Microtubule-dependent Plus- and Minus End–directed Motilities Are Competing Processes for Nuclear Targeting of Adenovirus

Abstract

Adenovirus (Ad) enters target cells by receptor-mediated endocytosis, escapes to the cytosol, and then delivers its DNA genome into the nucleus. Here we analyzed the trafficking of fluorophore-tagged viruses in HeLa and TC7 cells by time-lapse microscopy. Our results show that native or taxol-stabilized microtubules (MTs) support alternating minus- and plus end–directed movements of cytosolic virus with elementary speeds up to 2.6 μm/s. No directed movement was observed in nocodazole-treated cells. Switching between plus- and minus end–directed elementary speeds at frequencies up to 1 Hz was observed in the periphery and near the MT organizing center (MTOC) after recovery from nocodazole treatment. MT-dependent motilities allowed virus accumulation near the MTOC at population speeds of 1–10 μm/min, depending on the cell type. Overexpression of p50/dynamitin, which is known to affect dynein-dependent minus end–directed vesicular transport, significantly reduced the extent and the frequency of minus end–directed migration of cytosolic virus, and increased the frequency, but not the extent of plus end–directed motility. The data imply that a single cytosolic Ad particle engages with two types of MT-dependent motor activities, the minus end– directed cytoplasmic dynein and an unknown plus end– directed activity.

Virus trafficking – learning from single-virus tracking

“…2). This was observed for Reovirus, Adenovirus, HSV, influenza virus and HIV 5,[42][43][44][45] . Viral transport is often highly regulated as with, for example, HSV in neurons.…”

“…Important signalling events might occur during viral transport. During its dyneindependent motion along microtubules, Adenoviruses transiently activate distinct signalling cascades, such as cAMP dependent protein kinase A, p38/mitogen activated protein kinase (MAPK) and the downstream MAPK activated protein kinase 2, to balance nuclear targeting and enhance infection 42,54 .…”

“…An increasing number of CLs with different characteristics can be either covalently attached or non-covalently associated with the target protein 8 . For example, capsids of purified non-enveloped viruses can be covalently labelled with aminoreactive dyes (for example, cynine or Alexa dyes) 6,18,42 . The outer membrane of purified enveloped viruses can be labeled by the incorporation of lipophilic dyes for example, DiD, DiI and analogues5.…”

Adenoviral capsid modulates secretory compartment organization and function in acinar epithelial cells from rabbit lacrimal gland

“…Ad capsid proteins are known to interact with MTs and cytoplasmic dynein during transit to the nucleus. 40,41 We have previously shown that inhibition of cytoplasmic dynein activity in lacrimal acini severely impairs the formation of rab3D-enriched secretory vesicles. 27 However, direct dynein inhibition also prevents the recruitment of VAMP2-enriched secretory transport vesicles to the apical membrane in response to CCH, while this response is unaffected by Ad (data not shown), suggesting that some dynein-mediated traffic to the apical membrane can proceed normally in Ad-treated acini.…”

PCI-enhanced adenoviral transduction employs the known uptake mechanism of adenoviral particles

“…Other pathways have, however, been suggested, 40,41 and adenoviral particles have been observed in vesicles without clathrin coating and in macropinosomes. The enhanced gene transfer after photochemical treatment could be due to the release of the viral particles from any of these vesicles.…”