title: HIV integration sites in latently infected cell lines: evidence of ongoing replication.
abstract: BACKGROUND
  Assessing the location and frequency of HIV integration sites in latently infected cells can potentially inform our understanding of how HIV persists during combination antiretroviral therapy. We developed a novel high throughput sequencing method to evaluate HIV integration sites in latently infected cell lines to determine whether there was virus replication or clonal expansion in these cell lines observed as multiple integration events at the same position.
  
  
  RESULTS
  We modified a previously reported method using random DNA shearing and PCR to allow for high throughput robotic processing to identify the site and frequency of HIV integration in latently infected cell lines. Latently infected cell lines infected with intact virus demonstrated multiple distinct HIV integration sites (28 different sites in U1, 110 in ACH-2 and 117 in J1.1 per 150,000 cells). In contrast, cell lines infected with replication-incompetent viruses (J-Lat cells) demonstrated single integration sites. Following in vitro passaging of the ACH-2 cell line, we observed a significant increase in the frequency of unique HIV integration sites and there were multiple mutations and large deletions in the proviral DNA. When the ACH-2 cell line was cultured with the integrase inhibitor raltegravir, there was a significant decrease in the number of unique HIV integration sites and a transient increase in the frequency of 2-LTR circles consistent with virus replication in these cells.
  
  
  CONCLUSIONS
  Cell lines latently infected with intact HIV demonstrated multiple unique HIV integration sites indicating that these cell lines are not clonal and in the ACH-2 cell line there was evidence of low level virus replication. These findings have implications for the use of latently infected cell lines as models of HIV latency and for the use of these cells as standards.
authors.affiliation: The Peter Doherty Institute for Infection and Immunity, University of Melbourne and Royal Melbourne Hospital, 792 Elizabeth St, Melbourne, VIC, 3000, Australia.
authors.forename: Jori
authors.initials: J
authors.lastname: Symons
authors.affiliation: Institute for Immunology and Infectious Diseases (IIID), Murdoch University, Murdoch, WA, Australia.
authors.forename: Abha
authors.initials: A
authors.lastname: Chopra
authors.affiliation: HIV Translational Research Unit, Department of Internal Medicine, Faculty of Medicine and Health Sciences, Ghent University Hospital, Ghent University, Ghent, Belgium.
authors.forename: Eva
authors.initials: E
authors.lastname: Malatinkova
authors.affiliation: HIV Translational Research Unit, Department of Internal Medicine, Faculty of Medicine and Health Sciences, Ghent University Hospital, Ghent University, Ghent, Belgium.
authors.forename: Ward
authors.initials: W
authors.lastname: De Spiegelaere
authors.affiliation: Institute for Immunology and Infectious Diseases (IIID), Murdoch University, Murdoch, WA, Australia.
authors.forename: Shay
authors.initials: S
authors.lastname: Leary
authors.affiliation: Institute for Immunology and Infectious Diseases (IIID), Murdoch University, Murdoch, WA, Australia.
authors.forename: Don
authors.initials: D
authors.lastname: Cooper
authors.affiliation: Department of Pathology, Microbiology and Immunology, Vanderbilt University, Nashville, TN, 37232, USA.
authors.forename: Chike O
authors.initials: CO
authors.lastname: Abana
authors.affiliation: The Peter Doherty Institute for Infection and Immunity, University of Melbourne and Royal Melbourne Hospital, 792 Elizabeth St, Melbourne, VIC, 3000, Australia.
authors.forename: Ajantha
authors.initials: A
authors.lastname: Rhodes
authors.affiliation: The Peter Doherty Institute for Infection and Immunity, University of Melbourne and Royal Melbourne Hospital, 792 Elizabeth St, Melbourne, VIC, 3000, Australia.
authors.forename: Simin D
authors.initials: SD
authors.lastname: Rezaei
authors.affiliation: HIV Translational Research Unit, Department of Internal Medicine, Faculty of Medicine and Health Sciences, Ghent University Hospital, Ghent University, Ghent, Belgium.
authors.forename: Linos
authors.initials: L
authors.lastname: Vandekerckhove
authors.affiliation: Institute for Immunology and Infectious Diseases (IIID), Murdoch University, Murdoch, WA, Australia.|Department of Pathology, Microbiology and Immunology, Vanderbilt University, Nashville, TN, 37232, USA.
authors.forename: Simon
authors.initials: S
authors.lastname: Mallal
authors.affiliation: The Peter Doherty Institute for Infection and Immunity, University of Melbourne and Royal Melbourne Hospital, 792 Elizabeth St, Melbourne, VIC, 3000, Australia.|Department of Infectious Diseases, Alfred Hospital and Monash University, Melbourne, Australia.
authors.forename: Sharon R
authors.initials: SR
authors.lastname: Lewin
authors.affiliation: The Peter Doherty Institute for Infection and Immunity, University of Melbourne and Royal Melbourne Hospital, 792 Elizabeth St, Melbourne, VIC, 3000, Australia. paul.cameron@unimelb.edu.au.|Department of Infectious Diseases, Alfred Hospital and Monash University, Melbourne, Australia. paul.cameron@unimelb.edu.au.
authors.forename: Paul U
authors.initials: PU
authors.lastname: Cameron
mesh_terms: D002460:Cell Line; D006678:HIV / Q000502:physiology*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D016662:Virus Integration; D017735:Virus Latency; D014779:Virus Replication