Pseudorabies Virus US3-Induced Tunneling Nanotubes Contain Stabilized Microtubules, Interact with Neighboring Cells via Cadherins, and Allow Intercellular Molecular Communication

Abstract

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. 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.

Tunneling Nanotubes as a Novel Route of Cell-to-Cell Spread of Herpesviruses

“…This phenomenon might also function as a working hypothesis in the case of viral transport mediated by TNTs: while examining the dual-color mutant trafficking in live cells, we did not directly detect two-color structures inside TNTs (although that may have been the result of how the viral particle was positioned in the tube), and the two signals appeared to travel separately in the nanotubes. However, the presence of intact virions in nanotubes was reported for projections formed by pseudorabies virus (PRV)-infected cells (53).…”

“…PRV studies have indicated for the first time that Us3 protein kinase, a protein that is highly conserved among herpesviruses, stimulates the formation of long intercellular projections (54). While this manuscript was being edited for submission, another report about the possible role of tunneling nanotubes in alphaherpesvirus infection was published (53). The authors demonstrated that cells infected with pseudorabies virus or transfected with PRV Us3 produced TNT-type projections involved in the transport of viral proteins and viral particles.…”

Tumor microtubes connect pancreatic cancer cells in an Arp2/3 complex-dependent manner

“…TMTs in our DHPC-018 cells also contain microtubules and intermediate filaments. While microtubules are absent from some TNTs (Rustom et al, 2004;Sowinski et al, 2008;Wang et al, 2010;Desir et al, 2018;Kretschmer et al, 2019), they are present in others (Önfelt et al, 2006;Osswald et al, 2015;Jansens et al, 2017;Sáenz-de-Santa-María et al, 2017;Resnik et al, 2018;Zhang et al, 2019). Where examined, the microtubule-containing structures are wider than those lacking microtubules (Önfelt et al, 2006).…”

Cell-to-cell transport in viral families: faster than usual

“…SuHV-1 infected cells formed nanotubes and the presence of intact virions was clearly visible inside nanotubes. The nanotubes formed by SuHV-1 infected cells were remarkably stable compared to most TNTs described (Favoreel et al, 2005;Jansens et al, 2017). The Us3 induced TNTs contained stabilized microtubules and the virus particles were individually transported in membrane-bound vesicles and released along the TNTs and at the contact area between a TNT and adjacent cell.…”

“…The Us3 induced TNTs contained stabilized microtubules and the virus particles were individually transported in membrane-bound vesicles and released along the TNTs and at the contact area between a TNT and adjacent cell. Contact between Us3-induced TNTs and acceptor cells was very stable and rich for components of adherens junctions such as beta-catenin or E-cadherin at the contact area (Jansens et al, 2017) (Fig. 1).…”