DNA Breaks and Gaps Target Retroviral Integration

Integration into a host genome is essential for retrovirus infection and is catalyzed by a nucleoprotein complex (Intasome) containing the virus-encoded integrase (IN) and the reverse transcribed (RT) virus copy DNA (cDNA). Previous studies suggested that integration was limited by intasome-host DNA recognition progressions. Using single molecule Forster resonance energy transfer (smFRET) we show that PFV intasomes pause at nicked and gapped DNA, which targeted site-directed integration without inducing significant intasome conformational alterations. Base excision repair (BER) components that affect retroviral integration in vivo produce similar nick/gap intermediates during DNA lesion processing. Intasome pause dynamics was modified by the 5′-nick-gap chemistry, while an 8-oxo-guanine lesion, a mismatch, or a nucleotide insertion that induce backbone flexibility and/or static bends had no effect. These results suggest that dynamic often non-productive intasome-DNA interactions may be modulated to target retroviral integration.

Targeting the Integrated Stress Response Kinase GCN2 to Modulate Retroviral Integration

Multiple viral targets are now available in the clinic to fight HIV infection. Even if this targeted therapy is highly effective at suppressing viral replication, caregivers are facing growing therapeutic failures in patients due to resistance, with or without treatment-adherence glitches. Accordingly, it is important to better understand how HIV and other retroviruses replicate in order to propose alternative antiviral strategies. Recent studies have shown that multiple cellular factors are implicated during the integration step and, more specifically, that integrase can be regulated through post-translational modifications. We have shown that integrase is phosphorylated by GCN2, a cellular protein kinase of the integrated stress response, leading to a restriction of HIV replication. In addition, we found that this mechanism is conserved among other retroviruses. Accordingly, we developed an in vitro interaction assay, based on the AlphaLISA technology, to monitor the integrase-GCN2 interaction. From an initial library of 133 FDA-approved molecules, we identified nine compounds that either inhibited or stimulated the interaction between GCN2 and HIV integrase. In vitro characterization of these nine hits validated this pilot screen and demonstrated that the GCN2-integrase interaction could be a viable solution for targeting integrase out of its active site.

Investigating site-selection mechanisms of retroviral integration in supercoiled DNA braids

We theoretically study the integration of short viral DNA in a DNA braid made up by two entwined double-stranded DNA molecules. We show that the statistics of single integration events substantially differ in the straight and buckled, or plectonemic, phase of the braid and are more likely in the latter. We further discover that integration is most likely close to plectoneme tips, where the larger bending energy helps overcome the associated energy barrier and that successive integration events are spatio-temporally correlated, suggesting a potential mechanistic explanation of clustered integration sites in host genomes. The braid geometry we consider provides a novel experimental set-up to quantify integration in a supercoiled substrate in vitro , and to better understand the role of double-stranded DNA topology during this process.

Predicted Cellular Interactors of the Endogenous Retrovirus-K Integrase Enzyme

Integrase (IN) enzymes are found in all retroviruses and are crucial in the retroviral integration process. Many studies have revealed how exogenous IN enzymes, such as the human immunodeficiency virus (HIV) IN, contribute to altered cellular function. However, the same consideration has not been given to viral IN originating from symbionts within our own DNA. Endogenous retrovirus-K (ERVK) is pathologically associated with neurological and inflammatory diseases along with several cancers. The ERVK IN interactome is unknown, and the question of how conserved the ERVK IN protein–protein interaction motifs are as compared to other retroviral integrases is addressed in this paper. The ERVK IN protein sequence was analyzed using the Eukaryotic Linear Motif (ELM) database, and the results are compared to ELMs of other betaretroviral INs and similar eukaryotic INs. A list of putative ERVK IN cellular protein interactors was curated from the ELM list and submitted for STRING analysis to generate an ERVK IN interactome. KEGG analysis was used to identify key pathways potentially influenced by ERVK IN. It was determined that the ERVK IN potentially interacts with cellular proteins involved in the DNA damage response (DDR), cell cycle, immunity, inflammation, cell signaling, selective autophagy, and intracellular trafficking. The most prominent pathway identified was viral carcinogenesis, in addition to select cancers, neurological diseases, and diabetic complications. This potentiates the role of ERVK IN in these pathologies via protein–protein interactions facilitating alterations in key disease pathways.

