Directed Molecular Evolution of an Engineered Gammaretroviral Envelope Protein with Dual Receptor Use Shows Stable Maintenance of Both Receptor Specificities.

UNLABELLED: We have previously reported the construction of a murine leukemia virus-based replication-competent gammaretrovirus (SL3-AP) capable of utilizing the human G protein-coupled receptor APJ (hAPJ) as its entry receptor and its natural receptor, the murine Xpr1 receptor, with equal affinities. The apelin receptor has previously been shown to function as a coreceptor for HIV-1, and thus, adaptation of the viral vector to this receptor is of significant interest. Here, we report the molecular evolution of the SL3-AP envelope protein when the virus is cultured in cells harboring either the Xpr1 or the hAPJ receptor. Interestingly, the dual receptor affinity is maintained even after 10 passages in these cells. At the same time, the chimeric viral envelope protein evolves in a distinct pattern in the apelin cassette when passaged on D17 cells expressing hAPJ in three separate molecular evolution studies. This pattern reflects selection for reduced ligand-receptor interaction and is compatible with a model in which SL3-AP has evolved not to activate hAPJ receptor internalization. IMPORTANCE: Few successful examples of engineered retargeting of a retroviral vector exist. The engineered SL3-AP envelope is capable of utilizing either the murine Xpr1 or the human APJ receptor for entry. In addition, SL3-AP is the first example of an engineered retrovirus retaining its dual tropism after several rounds of passaging on cells expressing only one of its receptors. We demonstrate that the virus evolves toward reduced ligand-receptor affinity, which sheds new light on virus adaptation. We provide indirect evidence that such reduced affinity leads to reduced receptor internalization and propose a novel model in which too rapid receptor internalization may decrease virus entry.

The Effects of Dithiothreitol on DNA.

With the novel possibilities for detecting molecules of interest with extreme sensitivity also comes the risk of encountering hitherto negligible sources of error. In life science, such sources of error might be the broad variety of additives such as dithiothreitol (DTT) used to preserve enzyme stability during in vitro reactions. Using two different assays that can sense strand interruptions in double stranded DNA, we here show that DTT is able to introduce nicks in the DNA backbone. DTT was furthermore shown to facilitate the immobilization of fluorescent DNA on an NHS-ester functionalized glass surface. Such reactions may in particular impact the readout from single molecule detection studies and other ultrasensitive assays. This was highlighted by the finding that DTT markedly decreased the signal to noise ratio in a DNA sensor based assay with single molecule resolution.

Human endogenous retroviruses sustain complex and cooperative regulation of gene-containing loci and unannotated megabase-sized regions.

BACKGROUND: Evidence suggests that some human endogenous retroviruses and endogenous retrovirus-like repeats (here collectively ERVs) regulate the expression of neighboring genes in normal and disease states; e.g. the human globin locus is regulated by an ERV9 that coordinates long-range gene switching during hematopoiesis and activates also intergenic transcripts. While complex transcription regulation is associated with integration of certain exogenous retroviruses, comparable regulation sustained by ERVs is less understood. FINDINGS: We analyzed ERV transcription using ERV9 consensus sequences and publically available RNA-sequencing, chromatin immunoprecipitation with sequencing (ChIP-seq) and cap analysis gene expression (CAGE) data from ENCODE. We discovered previously undescribed and advanced transcription regulation mechanisms in several human reference cell lines. We show that regulation by ERVs involves long-ranging activations including complex RNA splicing patterns, and transcription of large unannotated regions ranging in size from several hundred kb to around 1 Mb. Moreover, regulation was found to be cooperatively sustained in some loci by multiple ERVs and also non-LTR repeats. CONCLUSION: Our analyses show that endogenous retroviruses sustain advanced transcription regulation in human cell lines, which shows similarities to complex insertional mutagenesis effects exerted by exogenous retroviruses. By exposing previously undescribed regulation effects, this study should prove useful for understanding fundamental transcription mechanisms resulting from evolutionary acquisition of retroviral sequence in the human genome.

Novel principles of gamma-retroviral insertional transcription activation in murine leukemia virus-induced end-stage tumors.

