Integration Site Selection Research Articles

Loops are geometric catalysts for DNA integration.

The insertion of DNA elements within genomes underpins both genetic diversity and disease when unregulated. Most of DNA insertions are not random and the physical mechanisms underlying the integration site selection are poorly understood. Here, we perform Molecular Dynamics simulations to study the insertion of DNA elements, such as viral DNA or transposons, into naked DNA or chromatin substrates. More specifically, we explore the role of loops within the polymeric substrate and discover that they act as 'geometric catalysts' for DNA integration by reducing the energy barrier for substrate deformation. Additionally, we discover that the 1D pattern and 3D conformation of loops have a marked effect on the distribution of integration sites. Finally, we show that loops may compete with nucleosomes to attract DNA integrations. These results may be tested in vitro and they may help to understand patterns of DNA insertions with implications in genome evolution and engineering.

Characterization of Nuclear HIV-Induced Membraneless Organelles Through Fluorescence Microscopy.

The postnuclear entry steps of HIV-1 involve reverse transcription, uncoating, and integration into the host genome. The differential regulation of these steps has a significant impact on HIV overall replication, including integration site selection and viral gene expression. Recently, another important phenomenon has been uncovered as part of HIV interplay with the nuclear environment, specifically involving the cleavage and polyadenylation specific factor 6 (CPSF6) protein. This phenomenon is the formation of nuclear HIV-induced membraneless organelles (HIV-1 MLOs). In this article, we will describe the methods used to assess the composition and liquid-liquid phase separation (LLPS) properties of these organelles using fluorescence microscopy. The study of HIV-1 MLOs represents a new frontier that may reveal previously unknown key players in the fate of HIV-infected cells.

Unraveling the palindromic and nonpalindromic motifs of retroviral integration site sequences by statistical mixture models.

A weak palindromic nucleotide motif is the hallmark of retroviral integration site alignments. Given that the majority of target sequences are not palindromic, the current model explains the symmetry by an overlap of the nonpalindromic motif present on one of the half-sites of the sequences. Here, we show that the implementation of multicomponent mixture models allows for different interpretations consistent with the existence of both palindromic and nonpalindromic submotifs in the sets of integration site sequences. We further show that the weak palindromic motifs result from freely combined site-specific submotifs restricted to only a few positions proximal to the site of integration. The submotifs are formed by either palindrome-forming nucleotide preference or nucleotide exclusion. Using the mixture models, we also identify HIV-1-favored palindromic sequences in Alu repeats serving as local hotspots for integration. The application of the novel statistical approach provides deeper insight into the selection of retroviral integration sites and may prove to be a valuable tool in the analysis of any type of DNA motifs.

Review and future perspectives on the integration characteristics for equine lentivirus in the host genome.

Despite over 40 years of research on the human immunodeficiency virus type 1 (HIV-1) vaccine, we still lack a considerable progress. Equine infectious anemia virus (EIAV) is a lentivirus in the Retroviridae family, akin to HIV-1 in genome structure and antigenicity. EIA is an important infectious disease in equids, characterized by anemia, persistent infection, and repeated fevers. The EIAV attenuated vaccine in China is the only lentiviral vaccine used on a large scale. Elucidating the mechanism of waning and induction of protective immunity from this attenuated vaccine strain will provide a critical theoretical basis and reference point for vaccine research, particularly in the development of lentivirus vaccines, with far-reaching scientific value and social significance. In this paper, we summarize the information related to EIAV integration site selection, particularly for the Chinese EIAV attenuated vaccine strains on the equine genome. This may improve our mechanistic understanding of EIAV virulence reduction at the host genome level. The obtained data may help elucidate the biological characteristics of EIAV, particularly the Chinese attenuated EIAV vaccine strain, and provide valuable information regarding retroviral infections, particularly lentiviral infection and associated therapeutic vectors.

