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Review
. 2021 Dec 10;13(12):2475.
doi: 10.3390/v13122475.

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

Sean C Patro  1 , Aurelie Niyongabo  1 , Frank Maldarelli  1 , Mary F Kearney  1
Affiliations
Review

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

Sean C Patro et al. Viruses. .

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.

Keywords: HIV reservoir; clonal expansion; intact proviral genomes; integration sites analysis; multiple displacement amplification; near full-length genome amplification.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Workflow of MDA-supplemented HIV proviral characterization. (Left) HIV (+) PBMC or other material can be CD4-enriched or cell sorted to target specific CD4+ memory subsets or phenotypes such as activation state or antigen reactivity. Genomic DNA or sorted cells are diluted to a single proviral endpoint and used for multiple displacement amplification (MDA). (Center) MDA reactions are screened for HIV-1 proviruses by amplification and sequencing of a sub-genomic region of HIV (e.g., LTR, gag, int, env), qPCR/ddPCR targeting specific genome elements, or size selection of the near full-length (NFL) HIV amplicon. Red circles designate candidate proviruses for follow-up. (Right) Multi-parametric HIV proviral analysis including full-length (NFL) HIV amplification and sequencing, integration site analysis, host-HIV amplification and sequencing, and/or CD4+ TCR sequencing.
Figure 2
Figure 2
Neighbor-joining trees of MDA-amplified SGS. (A) Single-genome sequencing of the P6-PR-RT region in PBMC from Patient 1 [5,8] showing integration-site identical proviruses within expanded CD4+ T cell clones. CD4+ memory subsets from which sequences were recovered are indicated by white (central/transitional memory) or black (effector memory) triangles. Integration site details (gene/nearest gene, chromosome, hg19 location, proviral orientation relative to gene (+/−, with/against, respectively), and number of observations) indicated for each group of identical sequences. (B) CD4+ memory T cell subset distribution of identical proviral integration sites within expanded cell clones. Cluster ID designated in coordination with phylogenetic tree (2A).
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