# Development and validation of HIV SMRTcap for the characterization of HIV-1 reservoirs across tissues and subtypes

## TL;DR

A new method called HIV SMRTcap is developed to better understand where HIV hides in the body, which could help in finding a cure.

## Contribution

HIV SMRTcap is a novel pipeline that simultaneously identifies HIV integration sites and proviral integrity using long-read sequencing.

## Key findings

- HIV SMRTcap enables comprehensive characterization of HIV-1 reservoirs across major global subtypes.
- The method works with both cell- and tissue-derived inputs, including samples from individuals on antiretroviral therapy.
- HIV SMRTcap provides high sensitivity for detecting HIV in various contexts and tissues.

## Abstract

Human Immunodeficiency Virus type 1 (HIV-1) is responsible for the global HIV/AIDS epidemic and the establishment of an integrated HIV-1 reservoir remains the primary obstacle to cure. Upon therapy interruption, reactivation of the persistent HIV-1 reservoir propagates viral rebound and mediates continued immunological decline. While furthering understanding of the HIV-1 reservoir is essential for HIV-1 cure, commonly used sequencing strategies are often limited by the reliance on short-read sequencing across separate assays to determine integration sites and proviral integrity – something that does not always adequately resolve complex human genomic repeats or low complexity regions. Simultaneous identification of proviral integration sites and proviral integrity at the single molecule level would enable HIV-1 reservoir characterization with minimal imputation or bioinformatic reconstruction. Here we present HIV Single Molecule Real Time Capture (HIV SMRTcap), a novel molecular and computational pipeline that directly and simultaneously identifies HIV-1 integration sites, defines proviral integrity, and characterizes clonal expansion of HIV-1 provirus-containing cells with single molecule resolution. In combination with long-read, single-molecule, real-time (SMRT) sequencing and custom analytic pipelines, HIV SMRTcap enables a highly comprehensive characterization of HIV-1 reservoirs. Moreover, we demonstrate here that HIV SMRTcap performs robustly across the major global subtypes (HIV-1 subtype A, B, C, D and A/D recombinant viruses), and can use both cell- and tissue-derived inputs, including samples from antiretroviral therapy (ART) treated individuals with undetectable viral loads. Our results demonstrate that HIV SMRTcap serves as a comprehensive, robust method for unbiased HIV-1 reservoir characterization. Used alone, or in combination with single-cell based methods, HIV SMRTcap will enable novel exploration of viral reservoirs across subtypes and in tissue-specific compartments, providing critical information needed to inform HIV-1 cure.

Human Immunodeficiency Virus (HIV) remains a persistent global and lifelong infection because the virus integrates into the genome of infected cells and is then protected by host mechanisms. Characterization of these virus-harboring cells (“reservoir”) has proven difficult due to a multitude of technical limitations, but remains critical as this viral reservoir reestablishes active infection upon therapy interruption. Understanding HIV integration patterns with regards to preferred genomic loci, prevalence of reservoir-containing cells within tissues, and profiling of internal deletions within the integrated viruses (proviruses) is pivotally important for vaccine and therapy design. In this work, we describe HIV Single Molecule Real Time Capture (HIV SMRTcap), an innovative pipeline that leverages long-read sequencing to simultaneously resolve proviral integration site(s) and integrity. This strategy enriches HIV-containing genomic DNA prior to single molecule sequencing and analyses using a custom informatic suite, ultimately capturing HIV at high sensitivity in contexts with and without antiretroviral therapy suppression, from primary tissue sources, and across HIV subtypes. We aim to describe HIV SMRTcap and its paired analysis tools in detail to enable wide adoption and encourage a pansubtype approach for equitable HIV research.