The HIV-2 OGH double reporter virus shows that HIV-2 is less cytotoxic and less sensitive to reactivation from latency than HIV-1 in cell culture.

A better understanding of HIV-1 latency is a research priority in HIV cure research. Conversely, little is known about the latency characteristics of HIV-2, the closely related human lentivirus. Though both viruses cause AIDS, HIV-2 infection progresses more slowly with significantly lower viral loads, even when corrected for CD4+ T cell counts. Hence a direct comparison of latency characteristics between HIV-1 and HIV-2 could provide important clues towards a functional cure. Transduction of SupT1 cells with single-round HIV-1 and HIV-2 viruses with an enhanced green fluorescent protein (eGFP) reporter showed higher levels of eGFP expression for HIV-2 than HIV-1, while HIV-1 expression appeared more cytotoxic. To compare HIV-1 and HIV-2 gene expression, latency and reactivation in more detail, we have generated HIV-2 OGH, a replication deficient, near full- length, double reporter virus that discriminates latently and productively infected cells in cell culture. This construct is based on HIV-1 OGH, and to our knowledge, first of its kind for HIV-2. Using this construct we have observed a higher eGFP expression for HIV-2, but higher losses of HIV-1 transduced cells in SupT1 and Jurkat cells and a reduced sensitivity of HIV-2 for reactivation with TNF-α. In addition, we have analysed HIV-2 integration sites and their epigenetic environment. HIV-1 and HIV-2 share a preference for actively transcribed genes in gene-dense regions and favor active chromatin marks while disfavoring methylation markers associated with heterochromatin. In conclusion the HIV-2 OGH construct provides an interesting tool for studying HIV-2 expression, latency and reactivation. As simian immunodeficiency virus (SIV) and HIV-2 have been proposed to model a functional HIV cure, a better understanding of the mechanisms governing HIV-2 and SIV latency will be important to move forward. Further research is needed to investigate if HIV-2 uses similar mechanisms as HIV-1 to achieve its integration site selectivity.

GS-9822, a preclinical LEDGIN candidate, displays a block-and-lock phenotype in cell culture.

The ability of HIV to integrate into the host genome and establish latent reservoirs is the main hurdle preventing an HIV cure. LEDGINs are small-molecule integrase inhibitors that target the binding pocket of LEDGF/p75, a cellular cofactor that substantially contributes to HIV integration site selection. They are potent antivirals that inhibit HIV integration and maturation. In addition, they retarget residual integrants away from transcription units and towards a more repressive chromatin environment. As a result, treatment with the LEDGIN CX14442 yielded residual provirus that proved more latent and more refractory to reactivation, supporting the use of LEDGINs as research tools to study HIV latency and a functional cure strategy. In this study we compared GS-9822, a potent, pre-clinical lead compound, with CX14442 with respect to antiviral potency, integration site selection, latency and reactivation. GS-9822 was more potent than CX14442 in most assays. For the first time, the combined effects on viral replication, integrase-LEDGF/p75 interaction, integration sites, epigenetic landscape, immediate latency and latency reversal was demonstrated at nanomolar concentrations achievable in the clinic. GS-9822 profiles as a preclinical candidate for future functional cure research.

The chromatin landscape at the HIV-1 provirus integration site determines viral expression.

HIV-1 persists lifelong in memory cells of the immune system as latent provirus that rebounds upon treatment interruption. Therefore, the latent reservoir is the main target for an HIV cure. Here, we studied the direct link between integration site and transcription using LEDGINs and Barcoded HIV-ensembles (B-HIVE). LEDGINs are antivirals that inhibit the interaction between HIV-1 integrase and the chromatin-tethering factor LEDGF/p75. They were used as a tool to retarget integration, while the effect on HIV expression was measured with B-HIVE. B-HIVE tracks insert-specific HIV expression by tagging a unique barcode in the HIV genome. We confirmed that LEDGINs retarget integration out of gene-dense and actively transcribed regions. The distance to H3K36me3, the marker recognized by LEDGF/p75, clearly increased. LEDGIN treatment reduced viral RNA expression and increased the proportion of silent provirus. Finally, silent proviruses obtained after LEDGIN treatment were located further away from epigenetic marks associated with active transcription. Interestingly, proximity to enhancers stimulated transcription irrespective of LEDGIN treatment, while the distance to H3K36me3 only changed after treatment with LEDGINs. The fact that proximity to these markers are associated with RNA expression support the direct link between provirus integration site and viral expression.

Block-And-Lock Strategies to Cure HIV Infection.

