Protein expression from unintegrated HIV-1 DNA introduces bias in primary in vitro post-integration latency models

Abstract

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.

Disrupting HIV ‐1 capsid formation causes cGAS sensing of viral DNA

“…Importantly, GFP expression was suppressed by raltegravir in parallel infection of PMA‐treated THP‐1 cells (Fig EV3D and E) confirming that integration was indeed inhibited by 10 μM raltegravir or D116N integrase mutation. We propose that the GFP positivity observed in monocytic THP‐1 cells, in the presence of raltegravir, or by ∆CA‐SP1 INT D116N, is due to expression from 2′‐LTR circles as has been observed in other cell types (Van Loock et al , ; Bonczkowski et al , ).…”

Antigen Production After Latency Reversal and Expression of Inhibitory Receptors in CD8+ T Cells Limit the Killing of HIV-1 Reactivated Cells

“…At this point, we obtained a RELI population enriched in uninfected and non-productively HIV-1-infected cells. RELI cells were then washed, treated with the HIV-1 integrase inhibitor raltegravir (RAL) to avoid multiple rounds of infection, and exposed to LRA shock for 48 h. We also treated RELI cells with azidothymidine (AZT) to avoid the formation of unintegrated episomal forms that may contribute to the expression of viral proteins (data not shown) (33). LRA-treated cells were then extensively washed to uncouple the LRA effect from CD8+ T-cell function against reactivated cells (24), and co-cultured with HLA-class I–matched HIV-1-specific CTLs for 20 h (kill) (Figure 1A).…”

Complex decay dynamics of HIV virions, intact and defective proviruses, and 2LTR circles following initiation of antiretroviral therapy

“…The initial phase has a t 1/2 of 24 d. It is not clear whether this represents degradation of the circles, virus-independent T cell turnover, or virus-dependent elimination of cells carrying circles. Although 2LTR circles are replication-defective, gene expression from 2LTR circles has been detected and could allow recognition by CTL ( 87 , 88 ). The next phase of decay of 2LTR circles has a t 1/2 of ∼15 mo.…”