BACKGROUND: Insertional mutagenesis screens of retrovirus-induced mouse tumors have proven valuable in human cancer research and for understanding adverse effects of retroviral-based gene therapies. In previous studies, the assignment of mouse genes to individual retroviral integration sites has been based on close proximity and expression patterns of annotated genes at target positions in the genome. We here employed next-generation RNA sequencing to map retroviral-mouse chimeric junctions genome-wide, and to identify local patterns of transcription activation in T-lymphomas induced by the murine leukemia gamma-retrovirus SL3-3. Moreover, to determine epigenetic integration preferences underlying long-range gene activation by retroviruses, the colocalization propensity with common epigenetic enhancer markers (H3K4Me1 and H3K27Ac) of 6,117 integrations derived from end-stage tumors of more than 2,000 mice was examined. RESULTS: We detected several novel mechanisms of retroviral insertional mutagenesis: bidirectional activation of mouse transcripts on opposite sides of a provirus including transcription of unannotated mouse sequence; sense/antisense-type activation of genes located on opposite DNA strands; tandem-type activation of distal genes that are positioned adjacently on the same DNA strand; activation of genes that are not the direct integration targets; combination-type insertional mutagenesis, in which enhancer activation, alternative chimeric splicing and retroviral promoter insertion are induced by a single retrovirus. We also show that irrespective of the distance to transcription start sites, the far majority of retroviruses in end-stage tumors colocalize with H3K4Me1 and H3K27Ac-enriched regions in murine lymphoid tissues. CONCLUSIONS: We expose novel retrovirus-induced host transcription activation patterns that reach beyond a single and nearest annotated gene target. Awareness of this previously undescribed layer of complexity may prove important for elucidation of adverse effects in retroviral-based gene therapies. We also show that wild-type gamma-retroviruses are frequently positioned at enhancers, suggesting that integration into regulatory regions is specific and also subject to positive selection for sustaining long-range gene activation in end-stage tumors. Altogether, this study should prove useful for extrapolating adverse outcomes of retroviral vector therapies, and for understanding fundamental cellular regulatory principles and retroviral biology.

Epigenetic marking and repression of porcine endogenous retroviruses.

Endogenous retroviruses (ERVs) are remnants of retroviral germ line infections and have been identified in all mammals investigated so far. Although the majority of ERVs are degenerated, some mammalian species, such as mice and pigs, carry replication-competent ERVs capable of forming infectious viral particles. In mice, ERVs are silenced by DNA methylation and histone modifications and some exogenous retroviruses were shown to be transcriptionally repressed after integration by a primer-binding site (PBS) targeting mechanism. However, epigenetic repression of porcine ERVs (PERVs) has remained largely unexplored so far. In this study, we screened the pig genome for PERVs using LTRharvest, a tool for de novo detection of ERVs, and investigated various aspects of epigenetic repression of three unrelated PERV families. We found that these PERV families are differentially up- or downregulated upon chemical inhibition of DNA methylation and histone deacetylation in cultured porcine cells. Furthermore, chromatin immunoprecipitation analysis revealed repressive histone methylation marks at PERV loci in primary porcine embryonic germ cells and immortalized embryonic kidney cells. PERV elements belonging to the PERV-γ1 family, which is the only known PERV family that has remained active up to the present, were marked by significantly higher levels of histone methylations than PERV-γ2 and PERV-ß3 proviruses. Finally, we tested three PERV-associated PBS sequences for repression activity in murine and porcine cells using retroviral transduction experiments and showed that none of these PBS sequences induced immediate transcriptional silencing in the tested primary porcine cells.

Construction of a gammaretrovirus with a novel tropism and wild-type replication kinetics capable of using human APJ as entry receptor.

