Additional Cellular Factors Research Articles

Cryo-EM structures of Gid12-bound GID E3 reveal steric blockade as a mechanism inhibiting substrate ubiquitylation

Protein degradation, a major eukaryotic response to cellular signals, is subject to numerous layers of regulation. In yeast, the evolutionarily conserved GID E3 ligase mediates glucose-induced degradation of fructose-1,6-bisphosphatase (Fbp1), malate dehydrogenase (Mdh2), and other gluconeogenic enzymes. “GID” is a collection of E3 ligase complexes; a core scaffold, RING-type catalytic core, and a supramolecular assembly module together with interchangeable substrate receptors select targets for ubiquitylation. However, knowledge of additional cellular factors directly regulating GID-type E3s remains rudimentary. Here, we structurally and biochemically characterize Gid12 as a modulator of the GID E3 ligase complex. Our collection of cryo-EM reconstructions shows that Gid12 forms an extensive interface sealing the substrate receptor Gid4 onto the scaffold, and remodeling the degron binding site. Gid12 also sterically blocks a recruited Fbp1 or Mdh2 from the ubiquitylation active sites. Our analysis of the role of Gid12 establishes principles that may more generally underlie E3 ligase regulation.

Cross-Activation of Two Nitrogenase Gene Clusters by CnfR1 or CnfR2 in the Cyanobacterium Anabaena variabilis.

ABSTRACTIn Anabaena variabilis, the nif1 genes, which are activated by CnfR1, produce a Mo-nitrogenase that is expressed only in heterocysts. Similarly, the nif2 genes, which are activated by CnfR2, make a Mo-nitrogenase that is expressed only in anaerobic vegetative cells. However, CnfR1, when it was expressed in anaerobic vegetative cells under the control of the cnfR2 promoter or from the Co2+-inducible coaT promoter, activated the expression of both nifB1 and nifB2. Activation of nifB2, but not nifB1, by CnfR1 required NtcA. Thus, expression of the nif1 system requires no heterocyst-specific factor other than CnfR1. In contrast, CnfR2, when it was expressed in heterocysts under the control of the cnfR1 promoter or from the coaT promoter, did not activate the expression of nifB1 or nifB2. Thus, activation of the nif2 system in anaerobic vegetative cells by CnfR2 requires additional factors absent in heterocysts. CnfR2 made from the coaT promoter activated nifB2 expression in anaerobic vegetative cells grown with fixed nitrogen; however, oxygen inhibited CnfR2 activation of nifB2 expression. In contrast, activation of nifB1 and nifB2 by CnfR1 was unaffected by oxygen. CnfR1, which does not activate the nifB2 promoter in heterocysts, activated the expression of the entire nif2 gene cluster from a nifB2::nifB1::nifB2 hybrid promoter in heterocysts, producing functional Nif2 nitrogenase in heterocysts. However, activity was poor compared to the normal Nif1 nitrogenase. Expression of the nif2 cluster in anaerobic vegetative cells of Nostoc sp. PCC 7120, a strain lacking the nif2 nitrogenase, resulted in expression of the nif2 genes but weak nitrogenase activity.IMPORTANCE Cyanobacterial nitrogen fixation is important in the global nitrogen cycle, in oceanic productivity, and in many plant and fungal symbioses. While the proteins that mediate nitrogen fixation have been well characterized, the regulation of this complex and expensive process is poorly understood in cyanobacteria. Using a genetic approach, we have characterized unique and overlapping functions for two homologous transcriptional activators CnfR1 and CnfR2 that activate two distinct nitrogenases in a single organism. We found that CnfR1 is promiscuous in its ability to activate both nitrogenase systems, whereas CnfR2 depends on additional cellular factors; thus, it activates only one nitrogenase system.

