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

62. Proteasome Activity Restricts Lentiviral Gene Transfer into Hematopoietic Stem Cells and Is Down-Regulated by Cytokines That Enhance Transduction

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Molecular Evidence of Lentiviral Vector-Mediated Gene Transfer into Human Self-Renewing, Multi-potent, Long-Term NOD/SCID Repopulating Hematopoietic Cells

A major challenge in gene therapy is to achieve efficient transduction of hematopoietic stem cells (HSC). It has previously been shown that lentiviral vectors (LV) transduce efficiently human cord blood-derived NOD/SCID mouse repopulating cells (SRC). Here we studied the effect of cytokines during the short ex vivo incubation with vector. Although SRC transduction was efficient without stimulation, the presence of cytokines significantly improved it. The treatment did not affect the engraftment level or the SRC frequency, but seemed to enhance SRC susceptibility to LV. SRC transduced in both conditions repopulated primary and secondary recipients, maintaining stable multi-lineage transgene expression. Using linear amplification-mediated PCR, we then analyzed vector integration in the bone marrow and CFC of the engrafted mice to monitor the clonal activity of the transduced SRC in vivo. We showed polyclonal engraftment, multi-lineage differentiation, and propagation to secondary recipients of individual SRC. We observed multiple integrations in most clones. These results provide the first formal demonstration that primitive human HSC with self-renewal and multi-lineage repopulation capacities were transduced by LV. Our findings are relevant for the design of clinical protocols that exploit this system to reach significant engraftment by genetically modified HSC in the absence of in vivo selection or strong conditioning regimens

Promoter trapping reveals significant differences in integration site selection between MLV and HIV vectors in primary hematopoietic cells

Recent reports have indicated that human immunodeficiency virus (HIV) and murine leukemia virus (MLV) vectors preferentially integrate into active genes. Here, we used a novel approach based on genetic trapping to rapidly score several thousand integration sites and found that MLV vectors trapped cellular promoters more efficiently than HIV vectors. Remarkably, 1 in 5 MLV integrations trapped an active promoter in different cell lines and primary hematopoietic cells. Such frequency was even higher in growth-stimulated lymphocytes. We show that the different behavior of MLV and HIV vectors was dependent on a different integration pattern within transcribed genes. Whereas MLV-based traps showed a strong bias for promoter-proximal integration leading to efficient reporter expression, HIV-based traps integrated throughout transcriptional units and were limited for expression by the distance from the promoter and the reading frame of the targeted gene. Our results indicate a strong propensity of MLV to establish transcriptional interactions with cellular promoters, a behavior that may have evolved to enhance proviral expression and may increase the insertional mutagenesis risk. Promoter trapping efficiency provides a convenient readout to assess transcriptional interactions between the vector and its flanking genes at the integration site and to compare integration site selection among different cell types and in different growth conditions

Global and stage specific patterns of Krüppel-associated-box zinc finger protein gene expression in murine early embryonic cells.

Highly coordinated transcription networks orchestrate the self-renewal of pluripotent stem cell and the earliest steps of mammalian development. KRAB-containing zinc finger proteins represent the largest group of transcription factors encoded by the genomes of higher vertebrates including mice and humans. Together with their putatively universal cofactor KAP1, they have been implicated in events as diverse as the silencing of endogenous retroelements, the maintenance of imprinting and the pluripotent self-renewal of embryonic stem cells, although the genomic targets and specific functions of individual members of this gene family remain largely undefined. Here, we first generated a list of Ensembl-annotated KRAB-containing genes encoding the mouse and human genomes. We then defined the transcription levels of these genes in murine early embryonic cells. We found that the majority of KRAB-ZFP genes are expressed in mouse pluripotent stem cells and other early progenitors. However, we also identified distinctively cell- or stage-specific patterns of expression, some of which are pluripotency-restricted. Finally, we determined that individual KRAB-ZFP genes exhibit highly distinctive modes of expression, even when grouped in genomic clusters, and that these cannot be correlated with the presence of prototypic repressive or activating chromatin marks. These results pave the way to delineating the role of specific KRAB-ZFPs in early embryogenesis

Experimental approach.

<p>A, CD4+ cells were purified from human peripheral blood (resting), exposed to high-dose IL2 and PHA for two days (activated) and then maintained for 11–14 days in low-dose IL2 (resting post-act.). B, the three cell populations were analyzed for expression of activation markers expression (CD25, CD69) (left) and GFP production 3 days after infection with a GFP-containing HIV1 derivative.</p

KAP1 regulates gene networks controlling mouse B-lymphoid cell differentiation and function

Chromatin remodeling is fundamental for B-cell differentiation. In the present study, we explored the role of KAP1, the cofactor of KRAB-ZFP transcriptional repressors, in this process. B-lymphoid-specific Kap1-KO mice displayed reduced numbers of mature B cells, lower steady-state levels of Abs, and accelerated rates of decay of neutralizing Abs after viral immunization. Transcriptome analyses of Kap1-deleted B splenocytes revealed an up-regulation of PTEN, the enzymatic counteractor of PIK3 signaling, and of genes encoding DNA-damage response factors, cell-cycle regulators, and chemokine receptors. ChIP/seq studies established that KAP1 bound at or close to several of these genes and controlled chromatin status at their promoters. Genome wide, KAP1 binding sites lacked active B cell-specific enhancers and were enriched in repressive histone marks, further supporting a role for this molecule in gene silencing in vivo. Likely responsible for tethering KAP1 to at least some of these targets, a discrete subset of KRAB-ZFPs is enriched in B lymphocytes. Our results therefore reveal the role of KRAB/KAP1-mediated epigenetic regulation in B-cell development and homeostasis

Mouse and human gene families encoding for KRAB-containing proteins and their conservation.

<p>A) Summary table of the mouse and human families. B) Venn diagram representing the comparison between mouse (left panel) and human (right panel) KRAB-ZFP gene lists generated in this work and in previously published studies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056721#pone.0056721-Emerson1" target="_blank">[1]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056721#pone.0056721-Tadepally1" target="_blank">[5]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056721#pone.0056721-Nowick1" target="_blank">[42]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056721#pone.0056721-Thomas3" target="_blank">[48]</a>. Numbers indicates the KRAB-ZFP genes identified in the different studies and the overlap among the different lists. C) Alignment of the C2H2 ZF domain amino acid sequences (excluding spacer sequences) of three mouse and human KRAB-ZFPs generated with Clustal Omega. Dark purple boxes indicate conserved residues. Light purple boxes indicate non-conserved residues. Dashes indicate gaps introduced by the alignment tool. Red residues highlighted in the consensus sequence point to the conserved cysteine and histidine residues of each C2H2 ZF domain. Human ZNF746 and mouse ZNF746 show 100% homology at the level of their C2H2 ZF domains. Human RBAK and mouse ZFP12 show 74.1% homology between their C2H2 ZF domains. Human ZFP57 and mouse ZFP57 show only 30.5% global homology between their C2H2 ZF domains, but the human third and fourth C2H2 ZF domains are homologous to the mouse first and second C2H2 ZF domains that are necessary and sufficient for the recognition of the conserved target sequence contained in ICRs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056721#pone.0056721-Quenneville1" target="_blank">[39]</a>.</p