We have constructed a replication-competent gammaretrovirus (SL3-AP) capable of using the human G-protein-coupled receptor hAPJ as its entry receptor. The envelope protein of the virus was made by insertion of the 13-amino-acid peptide ligand for hAPJ, flanked by linker sequences, into one of the variable loops of the receptor binding domain of SL3-2, a murine leukemia virus (MLV) that uses the xenotropic-polytropic virus receptor Xpr1 and which has a host range limited to murine cells. This envelope protein can utilize hAPJ as well as murine Xpr1 for entry into host cells with equal efficiencies. In addition, the SL3-AP virus replicates in cells expressing either of its receptors, hAPJ and murine Xpr1, and causes resistance to superinfection and downregulation of hAPJ in infected cells. Thus, SL3-AP is the first example of a retargeted replication-competent retrovirus, with replication characteristics and receptor interference properties similar to those of natural isolates.

Septin9 is involved in T-cell development and CD8+ T-cell homeostasis.

SEPTIN9 (SEPT9) is a filament-forming protein involved in numerous cellular processes. We have used a conditional knock out allele of Sept9 to specifically delete Sept9 in T-cells. As shown by fluorescence-activated cell sorting, loss of Sept9 at an early thymocyte stage in the thymus results in increased numbers of double-negative cells indicating that SEPT9 is involved in the transition from the double-negative stage during T-cell development. Accordingly, the relative numbers of mature T-cells in the periphery are decreased in mice with a T-cell-specific deletion of Sept9. Proliferation of Sept9-deleted CD8(+) T-cells from the spleen is decreased upon stimulation in culture. The altered T-cell homeostasis caused by the loss of Sept9 results in an increase of CD8(+) central memory T-cells.

Proximity ligation assay combined with flow cytometry is a powerful tool for the detection of cytokine receptor dimerization.

Many cytokine receptors are cell surface proteins that promiscuously combine to form active signalling homo- or heterodimers. Thus, receptor chain dimerization can be viewed as a direct measure of a high probability of intracellular signalling by specific cytokines. Proximity ligation assay (PLA) is an antibody-based method for selective and highly sensitive detection of protein interactions by microscopy. As proof of concept, the aim of this study was to combine antibodies towards interleukin 7 receptor alpha (IL-7Rα) and the common gamma chain (γc) with PLA and flow cytometry to enable the detection of IL-7 receptor heterodimers. The presence of IL-7 receptor heterodimers on the surface of the HPB-ALL T cell line was detected by PLA and microscopy with a resolution of one complex per cell. Optimisation of the PLA reaction on cell suspensions identified buffer effects with critical importance for the flow cytometric outcome. In addition, blocking, fixation and incubation conditions were optimised to prevent unspecific antibody binding. PLA combined with flow cytometry very sensitively detected receptor heterodimers on the cell surface. Thus, the method is a powerful tool for the investigation of cytokine receptor dimerization.

Silencing of endogenous envelope genes in human choriocarcinoma cells shows that envPb1 is involved in heterotypic cell fusions.

Syncytin-1 and envPb1 are two conserved envelope genes in the human genome encoded by single loci from the HERV-W and -Pb families, respectively. To characterize the role of these envelope proteins in cell-cell fusion, we have developed lentiviral vectors that express short hairpin RNAs for stable knockdown of syncytin-1 and envPb1. Analysis of heterotypic fusion activity between trophoblast-derived choriocarcinoma BeWo cells, in which syncytin-1 and envPb1 are specifically silenced, and endothelial cells demonstrated that both syncytin-1 and envPb1 are important to fusion. The ability to fuse cells makes syncytin-1 and envPb1 attractive candidate molecules in therapy against cancer. Our available vectors may help eventually to decipher roles for these genes in human health and/or disease.

A unique, thermostable dimer linkage structure of RD114 retroviral RNA.

