title: 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 TCM(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.
  
  
  OBJECTIVE
  Despite 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.
authors.affiliation: Division of Infectious Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Sara
authors.initials: S
authors.lastname: Sunshine
authors.affiliation: Bioinformatics Core, Harvard T. H. Chan School of Public Health, Boston, Massachusetts, USA.
authors.forename: Rory
authors.initials: R
authors.lastname: Kirchner
authors.affiliation: Partners HealthCare Personalized Medicine, Cambridge, Massachusetts, USA.
authors.forename: Sami S
authors.initials: SS
authors.lastname: Amr
authors.affiliation: Partners HealthCare Personalized Medicine, Cambridge, Massachusetts, USA.
authors.forename: Leandra
authors.initials: L
authors.lastname: Mansur
authors.affiliation: Partners HealthCare Personalized Medicine, Cambridge, Massachusetts, USA.
authors.forename: Rimma
authors.initials: R
authors.lastname: Shakhbatyan
authors.affiliation: Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.|Howard Hughes Medical Institute, Chevy Chase, Maryland, USA.
authors.forename: Michelle
authors.initials: M
authors.lastname: Kim
authors.affiliation: Department of Pathology, University of Utah, Salt Lake City, Utah, USA.
authors.forename: Alberto
authors.initials: A
authors.lastname: Bosque
authors.affiliation: Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.|Howard Hughes Medical Institute, Chevy Chase, Maryland, USA.
authors.forename: Robert F
authors.initials: RF
authors.lastname: Siliciano
authors.affiliation: Department of Pathology, University of Utah, Salt Lake City, Utah, USA.
authors.forename: Vicente
authors.initials: V
authors.lastname: Planelles
authors.affiliation: Bioinformatics Core, Harvard T. H. Chan School of Public Health, Boston, Massachusetts, USA.
authors.forename: Oliver
authors.initials: O
authors.lastname: Hofmann
authors.affiliation: Bioinformatics Core, Harvard T. H. Chan School of Public Health, Boston, Massachusetts, USA.
authors.forename: Shannan
authors.initials: S
authors.lastname: Ho Sui
authors.affiliation: Division of Infectious Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA Jli22@partners.org.
authors.forename: Jonathan Z
authors.initials: JZ
authors.lastname: Li
mesh_terms: D002460:Cell Line; D002478:Cells, Cultured; D002874:Chromosome Mapping; D019295:Computational Biology / Q000379:methods; D015342:DNA Probes; D006678:HIV / Q000502:physiology*; D015658:HIV Infections / Q000235:genetics* / Q000821:virology*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D017422:Sequence Analysis, DNA; D016662:Virus Integration; D017735:Virus Latency
chemical_list: D015342:DNA Probes