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

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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. Conclusion: 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.
Keywords
HUMAN-IMMUNODEFICIENCY-VIRUS, ACTIVE ANTIRETROVIRAL THERAPY, CD4(+) T-CELLS, POSTINTEGRATION LATENCY, IN-VITRO, EXPRESSION, GENE, SELECTION, RESERVOIR, CLONE

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MLA
Symons, Jori et al. “HIV Integration Sites in Latently Infected Cell Lines : Evidence of Ongoing Replication.” RETROVIROLOGY 14 (2017): n. pag. Print.
APA
Symons, J., Chopra, A., Malatinková, E., De Spiegelaere, W., Leary, S., Cooper, D., Abana, C. O., et al. (2017). HIV integration sites in latently infected cell lines : evidence of ongoing replication. RETROVIROLOGY, 14.
Chicago author-date
Symons, Jori, Abha Chopra, Eva Malatinková, Ward De Spiegelaere, Shay Leary, Don Cooper, Chike O Abana, et al. 2017. “HIV Integration Sites in Latently Infected Cell Lines : Evidence of Ongoing Replication.” Retrovirology 14.
Chicago author-date (all authors)
Symons, Jori, Abha Chopra, Eva Malatinková, Ward De Spiegelaere, Shay Leary, Don Cooper, Chike O Abana, Ajantha Rhodes, Simin D Rezaei, Linos Vandekerckhove, Simon Mallal, Sharon R Lewin, and Paul U Cameron. 2017. “HIV Integration Sites in Latently Infected Cell Lines : Evidence of Ongoing Replication.” Retrovirology 14.
Vancouver
1.
Symons J, Chopra A, Malatinková E, De Spiegelaere W, Leary S, Cooper D, et al. HIV integration sites in latently infected cell lines : evidence of ongoing replication. RETROVIROLOGY. 2017;14.
IEEE
[1]
J. Symons et al., “HIV integration sites in latently infected cell lines : evidence of ongoing replication,” RETROVIROLOGY, vol. 14, 2017.
@article{8502251,
  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.
Conclusion: 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.},
  articleno    = {2},
  author       = {Symons, Jori and Chopra, Abha and Malatinková, Eva and De Spiegelaere, Ward and Leary, Shay and Cooper, Don and Abana, Chike O and Rhodes, Ajantha and Rezaei, Simin D and Vandekerckhove, Linos and Mallal, Simon and Lewin, Sharon R and Cameron, Paul U},
  issn         = {1742-4690},
  journal      = {RETROVIROLOGY},
  keywords     = {HUMAN-IMMUNODEFICIENCY-VIRUS,ACTIVE ANTIRETROVIRAL THERAPY,CD4(+) T-CELLS,POSTINTEGRATION LATENCY,IN-VITRO,EXPRESSION,GENE,SELECTION,RESERVOIR,CLONE},
  language     = {eng},
  pages        = {11},
  title        = {HIV integration sites in latently infected cell lines : evidence of ongoing replication},
  url          = {http://dx.doi.org/10.1186/s12977-016-0325-2},
  volume       = {14},
  year         = {2017},
}

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