Abstract
Development of potential HIV-1 curative interventions requires accurate characterization of the proviral reservoir, defined as host-integrated viral DNA genomes that drive rebound of viremia upon halting ART (antiretroviral therapy). Evaluation of such interventions necessitates methods capable of pinpointing the rare, genetically intact, replication-competent proviruses within a background of defective proviruses. This evaluation can be achieved by identifying the distinct integration sites of intact proviruses within host genomes and monitoring the dynamics of these proviruses and host cell lineages over longitudinal sampling. Until recently, molecular genetic approaches at the single proviral level have been generally limited to one of a few metrics, such as proviral genome sequence/intactness, host-proviral integration site, or replication competency. New approaches, taking advantage of MDA (multiple displacement amplification) for WGA (whole genome amplification), have enabled multiparametric proviral characterization at the single-genome level, including proviral genome sequence, host-proviral integration site, and phenotypic characterization of the host cell lineage, such as CD4 memory subset and antigen specificity. In this review, we will examine the workflow of MDA-augmented molecular genetic approaches to study the HIV-1 reservoir, highlighting technical advantages and flexibility. We focus on a collection of recent studies in which investigators have used these approaches to comprehensively characterize intact and defective proviruses from donors on ART, investigate mechanisms of elite control, and define cell lineage identity and antigen specificity of infected CD4+ T cell clones. The highlighted studies exemplify how these approaches and their future iterations will be key in defining the targets and evaluating the impacts of HIV curative interventions.
Highlights
Author’s contribution to the Work was done as part of the Author’s official duties as a NIH employee and is aWork of the United States Government.copyright may not be established in the United States. 17 U.S.C.§ 105
Due to the maintenance of a stable HIV reservoir, upon cessation of antiretroviral therapy (ART), viremia rebounds to pretherapy levels in most individuals [1,2,3,4]
The HIV reservoir is defined as the viral genomes integrated into the DNA of host cells [5,6,7] that propagate the recrudescence of viremia upon cessation of ART [5,8,9,10,11,12,13,14]
Summary
Author’s contribution to the Work was done as part of the Author’s official duties as a NIH employee and is a. Have been guided by quantitative viral outgrowth assays (VOA) [26,30,31,32,33,34] that detect and quantify intact proviruses in cell samples by inducing them to express virus particles in limiting-dilution culture wells ex vivo or mice in vivo [35] While laborious, these approaches are the gold standard of HIV reservoir quantification. These MDA-augmented approaches maintain the original donor genetic material (e.g., DNA from blood, lymph node, gut biopsy), allowing for parallel downstream characterization of single proviruses and infected cell clones. Proviral information derived from MDA approaches provides bookmarks for tracking dynamics of infected cell clones, which is vital to the development and evaluation of current and future curative strategies
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