Antiretroviral APOBEC3 cytidine deaminases alter HIV-1 provirus integration site profiles

APOBEC3 (A3) proteins are host-encoded deoxycytidine deaminases that provide an innate immune barrier to retroviral infection, notably against HIV-1. Low levels of deamination are believed to contribute to the genetic evolution of HIV-1, while intense catalytic activity of these proteins can induce catastrophic hypermutation in proviral DNA leading to near-total HIV-1 restriction. So far, little is known about how A3 cytosine deaminases might impact HIV-1 proviral DNA integration sites in human chromosomal DNA. Using a deep sequencing approach, we analyze the influence of catalytic active and inactive APOBEC3F and APOBEC3G on HIV-1 integration site selections. Here we show that DNA editing is detected at the extremities of the long terminal repeat regions of the virus. Both catalytic active and non-catalytic A3 mutants decrease insertions into gene coding sequences and increase integration sites into SINE elements, oncogenes and transcription-silencing non-B DNA features. Our data implicates A3 as a host factor influencing HIV-1 integration site selection and also promotes what appears to be a more latent expression profile.

Determinants of Retroviral Integration and Implications for Gene Therapeutic MLV-Based Vectors and for a Cure for HIV-1 Infection.

To complete their replication cycle, retroviruses need to integrate a DNA copy of their RNA genome into a host chromosome. Integration site selection is not random and is driven by multiple viral and cellular host factors specific to different classes of retroviruses. Today, overwhelming evidence from cell culture, animal experiments and clinical data suggests that integration sites are important for retroviral replication, oncogenesis and/or latency. In this review, we will summarize the increasing knowledge of the mechanisms underlying the integration site selection of the gammaretrovirus MLV and the lentivirus HIV-1. We will discuss how host factors of the integration site selection of retroviruses may steer the development of safer viral vectors for gene therapy. Next, we will discuss how altering the integration site preference of HIV-1 using small molecules could lead to a cure for HIV-1 infection.

G-Quadruplex DNA and Other Non-Canonical B-Form DNA Motifs Influence Productive and Latent HIV-1 Integration and Reactivation Potential.

The integration of the HIV-1 genome into the host genome is an essential step in the life cycle of the virus and it plays a critical role in the expression, long-term persistence, and reactivation of HIV expression. To better understand the local genomic environment surrounding HIV-1 proviruses, we assessed the influence of non-canonical B-form DNA (non-B DNA) on the HIV-1 integration site selection. We showed that productively and latently infected cells exhibit different integration site biases towards non-B DNA motifs. We identified a correlation between the integration sites of the latent proviruses and non-B DNA features known to potently influence gene expression (e.g., cruciform, guanine-quadruplex (G4), triplex, and Z-DNA). The reactivation potential of latent proviruses with latency reversal agents also correlated with their proximity to specific non-B DNA motifs. The perturbation of G4 structures in vitro using G4 structure-destabilizing or -stabilizing ligands resulted in a significant reduction in integration within 100 base pairs of G4 motifs. The stabilization of G4 structures increased the integration within 300-500 base pairs from G4 motifs, increased integration near transcription start sites, and increased the proportion of latently infected cells. Moreover, we showed that host lens epithelium-derived growth factor (LEDGF)/p75 and cleavage and polyadenylation specificity factor 6 (CPSF6) influenced the distribution of integration sites near several non-B DNA motifs, especially G4 DNA. Our findings identify non-B DNA motifs as important factors that influence productive and latent HIV-1 integration and the reactivation potential of latent proviruses.

BET-Independent Murine Leukemia Virus Integration Is Retargeted In Vivo and Selects Distinct Genomic Elements for Lymphomagenesis.

