Brief Histories of Retroviral Integration Research and Associated International Conferences.

The field of retroviral integration research has a long history that started with the provirus hypothesis and subsequent discoveries of the retroviral reverse transcriptase and integrase enzymes. Because both enzymes are essential for retroviral replication, they became valued targets in the effort to discover effective compounds to inhibit HIV-1 replication. In 2007, the first integrase strand transfer inhibitor was licensed for clinical use, and subsequently approved second-generation integrase inhibitors are now commonly co-formulated with reverse transcriptase inhibitors to treat people living with HIV. International meetings specifically focused on integrase and retroviral integration research first convened in 1995, and this paper is part of the Viruses Special Issue on the 7th International Conference on Retroviral Integration, which was held in Boulder Colorado in the summer of 2023. Herein, we overview key historical developments in the field, especially as they pertain to the development of the strand transfer inhibitor drug class. Starting from the mid-1990s, research advancements are presented through the lens of the international conferences. Our overview highlights the impact that regularly scheduled, subject-specific international meetings can have on community-building and, as a result, on field-specific collaborations and scientific advancements.

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

Moloney 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.

Spatial and Genomic Correlates of HIV-1 Integration Site Targeting.

HIV-1 integrase and capsid proteins interact with host proteins to direct preintegration complexes to active transcription units within gene-dense regions of chromosomes for viral DNA integration. Analyses of spatially-derived genomic DNA coordinates, such as nuclear speckle-associated domains, lamina-associated domains, super enhancers, and Spatial Position Inference of the Nuclear (SPIN) genome states, have further informed the mechanisms of HIV-1 integration targeting. Critically, however, these different types of genomic coordinates have not been systematically analyzed to synthesize a concise description of the regions of chromatin that HIV-1 prefers for integration. To address this informational gap, we have extensively correlated genomic DNA coordinates of HIV-1 integration targeting preferences. We demonstrate that nuclear speckle-associated and speckle-proximal chromatin are highly predictive markers of integration and that these regions account for known HIV biases for gene-dense regions, highly transcribed genes, as well as the mid-regions of gene bodies. In contrast to a prior report that intronless genes were poorly targeted for integration, we find that intronless genes in proximity to nuclear speckles are more highly targeted than are spatially-matched intron-containing genes. Our results additionally highlight the contributions of capsid and integrase interactions with respective CPSF6 and LEDGF/p75 host factors in these HIV-1 integration targeting preferences.

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.

Designing Lentiviral Vectors for Gene Therapy of Genetic Diseases.

Lentiviral vectors are the most frequently used tool to stably transfer and express genes in the context of gene therapy for monogenic diseases. The vast majority of clinical applications involves an ex vivo modality whereby lentiviral vectors are used to transduce autologous somatic cells, obtained from patients and re-delivered to patients after transduction. Examples are hematopoietic stem cells used in gene therapy for hematological or neurometabolic diseases or T cells for immunotherapy of cancer. We review the design and use of lentiviral vectors in gene therapy of monogenic diseases, with a focus on controlling gene expression by transcriptional or post-transcriptional mechanisms in the context of vectors that have already entered a clinical development phase.

Allosteric HIV Integrase Inhibitors Promote Formation of Inactive Branched Polymers via Homomeric Carboxy-Terminal Domain Interactions.

The major effect of allosteric HIV integrase (IN) inhibitors (ALLINIs) is observed during virion maturation, where ALLINI treatment interrupts IN-RNA interactions via drug-induced IN aggregation, leading to the formation of aberrant virions. To understand the structural changes that accompany drug-induced aggregation, we determined the soft matter properties of ALLINI-induced IN aggregates. Using small-angle neutron scattering, SEM, and rheology, we have discovered that the higher-order aggregates induced by ALLINIs have the characteristics of weak three-dimensional gels with a fractal-like character. Their formation is inhibited by the host factor LEDGF/p75, as well as ex vivo resistance substitutions. Mutagenesis and biophysical analyses reveal that homomeric carboxy-terminal domain interactions are required to achieve the branched-polymer nature of the ALLINI-induced aggregates. These studies provide key insight into the mechanisms of ALLINI action and resistance in the context of the crowded virion environment where ALLINIs exert their effect.

[Role of Cellular DNA Repair Systems in HIV-1 Replication].