Strategies for Targeting Retroviral Integration for Safer Gene Therapy: Advances and Challenges

Retroviruses are obligate intracellular parasites that must integrate a copy of the viral genome into the host DNA. The integration reaction is performed by the viral enzyme integrase in complex with the two ends of the viral cDNA genome and yields an integrated provirus. Retroviral vector particles are attractive gene therapy delivery tools due to their stable integration. However, some retroviral integration events may dysregulate host oncogenes leading to cancer in gene therapy patients. Multiple strategies to target retroviral integration, particularly to genetic safe harbors, have been tested with limited success. Attempts to target integration may be limited by the multimerization of integrase or the presence of host co-factors for integration. Several retroviral integration complexes have evolved a mechanism of tethering to chromatin via a host protein. Integration host co-factors bind chromatin, anchoring the complex and allowing integration. The tethering factor allows for both close proximity to the target DNA and specificity of targeting. Each retrovirus appears to have distinct preferences for DNA sequence and chromatin features at the integration site. Tethering factors determine the preference for chromatin features, but do not affect the subtle sequence preference at the integration site. The sequence preference is likely intrinsic to the integrase protein. New developments may uncouple the requirement for a tethering factor and increase the ability to redirect retroviral integration.

Analysis of the intrinsic chromatin binding property of HIV-1 integrase and its regulation by LEDGF/p75 using human chromosomes spreads

ABSTRACTRetroviral integration requires the stable insertion of the viral genome into the host chromosomes. During this process, the functional integration complex must associate with cellular chromatin via the interaction between retroviral integrase and nucleosomes. The final association between the HIV-1 integration complex and the nucleosomal target DNA remains unclear and may involve the chromatin-binding properties of both the retroviral integrase and its cellular cofactor LEDGF/p75. To date, there is no experimental system allowing the direct monitoring of this protein association with chromatin to depict the molecular mechanism of this process fully. To investigate this and understand the LEDGF/p75-mediated chromatin tethering of HIV-1 integrase further, we used both biochemical approaches and an unedited chromosome-binding assays. Our study revealed that retroviral IN has an intrinsic ability to bind and recognize specific chromatin regions even in the absence of its cofactor. We also showed that this integrase chromatin-binding property was modulated by the interaction with its cofactor LEDGF/p75, which redirected the enzyme to alternative chromatin regions. Using these approaches, we also better determined the chronology of efficient LEDGF/p75-mediated targeting of HIV-1 integrase to chromatin. In addition to supporting a chromatin-binding function of the integrase protein acting in concert with LEDGF/p75 for the optimal association with the nucleosomal substrate, our work precisely elucidates the mechanism of action of LEDGF/p75 in this crucial integration step.

Factors that mold the nuclear landscape of HIV-1 integration

The integration of retroviral reverse transcripts into the chromatin of the cells that they infect is required for virus replication. Retroviral integration has far-reaching consequences, from perpetuating deadly human diseases to molding metazoan evolution. The lentivirus human immunodeficiency virus 1 (HIV-1), which is the causative agent of the AIDS pandemic, efficiently infects interphase cells due to the active nuclear import of its preintegration complex (PIC). To enable integration, the PIC must navigate the densely-packed nuclear environment where the genome is organized into different chromatin states of varying accessibility in accordance with cellular needs. The HIV-1 capsid protein interacts with specific host factors to facilitate PIC nuclear import, while additional interactions of viral integrase, the enzyme responsible for viral DNA integration, with cellular nuclear proteins and nucleobases guide integration to specific chromosomal sites. HIV-1 integration favors transcriptionally active chromatin such as speckle-associated domains and disfavors heterochromatin including lamina-associated domains. In this review, we describe virus-host interactions that facilitate HIV-1 PIC nuclear import and integration site targeting, highlighting commonalities among factors that participate in both of these steps. We moreover discuss how the nuclear landscape influences HIV-1 integration site selection as well as the establishment of active versus latent virus infection.

CPSF6-Dependent Targeting of Speckle-Associated Domains Distinguishes Primate from Nonprimate Lentiviral Integration

