Johanna A. Smith

Integration site selection by retroviral vectors: molecular mechanism and clinical consequences.

Retroviral DNA integration into the host cell genome is an essential feature of the retroviral life cycle. The ability to integrate their DNA into the DNA of infected cells also makes retroviruses attractive vectors for delivery of therapeutic genes into the genome of cells carrying adverse mutations in their cellular DNA. Sequencing of the entire human genome has enabled identification of integration site preferences of both replication-competent retroviruses and retroviral vectors. These results, together with the unfortunate outcome of a gene therapy trial, in which integration of a retroviral vector in the vicinity of a protooncogene was associated with the development of leukemia, have stimulated efforts to elucidate the molecular mechanism underlying integration site selection by retroviral vectors, as well as the development of methods to direct integration to specific DNA sequences and chromosomal regions. This review outlines our current knowledge of the mechanism of integration site selection by retroviruses in vitro, in cultured cells, and in vivo; the outcome of several of the more recent gene therapy trials, which employed these vectors; and the efforts of several laboratories to develop vectors that integrate at predetermined sites in the human genome.

Modification of Integration Site Preferences of an HIV-1-Based Vector by Expression of a Novel Synthetic Protein

HIV-1-based lentiviral vectors are a promising tool for gene therapy. However, integration of a lentiviral vector into host cell genes may lead to the development of cancer. Therefore, control of integration site selection is critical to the successful outcome of gene therapy approaches that use these vectors. The discovery that integration site selection by HIV-1 and HIV-1-based vectors is controlled by the LEDGF/p75 protein has presented new opportunities to control integration site selection. In this study, we tested the hypothesis that a fusion protein containing the C-terminal HIV integrase-binding portion of LEDGF/p75, and the N-terminal chromodomain of heterochromatin protein-1alpha (HP1alpha), can target HIV-1 vector DNA outside of genes. We show that this fusion protein, termed TIHPLE, associates with the heterochromatin hallmark trimethylated Lys-9 of histone H3 (H3K9me3). Transient overexpression of TIHPLE alters integration site selection by an HIV-1-based vector and decreases the number of integration events that occur in genes. This change in integration site selection was achieved without a reduction in overall integration efficiency. Furthermore, we show that TIHPLE increases integration in the vicinity of H3K9me3 and in repetitive DNA sequences. These data provide a novel approach to address the problem of the tendency of retroviral vectors to integrate at undesirable sites of the human genome.

Pyruvate kinase expression (PKM1 and PKM2) in cancer-associated fibroblasts drives stromal nutrient production and tumor growth.

We have previously demonstrated that enhanced aerobic glycolysis and/or autophagy in the tumor stroma supports epithelial cancer cell growth and aggressive behavior, via the secretion of high-energy metabolites. These nutrients include lactate and ketones, as well as chemical building blocks, such as amino acids (glutamine) and nucleotides. Lactate and ketones serve as fuel for cancer cell oxidative metabolism, and building blocks sustain the anabolic needs of rapidly proliferating cancer cells. We have termed these novel concepts the “Reverse Warburg Effect,” and the “Autophagic Tumor Stroma Model of Cancer Metabolism.” We have also identified a loss of stromal caveolin-1 (Cav-1) as a marker of stromal glycolysis and autophagy. The aim of the current study was to provide genetic evidence that enhanced glycolysis in stromal cells favors tumorigenesis. To this end, normal human fibroblasts were genetically-engineered to express the two isoforms of pyruvate kinase M (PKM1 and PKM2), a key enzyme in the glycolytic pathway. In a xenograft model, fibroblasts expressing PKM1 or PKM2 greatly promoted the growth of co-injected MDA-MB-231 breast cancer cells, without an increase in tumor angiogenesis. Interestingly, PKM1 and PKM2 promoted tumorigenesis by different mechanism(s). Expression of PKM1 enhanced the glycolytic power of stromal cells, with increased output of lactate. Analysis of tumor xenografts demonstrated that PKM1 fibroblasts greatly induced tumor inflammation, as judged by CD45 staining. In contrast, PKM2 did not lead to lactate accumulation, but triggered a “pseudo-starvation” response in stromal cells, with induction of an NFκB-dependent autophagic program, and increased output of the ketone body 3-hydroxy-buryrate. Strikingly, in situ evaluation of Complex IV activity in the tumor xenografts demonstrated that stromal PKM2 expression drives mitochondrial respiration specifically in tumor cells. Finally, immuno-histochemistry analysis of human breast cancer samples lacking stromal Cav-1 revealed PKM1 and PKM2 expression in the tumor stroma. Thus, our data indicate that a subset of human breast cancer patients with a loss of stromal Cav-1 show profound metabolic changes in the tumor microenvironment. As such, this subgroup of patients may benefit therapeutically from potent inhibitors targeting glycolysis, autophagy and/or mitochondrial activity (such as metformin).