Effects of DNA Supercoil Handedness on Catenation by Type II Topoisomerases

Type II topoisomerases interact differently with DNA substrates of opposite supercoil handedness: although the genome is globally unwound, DNA ahead of replication or transcription complexes is overwound, and therefore DNA geometry can influence where the enzyme acts during nucleic acid processes. Human topoisomerase IIa and bacterial topoisomerase IV relax positively supercoiled [(+)SC] DNA faster than negatively supercoiled [(-)SC] molecules, while human topoisomerase IIb shows no preference. An important function of topoisomerase IIa and topoisomerase IV is the decatenation of DNA prior to mitosis. Cellular studies in yeast and bacteria indicate that DNA becomes positively supercoiled through interaction with cellular factors prior to decatenation, but it is not yet known whether the enzyme displays an intrinsic preference for decatenating (+)SC DNA or if these additional cellular factors accelerate the reaction. To determine whether type II topoisomerases possess an intrinsic ability to recognize DNA supercoil handedness during catenation and decatenation, we have carried out a series of in vitro experiments. As these two reactions are in equilibrium, it is important to study the effects of DNA supercoil handedness on both of them. Our studies focused on the effects of (-)SC, (+)SC, and relaxed DNA on catenation by type II topoisomerases. Topoisomerase IIa catenated relaxed DNA ~10-fold faster than (-)SC DNA, and ~45-fold faster than (+)SC DNA. The C-terminal domain of topoisomerase IIa has been previously shown to confer the ability of the enzyme to preferentially relax (+)SC DNA over (-)SC DNA. A deletion construct of topoisomerase IIa that lacked the C-terminal domain showed that this enzyme domain is also required for the preferential catenation of relaxed and (-)SC DNA. Catenation assays with (+)SC substrates did not go to completion, suggesting that chromosomes are less likely to be catenated following positive supercoiling. Additional studies performed with topoisomerase IIa indicated that (-)SC DNA does not relax prior to catenation and, at high concentrations, the enzyme can catenate DNA with very low concentrations of ATP as compared to other strand passage reactions. Topoisomerase IIb displayed slower rates of catenation than topoisomerase IIa and did not discriminate between different supercoil handedness states. In contrast, topoisomerase IV preferentially catenated (+)SC DNA over (-)SC and relaxed DNA. Results show that topoisomerase IIa and topoisomerase IV can distinguish supercoil handedness during catenation and may help determine whether (+)SC decatenation uses a different control mechanism.

Co-culture of bone marrow stromal cells and chondrocytes in vivo for the repair of the goat condylar cartilage defects.

This study explored the feasibility of inducing the differentiation of BMSCs into chondrocytes through co-culture with chondrocytes in hydrogel constructs (Pluronic F-127 gel) in vivo for the repair of goat mandibular condylar cartilage defects. Chondrocytes and BMSCs were isolated from goat auricular cartilage and bone marrow, respectively, and were mixed at a ratio of 3:7. BMSCs were labelled with green fluorescence protein (GFP) using a retrovirus vector for tracing. Mixed cells were re-suspended in 30% Pluronic F-127 at a concentration of 5×107 cells/ml to form a gel-cell complex. The gel-cell complex was implanted into the temporomandibular joint condylar articular cartilage defects. The whole temporomandibular joint and adjacent tissues were harvested at 4, 8, and 12 weeks after surgery, and gross observation, histology and collagen II expression were evaluated. In the co-culture group, cartilage-like tissues were formed, and abundant type II collagen could be detected by immunohistochemistry in the condylar cartilage defects. Confocal microscopy revealed that implanted GFP-labelled BMSCs were embedded in cartilage-like tissues. The co-culture system described herein provides a chondrogenic microenvironment to induce the chondrogenic differentiation of BMSCs in vivo without any additional cellular factors.

SMARCA2-regulated host cell factors are required for MxA restriction of influenza A viruses

The human interferon (IFN)-induced MxA protein is a key antiviral host restriction factor exhibiting broad antiviral activity against many RNA viruses, including highly pathogenic avian influenza A viruses (IAV) of the H5N1 and H7N7 subtype. To date the mechanism for how MxA exerts its antiviral activity is unclear, however, additional cellular factors are believed to be essential for this activity. To identify MxA cofactors we performed a genome-wide siRNA-based screen in human airway epithelial cells (A549) constitutively expressing MxA using an H5N1 reporter virus. These data were complemented with a proteomic screen to identify MxA-interacting proteins. The combined data identified SMARCA2, the ATPase subunit of the BAF chromatin remodeling complex, as a crucial factor required for the antiviral activity of MxA against IAV. Intriguingly, our data demonstrate that although SMARCA2 is essential for expression of some IFN-stimulated genes (ISGs), and the establishment of an antiviral state, it is not required for expression of MxA, suggesting an indirect effect on MxA activity. Transcriptome analysis of SMARCA2-depleted A549-MxA cells identified a small set of SMARCA2-regulated factors required for activity of MxA, in particular IFITM2 and IGFBP3. These findings reveal that several virus-inducible factors work in concert to enable MxA restriction of IAV.

