Preliminary characterisation of nanotubes connecting T-cells and their use by HIV-1

Background Information Cells, especially those of the immune system, can form long and thin connections termed tunnelling nanotubes (TNTs). These structures can reach >100 µm in length and, in T‐cells, contain actin but no tubulin and are not open ended. T‐cell TNTs were found to form following cell contact and to enable the transfer of HIV‐1 from an infected‐ to a connected‐T‐cell. TNTs are poorly characterised at molecular level. Results We found Rab11 and tetraspanins, especially CD81, all along T‐cells TNTs, whereas Rab4 and Rab35 were absent from these structures. Regarding actin cytoskeleton regulators, Exo70, N‐WASP and especially ezrin accumulated at the level of the TNT tip that contacts the connected cell. Phosphoinositides such as PI(4,5)P2 were also concentrated at this level together with HIV‐1 Gag. Gag spots on cells and TNTs were essentially immobile, and likely correspond to area of Gag multimerisation for budding to form virus‐like particles. Mobility of PHPLCδ, a specific probe for PI(4,5)P2, was reduced > threefold at the level of TNT basis or tip compared with the cell body. Conclusion Our study identified the TNT tip as an active zone of actin cytoskeleton reorganisation with the presence of ezrin, Exo70, N‐WASP and PI(4,5)P2. The latter is also known to enable HIV‑1 Gag recruitment for viral budding, and the presence of Gag at this level, contacting the connected cell, indicates that the TNT tip is also a favourite place for HIV‐1 assembly and budding. Research article We characterised tunnelling nanotubes (TNTs) connecting T‐cells. We found that their tip is enriched in actin regulators such as PI(4,5)P2, ezrin and Exo70. HIV‐1 Gag is also recruited by PI(4,5)P2 and accumulated at the TNT apex that seems to be a preferential virus assembly platform for budding in close proximity with the connected cell to enable efficient virus transmission. Tetraspanins, especially CD81, were enriched in TNTs but did not show any specific accumulation at their tip.