HIV-1 Tat and AIDS-associated cancer: targeting the cellular anti-cancer barrier?

The acquired immunodeficiency syndrome (AIDS) is accompanied by a significant increase in the incidence of neoplasms. Several causative agents have been proposed for this phenomenon. These include immunodeficiency and oncogenic DNA viruses and the HIV-1 protein Tat. Cancer in general is closely linked to genomic instability and DNA repair mechanisms. The latter maintains genomic stability and serves as a cellular anti-cancer barrier. Defects in DNA repair pathway are associated with carcinogenesis.This review focuses on newly discovered connections of the HIV-1 protein Tat, as well as cellular co-factors of Tat, to double-strand break DNA repair. We propose that the Tat-induced DNA repair deficiencies may play a significant role in the development of AIDS-associated cancer.

Evidence that the Nijmegen breakage syndrome protein, an early sensor of double-strand DNA breaks (DSB), is involved in HIV-1 post-integration repair by recruiting the ataxia telangiectasia-mutated kinase in a process similar to, but distinct from, cellular DSB repair.

Retroviral transduction involves integrase-dependent linkage of viral and host DNA that leaves an intermediate that requires post-integration repair (PIR). We and others proposed that PIR hijacks the host cell double-strand DNA break (DSB) repair pathways. Nevertheless, the geometry of retroviral DNA integration differs considerably from that of DSB repair and so the precise role of host-cell mechanisms in PIR remains unclear. In the current study, we found that the Nijmegen breakage syndrome 1 protein (NBS1), an early sensor of DSBs, associates with HIV-1 DNA, recruits the ataxia telangiectasia-mutated (ATM) kinase, promotes stable retroviral transduction, mediates efficient integration of viral DNA and blocks integrase-dependent apoptosis that can arise from unrepaired viral-host DNA linkages. Moreover, we demonstrate that the ATM kinase, recruited by NBS1, is itself required for efficient retroviral transduction. Surprisingly, recruitment of the ATR kinase, which in the context of DSB requires both NBS1 and ATM, proceeds independently of these two proteins. A model is proposed emphasizing similarities and differences between PIR and DSB repair. Differences between the pathways may eventually allow strategies to block PIR while still allowing DSB repair.

Pentoxifylline suppresses transduction by HIV-1-based vectors.

Pentoxifylline, a caffeine-related compound, was shown to suppress human immunodeficiency virus type 1 (HIV-1) replication. This effect is thought to be mediated by inhibition of tumor necrosis factor-alpha (TNFα)-mediated long-terminal repeat (LTR)-driven expression. We now demonstrate that pentoxifylline efficiently inhibits transduction by HIV-1-based vectors. This latter effect is independent of LTR-driven expression, and correlates with a reduced efficiency of the completion of the integration process in infected cells. Finally, the effect of pentoxifylline is dramatically reduced in cells expressing a dominant negative ATR protein, and in primary human cells that exhibit low level of ATR activity, suggesting that the effect of pentoxifylline on HIV-1 transduction and replication is at least partly mediated by suppression of the ATR kinase.

A role for the Werner syndrome protein in epigenetic inactivation of the pluripotency factor Oct4.