Retroviruses package their genome as RNA dimers linked together primarily by base-pairing between palindromic stem-loop (psl) sequences at the 5' end of genomic RNA. Retroviral RNA dimers usually melt in the range of 55 degrees C-70 degrees C. However, RNA dimers from virions of the feline endogenous gammaretrovirus RD114 were reported to melt only at 87 degrees C. We here report that the high thermal stability of RD114 RNA dimers generated from in vitro synthesized RNA is an effect of multiple dimerization sites located in the 5' region from the R region to sequences downstream from the splice donor (SD) site. By antisense oligonucleotide probing we were able to map at least five dimerization sites. Computational prediction revealed a possibility to form stems with autocomplementary loops for all of the mapped dimerization sites. Three of them were located upstream of the SD site. Mutant analysis supported a role of all five loop sequences in the formation and thermal stability of RNA dimers. Four of the five psls were also predicted in the RNA of two baboon endogenous retroviruses proposed to be ancestors of RD114. RNA fragments of the 5' R region or prolonged further downstream could be efficiently dimerized in vitro. However, this was not the case for the 3' R region linked to upstream U3 sequences, suggesting a specific mechanism of negative regulation of dimerization at the 3' end of the genome, possibly explained by a long double-stranded RNA region at the U3-R border. Altogether, these data point to determinants of the high thermostability of the dimer linkage structure of the RD114 genome and reveal differences from other retroviruses.

DNA Origami Calibrators for Counting Fluorophores on Single Particles by Flow Cytometry.

Flow cytometry (FCM) is a high-throughput fluorescence-based technique for multiparameter analysis of individual particles, including cells and nanoparticles. Currently, however, FCM does in many cases not permit proper counting of fluorophore-tagged markers on individual particles, due to a lack of tools for translating FCM output intensities into accurate numbers of fluorophores. This lack hinders derivation of detailed biologic information and comparison of data between experiments with FCM. To address this technological void, the authors here use DNA nanotechnology to design and construct barrel-shaped DNA-origami nanobeads for fluorescence/antigen quantification in FCM. Each bead contains a specific number of calibrator fluorophores and a fluorescent trigger domain with an alternative fluorophore for proper detection in FCM. Using electron microscopy, single-particle fluorescence microscopy, and FCM, the design of each particle is verified. To validate that the DNA bead-based FCM calibration enabled the authors to determine the number of antigens on a biological particle, the uniform and well-characterized murine leukemia virus (MLV) is studied. 48 ± 11 envelope surface protein (Env) trimers per MLV is obtained, which is consistent with reported numbers that relied on low-throughput imaging. Thus, the authors' DNA-beads should accelerate quantitative studies of the biology of individual particles with FCM.

An albumin-angiotensin converting enzyme 2-based SARS-CoV-2 decoy with FcRn-driven half-life extension.

The emergence of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutants and breakthrough infections despite available coronavirus disease 2019 (COVID-19) vaccines calls for antiviral therapeutics. The application of soluble angiotensin converting enzyme 2 (ACE2) as a SARS-CoV-2 decoy that reduces cell bound ACE2-mediated virus entry is limited by a short plasma half-life. This work presents a recombinant human albumin ACE2 genetic fusion (rHA-ACE2) to increase the plasma half-life by an FcRn-driven cellular recycling mechanism, investigated using a wild type (WT) albumin sequence and sequence engineered with null FcRn binding (NB). Binding of rHA-ACE2 fusions to SARS-CoV-2 spike protein subdomain 1 (S1) was demonstrated (WT-ACE2 KD = 32.8 nM and NB-ACE2 KD = 31.7 nM) using Bio-Layer Interferometry and dose-dependent in vitro inhibition of host cell infection of pseudotyped viruses displaying surface SARS-CoV-2 spike (S) protein. FcRn-mediated in vitro recycling was translated to a five times greater plasma half-life of WT-ACE2 (t½ ß = 13.5 h) than soluble ACE2 (t½ ß = 2.8 h) in humanised FcRn/albumin double transgenic mice. The rHA-ACE2-based SARS-CoV-2 decoy system exhibiting FcRn-driven circulatory half-life extension introduced in this work offers the potential to expand and improve the anti-COVID-19 anti-viral drug armoury. STATEMENT OF SIGNIFICANCE: The COVID-19 pandemic has highlighted the need for rapid development of efficient antiviral therapeutics to combat SARS-CoV-2 and new mutants to lower morbidity and mortality in severe cases, and for people that are unable to receive a vaccine. Here we report a therapeutic albumin ACE2 fusion protein (rHA-ACE2), that can bind SARS-CoV-2 S protein decorated virus-like particles to inhibit viral infection, and exhibits extended in vivo half-life compared to ACE2 alone. Employing ACE2 as a binding decoy for the virus is expected to efficiently inhibit all SARS-CoV-2 mutants as they all rely on binding with endogenous ACE2 for viral cell entry and, therefore, rHA-ACE2 constitutes a versatile addition to the therapeutic arsenal for combatting COVID-19.

