Gene therapy: too much splice can spoil the dish

The use of integrating vectors for gene therapy - required for stable correction of gene expression - carries the risk of insertional mutagenesis, which can lead to activation of a tumorigenic program. In this issue of the JCI, Moiani et al. and Cesana et al. investigate how viral vectors can induce aberrant splicing, resulting in chimeric cellular-viral transcripts. The finding that this is a general phenomenon is concerning, but some of their results do suggest approaches for the development of safeguards in gene therapy vector design

APOBEC3G-Depleted Resting CD4+ T Cells Remain Refractory to HIV1 Infection

Background: CD4+ T lymphocytes are the primary targets of HIV1 but cannot be infected when fully quiescent, due to a post-entry block preventing the completion of reverse transcription. Chiu et al. recently proposed that this restriction reflects the action of APOBEC3G (A3G). They further suggested that T cell activation abrogates the A3G-mediated block by directing this protein to a high molecular mass complex. Methodology/Principal Findings: In the present work, we sought to explore further this model. However, we found that effective suppression of A3G by combined RNA interference and expression of HIV1 Vif does not relieve the restrictive phenotype of post-activation resting T cells. We also failed to find a correlation between HIV resistance and the presence of A3G in a low molecular complex in primary T cells. Conclusions/Significance: We conclude that A3G is unlikely to play a role in the HIV restrictive phenotype of quiescent T lymphocytes

Lentiviral vectors and antiretroviral intrinsic immunity

Multicellular organisms have evolved under relentless attacks from pathogens, and as a consequence have spiked their genomes with numerous genes that serve to thwart these threats, notably through the building of the innate and adaptive arms of the immune system. The innate immune system is by far the most ancient, being found as widely as in plants and Drosophila, while adaptive immunity arose with the emergence of cartilaginous fishes. Innate immunity enters rapidly into the game during the course of an infection and generally involves the recognition by specific cellular receptors of common pathogen-associated patterns to elicit broad defensive responses, mediated in humans by interferons, macrophages, and natural killer cells, amongst others. When innate immunity fails to eradicate the infection quickly, adaptive immune responses enter into play, to generate exquisitely specific defenses to virtually any pathogen, thanks to a quasi-infinite repertoire of nonself receptors and effectors. A specific form of innate immunity, coined "intrinsic immunity," completes this protection by providing a constant, always-on, line of defense, generally through intracellular obstacles to the replication of pathogens. This component of the immune system has gained much attention as it was discovered that it is a cornerstone of the resistance of mammals against retroviruses. One of these newly discovered intracellular molecular weapons, the APOBEC family of proteins, is active against several classes of retroelements. We present here the current state of knowledge on this rapidly evolving field and discuss implications for gene therapy

The integrase interactor 1 (INI1) proteins facilitate Tat-mediated human immunodeficiency virus type 1 transcription

Integration of human immunodeficiency virus type 1 (HIV-1) into the host genome is catalyzed by the viral integrase (IN) and preferentially occurs within transcriptionally active genes. During the early phase of HIV-1 infection, the incoming viral preintegration complex (PIC) recruits the integrase interactor 1 (INI1)/hSNF5, a chromatin remodeling factor which directly binds to HIV-1 IN. The impact of this event on viral replication is so far unknown, although it has been hypothesized that it could tether the preintegration complex to transcriptionally active genes, thus contributing to the bias of HIV integration for these regions of the genome. Here, we demonstrate that while INI1 is dispensable for HIV-1 transduction, it can facilitate HIV-1 transcription by enhancing Tat function. INI1 bound to Tat and both the repeat (Rpt) 1 and Rpt 2 domains of INI1 were required for efficient activation of Tat-mediated transcription. These results suggest that the incoming PICs might recruit INI1 to facilitate proviral transcription

KRAB zinc finger proteins

Kruppel-associated box domain zinc finger proteins (KRAB-ZFPs) are the largest family of transcriptional regulators in higher vertebrates. Characterized by an N-terminal KRAB domain and a C-terminal array of DNA-binding zinc fingers, they participate, together with their co-factor KAP1 (also known as TRIM28), in repression of sequences derived from transposable elements (TEs). Until recently, KRAB-ZFP/KAP1-mediated repression of TEs was thought to lead to irreversible silencing, and the evolutionary selection of KRAB-ZFPs was considered to be just the host component of an arms race against TEs. However, recent advances indicate that KRAB-ZFPs and their TE targets also partner up to establish species-specific regulatory networks. Here, we provide an overview of the KRAB-ZFP gene family, highlighting how its evolutionary history is linked to that of TEs, and how KRAB-ZFPs influence multiple aspects of development and physiology

Characterization of APOBEC3G binding to 7SL RNA

Human APOBEC3 proteins are editing enzymes that can interfere with the replication of exogenous retroviruses such as human immunodeficiency virus (HIV), hepadnaviruses such as hepatitis B virus (HBV), and with the retrotransposition of endogenous retroelements such as long-interspersed nuclear elements (LINE) and Alu. Here, we show that APOBEC3G, but not other APOBEC3 family members, binds 7SL RNA, the common ancestor of Alu RNAs that is specifically recruited into HIV virions. Our data further indicate that APOBEC3G recognizes 7SL RNA and Alu RNA by its common structure, the Alu domain, suggesting a mechanism for APOBEC3G- mediated inhibition of Alu retrotransposition. However, we also demonstrate that APOBEC3F and APOBEC3G are normally recruited into and inhibit the infectivity of ΔVif HIV1 virions when 7SLRNA is prevented from accessing particles by RNA interference against SRP14 or by over expression of SRP19, both components of the signal recognition particle. We thus conclude that 7SL RNA is not an essential mediator of the virion packaging of these antiviral cytidine deaminases

