Pseudorabies virus US3-induced tunneling nanotubes contain stabilized microtubules, interact with neighbouring cells via cadherins and allow intercellular molecular communication.

Tunneling nanotubes (TNTs) are long bridge-like structures that connect eukaryotic cells and mediate intercellular communication. We found earlier that the conserved alphaherpesvirus US3 protein kinase induces long cell projections that contact distant cells and promote intercellular virus spread. In this report, we show that the US3-induced cell projections constitute TNTs. In addition, we report that US3-induced TNTs mediate intercellular transport of information (i.e. GFP) in the absence of other viral proteins. US3-induced TNTs are remarkably stable compared to most TNTs described in literature. In line with this, US3-induced TNTs were found to contain stabilized (acetylated and detyrosinated) microtubules. Transmission electron microscopy showed that virus particles are individually transported in membrane-bound vesicles in US3-induced TNTs and are released along the TNT and at the contact area between TNT and adjacent cell. Contact between US3-induced TNTs and acceptor cells is very stable, which correlated with a marked enrichment in adherens junction components beta-catenin and E-cadherin at the contact area. These data provide new structural insights in US3-induced TNTs and how they may contribute to intercellular communication and alphaherpesvirus spread. IMPORTANCE Tunneling nanotubes (TNT) represent an important, yet still poorly understood mode of long distance intercellular communication. We and others reported earlier that the conserved alphaherpesvirus US3 protein kinase induces long cellular protrusions in infected and transfected cells. Here, we show that US3-induced cell projections constitute TNTs, based on structural properties and transport of biomolecules. In addition, we report on different particular characteristics of US3-induced TNTs that aid to explain their remarkable stability compared to physiological TNTs. In addition, transmission electron microscopy assays indicate that, in infected cells, virions travel in the US3-induced TNTs in membranous transport vesicles, and leave the TNT via exocytosis. These data generate new fundamental insights in the biology of (US3-induced) TNTs, and how they may contribute to intercellular virus spread and communication.