Antisense transcription in gammaretroviruses as a mechanism of insertional activation of host genes.

Transcription of retroviruses is initiated at the U3-R region boundary in the integrated provirus and continues unidirectionally to produce genomic and mRNA products of positive polarity. Several studies have recently demonstrated the existence of naturally occurring protein-encoding transcripts of negative polarity in complex retroviruses. We report here on the identification of transcripts of negative polarity in simple murine leukemia virus (MLV). In T-cell and B-cell lymphomas induced by SL3-3 and Akv MLV, antisense transcripts initiated in the U3 region of the proviral 5' long terminal repeat (LTR) and continued into the cellular proto-oncogenes Jdp2 and Bach2 to create chimeric transcripts consisting of viral and host sequence. The phenomenon was validated in vivo using a knock-in mouse model homozygous for a single LTR at a position known to activate Nras in B-cell lymphomas. A 5' rapid amplification of cDNA ends (RACE) analysis indicated a broad spectrum of initiation sites within the U3 region of the 5' LTR. Our data show for the first time transcriptional activity of negative polarity initiating in the U3 region of simple retroviruses and suggest a novel mechanism of insertional activation of host genes. Elucidation of the nature and potential regulatory role of 5' LTR antisense transcription will be relevant to the design of therapeutic vectors and may contribute to the increasing recognition of pervasive eukaryotic transcription.

Activation of alternative Jdp2 promoters and functional protein isoforms in T-cell lymphomas by retroviral insertion mutagenesis.

Retroviral insertional mutagenesis has been instrumental for the identification of genes important in cancer development. The molecular mechanisms involved in retroviral-mediated activation of proto-oncogenes influence the distribution of insertions within specific regions during tumorigenesis and hence may point to novel gene structures. From a retroviral tagging screen on tumors of 1767 SL3-3 MLV-infected BALB/c mice, intron 2 of the AP-1 repressor Jdp2 locus was found frequently targeted by proviruses resulting in upregulation of non-canonical RNA subspecies. We identified several promoter regions within 1000 bp upstream of exon 3 that allowed for the production of Jdp2 protein isoforms lacking the histone acetylase inhibitory domain INHAT present in canonical Jdp2. The novel Jdp2 isoforms localized to the nucleus and over-expression in murine fibroblast cells induced cell death similar to canonic Jdp2. When expressed in the context of oncogenic NRAS both full length Jdp2 and the shorter isoforms increased anchorage-independent growth. Our results demonstrate a biological function of Jdp2 lacking the INHAT domain and suggest a post-genomic application for the use of retroviral tagging data in identifying new gene products with a potential role in tumorigenesis.

A STING antagonist modulating the interaction with STIM1 blocks ER-to-Golgi trafficking and inhibits lupus pathology.

BACKGROUND: Nucleic acids are potent stimulators of type I interferon (IFN-I) and antiviral defense, but may also promote pathological inflammation. A range of diseases are characterized by elevated IFN-I, including systemic lupus erythematosus (lupus). The DNA-activated cGAS-STING pathway is a major IFN-I-inducing pathway, and activation of signaling is dependent on trafficking of STING from the ER to the Golgi. METHODS: Here we used cell culture systems, a mouse lupus model, and material from lupus patients, to explore the mode of action of a STING antagonistic peptide, and its ability to modulate disease processes. FINDINGS: We report that the peptide ISD017 selectively inhibits all known down-stream activities of STING, including IFN-I, inflammatory cytokines, autophagy, and apoptosis. ISD017 blocks the essential trafficking of STING from the ER to Golgi through a mechanism dependent on the STING ER retention factor STIM1. Importantly, ISD017 blocks STING activity in vivo and ameliorates disease development in a mouse model for lupus. Finally, ISD017 treatment blocks pathological cytokine responses in cells from lupus patients with elevated IFN-I levels. INTERPRETATION: These data hold promise for beneficial use of STING-targeting therapy in lupus. FUNDING: The Novo Nordisk Foundation, The European Research Council, The Lundbeck Foundation, European Union under the Horizon 2020 Research, Deutsche Forschungsgemeinschaft, Chulalongkorn University.