Interfering Residues Narrow the Spectrum of MLV Restriction by Human TRIM5α

TRIM5α is a restriction factor that limits infection of human cells by so-called N- but not B- or NB-tropic strains of murine leukemia virus (MLV). Here, we performed a mutation-based functional analysis of TRIM5α-mediated MLV restriction. Our results reveal that changes at tyrosine336 of human TRIM5α, within the variable region 1 of its C-terminal PRYSPRY domain, can expand its activity to B-MLV and to the NB-tropic Moloney MLV. Conversely, we demonstrate that the escape of MLV from restriction by wild-type or mutant forms of huTRIM5α can be achieved through interdependent changes at positions 82, 109, 110, and 117 of the viral capsid. Together, our results support a model in which TRIM5α-mediated retroviral restriction results from the direct binding of the antiviral PRYSPRY domain to the viral capsid, and can be prevented by interferences exerted by critical residues on either one of these two partners

DUX-family transcription factors regulate zygotic genome activation in placental mammals

In animal embryos, transcription is mostly silent for several cell divisions, until the release of the first major wave of embryonic transcripts through so-called zygotic genome activation (ZGA). Maternally provided ZGA-triggering factors have been identified in Drosophila melanogaster and Danio rerio, but their mammalian homologs are still undefined. Here, we provide evidence that the DUX family of transcription factors is essential to this process in mice and potentially in humans. First, human DUX4 and mouse Dux are both expressed before ZGA in their respective species. Second, both orthologous proteins bind the promoters of ZGA-associated genes and activate their transcription. Third, Dux knockout in mouse embryonic stem cells (mESCs) prevents the cells from cycling through a 2-cell-like state. Finally, zygotic depletion of Dux leads to impaired early embryonic development and defective ZGA. We conclude that DUX-family proteins are key inducers of zygotic genome activation in placental mammals

Individual retrotransposon integrants are differentially controlled by KZFP/KAP1-dependent histone methylation, DNA methylation and TET-mediated hydroxymethylation in naïve embryonic stem cells

Abstract BACKGROUND: The KZFP/KAP1 (KRAB zinc finger proteins/KRAB-associated protein 1) system plays a central role in repressing transposable elements (TEs) and maintaining parent-of-origin DNA methylation at imprinting control regions (ICRs) during the wave of genome-wide reprogramming that precedes implantation. In naïve murine embryonic stem cells (mESCs), the genome is maintained highly hypomethylated by a combination of TET-mediated active demethylation and lack of de novo methylation, yet KAP1 is tethered by sequence-specific KZFPs to ICRs and TEs where it recruits histone and DNA methyltransferases to impose heterochromatin formation and DNA methylation. RESULTS: Here, upon removing either KAP1 or the cognate KZFP, we observed rapid TET2-dependent accumulation of 5hmC at both ICRs and TEs. In the absence of the KZFP/KAP1 complex, ICRs lost heterochromatic histone marks and underwent both active and passive DNA demethylation. For KAP1-bound TEs, 5mC hydroxylation correlated with transcriptional reactivation. Using RNA-seq, we further compared the expression profiles of TEs upon Kap1 removal in wild-type, Dnmt and Tet triple knockout mESCs. While we found that KAP1 represents the main effector of TEs repression in all three settings, we could additionally identify specific groups of TEs further controlled by DNA methylation. Furthermore, we observed that in the absence of TET proteins, activation upon Kap1 depletion was blunted for some TE integrants and increased for others. CONCLUSIONS: Our results indicate that the KZFP/KAP1 complex maintains heterochromatin and DNA methylation at ICRs and TEs in naïve embryonic stem cells partly by protecting these loci from TET-mediated demethylation. Our study further unveils an unsuspected level of complexity in the transcriptional control of the endovirome by demonstrating often integrant-specific differential influences of histone-based heterochromatin modifications, DNA methylation and 5mC oxidation in regulating TEs expression

DPPA2 and DPPA4 are necessary to establish a 2C‐like state in mouse embryonic stem cells

After fertilization of the transcriptionally silent oocyte, expression from both parental chromosomes is launched through zygotic genome activation (ZGA), occurring in the mouse at the 2-cell (2C) stage. Among the first elements to be transcribed are the Dux gene, the product of which induces a wide array of ZGA genes, and a subset of evolutionary recent LINE-1 retrotransposons that regulate chromatin accessibility in the early embryo. The maternally inherited factors that activate Dux and LINE-1 transcription have so far remained unknown. Mouse embryonic stem cells (mESCs) recapitulate some aspects of ZGA in culture, owing to their ability to cycle through a 2C-like stage when Dux, its target genes, and LINE-1 integrants are expressed. Here, we identify the paralog proteins DPPA2 and DPPA4 as necessary for the activation of Dux and LINE-1 expression in mESCs. Since their encoding RNAs are maternally transmitted to the zygote, it is likely that these factors are important upstream mediators of murine ZGA