BACKGROUND: The origin of viral rebound remains elusive as only a few links between proviral sequences and rebound plasma viruses have been described. Here we characterized the translation- and replication-competent reservoirs of three individuals under ART and compared it to rebound plasma viruses detected during analytical treatment interruption (ATI).
METHODS: Peripheral blood CD4 T cells were collected from 3 ART-treated individuals right before ATI. P24-expressing cells were single-cell sorted following latency reversal and near full-length proviral sequencing was performed. Replication-competent viral sequences were isolated from supernatant of positive quantitative viral outgrowth assay (qVOA) wells. During ATI, plasma was collected at first detectable viral load (>1000 copies/mL) and either 5’- or 3’-half viral RNA genomes were isolated through single genome amplification.
RESULTS: We retrieved 20 sequences from positive qVOA wells, 30 proviral genomes from p24+ cells, 89 (median=32) 5’-half and 94 (median=32) 3’-half rebound plasma sequences acquired during ATI. Among distinct sequences retrieved from p24+ cells, 88% displayed defects in the packaging signal (PSI) region and/or major splice donor (MSD) site. Among qVOA and rebound sequences, none had PSI/MSD defects, suggesting their minor role in viral rebound and replication. One overlap between the translation- and replication-competent reservoir was observed, notably between the only p24+ cell with an intact PSI/MSD and one genome-intact qVOA sequence. Moreover, two overlaps were observed between viruses from qVOA wells and 5’-half rebound plasma sequences.

CONCLUSIONS: The direct origin of rebounding plasma virus remains hard to identify as only few overlaps were detected by comparing the translation and replication-competent fractions to plasma viruses collected during ATI, with no overlap between the three datasets. Yet, we report an overlap between an intact qVOA sequence and a provirus from a p24-expressing cell, confirming that some of the translation-competent proviruses are capable of inducing new cycles of replication.

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