HIV Integration Site Analysis of Cellular Models of HIV Latency with a Probe-Enriched Next-Generation Sequencing Assay

Abstract

Antiretroviral therapy (ART) is successful in the suppression of HIV but cannot target and eradicate the latent proviral reservoir. The location of retroviral integration into the human genome is thought to play a role in the clonal expansion of infected cells and HIV persistence. We developed a high-throughput targeted sequence capture assay that uses a pool of HIV-specific probes to enrich Illumina libraries prior to deep sequencing. Using an expanded clonal population of ACH-2 cells, we demonstrate that this sequence capture assay has an extremely low false-positive rate. This assay assessed four cellular models commonly used to study HIV latency and latency-reversing agents: ACH-2 cells, J-Lat cells, the Bcl-2-transduced primary CD4؉ model, and the cultured T CM (central memory) CD4؉ model. HIV integration site characteristics and genes were compared between these cellular models and to previously reported patient data sets. Across these cellular models, there were significant differences in integration site characteristics, including orientation relative to that of the host gene, the proportion of clonally expanded sites, and the proportion located within genic regions and exons. Despite a greater diversity of minority integration sites than expected in ACH-2 cells, their integration site characteristics consistently differed from those of the other models and from the patient samples. Gene ontology analysis of highly represented genes from the patient samples found little overlap with HIV-containing genes from the cell lines. These findings show that integration site differences exist among the commonly used cellular models of HIV latency and in comparison to integration sites found in patient samples. IMPORTANCEDespite the success of ART, currently there is no successful therapy to eradicate integrated proviruses. Cellular models of HIV latency are used to test the efficacy of latency-reversing agents, but it is unclear how well these models reflect HIV integration into the human genome in vivo. We have developed a novel probe-based sequence enrichment assay to sequence and analyze integrated HIV. We compared HIV integration site characteristics between four cellular models and to previously described patient data sets. Significant differences were detected in the distribution of HIV integration sites between cellular models of HIV latency and compared to data sets from patient samples. The results from this study have implications for how well these cellular models of HIV infection truly reflect HIV integration in vivo and their applicability in drug discovery for novel latency-reversing agents.

HIV integration sites in latently infected cell lines: evidence of ongoing replication

“…In the ACH-2 cell line, the major HIV integration site was detected in chromosome 7 in the cytosolic 5′-nucleotidase 3A ( NT5C3A ) gene as previously reported [17, 23]. We also detected an additional integration site in chromosome 9 which was always detected in high frequency in the solute carrier family 25 antisense RNA ( SLC25A25 - AS ) noncoding RNA gene as recently reported [17].…”

“…We also detected an additional integration site in chromosome 9 which was always detected in high frequency in the solute carrier family 25 antisense RNA ( SLC25A25 - AS ) noncoding RNA gene as recently reported [17]. Additionally, we detected two sites with multiple integration events intermittently, one in chromosome 2 in the ethanolaminephosphotransferase 1 ( EPT1 ) gene, the other in chromosome 17 in the nuclear protein localization 4 ( NPLOC4 ) gene (Table 2).…”

“…In addition, HIV latency is established by a point mutation in TAR (C37T), whereby this mutation impairs the response of the LTR to Tat [26]. Low level transcription of multiply spliced transcripts persist in ACH-2 cells in the absence of stimulation [27], and here, we show that ACH-2 cells harbour multiple integration sites in addition to the major site in chromosome 7, as recently demonstrated by others [17]. …”

“…Strategies to determine sites of HIV integration include sequencing and cloning [15, 16] or bulk sequencing [5, 12, 13, 17]. Most bulk sequencing approaches use restriction enzymes or random shearing of genomic DNA followed by PCR, using primers in the long terminal repeat (LTR) and a linker [5, 12, 13, 17].…”

“…Most bulk sequencing approaches use restriction enzymes or random shearing of genomic DNA followed by PCR, using primers in the long terminal repeat (LTR) and a linker [5, 12, 13, 17]. Random shearing leads to different sized PCR products.…”