Vailable at the end of the articleof HIV latency have beenVailable at the end of

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Vailable at the end of the articleof HIV latency have been developed including latently infected cells lines and primary T-cells [4]. Understanding the location and frequency of HIV integration in the host genome in models of HIV latency as well as resting CD4+ T-cells from HIV-infected individuals on cART can potentially provide insights into the origin of infection, clonal expansion and potentially the response to latency reversing agents [5]. Latently infected cell lines are established following infection with either intact, replication-competent virus or mutated, replication-defective viruses. Examples of cell lines infected with replication competent virus include U1, ACH-2 and J1.1 cells [6?] and with replication defective virus include J-Lat, where the cell lines?The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://Necrosulfonamide site creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Symons et al. Retrovirology (2017) 14:Page 2 ofare monoclonal and harbour a single integration site [10, 11]. In CD4+ T-cells from HIV-infected individuals on cART, several groups have recently shown a significant expansion of latently infected cells with a distinct site of integration, consistent with clonal expansion in vivo [5, 12?4]. Understanding whether similar patterns of integration occur in in vitro models of HIV latency and in patient derived cells is important, if these models are to be used to study the establishment, maintenance and reversal of latency. 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]. Random shearing leads to different sized PCR products. Therefore, if an identical HIV integration site is detected but the length of the PCR product is different, it is most likely that this HIV integration sites was derived from a clonally expanded cell. Another method of determining the frequency of HIV integration sites is by limiting dilution of genomic DNA based on the estimated copies PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27488460 of HIV integrated DNA followed by loop amplification, and sequencing using primers located in the LTR [14]. Here, we describe a method to significantly streamline the assessment of HIV integration sites using robotic processing. Using this method, we evaluated HIV integration sites in commonly used latently infected cell lines and demonstrated that multiple cell lines that are traditionally used to study latency have evidence of productive infection.Table 1 Cell lines analysed in this study from NIH AIDS reagent programCell line Replication Site of competent mutation virus Yes Yes Yes No No No No No No No Proviral Reference copies reported per cell [6] [7, 8] [9] PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27488460 [10] [10] [10] [10] [10, 11] [10, 11] [10, 11] [10, 11]U1 ACH-2 J1.1 J-Lat 8.4 J-Lat 9.2 J-Lat 10.6 J-Lat 15.4 J-Lat tat-GFP 8.2 J-Lat tat-GFP A1 J-Lat tat-GFP HMutation i.