- Open Access
Replication competent virus as an important source of bias in HIV latency models utilizing single round viral constructs
© Bonczkowski et al.; licensee BioMed Central Ltd. 2014
- Received: 26 June 2014
- Accepted: 31 July 2014
- Published: 21 August 2014
The central memory T cell (TCM) model forms a unique HIV-1 latency model based on primary cells that closely resemble in vivo TCM. The virus employed in this model is based on an engineered vector incapable of replication after initial infection. We show that despite this strategy, replication competent viral particles are released into the culture medium due to recombination between overlapping sequences of the env deleted HIV genome that is co-transfected with intact env. This finding emphasizes the need for careful data analysis and interpretation if similar constructs are employed and urges for additional caution during laboratory work.
- Single round HIV
- Latency models
- Central memory T cells
- Non-polarized T cells
- HIV latency
Replication competent viruses generated in simple co-transfection systems may impact biosafety and bias research results. Researchers should provide evidence proving the replication incompetence of new constructs. The findings impact earlier notions arising from the TCM model and therefore the virus initially included as part of this model cannot be used as originally described . However, the model may still be suitable to understand HIV-1 latency provided that specific modifications are introduced. There are two possible options here. Efforts can be made to avoid recombination by implementation of envelope genes originating from non-HIV species (e.g. VSV-G) , by utilizing split vector systems such as these used in gene therapy or more elaborate co-transfection systems with a proven lack of recombination. Alternatively, the model can be used with replication competent viruses in combination with antiretroviral drugs in order to limit the spreading infection. Finally, we would like to state for the record that we are confident that cultured TCM generated as described by Bosque and Planelles  continue to be a relevant and suitable cell model for investigations of HIV infection and latency/reactivation, and that the findings described herein do not impact the methods for the generation of such cells or their use in the laboratory.
PB, WDS, AB and LV conceived and designed experiments. PB, AB, EM, MK and AVN performed the experiments. PB, LV, WDS, AB, BV and VP analyzed the data. CHW and CHW generated and analyzed NGS data. LV, WDS, AVN, WT, JV, WW and BV provided technical expertise throughout the experiment. PB, WDS and LV wrote the manuscript. All authors read and approved the final manuscript.
Pawel Bonczkowski and Eva Malatinkova are supported by the Agency for Innovation by Science and Technology in Flanders (IWT; Grant nr: 111393 & 111286). Prof. Linos Vandekerckhove is supported by the Research Foundation - Flanders (FWO; Grant 1.8.020.09.N.00). Maja Kiselinova is supported by a `Special Research Grant - BOF grant’ of Ghent University (Grant nr: 01 N02712). This work was also supported by a travel grant of the Research Foundation - Flanders to Ward De Spiegelaere (FWO V410513N), HIV-ERA/SBO-IWT (Grant 130442), Bill & Melinda Gates Foundation, Grant ID # OPP1035848 and King Baudouin Foundation, Grant 2010-R20640-003. Jolien Vermeire and Wojciech Witkowski are PhD fellows and Bruno Verhasselt is a Senior Clinical Investigator of the FWO supported by Research Foundation - Flanders (FWO). The work was supported by grants to Bruno Verhasselt: Ghent University grant BOF11/GOA/013 and grants from the Research Foundation - Flanders (FWO 007414 N), and HIV-ERA (IRIFCURE).
Additionally, this work was performed with the support of the Collaboratory of AIDS Researchers for Eradication (CARE, AI096113), the Genomics Core at the UCSD CFAR (AI036214), equipment from the James B. Pendleton Foundation, the San Diego Veterans Medical Research Foundation, and National Institutes of Health research grants (AI104282 and AI087508). Total RNA-Seq was performed by Expression Analysis Inc.
