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 (e.g., green fluorescent protein [GFP]) in the absence of other viral proteins. US3-induced TNTs are remarkably stable compared to most TNTs described in the 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 a TNT and the 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 into US3-induced TNTs and how they may contribute to intercellular communication and alphaherpesvirus spread.

IMPORTANCE Tunneling nanotubes (TNT) represent an important and 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 help 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 into the biology of (US3-induced) TNTs and into how they may contribute to intercellular virus spread and communication.

隧道纳米管（TNT）是连接真核细胞并介导细胞间通讯的长桥状结构。我们较早地发现，保守的α疱疹病毒US3蛋白激酶可诱导长细胞投射，该投射会与远处的细胞接触并促进细胞间病毒的传播。在此报告中，我们显示了US3诱导的细胞投射构成TNT。另外，我们报道在没有其他病毒蛋白的情况下，US3诱导的TNTs介导信息的细胞间运输（例如，绿色荧光蛋白[GFP]）。与文献中描述的大多数TNT相比，US3诱导的TNT非常稳定。与此相一致，发现US3诱导的TNTs含有稳定的（乙酰化和去酪氨酸化的）微管。透射电子显微镜显示，病毒颗粒在US3诱导的TNTs中的膜结合囊泡中单独运输，并沿着TNT以及在TNT与相邻细胞之间的接触区域释放。US3诱导的TNT与受体细胞之间的接触非常稳定，这与接触区域粘附连接成分β-catenin和E-cadherin的显着富集相关。这些数据为US3诱导的TNT及其如何促进细胞间通讯和α疱疹病毒的传播提供了新的结构见解。这与接触区域粘附连接蛋白β-catenin和E-cadherin的显着富集有关。这些数据为US3诱导的TNT及其如何促进细胞间通讯和α疱疹病毒的传播提供了新的结构见解。这与接触区域粘附连接成分β-catenin和E-cadherin的显着富集有关。这些数据为US3诱导的TNT及其如何促进细胞间通讯和α疱疹病毒的传播提供了新的结构见解。

重要性隧穿纳米管（TNT）代表了一种重要的但仍知之甚少的长距离细胞间通讯模式。我们和其他人先前报道，保守的α疱疹病毒US3蛋白激酶在感染和转染的细胞中诱导长细胞突起。在这里，我们显示US3诱导的细胞投射构成TNT，基于结构性质和生物分子的运输。此外，我们报道了US3诱导的TNT的不同特殊特征，这有助于解释它们与生理TNT相比的显着稳定性。另外，透射电子显微镜分析表明，在被感染的细胞中，病毒体在膜转运转运囊泡中的US3诱导的TNT中行进，并通过胞吐作用离开TNT。