A retroviral mutagenesis screen identifies Cd74 as a common insertion site in murine B-lymphomas and reveals the existence of a novel IFNgamma-inducible Cd74 isoform.

BACKGROUND: Insertional mutagenesis screens in the mouse are an acknowledged approach to identify genes involved in the pathogenesis of cancer. The potential of these screens to identify genes causally involved in tumorigenesis is not only limited to the murine host, but many of these genes have also been proven to be involved in the oncogenic process in man. RESULTS: Through an insertional mutagenesis screen applying murine leukemia viruses in mouse, we found that Cd74 was targeted by proviral insertion in tumors of B-cell origin. This locus encodes a protein playing crucial roles in antigen presentation and B-cell homeostasis, and its deregulation is often associated with cancer in man. The distribution of insertions within the Cd74 locus prompted the identification of an alternative transcript initiated in intron 1 of Cd74 encoding an N-terminally truncated Cd74 isoform in tissues from un-infected mice, and transcriptional activation assays revealed a positive effect on the novel intronic promoter by a formerly described intronic enhancer in the Cd74 locus. Furthermore, we show that the new Cd74 isoform is IFNgamma inducible and that its expression is differentially regulated from the canonical Cd74 isoform at the transcriptional level. CONCLUSIONS: We here identify Cd74 as a common insertion site in murine B-lymphomas and describe a novel IFNgamma-inducible murine Cd74 isoform differentially regulated from the canonical isoform and expressed under the control of an intronic promoter. The distribution and orientation of proviral insertion sites within the Cd74 locus underscores the causal involvement of the isoforms in the murine B-lymphomagenic process.

Coupling of receptor interference and a host-dependent post-binding entry deficiency in a gammaretroviral envelope protein.

BACKGROUND: SL3-2 is a unique polytropic murine gammaretroviral isolate that is only able to infect murine cells. We have previously shown that two mutations R212G and T213I located on the surface of the receptor binding domain in a region designated the VR3 loop can alter the species tropism of this envelope protein. This location suggests that the VR3 loop composition has an influence on receptor interaction and thereby affects binding as well as superinfection resistance. In order to investigate this further, we have studied the binding and interference patterns of the SL3-2 envelope and its mutants. RESULTS: We find unexpectedly that wild type SL3-2 envelope binds equally well to both permissive and non-permissive cells, indicating a post binding defect when interacting with the human Xpr1. Using replication competent viruses containing envelopes from SL3-2 or its mutants we find that the same amino acid mutations can dramatically alter the interference profile of this polytropic ENV, suggesting that the same amino acid changes that cause the post binding defect also influence interaction with the receptor. CONCLUSIONS: The envelope protein of SL3-2 MLV shows an entry defect on non-murine cells. This is coupled to a dramatically reduced ability to interfere with entry of other polytropic viruses. Two point mutations in the VR3 loop of the receptor binding domain of this envelope result both in a much increased interference ability and in removing the post-binding defect on non-murine cells, suggesting that both of these phenotypes are a consequence of insufficient interaction between the envelope and the receptor.

Control of pathogenicity and disease specificity of a T-lymphomagenic gammaretrovirus by E-box motifs but not by an overlapping glucocorticoid response element.

