Role of Tunneling Nanotube–like Structures during the Early Events of HIV Infection: Novel Features of Tissue Compartmentalization and Mechanism of HIV Spread

Abstract

HIV has become a chronic disease despite the effective use of antiretroviral therapy (ART). However, the mechanisms of tissue colonization, viral evolution, generation of viral reservoirs, and compartmentalization are still a matter of debate due to the challenges involved in examining early events of infection at the cellular and molecular level. Thus, there is still an urgent need to explore these areas to develop effective HIV cure strategies. In this study, we describe the early events of tissue colonization and compartmentalization as well as the role of tunneling nanotube-like structures during viral spread in the presence and absence of effective antiretroviral treatment. To examine these mechanisms, NOD/SCID IL-2 RG 2/2 humanized mice were either directly infected with HIV ADA or with low numbers of HIV ADA -infected leukocytes to limit tissue colonization in the presence and absence of TAK779, an effective CCR5 blocker of HIV entry. We identify that viral seeding in tissues occurs early in a tissue-and cell typespecific manner (24-72 h). Reduction in systemic HIV replication by TAK779 treatment did not affect tissue seeding or spreading, despite reduced systemic viral replication. Tissue-associated HIV-infected cells had different properties than cells in the circulation because the virus continues to spread in tissues in a tunneling nanotube-like structure-dependent manner, despite ART. Thus, understanding these mechanisms can provide new approaches to enhance the efficacy of existing ART and HIV infection cure strategies.

Tunneling nanotubes, TNT, communicate glioblastoma with surrounding non-tumor astrocytes to adapt them to hypoxic and metabolic tumor conditions

“…Several groups, including ours, have demonstrated that TNT formation is induced under several pathogenic conditions, including carcinogenesis 3 , 4 , 9 , 13 , 23 , 47 – 49 . In contrast, healthy adult cells express low to undetectable TNT levels 3 , 21 .…”

“…Normally, TNT formation and associated intercellular communication participate in key biological processes, including development, signaling, and the immune response, but TNT formation during healthy conditions is minimal 1 – 8 . We and others demonstrated that TNT formation participates in the pathogenesis of several diseases, including HIV 9 – 13 . However, the role of TNT or tumor microtubes in glioblastoma (GBM) have only recently been explored 14 – 23 .…”

“…Third, TNT communicate the cytoplasm of two or more cells at a minimal distance of 30 µm with open-ended and non-open-ended processes. Fourth, the TNT process can branch and reach distances up to 500 µm in vivo and in vitro 13 , 22 , 23 , 105 , 107 . Fifth, TNT can transport organelles, vesicular structures, and small molecules between TNT-connected cells.…”

“…Cells were incubated in a blocking solution for 30 min at room temperature and then in primary antibody overnight at 4 °C. Cells were washed several times with PBS at room temperature and incubated with phalloidin conjugated to Texas Red to identify actin filaments and/or the appropriate secondary antibody conjugated to FITC (Sigma, St. Louis, MO) for 1 h at room temperature, followed by another wash in PBS for 1 h. Coverslips were then mounted using an anti-fade reagent with DAPI, and cells were examined by confocal microscopy using an A1R Nikon confocal microscope with spectral detection (Tokyo, Japan) as we described 3 , 13 , 23 .…”

“…The statistical tests were chosen according to the following: two-tailed paired or unpaired t-test was applied on datasets with a normal distribution (Kolmogorov–Smirnov test), whereas two-tailed Mann–Whitney (unpaired test) or Wilcoxon matched-paired signed-rank tests were used otherwise. p < 0.05 was considered as the level of statistical significance as described for other TNT manuscripts 3 , 13 , 23 .…”