Clonally expanded HIV-1 proviruses with 5′-leader defects can give rise to nonsuppressible residual viremia
Jennifer A. White, … , Janet D. Siliciano, Francesco R. Simonetti
Jennifer A. White, … , Janet D. Siliciano, Francesco R. Simonetti
Published January 5, 2023
Citation Information: J Clin Invest. 2023;133(6):e165245. https://doi.org/10.1172/JCI165245.
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Clinical Research and Public Health AIDS/HIV

Clonally expanded HIV-1 proviruses with 5′-leader defects can give rise to nonsuppressible residual viremia

Abstract

Background Antiretroviral therapy (ART) halts HIV-1 replication, decreasing viremia to below the detection limit of clinical assays. However, some individuals experience persistent nonsuppressible viremia (NSV) originating from CD4+ T cell clones carrying infectious proviruses. Defective proviruses represent over 90% of all proviruses persisting during ART and can express viral genes, but whether they can cause NSV and complicate ART management is unknown.Methods We undertook an in-depth characterization of proviruses causing NSV in 4 study participants with optimal adherence and no drug resistance. We investigated the impact of the observed defects on 5′-leader RNA properties, virus infectivity, and gene expression. Integration-site specific assays were used to track these proviruses over time and among cell subsets.Results Clones carrying proviruses with 5′-leader defects can cause persistent NSV up to approximately 103 copies/mL. These proviruses had small, often identical deletions or point mutations involving the major splicing donor (MSD) site and showed partially reduced RNA dimerization and nucleocapsid binding. Nevertheless, they were inducible and produced noninfectious virions containing viral RNA, but lacking envelope.Conclusion These findings show that proviruses with 5′-leader defects in CD4+ T cell clones can give rise to NSV, affecting clinical care. Sequencing of the 5′-leader can help in understanding failure to completely suppress viremia.Funding Office of the NIH Director and National Institute of Dental and Craniofacial Research, NIH; Howard Hughes Medical Institute; Johns Hopkins University Center for AIDS Research; National Institute for Allergy and Infectious Diseases (NIAID), NIH, to the PAVE, BEAT-HIV, and DARE Martin Delaney collaboratories.

Authors

Jennifer A. White, Fengting Wu, Saif Yasin, Milica Moskovljevic, Joseph Varriale, Filippo Dragoni, Angelica Camilo-Contreras, Jiayi Duan, Mei Y. Zheng, Ndeh F. Tadzong, Heer B. Patel, Jeanelle Mae C. Quiambao, Kyle Rhodehouse, Hao Zhang, Jun Lai, Subul A. Beg, Michael Delannoy, Christin Kilcrease, Christopher J. Hoffmann, Sébastien Poulin, Frédéric Chano, Cécile Tremblay, Jerald Cherian, Patricia Barditch-Crovo, Natasha Chida, Richard D. Moore, Michael F. Summers, Robert F. Siliciano, Janet D. Siliciano, Francesco R. Simonetti

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Figure 4

5′-L-defective proviruses are inducible and their genomic RNA is packaged and can use alternative splicing donors.

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5′-L-defective proviruses are inducible and their genomic RNA is package...
(A) CD4+ T cells from P1 and P2 were cultured for 48 hours in the presence of dolutegravir (DTG) and anti-CD3/CD28 beads. Cells and supernatants were collected at 0, 24, and 48 hours. Right panel shows the location of primers and probes used to quantify viral RNA. Shaded areas indicate 5′-L deletion used to measure provirus-specific transcription. (B) Mean levels of read-through, total, and provirus-specific RNA detected in cells and supernatant upon T cell activation. Gray circles represent digital PCR replicate reactions. Error bars indicate SEM. (C) Singly and multiply spliced transcripts amplified at limiting dilution from cells at 48 hours. Arrows indicate primer locations. Ticks indicate the locations of splicing donors and acceptors in the HIV-1 genome. Major and alternative splicing donor sites are labeled in black. Mapped splicing junctions are underlined and in bold; 22 nt deletion is represented by dashed lines. Numbers in parentheses indicate the number of sequences recovered for each type of spliced variant.