EBF1 binds to EBNA2 and promotes the assembly of EBNA2 chromatin complexes in B cells

Epstein-Barr virus (EBV) infection converts resting human B cells into permanently proliferating lymphoblastoid cell lines (LCLs). The Epstein-Barr virus nuclear antigen 2 (EBNA2) plays a key role in this process. It preferentially binds to B cell enhancers and establishes a specific viral and cellular gene expression program in LCLs. The cellular DNA binding factor CBF1/CSL serves as a sequence specific chromatin anchor for EBNA2. The ubiquitous expression of this highly conserved protein raises the question whether additional cellular factors might determine EBNA2 chromatin binding selectively in B cells. Here we used CBF1 deficient B cells to identify cellular genes up or downregulated by EBNA2 as well as CBF1 independent EBNA2 chromatin binding sites. Apparently, CBF1 independent EBNA2 target genes and chromatin binding sites can be identified but are less frequent than CBF1 dependent EBNA2 functions. CBF1 independent EBNA2 binding sites are highly enriched for EBF1 binding motifs. We show that EBNA2 binds to EBF1 via its N-terminal domain. CBF1 proficient and deficient B cells require EBF1 to bind to CBF1 independent binding sites. Our results identify EBF1 as a co-factor of EBNA2 which conveys B cell specificity to EBNA2.

Transcriptomic Determinants of Scrapie Prion Propagation in Cultured Ovine Microglia

Susceptibility to infection by prions is highly dependent on the amino acid sequence and host expression of the cellular prion protein (PrPC); however, cellular expression of a genetically susceptible PrPC is insufficient. As an example, it has been shown in cultured cells that permissive and resistant sublines derived from the same parental population often have similar expression levels of PrPC. Thus, additional cellular factors must influence susceptibility to prion infection. The aim of this study was to elucidate the factors associated with relative permissiveness and resistance to scrapie prions in cultured cells derived from a naturally affected species. Two closely related ovine microglia clones with different prion susceptibility, but no detectable differences in PrPC expression levels, were inoculated with either scrapie-positive or scrapie-negative sheep brainstem homogenates. Five passages post-inoculation, the transcriptional profiles of mock and infected clones were sequenced using Illumina technology. Comparative transcriptional analyses identified twenty-two differentially transcribed genes, most of which were upregulated in poorly permissive microglia. This included genes encoding for selenoprotein P, endolysosomal proteases, and proteins involved in extracellular matrix remodeling. Furthermore, in highly permissive microglia, transforming growth factor β–induced, retinoic acid receptor response 1, and phosphoserine aminotranspherase 1 gene transcripts were upregulated. Gene Set Enrichment Analysis identified proteolysis, translation, and mitosis as the most affected pathways and supported the upregulation trend of several genes encoding for intracellular proteases and ribosomal proteins in poorly permissive microglia. This study identifies new genes potentially involved in scrapie prion propagation, corroborates results from other studies, and extends those results into another cell culture model.

Identification, molecular cloning, and analysis of full-length hepatitis C virus transmitted/founder genotypes 1, 3, and 4.

ABSTRACTHepatitis C virus (HCV) infection is characterized by persistent replication of a complex mixture of viruses termed a “quasispecies.” Transmission is generally associated with a stringent population bottleneck characterized by infection by limited numbers of “transmitted/founder” (T/F) viruses. Characterization of T/F genomes of human immunodeficiency virus type 1 (HIV-1) has been integral to studies of transmission, immunopathogenesis, and vaccine development. Here, we describe the identification of complete T/F genomes of HCV by single-genome sequencing of plasma viral RNA from acutely infected subjects. A total of 2,739 single-genome-derived amplicons comprising 10,966,507 bp from 18 acute-phase and 11 chronically infected subjects were analyzed. Acute-phase sequences diversified essentially randomly, except for the poly(U/UC) tract, which was subject to polymerase slippage. Fourteen acute-phase subjects were productively infected by more than one genetically distinct virus, permitting assessment of recombination between replicating genomes. No evidence of recombination was found among 1,589 sequences analyzed. Envelope sequences of T/F genomes lacked transmission signatures that could distinguish them from chronic infection viruses. Among chronically infected subjects, higher nucleotide substitution rates were observed in the poly(U/UC) tract than in envelope hypervariable region 1. Fourteen full-length molecular clones with variable poly(U/UC) sequences corresponding to seven genotype 1a, 1b, 3a, and 4a T/F viruses were generated. Like most unadapted HCV clones, T/F genomes did not replicate efficiently in Huh 7.5 cells, indicating that additional cellular factors or viral adaptations are necessary for in vitro replication. Full-length T/F HCV genomes and their progeny provide unique insights into virus transmission, virus evolution, and virus-host interactions associated with immunopathogenesis.