ABSTRACTMoloney murine leukemia virus (MLV) infects BALB/c mice and induces T-cell lymphoma in mice. Retroviral integration is mediated by the interaction of the MLV integrase (IN) with members of the bromodomain and extraterminal motif (BET) protein family (BRD2, BRD3, and BRD4). The introduction of the W390A mutation into MLV IN abolishes the BET interaction. Here, we compared the replication of W390A MLV to that of wild-type (WT) MLV in adult BALB/c mice to study the role of BET proteins in replication, integration, and tumorigenesis in vivo. Comparing WT and W390A MLV infections revealed similar viral loads in the blood, thymus, and spleen cells. Interestingly, W390A MLV integration was retargeted away from GC-enriched genomic regions. However, both WT MLV- and W390A MLV-infected mice developed T-cell lymphoma after similar latencies represented by an enlarged thymus and spleen and multiorgan tumor infiltration. Integration site sequencing from splenic tumor cells revealed clonal expansion in all WT MLV- and W390A MLV-infected mice. However, the integration profiles of W390A MLV and WT MLV differed significantly. Integrations were enriched in enhancers and promoters, but compared to the WT, W390A MLV integrated less frequently into enhancers and more frequently into oncogene bodies such as Notch1 and Ppp1r16b. We conclude that host factors direct MLV in vivo integration site selection. Although BET proteins target WT MLV integration preferentially toward enhancers and promoters, insertional lymphomagenesis can occur independently from BET, likely due to the intrinsically strong enhancer/promoter of the MLV long terminal repeat (LTR).IMPORTANCE In this study, we have shown that the in vivo replication of murine leukemia virus happens independently of BET proteins, which are key host determinants involved in retroviral integration site selection. This finding opens a new research line in the discovery of alternative viral or host factors that may complement the dominant host factor. In addition, our results show that BET-independent murine leukemia virus uncouples insertional mutagenesis from gene enhancers, although lymphomagenesis still occurs despite the lack of an interaction with BET proteins. Our findings also have implications for the engineering of BET-independent MLV-based vectors for gene therapy, which may not be a safe alternative.

QRIS: a machine learning framework to investigate the determinants of retroviral integration specificity

<p indent="0mm">Retroviral activity is linked with various diseases. Retrovirus can integrate a copy of its genomic DNA into the host genome and store it as the provirus. Thus, it threatens the host cell by interrupting the host genome architecture and transcribing its provirus for detrimental expansion. However, depending on where the retrovirus integrates, the host has corresponding defense mechanisms to repress the transcription of the provirus and eliminate harm. Therefore, the integration site selection is a vital process for retrovirus’ fate. Many efforts have been made to understand its integration specificity and find diverse DNA motif preferences across retrovirus genera. However, the effect of genome-wide DNA structural properties, such as DNA shapes, on retroviral integration is unclear. Therefore, we systematically investigated this issue on six retroviruses that are representative of four genera. We devised QRIS (quantify the retrovirus integration specificity), a machine learning framework to assess the effect of DNA shape on large-scale retroviral integration sites. We observed that the DNA shape could independently or cooperatively work with the DNA motif to regulate retroviral integration. Based on this, we classified these retroviruses into three categories: StrongFavor retrovirus, WeakFavor retrovirus, and Strongshape retrovirus. Interestingly, the Strongshape retrovirus can gain specificity through DNA shapes without a particular DNA motif. Our qualitative and quantitative evaluation of DNA shape and motif revealed their diverse roles in regulating the retroviral integration specificity. Our findings may help control the lentivirus vector more precisely for gene therapy and disturb retroviral integration during pathogenesis.

The local integration preference of the Tf1 retrotransposon in Schizosaccharomyces pombe

Transposons are mobile DNAs that can move to different locations in host genomes. The integration site selection of transposons is critical for both themselves and host cells. Studies on the integration of retrotransposons and retroviruses have focused more on the global preference than on the local preference. The local preferences of retrotransposons are usually weak and of large diversity. Here, we analyzed hundreds of thousands of independent integration events of the Tf1 retrotransposon in Schizosaccharomyces pombe. The consensus sequence at the Tf1 integration sites shows a palindromic pattern, which can be divided into four sections, each of them contains one or more CGnTA units with a period of 10 base pairs, indicating interaction with subunits of the integrase oligomer in the pre-integration complex. Moreover, the analysis on the nucleosome occupancy flanking Tf1 target sites shows that Tf1 integration favors regions with one entire nucleosome depletion.