ABSTRACT Lentiviral DNA integration favors transcriptionally active chromatin. We previously showed that the interaction of human immunodeficiency virus type 1 (HIV-1) capsid with cleavage and polyadenylation specificity factor 6 (CPSF6) localizes viral preintegration complexes (PICs) to nuclear speckles for integration into transcriptionally active speckle-associated domains (SPADs). In the absence of the capsid-CPSF6 interaction, PICs uncharacteristically accumulate at the nuclear periphery and target heterochromatic lamina-associated domains (LADs) for integration. The integrase-binding protein lens epithelium-derived growth factor (LEDGF)/p75 in contrast to CPSF6 predominantly functions to direct HIV-1 integration to interior regions of transcription units. Though CPSF6 and LEDGF/p75 can reportedly interact with the capsid and integrase proteins of both primate and nonprimate lentiviruses, the extents to which these different viruses target SPADs versus LADs, as well as their dependencies on CPSF6 and LEDGF/p75 for integration targeting, are largely unknown. Here, we mapped 5,489,157 primate and nonprimate lentiviral integration sites in HEK293T and Jurkat T cells as well as derivative cells that were knocked out or knocked down for host factor expression. Despite marked preferences of all lentiviruses to target genes for integration, nonprimate lentiviruses only marginally favored SPADs, with corresponding upticks in LAD-proximal integration. While LEDGF/p75 knockout disrupted the intragenic integration profiles of all lentiviruses similarly, CPSF6 depletion specifically counteracted SPAD integration targeting by primate lentiviruses. CPSF6 correspondingly failed to appreciably interact with nonprimate lentiviral capsids. We conclude that primate lentiviral capsid proteins evolved to interact with CPSF6 to optimize PIC localization for integration into transcriptionally active SPADs. IMPORTANCE Integration is the defining step of the retroviral life cycle and underlies the inability to cure HIV/AIDS through the use of intensified antiviral therapy. The reservoir of latent, replication-competent proviruses that forms early during HIV infection reseeds viremia when patients discontinue medication. HIV cure research is accordingly focused on the factors that guide provirus formation and associated chromatin environments that regulate transcriptional reactivation, and studies of orthologous infectious agents such as nonprimate lentiviruses can inform basic principles of HIV biology. HIV-1 utilizes the integrase-binding protein LEDGF/p75 and the capsid interactor CPSF6 to target speckle-associated domains (SPADs) for integration. However, the extent to which these two host proteins regulate integration of other lentiviruses is largely unknown. Here, we mapped millions of retroviral integration sites in cell lines that were depleted for LEDGF/p75 and/or CPSF6. Our results reveal that primate lentiviruses uniquely target SPADs for integration in a CPSF6-dependent manner.

CRYO-EM STRUCTURE OF THE DELTA-RETROVIRAL INTASOME IN COMPLEX WITH THE PP2A REGULATORY SUBUNIT B56γ

The Retroviridae delta-retrovirus genus includes the most oncogenic pathogen– human T-cell lymphotropic virus type 1 (HTLV-1)(1). Many of the ~20 million people infected with HTLV-1 will develop severe leukaemia (2) or an ALS-like motor disease (3) unless a therapy becomes available. A key step in the establishment of infection is the integration of viral genetic material into the host genome, catalysed by the viral integrase (IN) enzyme. Here we used X-ray crystallography and single-particle cryo-electron microscopy to determine the structure of functional delta-retroviral IN assembled on viral DNA ends and bound the B56γ subunit of its human host factor, the protein phosphatase 2A (4). The structure reveals a tetrameric IN assembly bound to the phosphatase via a conserved short linear motif found within the extended linker connecting the catalytic core (CCD) and C-terminal (CTD) IN domains. Unexpectedly, all four IN subunits are involved in B56γ binding, taking advantage of the flexibility of the CCD-CTD linkers. Our results fill the current gap in the structural understanding of the delta-retroviral integration machinery. Insight into the interactions between the delta-retroviral intasome and the host will be crucial for understanding the pattern of integration events in infected individuals and therefore bears important clinical implications.

Specialized DNA structures act as genomic beacons for integration by evolutionarily diverse retroviruses

ABSTRACTRetroviral integration site targeting is not random and plays a critical role in expression and long-term survival of the integrated provirus. To better understand the genomic environment surrounding retroviral integration sites, we performed an extensive comparative analysis of new and previously published integration site data from evolutionarily diverse retroviruses from seven genera, including different HIV-1 subtypes. We showed that evolutionarily divergent retroviruses exhibited distinct integration site profiles with strong preferences for non-canonical B-form DNA (non-B DNA). Whereas all lentiviruses and most retroviruses integrate within or near genes and non-B DNA, MMTV and ERV integration sites were highly enriched in heterochromatin and transcription-silencing non-B DNA features (e.g. G4, triplex and Z-DNA). Compared to in vitro-derived HIV-1 integration sites, in vivo-derived sites are significantly more enriched in transcriptionally silent regions of the genome and transcription-silencing non-B DNA features. Integration sites from individuals infected with HIV-1 subtype A, C or D viruses exhibited different preferences for non-B DNA and were more enriched in transcriptionally active regions of the genome compared to subtype B virus. In addition, we identified several integration site hotspots shared between different HIV-1 subtypes with specific non-B DNA sequence motifs present at these hotspots. Together, these data highlight important similarities and differences in retroviral integration site targeting and provides new insight into how retroviruses integrate into genomes for long-term survival.Graphical AbstractSchematic comparing integration site profiles from evolutionarily diverse retroviruses. Upper left, heatmaps showing the fold-enrichment (blue) and fold-depletion (red) of integration sites near non-B DNA features (lower left). Lower right, circa plot showing integration site hotspots shared between HIV-1 subtype A, B, C and D virus.