Werner syndrome (WS) is an autosomal recessive disorder, the hallmarks of which are premature aging and early onset of neoplastic diseases ( Orren, 2006 ; Bohr, 2008 ). The gene, whose mutation underlies the WS phenotype, is called WRN. The protein encoded by the WRN gene, WRNp, has DNA helicase activity ( Gray et al., 1997 ; Orren, 2006 ; Bohr, 2008 ; Opresko, 2008 ). Extensive evidence suggests that WRNp plays a role in DNA replication and DNA repair ( Chen et al., 2003 ; Hickson, 2003 ; Orren, 2006 ; Turaga et al., 2007 ; Bohr, 2008 ). However, WRNp function is not yet fully understood. In this study, we show that WRNp is involved in de novo DNA methylation of the promoter of the Oct4 gene, which encodes a crucial stem cell transcription factor. We demonstrate that WRNp localizes to the Oct4 promoter during retinoic acid‐induced differentiation of human pluripotent cells and associates with the de novo methyltransferase Dnmt3b in the chromatin of differentiating pluripotent cells. Depletion of WRNp does not affect demethylation of lysine 4 of the histone H3 at the Oct4 promoter, nor methylation of lysine 9 of H3, but it blocks the recruitment of Dnmt3b to the promoter and results in the reduced methylation of CpG sites within the Oct4 promoter. The lack of DNA methylation was associated with continued, albeit greatly reduced, Oct4 expression in WRN‐deficient, retinoic acid‐treated cells, which resulted in attenuated differentiation. The presented results reveal a novel function of WRNp and demonstrate that WRNp controls a key step in pluripotent stem cell differentiation.

Human vaginal fluid contains exosomes that have an inhibitory effect on an early step of the HIV-1 life cycle.

Objective:Vaginal transmission is crucial to the spread of HIV-1 around the world. It is not yet clear what type (s) of innate defenses against HIV-1 infection are present in the vagina. Here, we aimed to determine whether human vaginal fluid contains exosomes that may possess anti-HIV-1 activity. Methods:The exosomal fraction was isolated from samples of vaginal fluids. The presence of exosomes was confirmed by flow cytometry and western blotting. The newly discovered exosomes were tested for their ability to block early steps of HIV-1 infection in vitro using established cell culture systems and real time PCR-based methods. Results:Vaginal fluid contains exosomes expressing CD9, CD63, and CD81 exosomal markers. The exosomal fraction of the fluid-reduced transmission of HIV-1 vectors by 60%, the efficiency of reverse transcription step by 58.4%, and the efficiency of integration by 47%. Exosomes had no effect on the entry of HIV-1 vectors. Conclusion:Human vaginal fluid exosomes are newly discovered female innate defenses that may protect women against HIV-1 infection.

Proliferating cell nuclear antigen is required for loading of the SMCX/KMD5C histone demethylase onto chromatin

BackgroundHistone methylation is regulated by a large number of histone methyltransferases and demethylases. The recently discovered SMCX/KMD5C demethylase has been shown to remove methyl residues from lysine 4 of histone H3 (H3K4), and constitutes an important component of the regulatory element-1-silencing transcription factor (REST) protein complex. However, little is known about the cellular mechanisms that control SMCX activity and intracellular trafficking.ResultsIn this study, we found that small interfering RNA-mediated knockdown of proliferating cell nuclear antigen (PCNA) resulted in the reduction of the chromatin-bound SMCX fraction. We identified a PCNA-interaction protein motif (PIP box) in the SMCX protein. Using site-directed mutagenesis, we found that the amino acids of the SMCX PIP box are involved in the association of SMCX with PCNA and its interaction with chromatin.ConclusionsOur data indicate that the intracellular trafficking of SMCX is controlled by its association with PCNA.

Up-regulation of HIV-1 transduction in nondividing cells by double-strand DNA break-inducing agents

Efficient HIV-1 transduction depends on a number of cellular co-factors. Cellular double-strand DNA break (DSB) repair proteins have been proposed, by ourselves and others, to be required for efficient HIV-1 transduction. Expression and/or activity of these DNA repair proteins can be induced by the introduction of DSBs into the host cell genome. HIV-1 transduction was up-regulated by treatment with DSB-inducing agents in both drug-arrested cells and differentiated neuronal cells. The presented data support the hypothesis that DSB repair proteins are involved in the early steps of the retroviral life-cycle.