HIV-1 integration sites in CD4+ T cells during primary, chronic, and late presentation of HIV-1 infection.

HIV-1 is capable of integrating its genome into that of its host cell. We examined the influence of the activation state of CD4+ T cells, the effect of antiretroviral therapy (ART), and the clinical stage of HIV-1 infection on HIV-1 integration site features and selection. HIV-1 integration sites were sequenced from longitudinally sampled resting and activated CD4+ T cells from 12 HIV-1–infected individuals. In total, 589 unique HIV-1 integration sites were analyzed: 147, 391, and 51 during primary, chronic, and late presentation of HIV-1 infection, respectively. As early as during primary HIV-1 infection and independent of the activation state of CD4+ T cells collected on and off ART, HIV-1 integration sites were preferentially detected in recurrent integration genes, genes associated with clonal expansion of latently HIV-1–infected CD4+ T cells, cancer-related genes, and highly expressed genes. The preference for cancer-related genes was more pronounced at late stages of HIV-1 infection. Host genomic features of HIV-1 integration site selection remained stable during HIV-1 infection in both resting and activated CD4+ T cells. In summary, characteristic HIV-1 integration site features are preestablished as early as during primary HIV-1 infection and are found in both resting and activated CD4+ T cells.

Towards a Functional Cure of HIV-1: Insight Into the Chromatin Landscape of the Provirus.

Despite potent combination antiretroviral therapy, HIV-1 infection persists due to irreversible integration of the virus in long-living cells of the immune system. The main focus of HIV-1 cure strategies has been on HIV-1 eradication, yet without great success so far. Therefore, HIV-1 remission or a functional cure, whereby the virus is silenced rather than eradicated, is considered as an alternative strategy. Elite controllers, individuals who spontaneously control HIV-1, may point us the way toward a functional HIV-1 cure. In order to achieve such a cure, a profound understanding of the mechanisms controlling HIV-1 expression and silencing is needed. In recent years, evidence has grown that the site of integration as well as the chromatin landscape surrounding the integration site affects the transcriptional state of the provirus. Still, at present, the impact of integration site selection on the establishment and maintenance of the HIV-1 reservoirs remains poorly understood. The discovery of LEDGF/p75 as a binding partner of HIV-1 integrase has led to a better understanding of integration site selection. LEDGF/p75 is one of the important determinants of integration site selection and targets integration toward active genes. In this review, we will provide an overview of the most important determinants of integration site selection. Secondly, we will discuss the chromatin landscape at the integration site and its implications on HIV-1 gene expression and silencing. Finally, we will discuss how interventions that affect integration site selection or modifications of the chromatin could yield a functional cure of HIV-1 infection.

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.

Epigenomic features revealed by ATAC‐seq impact transgene expression in CHO cells

Different regions of a mammalian genome have different accessibilities to transcriptional machinery. The integration site of a transgene affects how actively it is transcribed. Highly accessible genomic regions called super-enhancers have beenrecently described as strong regulatory elements that shape cell identity. Super-enhancers have beenidentified in Chinese hamster ovary (CHO) cells using the Assay for Transposase-Accessible Chromatin Sequencing (ATAC-seq). Genes near super-enhancer regions had high transcript levels and were enriched for oncogenic signaling and proliferation functions, consistent with an immortalized phenotype. Inaccessible regions in the genome with low ATAC signal also had low transcriptional activity. Genes in inaccessible regions were enriched for remote tissue functions such as taste, smell, and neuronal activation. A lentiviral reporter integration assay showed integration into super-enhancer regions conferred higher reporter expression than insertion into inaccessible regions. Targeted integration of an IgG vector into the Plec super-enhancer region yielded clones that expressed the immunoglobulin light chain gene mostly in the top 20% of all transcripts with the majority in the top 5%. The results suggest the epigenomic landscape of CHO cells can guide the selection of integration sites in the development of cell lines for therapeutic protein production.