A serious problem in the treatment of HIV infection is the emergence of drug-resistant forms of the virus. One promising approach to solving this problem is the development of inhibitors of the interaction between viral proteins with cellular co-factors. However, the development of this approach is hampered due to the lack of knowledge about the involvement of cellular proteins in the pathogenesis of HIV infection. In particular, it is known that the integration of viral DNA into the host genome generates numerous lesions in the cellular DNA, the repair of which is absolutely necessary for successful replication of the virus. However, it is still unknown which cellular proteins are involved in repairing this damage. In this review, we summarize what is known to date about the role of cellular repair systems in the replication of HIV-1 in general, and in the repair of damage that occurs during the integration of viral DNA into a cell's genome, in particular.

Modulation of the functional association between the HIV-1 intasome and the nucleosome by histone amino-terminal tails.

BACKGROUND: Stable insertion of the retroviral DNA genome into host chromatin requires the functional association between the intasome (integrase·viral DNA complex) and the nucleosome. The data from the literature suggest that direct protein-protein contacts between integrase and histones may be involved in anchoring the intasome to the nucleosome. Since histone tails are candidates for interactions with the incoming intasomes we have investigated whether they could participate in modulating the nucleosomal integration process. RESULTS: We show here that histone tails are required for an optimal association between HIV-1 integrase (IN) and the nucleosome for efficient integration. We also demonstrate direct interactions between IN and the amino-terminal tail of human histone H4 in vitro. Structure/function studies enabled us to identify amino acids in the carboxy-terminal domain of IN that are important for this interaction. Analysis of the nucleosome-binding properties of catalytically active mutated INs confirmed that their ability to engage the nucleosome for integration in vitro was affected. Pseudovirus particles bearing mutations that affect the IN/H4 association also showed impaired replication capacity due to altered integration and re-targeting of their insertion sites toward dynamic regions of the chromatin with lower nucleosome occupancy. CONCLUSIONS: Collectively, our data support a functional association between HIV-1 IN and histone tails that promotes anchoring of the intasome to nucleosomes and optimal integration into chromatin.

Modulation of chromatin structure by the FACT histone chaperone complex regulates HIV-1 integration.

BACKGROUND: Insertion of retroviral genome DNA occurs in the chromatin of the host cell. This step is modulated by chromatin structure as nucleosomes compaction was shown to prevent HIV-1 integration and chromatin remodeling has been reported to affect integration efficiency. LEDGF/p75-mediated targeting of the integration complex toward RNA polymerase II (polII) transcribed regions ensures optimal access to dynamic regions that are suitable for integration. Consequently, we have investigated the involvement of polII-associated factors in the regulation of HIV-1 integration. RESULTS: Using a pull down approach coupled with mass spectrometry, we have selected the FACT (FAcilitates Chromatin Transcription) complex as a new potential cofactor of HIV-1 integration. FACT is a histone chaperone complex associated with the polII transcription machinery and recently shown to bind LEDGF/p75. We report here that a tripartite complex can be formed between HIV-1 integrase, LEDGF/p75 and FACT in vitro and in cells. Biochemical analyzes show that FACT-dependent nucleosome disassembly promotes HIV-1 integration into chromatinized templates, and generates highly favored nucleosomal structures in vitro. This effect was found to be amplified by LEDGF/p75. Promotion of this FACT-mediated chromatin remodeling in cells both increases chromatin accessibility and stimulates HIV-1 infectivity and integration. CONCLUSIONS: Altogether, our data indicate that FACT regulates HIV-1 integration by inducing local nucleosomes dissociation that modulates the functional association between the incoming intasome and the targeted nucleosome.

High-Throughput Mapping and Clonal Quantification of Retroviral Integration Sites.

We describe here a method to identify the position of retroviral insertion sites and simultaneously to quantify the absolute abundance of each clone, i.e., the number of cells having the provirus inserted at a given place in the host genome. The method is based on random shearing of the host cell DNA, followed by a linker-mediated PCR to amplify the genomic regions flanking the proviruses, and high-throughput sequencing of the amplicons. The quantification of the abundance of each infected clone allowed us to develop two new metrics: i. the oligoclonality index, which quantifies the nonuniformity of the distribution of clone abundance, and ii. an estimator of the total number of clones in the body of the host. These new tools are valuable for the study of retroviral infections and can also be adapted for the tracking of gene-edited cells.