsids were positive for ICP0 and ICP4. Unexpectedly, nuclear C capsids and cytoplasmic capsids produced in vitro without any cytosolic viral proteins also scored positive for U L 36 and U L 37. Immunoelectron microscopy confirmed that these tegument proteins were closely associated with nuclear capsids. When cytosolic viral proteins were present in the in vitro assay, no additional tegument proteins were detected on the capsids. As previously reported, the tegument was sensitive to high-salt extraction but, surprisingly, was stabilized by exogenous proteins. Finally, some tegument proteins seemed partially lost during egress, while others possibly were added at multiple steps or modified along the way. Overall, an emerging picture hints at the early coating of capsids with up to 5 tegument proteins at the nuclear stage, the shedding of some viral proteins during nuclear egress, and the acquisition of others tegument proteins during reenvelopment.

Newly assembled herpesvirus capsids travel from the nucleus to the plasma membrane by a mechanism that is poorly understood. Furthermore, the contribution of cellular proteins to this egress has yet to be clarified. To address these issues, an in vitro nuclear egress assay that reproduces the exit of herpes simplex virus type 1 (HSV-1) capsids from nuclei isolated from infected cells was established. As expected, the assay has all the hallmarks of intracellular transport assays, namely, a dependence on time, energy, and temperature. Surprisingly, it is also dependent on cytosol and was slightly enhanced by infected cytosol, suggesting an implication of both host and viral proteins in the process. The capsids escaped these nuclei by budding through the inner nuclear membrane, accumulated as enveloped capsids between the two nuclear membranes, and were released in cytosol exclusively as naked capsids, exactly as in intact cells. This is most consistent with the view that the virus escapes by crossing the two nuclear membranes rather than through nuclear pores. Unexpectedly, nuclei isolated at the nonpermissive temperature from cells infected with a U L 26 thermosensitive protease mutant (V701) supported capsid egress. Although electron microscopy, biochemical, and PCR analyses hinted at a likely reconstitution of capsid maturation, DNA encapsidation could not be confirmed by a traditional SQ test. This assay should prove very useful for identification of the molecular players involved in HSV-1 nuclear egress.Herpes simplex virus type 1 (HSV-1) replicates and assembles its capsids in the nucleus. It must then find its way to the plasma membrane for release in the extracellular environment. Given the restrictive nature of the much smaller nuclear pores, it has been assumed that the capsids could escape the nuclei only by budding through the inner nuclear membrane. Numerous electron microscopy (EM) observations of enveloped virions in the gap between the two nuclear envelopes, herein called the perinuclear space, indeed support this view (11,23,49,72). Once in the perinuclear space, the virions must traverse the outer nuclear membrane. Given the continuity between the perinuclear space and the reticulum endoplasmic, a luminal model claims that the virions travel through the biosynthetic pathway (11,30). They would thus reach the Golgi, trans-Golgi network, and plasma membrane in transport vesicles in much the same way that secreted proteins do. In this scenario, naked capsids would be released only accidentally in the cytoplasm and would not reach the extracellular medium. A second, more widely accepted model suggests that the perinuclear virions rather fuse with the outer nuclear membrane, thereby releasing naked capsids in the cytoplasm (70, 72). These capsids would then be reenveloped later on, likely at the trans-Golgi network (22-24, 26, 35, 74, 78). Interestingly, this deenvelopment/reenvelopment model may be valid for all members of the herpesvirus family (15,43,44). Finally, a third model has recently been pr...

This study was funded by grants from Fondation Jean-Louis Lévesque (Canada), CIHR (MOP142334) and CFI (32711) to GF. JH is supported by Postdoctoral Fellowships from The Lalor Foundation and CIHR (146703). The authors have no conflict of interest.