- Bosque A, Planelles V: Induction of HIV-1 latency and reactivation in primary memory CD4(+) T cells. Blood. 2009, 113: 58-65. 10.1182/blood-2008-07-168393.PubMed CentralView ArticlePubMedGoogle Scholar
- Westerman KA, Ao Z, Cohen EA, Leboulch P: Design of a trans protease lentiviral packaging system that produces high titer virus. Retrovirology. 2007, 4: 1742-4690. 10.1186/1742-4690-4-96.View ArticleGoogle Scholar
- Naldini L, Blomer U, Gage FH, Trono D, Verma IM: Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proc Natl Acad Sci U S A. 1996, 93: 11382-11388. 10.1073/pnas.93.21.11382.PubMed CentralView ArticlePubMedGoogle Scholar
- Zufferey R, Nagy D, Mandel RJ, Naldini L, Trono D: Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat Biotechnol. 1997, 15: 871-875. 10.1038/nbt0997-871.View ArticlePubMedGoogle Scholar
- Kinoshita S, Chen BK, Kaneshima H, Nolan GP: Host control of HIV-1 parasitism in T cells by the nuclear factor of activated T cells. Cell. 1998, 95: 595-604. 10.1016/S0092-8674(00)81630-X.View ArticlePubMedGoogle Scholar
- Gavegnano C, Detorio M, Montero C, Bosque A, Planelles V, Schinazi RF: Ruxolitinib and tofacitinib are potent and selective inhibitors of HIV-1 replication and virus reactivation in vitro. Antimicrob Agents Chemother. 2014, 58: 1977-1986. 10.1128/AAC.02496-13.PubMed CentralView ArticlePubMedGoogle Scholar
- Duverger A, Wolschendorf F, Anderson JC, Wagner F, Bosque A, Shishido T, Jones J, Planelles V, Willey C, Cron RQ, Kutsch O: Kinase control of latent HIV-1 infection: PIM-1 kinase as a major contributor to HIV-1 reactivation. J Virol. 2014, 88: 364-376. 10.1128/JVI.02682-13.PubMed CentralView ArticlePubMedGoogle Scholar
- Bosque A, Planelles V: Studies of HIV-1 latency in an ex vivo model that uses primary central memory T cells. Methods. 2011, 53: 54-61. 10.1016/j.ymeth.2010.10.002.PubMed CentralView ArticlePubMedGoogle Scholar
- Sherrill-Mix S, Lewinski MK, Famiglietti M, Bosque A, Malani N, Ocwieja KE, Berry CC, Looney D, Shan L, Agosto LM, Pace MJ, Siliciano RF, O'Doherty U, Guatelli J, Planelles V, Bushman FD: HIV latency and integration site placement in five cell-based models. Retrovirology. 2013, 10: 90-10.1186/1742-4690-10-90.PubMed CentralView ArticlePubMedGoogle Scholar
- Bosque A, Famiglietti M, Weyrich AS, Goulston C, Planelles V: Homeostatic proliferation fails to efficiently reactivate HIV-1 latently infected central memory CD4+ T cells. PLoS Pathog. 2011, 7: e1002288-10.1371/journal.ppat.1002288.PubMed CentralView ArticlePubMedGoogle Scholar
- Vandergeeten C, Fromentin R, Dafonseca S, Lawani MB, Sereti I, Lederman MM, Ramgopal M, Routy JP, Sekaly RP, Chomont N: Interleukin-7 promotes HIV persistence during antiretroviral therapy. Blood. 2013, 15: 15-Google Scholar
- Novis C, Archin NM, Buzon MJ, Verdin E, Round J, Lichterfeld M, Margolis DM, Planelles V, Bosque A: Reactivation of latent HIV-1 in central memory CD4+ T cells through TLR-1/2 stimulation. Retrovirology. 2013, 10: 119-10.1186/1742-4690-10-119.PubMed CentralView ArticlePubMedGoogle Scholar
- Wei DG, Chiang V, Fyne E, Balakrishnan M, Barnes T, Graupe M, Hesselgesser J, Irrinki A, Murry JP, Stepan G, Stray KM, Tsai A, Yu H, Spindler J, Kearney M, Spina CA, McMahon D, Lalezari J, Sloan D, Mellors J, Geleziunas R, Cihlar T: Histone deacetylase inhibitor Romidepsin induces HIV expression in CD4 T cells from patients on suppressive antiretroviral therapy at concentrations achieved by clinical dosing. PLoS Pathog. 2014, 10: e1004071-10.1371/journal.ppat.1004071.PubMed CentralView ArticlePubMedGoogle Scholar
- Lusic M, Marini B, Ali H, Lucic B, Luzzati R, Giacca M: Proximity to PML nuclear bodies regulates HIV-1 latency in CD4+ T cells. Cell Host Microbe. 2013, 13: 665-677. 10.1016/j.chom.2013.05.006.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. 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.