Although transcription factors of the basic helix-loop-helix family have been shown to regulate enhancers of lymphomagenic gammaretroviruses through E-box motifs, the overlap of an E-box motif (Egre) with the glucocorticoid response element (GRE) has obscured their function in vivo. We report here that Egre, but not the GRE, affects disease induction by the murine T-lymphomagenic SL3-3 virus. Mutating all three copies of Egre prolonged the tumor latency period from 60 to 109 days. Further mutating an E-box motif (Ea/s) outside the enhancer prolonged the latency period to 180 days, suggesting that Ea/s works as a backup site for Egre. While wild-type SL3-3 and GRE and Ea/s mutants exclusively induced T-cell lymphomas with wild-type latencies mainly of the CD4(+) CD8(-) phenotype, Egre as well as the Egre and Ea/s mutants induced B-cell lymphomas and myeloid leukemia in addition to T-cell lymphomas. T-cell lymphomas induced by the two Egre mutants had the same phenotype as those induced by wild-type SL3-3, indicating the incomplete disruption of T-cell lymphomagenesis, which is in contrast to previous findings for a Runx site mutant of SL3-3. Mutating the Egre site or Egre and Ea/s triggered several tumor phenotype-associated secondary enhancer changes encompassing neighboring sites, none of which led to the regeneration of an E-box motif. Taken together, our results demonstrate a role for the E-box but not the GRE in T lymphomagenesis by SL3-3, unveil an inherent broader disease specificity of the virus, and strengthen the notion of selection for more potent enhancer variants of mutated viruses during tumor development.

Loss of MicroRNA targets in the 3' untranslated region as a mechanism of retroviral insertional activation of growth factor independence 1.

The non-oncogene-bearing retrovirus SL3-3 murine leukemia virus induces strictly T-cell lymphomas with a mean latency of 2 to 4 months in mice of the NMRI-inbred (NMRI-i) strain. By high-throughput sequencing of retroviral tags, we have identified the genomic region carrying the transcriptional repressor and oncogene growth factor independence 1 (Gfi1) as a frequent target for SL3-3 in the NMRI-i mouse genome. Twenty-four SL3-3 insertions were identified within a 1-kb window of the 3' untranslated region (3'UTR) of the Gfi1 gene, a clustering pattern unique for this lymphoma model. Expression analysis determined that the Gfi1 gene was transcriptionally activated by SL3-3 insertions, and an upregulation of Gfi1 protein expression was detected for tumors harboring insertions in the Gfi1 3'UTR. Here we provide data in support of a mechanism by which retroviral insertions in the Gfi1 3'UTR decouple microRNA-mediated posttranscriptional regulation.

Bestrophin-3 (vitelliform macular dystrophy 2-like 3 protein) is essential for the cGMP-dependent calcium-activated chloride conductance in vascular smooth muscle cells.

Although the biophysical fingerprints (ion selectivity, voltage-dependence, kinetics, etc) of Ca(2+)-activated Cl(-) currents are well established, their molecular identity is still controversial. Several molecular candidates have been suggested; however, none of them has been fully accepted. We have recently characterized a cGMP-dependent Ca(2+)-activated Cl(-) current with unique characteristics in smooth muscle cells. This novel current has been shown to coexist with a "classic" (cGMP-independent) Ca(2+)-activated Cl(-) current and to have characteristics distinct from those previously known for Ca(2+)-activated Cl(-) currents. Here, we suggest that a bestrophin, a product of the Best gene family, is responsible for the cGMP-dependent Ca(2+)-activated Cl(-) current based on similarities between the membrane currents produced by heterologous expressions of bestrophins and the cGMP-dependent Ca(2+)-activated Cl(-) current. This is supported by similarities in the distribution pattern of the cGMP-dependent Ca(2+)-activated Cl(-) current and bestrophin-3 (the product of Best-3 gene) expression in different smooth muscle. Furthermore, downregulation of Best-3 gene expression with small interfering RNA both in cultured cells and in vascular smooth muscle cells in vivo was associated with a significant reduction of the cGMP-dependent Ca(2+)-activated Cl(-) current, whereas the magnitude of the classic Ca(2+)-activated Cl(-) current was not affected. The majority of previous suggestions that bestrophins are a new Cl(-) channel family were based on heterologous expression in cell culture studies. Our present results demonstrate that at least 1 family member, bestrophin-3, is essential for a well-defined endogenous Ca(2+)-activated Cl(-) current in smooth muscles in the intact vascular wall.