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

ABSTRACT: 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.

SUBMITTER: Patro SC

PROVIDER: S-EPMC6925994 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Publications

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

Patro Sean C SC Brandt Leah D LD Bale Michael J MJ Halvas Elias K EK Joseph Kevin W KW Shao Wei W Wu Xiaolin X Guo Shuang S Murrell Ben B Wiegand Ann A Spindler Jonathan J Raley Castle C Hautman Christopher C Sobolewski Michele M Fennessey Christine M CM Hu Wei-Shau WS Luke Brian B Hasson Jenna M JM Niyongabo Aurelie A Capoferri Adam A AA Keele Brandon F BF Milush Jeff J Hoh Rebecca R Deeks Steven G SG Maldarelli Frank F Hughes Stephen H SH Coffin John M JM Rausch Jason W JW Mellors John W JW Kearney Mary F MF

Proceedings of the National Academy of Sciences of the United States of America 20191127 51

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 sequen ...[more]

PMID: 31776247

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Project description:Modern gene therapy methods have limited control over where a therapeutic viral vector inserts into the host genome. Vector integration can activate local gene expression, which can cause cancer if the vector inserts near an oncogene. Viral integration hot-spots or 'common insertion sites' (CIS) are scrutinized to evaluate and predict patient safety. CIS are typically defined by a minimum density of insertions (such as 2-4 within a 30-100 kb region), which unfortunately depends on the total number of observed VIS. This is problematic for comparing hot-spot distributions across data sets and patients, where the VIS numbers may vary.We develop two new methods for defining hot-spots that are relatively independent of data set size. Both methods operate on distributions of VIS across consecutive 1 Mb 'bins' of the genome. The first method 'z-threshold' tallies the number of VIS per bin, converts these counts to z-scores, and applies a threshold to define high density bins. The second method 'BCP' applies a Bayesian change-point model to the z-scores to define hot-spots. The novel hot-spot methods are compared with a conventional CIS method using simulated data sets and data sets from five published human studies, including the X-linked ALD (adrenoleukodystrophy), CGD (chronic granulomatous disease) and SCID-X1 (X-linked severe combined immunodeficiency) trials. The BCP analysis of the human X-linked ALD data for two patients separately (774 and 1627 VIS) and combined (2401 VIS) resulted in 5-6 hot-spots covering 0.17-0.251% of the genome and containing 5.56-7.74% of the total VIS. In comparison, the CIS analysis resulted in 12-110 hot-spots covering 0.018-0.246% of the genome and containing 5.81-22.7% of the VIS, corresponding to a greater number of hot-spots as the data set size increased. Our hot-spot methods enable one to evaluate the extent of VIS clustering, and formally compare data sets in terms of hot-spot overlap. Finally, we show that the BCP hot-spots from the repopulating samples coincide with greater gene and CpG island density than the median genome density.The z-threshold and BCP methods are useful for comparing hot-spot patterns across data sets of disparate sizes. The methodology and software provided here should enable one to study hot-spot conservation across a variety of VIS data sets and evaluate vector safety for gene therapy trials.

Dataset Information

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

Publications

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

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