Reconstitution of microtubule-based motility using cell extracts.

Long-range intracellular transport of organelles driven by kinesin and dynein motor proteins depends on additional cellular factors including adaptors and scaffolding proteins. While single-molecule studies of the motility of purified motor proteins have been a powerful approach, these assays are not fully representative of the complex interactions that occur in a cellular environment. To gain insights into the functioning of adaptor proteins that work in concert with motors proteins, motility assays in cell extracts have been developed. These assays are an attractive means to begin to dissect the roles of additional factors in motor-driven transport. Further, this system can be easily manipulated to study this process in different physiological environments. Here we describe in vitro reconstitution of motor-driven motility along microtubules in cell extracts, followed by considerations for data analysis and how these assays can be powerful in informing our understanding of basic cellular processes.

Histone H3.3 and cancer: A potential reader connection

The building block of chromatin is nucleosome, which consists of 146 base pairs of DNA wrapped around a histone octamer composed of two copies of histone H2A, H2B, H3, and H4. Significantly, the somatic missense mutations of the histone H3 variant, H3.3, are associated with childhood and young-adult tumors, such as pediatric high-grade astrocytomas, as well as chondroblastoma and giant-cell tumors of the bone. The mechanisms by which these histone mutations cause cancer are by and large unclear. Interestingly, two recent studies identified BS69/ZMYND11, which was proposed to be a candidate tumor suppressor, as a specific reader for a modified form of H3.3 (H3.3K36me3). Importantly, some H3.3 cancer mutations are predicted to abrogate the H3.3K36me3/BS69 interaction, suggesting that this interaction may play an important role in tumor suppression. These new findings also raise the question of whether H3.3 cancer mutations may lead to the disruption and/or gain of interactions of additional cellular factors that contribute to tumorigenesis.

Highlights of This Issue

Despite promising preclinical evidence, therapies targeting the insulin-like growth factor (IGF) fail for many Ewing sarcoma patients, suggesting the influence of additional cellular factors. Patel and colleagues performed a genomic analysis using FAIRE-seq, which unexpectedly revealed PTEN deletion and loss of PTEN expression in a large fraction of primary human tumors. PTEN deficiency was associated with hyperactivation of AKT signaling and a more aggressive cellular phenotype. Strikingly, PTEN loss decreased sensitivity to IGF1 receptor inhibitors but increased responsiveness to temsirolimus, an mTOR inhibitor, marked by increased autophagy and an associated decrease in viability. These data support PTEN status as a clinically relevant biomarker to guide patient treatment.Pediatric malignant rhabdoid tumors (MRT) generally arise from inactivation of SNF5 (SMARCB1/INI1), a subunit of the macromolecular SWI/SNF chromatin-remodeling complex. However, the mechanism by which SNF5 loss initiates MRT development remains unclear. Wei and colleagues used mass spectrometry-based proteomics to demonstrate that SNF5 loss in MRT alters the subunit composition of the SWI/SNF complex. Importantly, SNF5 restoration rescues SWI/SNF complex integrity. Therefore, SNF5 inactivation drives the appearance of MRTs by dramatically modifying the composition of SWI/SNF complexes and preventing formation of complexes required for cellular differentiation.As a synthetic lethality-based cancer therapeutic strategy, PARP inhibition selective for BRCA1/2 deficiency has shown promising effect; therefore, defining human tumors with wild-type BRCA1/2 genes that are also sensitive to PARP inhibitors is of great clinical interest. Liu and colleagues show that TGFβ, a multitasking cytokine elevated in tumor microenvironments, regulates DNA repair by simultaneously suppressing the gene expression of ATM, MSH2, and BRCA1, resulting in a “BRCAness” phenotype that includes impaired DNA repair efficiency and reduced genomic stability, as well as a synthetic lethality to PARP inhibition. This approach improves selection of breast cancer patients based on TGFβ pathway biomarkers for PARP-targeting therapy.Estrogen and its receptors, predominantly ERα, are important mediators and therapeutic targets in hormone-dependent breast cancer. Cellular and epidemiologic evidence indicates that the G protein-coupled estrogen receptor (GPR30/GPER) has tumorigenic potential. To directly determine the oncogenic role of GPER in breast tumorigenesis, Marjon and colleagues intercrossed GPER-null mice with mammary tumor–prone MMTV-PyMT transgenic mice. Mice lacking GPER displayed reduced tumors with lower mitotic index and histologic grade. Moreover, distant metastasis was greatly diminished in GPER-deficient mice. These findings reveal that GPER contributes to tumorigenesis and, importantly, to metastasis, suggesting GPER as a new diagnostic and therapeutic target in breast cancer.

