Opposite transcriptional regulation of integrated vs unintegrated HIV genomes by the NF-κB pathway (original) (raw)

Integration of HIV-1 linear DNA into host chromatin is required for high levels of viral expression, and constitutes a key therapeutic target. Unintegrated viral DNA (uDNA) can support only limited transcription but may contribute to viral propagation, persistence and/or treatment escape under specific situations. The molecular mechanisms involved in the differential expression of HIV uDNA vs integrated genome (iDNA) remain to be elucidated. Here, we demonstrate, for the first time, that the expression of HIV uDNA is mainly supported by 1-LTR circles, and regulated in the opposite way, relatively to iDNA, following NF-κB pathway modulation. Upon treatment activating the NF-κB pathway, NF-κB p65 and AP-1 (cFos/cJun) binding to HIV LTR iDNA correlates with increased iDNA expression, while uDNA expression decreases. On the contrary, inhibition of the NF-κB pathway promotes the expression of circular uDNA, and correlates with Bcl-3 and AP-1 binding to its LTR region. Finally, this study identifies NF-κB subunits and Bcl-3 as transcription factors binding the HIV promoter differently depending on viral genome topology, and opens new insights on the potential roles of episomal genomes during the HIV-1 latency and persistence. Integration of the HIV genome is an essential step of the retroviral cycle, supporting massive production of viral particles. Strikingly, integrated viral DNAs (iDNAs) only represent a minor part of reverse-transcribed genomes, that remains mainly under unintegrated viral forms (uDNAs) at early times post-infection as well as during untreated chronic infection 1,2. Unintegrated HIV genomes mainly include linear DNAs (DNA L) that are quickly degraded, and circular DNAs containing one or two long terminal repeats (1-LTRc and 2-LTRc, respectively) 3,4. Conversely, 1-LTRc and 2-LTRc episomal DNAs remain intrinsically stable and only diminish through cell death or division (e.g. following T cell activation) 4,5. Several studies have demonstrated the stability of uDNAs in non-dividing primary macrophages and resting CD4 T cells 6-9. This stability is supported by clinical trials highlighting the high levels and persistence of 2-LTRc in HIV-1 controllers 10,11. Until recently, uDNAs were considered as "dead-end products" of reverse transcription. However, several reports have now established that circular uDNAs can support low levels of HIV expression 7,8,12-15 , can constitute a reserve substrate for de novo integration 16 , and be a source of infectious virus 17-19 (for a review 20). Therefore, HIV uDNAs should be considered as potential reserve genomes that could be involved in persistence and treatment escape. Although expression of uDNAs can be several orders of magnitude lower than that of an integrated provirus 13,15 , it can lead to the expression of accessory proteins such as Nef and Tat 14,15. Importantly, this low level of Nef expression is sufficient to down-regulate CD4 expression on host cell surfaces and to induce T cell activation 7 , highlighting the importance of uDNA expression on HIV-host interaction. LTR-mediated expression from HIV iDNA is well documented. Host transcription factor mobilization and chromatin decondensation are required for robust HIV transcription, so that HIV post-integration latency is considered as a transcription factor restriction phenomenon 21,22. Notably, HIV-1 LTR contains binding sites for several inducible transcription factors, including NFκ B, NFAT or AP-1 (c-Jun/cFos family members) (for reviews 21,23,24). HIV-1 transcription is thus tightly coupled to cell type and activation status. After appropriate stimulation of