Dolutegravir-containing HIV therapy reversibly alters mitochondrial health and morphology in cultured human fibroblasts and peripheral blood mononuclear cells.

OBJECTIVES: Given the success of combination antiretroviral therapy (cART) in treating HIV viremia, drug toxicity remains an area of interest in HIV research. Despite newer integrase strand transfer inhibitors (InSTIs), such as dolutegravir (DTG) and raltegravir (RAL), having excellent clinical tolerance, there is emerging evidence of off-target effects and toxicities. Although limited in number, recent reports have highlighted the vulnerability of mitochondria to these toxicities. The aim of the present study is to quantify changes in cellular and mitochondrial health following exposure to current cART regimens at pharmacological concentrations. A secondary objective is to determine whether any cART-associated toxicities would be modulated by human telomerase reverse transcriptase (hTERT). METHODS: We longitudinally evaluated markers of cellular (cell count, apoptosis), and mitochondrial health [mitochondrial reactive oxygen species (mtROS), membrane potential (MMP) and mass (mtMass)] by flow cytometry in WI-38 human fibroblast with differing hTERT expression/localization and peripheral blood mononuclear cells. This was done after 9 days of exposure to, and 6 days following the removal of, seven current cART regimens, including three that contained DTG. Mitochondrial morphology was assessed by florescence microscopy and quantified using a recently developed deep learning-based pipeline. RESULTS: Exposure to DTG-containing regimens increased apoptosis, mtROS, mtMass, induced fragmented mitochondrial networks compared with non-DTG-containing regimens, including a RAL-based combination. These effects were unmodulated by telomerase expression. All effects were fully reversible following removal of drug pressure. CONCLUSION: Taken together, our observations indicate that DTG-containing regimens negatively impact cellular and mitochondrial health and may be more toxic to mitochondria, even among the generally well tolerated InSTI-based cART.

RNA-tethering assay and eIF4G:eIF4A obligate dimer design uncovers multiple eIF4F functional complexes.

Eukaryotic cellular mRNAs possess a 5' cap structure (m7GpppN) which plays a critical role in translation initiation mediated by eukaryotic initiation factor (eIF) 4F. The heterotrimeric eIF4F complex possesses several activities imparted by its subunits that include cap recognition (by eIF4E), RNA unwinding (eIF4A), and factor/ribosome recruitment (eIF4G). Mammalian cells have paralogs of all three eIF4F subunits and it remains an open question as to whether these all can participate in the process of ribosome recruitment. To query the activities of the eIF4F subunits in translation initiation, we adopted an RNA-tethering assay in which select subunits are recruited to a specific address on a reporter mRNA template. We find that all eIF4F subunits can participate in the initiation process. Based on eIF4G:eIF4A structural information, we also designed obligate dimer pairs to probe the activity of all combinations of eIF4G and eIF4A paralogs. We demonstrate that both eIF4GI and eIF4GII can associate with either eIF4A1 or eIF4A2 to recruit ribosomes to mRNA templates. In combination with eIF4E and eIF4E3, our results indicate the presence of up to eight eIF4F complexes that can operate in translation initiation.