Membrane deformation and scission by the HSV-1 nuclear egress complex.

The nuclear egress complex (NEC) of herpesviruses such as HSV-1 is essential for the exit of nascent capsids from the cell nucleus. The NEC drives nuclear envelope vesiculation in cells, but the precise budding mechanism and the potential involvement of cellular proteins are unclear. Here we report that HSV-1 NEC alone is sufficient for membrane budding in vitro and thus represents a complete membrane deformation and scission machinery. It forms ordered coats on the inner surface of budded vesicles, suggesting that it mediates scission by scaffolding the membrane bud and constricting the neck to the point of scission. The inward topology of NEC-mediated budding in vitro resembles capsid budding into the inner nuclear membrane during HSV-1 infection and nuclear envelope vesiculation in NEC-transfected cells. We propose that the NEC functions as minimal virus-encoded membrane-budding machinery during nuclear egress and does not require additional cellular factors.

Physicochemical analysis of rotavirus segment 11 supports a ‘modified panhandle’ structure and not the predicted alternative tRNA-like structure (TRLS)

Rotaviruses are a major cause of acute gastroenteritis, which is often fatal in infants. The viral genome consists of 11 double-stranded RNA segments, but little is known about their cis-acting sequences and structural elements. Covariation studies and phylogenetic analysis exploring the potential structure of RNA11 of rotaviruses suggested that, besides the previously predicted “modified panhandle” structure, the 5’ and 3’ termini of one of the isoforms of the bovine rotavirus UKtc strain may interact to form a tRNA-like structure (TRLS). Such TRLSs have been identified in RNAs of plant viruses, where they are important for enhancing replication and packaging. However, using tRNA mimicry assays (in vitro aminoacylation and 3’- adenylation), we found no biochemical evidence for tRNA-like functions of RNA11. Capping, synthetic 3’ adenylation and manipulation of divalent cation concentrations did not change this finding. NMR studies on a 5’- and 3’-deletion construct of RNA11 containing the putative intra-strand complementary sequences supported a predominant panhandle structure and did not conform to a cloverleaf fold despite the strong evidence for a predicted structure in this conserved region of the viral RNA. Additional viral or cellular factors may be needed to stabilise it into a form with tRNA-like properties.

RACK1 (receptor for activated C-kinase 1) interacts with FBW2 (F-box and WD-repeat domain-containing 2) to up-regulate GCM1 (glial cell missing 1) stability and placental cell migration and invasion

GCM1 (glial cell missing 1) is a short-lived transcription factor essential for placental development. The F-box protein, FBW2 (F-box and WD-repeat domain-containing 2), which contains five WD (tryptophan-aspartate) repeats, recognizes GCM1 and mediates its ubiquitination via the SCFFBW2 E3 ligase complex. Although the interaction between GCM1 and FBW2 is facilitated by GCM1 phosphorylation, it is possible that this interaction might be regulated by additional cellular factors. In the present study, we perform tandem-affinity purification coupled with MS analysis identifying RACK1 (receptor for activated C-kinase 1) as an FBW2-interacting protein. RACK1 is a multifaceted scaffold protein containing seven WD repeats. We demonstrate that the WD repeats in both RACK1 and FBW2 are required for the interaction of RACK1 and FBW2. Furthermore, RACK1 competes with GCM1 for FBW2 and thereby prevents GCM1 ubiquitination, which is also supported by the observation that GCM1 is destabilized in RACK1-knockdown BeWo placental cells. Importantly, RACK1 knockdown leads to decreased expression of the GCM1 target gene HTRA4 (high-temperature requirement protein A4), which encodes a serine protease crucial for cell migration and invasion. As a result, migration and invasion activities are down-regulated in RACK1-knockdown BeWo cells. The present study reveals a novel function for RACK1 to regulate GCM1 activity and placental cell migration and invasion.

