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The persistence of replication-competent HIV reservoirs in people living with HIV (PLWH) receiving antiretroviral therapy (ART) is a barrier to cure. Therefore, their accurate quantification is essential for evaluating the efficacy of new therapeutic interventions and orienting the decision to interrupt ART. Quantitative viral outgrowth assays (QVOAs) represent the “gold standard” for measuring the size of replication-competent HIV reservoirs. However, they require large numbers of cells and are technically challenging. This justifies the need for the development of novel simplified methods adapted for small biological samples. Herein, we sought to simplify the viral outgrowth procedure (VOP) by (i) using memory CD4+ T-cells, documented to be enriched in HIV reservoirs (ii) optimizing cell-culture conditions, and (iii) supplementing with all-trans retinoic acid (ATRA), a positive regulator of HIV replication. Memory CD4+ T-cells were sorted from the peripheral blood of ART-treated (HIV+ART; n = 14) and untreated (HIV+; n = 5) PLWH. The VOP was first performed with one original replicate of 1 × 106 cells/well in 48-well plates. Cells were stimulated via CD3/CD28 for 3 days, washed to remove residual CD3/CD28 Abs, split every 3 days for optimal cell density, and cultured in the presence or the absence of ATRA for 12 days. Soluble and intracellular HIV-p24 levels were quantified by ELISA and flow cytometry, respectively. Optimal cell-culture density achieved by splitting improved HIV outgrowth detection. ATRA promoted superior/accelerated detection of replication-competent HIV in all HIV+ART individuals tested, including those with low/undetectable viral outgrowth in the absence of ATRA. Finally, this VOP was used to design a simplified ATRA-based QVOA by including 4 and 6 original replicates of 1 × 106 cells/well in 48-well plates and 2 × 105 cells/well in 96-well plates, respectively. Consistently, the number of infectious units per million cells (IUPM) was significantly increased in the presence of ATRA. In conclusion, we demonstrate that memory CD4+ T-cell splitting for optimal density in culture and ATRA supplementation significantly improved the efficacy of HIV outgrowth in a simplified ATRA-based QVOA performed in the absence of feeder/target cells or indicator cell lines.

Cilia and flagella are conserved subcellular organelles, which arise from centrioles and play critical roles in development and reproduction of eukaryotes. Dysfunction of cilia leads to life-threatening ciliopathies. HYLS1 is an evolutionarily conserved centriole protein, which is critical for ciliogenesis, and its mutation causes ciliopathy–hydrolethalus syndrome. However, the molecular function of HYLS1 remains elusive. Here, we investigated the function of HYLS1 in cilia formation using the Drosophila model. We demonstrated that Drosophila HYLS1 is a conserved centriole and basal body protein. Deletion of HYLS1 led to sensory cilia dysfunction and spermatogenesis abnormality. Importantly, we found that Drosophila HYLS1 is essential for giant centriole/basal body elongation in spermatocytes and is required for spermatocyte centriole to efficiently recruit pericentriolar material and for spermatids to assemble the proximal centriole-like structure (the precursor of the second centriole for zygote division). Hence, by taking advantage of the giant centriole/basal body of Drosophila spermatocyte, we uncover previously uncharacterized roles of HYLS1 in centriole elongation and assembly.