Light and shadow on the mechanisms of integration site selection in yeast Ty retrotransposon families.

Transposable elements are ubiquitous in genomes. Their successful expansion depends in part on their sites of integration in their host genome. In Saccharomyces cerevisiae, evolution has selected various strategies to target the five Ty LTR-retrotransposon families into gene-poor regions in a genome, where coding sequences occupy 70% of the DNA. The integration of Ty1/Ty2/Ty4 and Ty3 occurs upstream and at the transcription start site of the genes transcribed by RNA polymerase III, respectively. Ty5 has completely different integration site preferences, targeting heterochromatin regions. Here, we review the history that led to the identification of the cellular tethering factors that play a major role in anchoring Ty retrotransposons to their preferred sites. We also question the involvement of additional factors in the fine-tuning of the integration site selection, with several studies converging towards an importance of the structure and organization of the chromatin.

Retroviral prototype foamy virus intasome binding to a nucleosome target does not determine integration efficiency

Retroviral integrases must navigate host DNA packaged as chromatin during integration of the viral genome. Prototype foamy virus (PFV) integrase (IN) forms a tetramer bound to two viral DNA (vDNA) ends in a complex termed an intasome. PFV IN consists of four domains: the amino terminal extension domain (NED), amino terminal domain (NTD), catalytic core domain (CCD), and carboxyl terminal domain (CTD). The domains of the two inner IN protomers have been visualized, as well as the CCDs of the two outer IN protomers. However, the roles of the amino and carboxyl terminal domains of the PFV intasome outer subunits during integration to a nucleosome target substrate are not clear. We used the well-characterized 601 nucleosome to assay integration activity as well as intasome binding. PFV intasome integration to 601 nucleosomes occurs in clusters at four independent sites. We find that the outer protomer NED and NTD domains have no significant effects on integration efficiency, site selection, or binding. The CTDs of the outer PFV intasome subunits dramatically affect nucleosome binding but have little effect on total integration efficiency. The outer PFV IN CTDs did significantly alter the integration efficiency at one site. Histone tails also significantly affect intasome binding, but have little impact on PFV integration efficiency or site selection. These results indicate that binding to nucleosomes does not correlate with integration efficiency and suggests most intasome-binding events are unproductive.

LEDGINs, Inhibitors of the Interaction Between HIV-1 Integrase and LEDGF/p75, Are Potent Antivirals with a Potential to Cure HIV Infection.

A permanent cure remains the greatest challenge in the field of HIV research. In order to reach this goal, a profound understanding of the molecular mechanisms controlling HIV integration and transcription is needed. Here we provide an overview of recent advances in the field. Lens epithelium-derived growth factor p75 (LEDGF/p75), a transcriptional coactivator, tethers and targets the HIV integrase into transcriptionally active regions of the chromatin through an interaction with the epigenetic mark H3K36me2/3. This finding prompted us to propose a "block-and-lock" strategy to retarget HIV integration into deep latency. A decade ago, we pioneered protein-protein interaction inhibitors for HIV and discovered LEDGINs. LEDGINs are small molecule inhibitors of the interaction between the integrase binding domain (IBD) of LEDGF/p75 and HIV integrase. They modify integration site selection and therefore might be molecules with a "block-and-lock" mechanism of action. Here we will describe how LEDGINs may become part in the future functional cure strategies.

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.

PP 1.14 - HIV integration site selection in the 3D genome: impact on viral and host gene expression

PP 1.14 - HIV integration site selection in the 3D genome: impact on viral and host gene expression

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

BackgroundPersistence 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).ResultsWe 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.ConclusionLEDGINs 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.