Anionic Lipids Are Required for Vesicular Stomatitis Virus G Protein-mediated Single Particle Fusion with Supported Lipid Bilayers

Viral glycoproteins mediate fusion between viral and cellular membranes upon binding to cognate receptors and/or experiencing low pH. Although activation of viral glycoproteins is thought to be necessary and sufficient for fusion, accumulating evidence suggests that additional cellular factors, including lipids, can modulate the fusion process. Understanding the role of lipids in virus entry via endocytosis is impeded by poor accessibility and the highly diverse nature of endosomes. Here we imaged fusion of single retroviral particles pseudotyped with the vesicular stomatitis virus (VSV) G protein with dextran-supported lipid bilayers. Incorporation of diffusible fluorescent labels into the viral membrane and the viral interior enabled detection of the lipid mixing (hemifusion) and content transfer (full fusion) steps of VSV G-mediated fusion at low pH. Although single virus fusion with supported bilayers made of zwitterionic lipids could not be detected, inclusion of anionic lipids, phosphatidylserine, and bis(monoacylglycero)phosphate (BMP), greatly enhanced the efficiency of hemifusion and permitted full fusion. Importantly, lipid mixing always preceded the opening of a fusion pore, demonstrating that VSV G-mediated fusion proceeds through a long-lived hemifusion intermediate. Kinetic analysis of lipid and content transfer showed that the lags between lipid and content mixing defining the lifetime of a hemifusion intermediate were significantly shorter for BMP-containing compared with PS-containing bilayers. The strong fusion-enhancing effect of BMP, a late endosome-resident lipid, is consistent with the model that VSV initiates fusion in early endosomes but releases its core into the cytosol after reaching late endosomal compartments.

Pathway of actin filament branch formation by Arp2/3 complex revealed by single-molecule imaging

Actin filament nucleation by actin-related protein (Arp) 2/3 complex is a critical process in cell motility and endocytosis, yet key aspects of its mechanism are unknown due to a lack of real-time observations of Arp2/3 complex through the nucleation process. Triggered by the verprolin homology, central, and acidic (VCA) region of proteins in the Wiskott-Aldrich syndrome protein (WASp) family, Arp2/3 complex produces new (daughter) filaments as branches from the sides of preexisting (mother) filaments. We visualized individual fluorescently labeled Arp2/3 complexes dynamically interacting with and producing branches on growing actin filaments in vitro. Branch formation was strikingly inefficient, even in the presence of VCA: only ~1% of filament-bound Arp2/3 complexes yielded a daughter filament. VCA acted at multiple steps, increasing both the association rate of Arp2/3 complexes with mother filament and the fraction of filament-bound complexes that nucleated a daughter. The results lead to a quantitative kinetic mechanism for branched actin assembly, revealing the steps that can be stimulated by additional cellular factors.

Distinct Mutational Behaviors Differentiate Short Tandem Repeats from Microsatellites in the Human Genome

A tandem repeat’s (TR) propensity to mutate increases with repeat number, and can become very pronounced beyond a critical boundary, transforming it into a microsatellite (MS). However, a clear understanding of the mutational behavior of different TR classes and motifs and related mechanisms is lacking, as is a consensus on the existence of a boundary separating short TRs (STRs) from MSs. This hinders our understanding of MSs’ mutational properties and their effective use as genetic markers. Using indel calls for 179 individuals from 1000 Genomes Pilot-1 Project, we determined polymorphism incidence for four major TR classes, and formalized its varying relationship with repeat number using segmented regression. We observed a biphasic regime with a transition from a faster to a slower exponential growth at 9, 5, 4, and 4 repeats for mono-, di-, tri-, and tetranucleotide TRs, respectively. We used an in vitro mutagenesis assay to evaluate the contribution of strand slippage errors to mutability. STRs and MSs differ in their absolute polymorphism levels, but more importantly in their rates of mutability growth. Although strand slippage is a major factor driving mononucleotide polymorphism incidence, dinucleotide polymorphism incidence is greater than that expected due to strand slippage alone, indicating that additional cellular factors might be driving dinucleotide mutability in the human genome. Leveraging on hundreds of human genomes, we present the first comprehensive, genome-wide analysis of TR mutational behavior, encompassing several motif sizes and compositions.

