New Approaches to Multi-Parametric HIV-1 Genetics Using Multiple Displacement Amplification: Determining the What, How, and Where of the HIV-1 Reservoir

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

Combined HIV-1 sequence and integration site analysis informs viral dynamics and allows reconstruction of replicating viral ancestors

Understanding HIV-1 persistence despite antiretroviral therapy (ART) is of paramount importance. Both single-genome sequencing (SGS) and integration site analysis (ISA) provide useful information regarding the structure of persistent HIV DNA populations; however, until recently, there was no way to link integration sites to their cognate proviral sequences. Here, we used multiple-displacement amplification (MDA) of cellular DNA diluted to a proviral endpoint to obtain full-length proviral sequences and their corresponding sites of integration. We applied this method to lymph node and peripheral blood mononuclear cells from 5 ART-treated donors to determine whether groups of identical subgenomic sequences in the 2 compartments are the result of clonal expansion of infected cells or a viral genetic bottleneck. We found that identical proviral sequences can result from both cellular expansion and viral genetic bottlenecks occurring prior to ART initiation and following ART failure. We identified an expanded T cell clone carrying an intact provirus that matched a variant previously detected by viral outgrowth assays and expanded clones with wild-type and drug-resistant defective proviruses. We also found 2 clones from 1 donor that carried identical proviruses except for nonoverlapping deletions, from which we could infer the sequence of the intact parental virus. Thus, MDA-SGS can be used for "viral reconstruction" to better understand intrapatient HIV-1 evolution and to determine the clonality and structure of proviruses within expanded clones, including those with drug-resistant mutations. Importantly, we demonstrate that identical sequences observed by standard SGS are not always sufficient to establish proviral clonality