To understand the persistence of latently HIV-1 infected cells in virally suppressed infected patients, a number of in vitro models of HIV latency have been developed. In an attempt to mimic the in vivo situation as closely as possible, several models use primary cells and replication-competent viruses in combination with antiretroviral compounds to prevent ongoing replication. Latency is subsequently measured by HIV RNA and/or protein production after cellular activation. To discriminate between pre- and post-integration latency, integrase inhibitors are routinely used, preventing novel integrations upon cellular activation. Here, we show that this choice of antiretrovirals may still cause a bias of pre-integration latency in these models, as unintegrated HIV DNA can form and directly contribute to the levels of HIV RNA and protein production. We further show that the addition of reverse transcriptase inhibitors effectively suppresses the levels of episomal HIV DNA (as measured by 2-LTR circles) and decreases the levels of HIV transcription. Consequently, we show that latency levels described in models that only use integrase inhibitors may be overestimated. The inclusion of additional control conditions, such as 2-LTR quantification and the addition of reverse transcriptase inhibitors, is crucial to fully elucidate the actual levels of post-integration latency.

HIV Cure Research Center Department of Internal Medicine Faculty of Medicine and Health Sciences Ghent University and Ghent University Hospital Ghent Belgium

HIV Cure Research Center Department of Internal Medicine Faculty of Medicine and Health Sciences Ghent University and Ghent University Hospital Ghent Belgium Department of Morphology Faculty of Veterinary Sciences Ghent University Merelbeke Belgium

Paul Ehrlich Institut Paul Ehrlich Strasse 51 59 63225 Langen Germany

Paul Ehrlich Institut Paul Ehrlich Strasse 51 59 63225 Langen Germany Department of Immunology and Microbiology 1st Medical Faculty Charles University Studnickova 7 128 00 Prague 2 Czech Republic

Paul Ehrlich Institut Paul Ehrlich Strasse 51 59 63225 Langen Germany Immunity and Pathogenesis Program Sanford Burnham Prebys Medical Discovery Institute La Jolla CA 92037 USA

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