Understanding lentiviral vector chromatin targeting: working to reduce insertional mutagenic potential for gene therapy

Replication-deficient retroviruses have been successfully utilized as vectors, offering an efficient, stable method of therapeutic gene delivery. Many examples exist proving this mode of integrative gene transfer is both effective and safe in cultured systems and clinical trials. Along with their success, severe side effects have occurred with early retroviral vectors causing a shift in the approach to vector design before further clinical testing. Several alternative delivery methods are available but lentiviral vectors (LV) are among the most favorable as they are already well understood. LV offer safer integration site selection profiles and a lower degree of genotoxicity, compared with γ-retroviral vectors. Following their introduction, development of the self-inactivating vector configuration was a huge step to this mode of therapy but did not confer full protection against insertional mutagenesis. As a result integration, modeling must be improved to eventually avoid this possibility. The cellular factor LEDGF/p75 seems to play an essential role in the process of LV site selection and its interactions with chromatin are being quickly resolved. LEDGF/p75 is at the center of one example directed integration effort where recombinant products bias the integration event, a step toward fully directed integration into pre-determined benign loci. A more accurate picture of the details of LEDGF/p75 in the natural integration process is emerging, including new binding specificities, chromatin interaction kinetics and additional cellular factors. Together with next-generation sequencing technology and bio-informatics to analyze integration patterns, these advancements will lead to highly focused directed integration, accelerating wide-spread acceptance of LV for gene therapy.

Membrane Fusion and Cell Entry of XMRV Are pH-Independent and Modulated by the Envelope Glycoprotein's Cytoplasmic Tail

Xenotropic murine leukemia virus-related virus (XMRV) is a gammaretrovirus that was originally identified from human prostate cancer patients and subsequently linked to chronic fatigue syndrome. Recent studies showed that XMRV is a recombinant mouse retrovirus; hence, its association with human diseases has become questionable. Here, we demonstrated that XMRV envelope (Env)-mediated pseudoviral infection is not blocked by lysosomotropic agents and cellular protease inhibitors, suggesting that XMRV entry is not pH-dependent. The full length XMRV Env was unable to induce syncytia formation and cell-cell fusion, even in cells overexpressing the viral receptor, XPR1. However, truncation of the C-terminal 21 or 33 amino acid residues in the cytoplasmic tail (CT) of XMRV Env induced substantial membrane fusion, not only in the permissive 293 cells but also in the nonpermissive CHO cells that lack a functional XPR1 receptor. The increased fusion activities of these truncations correlated with their enhanced SU shedding into culture media, suggesting conformational changes in the ectodomain of XMRV Env. Noticeably, further truncation of the CT of XMRV Env proximal to the membrane-spanning domain severely impaired the Env fusogenicity, as well as dramatically decreased the Env incorporations into MoMLV oncoretroviral and HIV-1 lentiviral vectors resulting in greatly reduced viral transductions. Collectively, our studies reveal that XMRV entry does not require a low pH or low pH-dependent host proteases, and that the cytoplasmic tail of XMRV Env critically modulates membrane fusion and cell entry. Our data also imply that additional cellular factors besides XPR1 are likely to be involved in XMRV entry.

Therapeutic Intervention by the Simultaneous Inhibition of DNA Repair and Type I or Type II DNA Topoisomerases: One Strategy, Many Outcomes

Many anticancer drugs reduce the integrity of DNA, forming strand breaks. This can cause mutations and cancer or cell death if the lesions are not repaired. Interestingly, DNA repair-deficient cancer cells (e.g., those with BRCA1/2 mutations) have been shown to exhibit increased sensitivity to chemotherapy. Based on this observation, a new therapeutic approach termed 'synthetic lethality' has been developed, in which radiation therapy or cytotoxic anticancer agents are employed in conjunction with selective inhibitors of poly(ADP-ribose)polymerase-1 (PARP-1). Such combinations can cause severe genomic instability in transformed cells resulting in cell death. The synergistic effects of combining PARP-1 inhibition with anticancer drugs have been demonstrated. However, the outcome of this therapeutic strategy varies significantly between cancer types, suggesting that synthetic lethality may be influenced by additional cellular factors. This review focuses on the outcomes of the combined action of PARP-1 inhibitors and agents that affect the activity of DNA topoisomerases.