Today HIV infection cannot be cured due to the presence of a reservoir of latently infected cells inducing a viral rebound upon treatment interruption. Hence, the latent reservoir is considered as the major barrier for an HIV cure. So far, efforts to completely eradicate the reservoir via a shock-and-kill approach have proven difficult and unsuccessful. Therefore, more research has been done recently on an alternative block-and-lock functional cure strategy. In contrast to the shock-and-kill strategy that aims to eradicate the entire reservoir, block-and-lock aims to permanently silence all proviruses, even after treatment interruption. HIV silencing can be achieved by targeting different factors of the transcription machinery. In this review, we first describe the underlying mechanisms of HIV transcription and silencing. Next, we give an overview of the different block-and-lock strategies under investigation.

Impact of LEDGIN treatment during virus production on residual HIV-1 transcription.

BACKGROUND: Persistence of latent, replication-competent provirus is the main impediment towards the cure of HIV infection. One of the critical questions concerning HIV latency is the role of integration site selection in HIV expression. Inhibition of the interaction between HIV integrase and its chromatin tethering cofactor LEDGF/p75 is known to reduce integration and to retarget residual provirus to regions resistant to reactivation. LEDGINs, small molecule inhibitors of the interaction between HIV integrase and LEDGF/p75, provide an interesting tool to study the underlying mechanisms. During early infection, LEDGINs block the interaction with LEDGF/p75 and allosterically inhibit the catalytic activity of IN (i.e. the early effect). When present during virus production, LEDGINs interfere with proper maturation due to enhanced IN oligomerization in the progeny virions (i.e. the late effect). RESULTS: We studied the effect of LEDGINs present during virus production on the transcriptional state of the residual virus. Infection of cells with viruses produced in the presence of LEDGINs resulted in a residual reservoir that was refractory to activation. Integration of residual provirus was less favored near epigenetic markers associated with active transcription. However, integration near H3K36me3 and active genes, both targeted by LEDGF/p75, was not affected. Also in primary cells, LEDGIN treatment induced a reservoir resistant to activation due to a combined early and late effect. CONCLUSION: LEDGINs present a research tool to study the link between integration and transcription, an essential question in retrovirology. LEDGIN treatment during virus production altered integration of residual provirus in a LEDGF/p75-independent manner, resulting in a reservoir that is refractory to activation.

Insight in HIV Integration Site Selection Provides a Block-and-Lock Strategy for a Functional Cure of HIV Infection.

Despite significant improvements in therapy, the HIV/AIDS pandemic remains an important threat to public health. Current treatments fail to eradicate HIV as proviral DNA persists in long-living cellular reservoirs, leading to viral rebound whenever treatment is discontinued. Hence, a better understanding of viral reservoir establishment and maintenance is required to develop novel strategies to destroy latently infected cells, and/or to durably silence the latent provirus in infected cells. Whereas the mechanism of integration has been well studied from a catalytic point of view, it remains unknown how integration site selection and transcription are linked. In recent years, evidence has grown that lens epithelium-derived growth factor p75 (LEDGF/p75) is the main determinant of HIV integration site selection and that the integration site affects the transcriptional state of the provirus. LEDGINs have been developed as small molecule inhibitors of the interaction between LEDGF/p75 and integrase. Recently, it was shown that LEDGIN treatment in cell culture shifts the residual integrated provirus towards the inner nuclear compartment and out of transcription units in a dose dependent manner. This LEDGIN-mediated retargeting increased the proportion of provirus with a transcriptionally silent phenotype and the residual reservoir proved refractory to reactivation in vitro. LEDGINs provide us with a research tool to study the link between integration and transcription, a quintessential question in retrovirology. LEDGIN-mediated retargeting of the residual reservoirs provides a novel potential "block-and-lock" strategy as a functional cure of HIV infection.

LEDGIN-mediated Inhibition of Integrase-LEDGF/p75 Interaction Reduces Reactivation of Residual Latent HIV.

Persistence of latent, replication-competent Human Immunodeficiency Virus type 1 (HIV-1) provirus is the main impediment towards a cure for HIV/AIDS (Acquired Immune Deficiency Syndrome). Therefore, different therapeutic strategies to eliminate the viral reservoirs are currently being explored. We here propose a novel strategy to reduce the replicating HIV reservoir during primary HIV infection by means of drug-induced retargeting of HIV integration. A novel class of integration inhibitors, referred to as LEDGINs, inhibit the interaction between HIV integrase and the LEDGF/p75 host cofactor, the main determinant of lentiviral integration site selection. We show for the first time that LEDGF/p75 depletion hampers HIV-1 reactivation in cell culture. Next we demonstrate that LEDGINs relocate and retarget HIV integration resulting in a HIV reservoir that is refractory to reactivation by different latency-reversing agents. Taken together, these results support the potential of integrase inhibitors that modulate integration site targeting to reduce the likeliness of viral rebound.