Nuclear-localized human respiratory syncytial virus NS1 protein modulates host gene transcription.

Human respiratory syncytial virus (RSV) is a common cause of lower respiratory tract infections in the pediatric, elderly, and immunocompromised individuals. RSV non-structural protein NS1 is a known cytosolic immune antagonist, but how NS1 modulates host responses remains poorly defined. Here, we observe NS1 partitioning into the nucleus of RSV-infected cells, including the human airway epithelium. Nuclear NS1 coimmunoprecipitates with Mediator complex and is chromatin associated. Chromatin-immunoprecipitation demonstrates enrichment of NS1 that overlaps Mediator and transcription factor binding within the promoters and enhancers of differentially expressed genes during RSV infection. Mutation of the NS1 C-terminal helix reduces NS1 impact on host gene expression. These data suggest that nuclear NS1 alters host responses to RSV infection by binding at regulatory elements of immune response genes and modulating host gene transcription. Our study identifies another layer of regulation by virally encoded proteins that shapes host response and impacts immunity to RSV.

H3K4me3 histone modification in baculovirus-infected silkworm cells.

Baculovirus infection modulates the chromatin states and gene expression of host insect cells. Here we performed chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) of H3 trimethylated at Lys4 (H3K4me3) histone modification in Bombyx mori nucleopolyhedrovirus-infected Bombyx mori cells. The ChIP-seq data revealed the changes of the genome-wide distribution and accumulation of euchromatic histone marks in host insect cells during the progression of baculovirus infection.

Epstein-Barr virus inactivates the transcriptome and disrupts the chromatin architecture of its host cell in the first phase of lytic reactivation.

Epstein-Barr virus (EBV), a herpes virus also termed HHV 4 and the first identified human tumor virus, establishes a stable, long-term latent infection in human B cells, its preferred host. Upon induction of EBV's lytic phase, the latently infected cells turn into a virus factory, a process that is governed by EBV. In the lytic, productive phase, all herpes viruses ensure the efficient induction of all lytic viral genes to produce progeny, but certain of these genes also repress the ensuing antiviral responses of the virally infected host cells, regulate their apoptotic death or control the cellular transcriptome. We now find that EBV causes previously unknown massive and global alterations in the chromatin of its host cell upon induction of the viral lytic phase and prior to the onset of viral DNA replication. The viral initiator protein of the lytic cycle, BZLF1, binds to >105 binding sites with different sequence motifs in cellular chromatin in a concentration dependent manner implementing a binary molar switch probably to prevent noise-induced erroneous induction of EBV's lytic phase. Concomitant with DNA binding of BZLF1, silent chromatin opens locally as shown by ATAC-seq experiments, while previously wide-open cellular chromatin becomes inaccessible on a global scale within hours. While viral transcripts increase drastically, the induction of the lytic phase results in a massive reduction of cellular transcripts and a loss of chromatin-chromatin interactions of cellular promoters with their distal regulatory elements as shown in Capture-C experiments. Our data document that EBV's lytic cycle induces discrete early processes that disrupt the architecture of host cellular chromatin and repress the cellular epigenome and transcriptome likely supporting the efficient de novo synthesis of this herpes virus.

HPV-CCDC106 integration alters local chromosome architecture and hijacks an enhancer by three-dimensional genome structure remodeling in cervical cancer.

Integration of human papillomavirus (HPV) DNA into the human genome is a reputed key driver of cervical cancer. However, the effects of HPV integration on chromatin structural organization and gene expression are largely unknown. We studied a cohort of 61 samples and identified an integration hot spot in the CCDC106 gene on chromosome 19. We then selected fresh cancer tissue that contained the unique integration loci at CCDC106 with no HPV episomal DNA and performed whole-genome, RNA, chromatin immunoprecipitation and high-throughput chromosome conformation capture (Hi-C) sequencing to identify the mechanisms of HPV integration in cervical carcinogenesis. Molecular analyses indicated that chromosome 19 exhibited significant genomic variation and differential expression densities, with correlation found between three-dimensional (3D) structural change and gene expression. Importantly, HPV integration divided one topologically associated domain (TAD) into two smaller TADs and hijacked an enhancer from PEG3 to CCDC106, with a decrease in PEG3 expression and an increase in CCDC106 expression. This expression dysregulation was further confirmed using 10 samples from our cohort, which exhibited the same HPV-CCDC106 integration. In summary, we found that HPV-CCDC106 integration altered local chromosome architecture and hijacked an enhancer via 3D genome structure remodeling. Thus, this study provides insight into the 3D structural mechanism underlying HPV integration in cervical carcinogenesis.

Endogenous retroviruses drive species-specific germline transcriptomes in mammals.

Gene regulation in the germline ensures the production of high-quality gametes, long-term maintenance of the species and speciation. Male germline transcriptomes undergo dynamic changes after the mitosis-to-meiosis transition and have been subject to evolutionary divergence among mammals. However, the mechanisms underlying germline regulatory divergence remain undetermined. Here, we show that endogenous retroviruses (ERVs) influence species-specific germline transcriptomes. After the mitosis-to-meiosis transition in male mice, specific ERVs function as active enhancers to drive germline genes, including a mouse-specific gene set, and bear binding motifs for critical regulators of spermatogenesis, such as A-MYB. This raises the possibility that a genome-wide transposition of ERVs rewired germline gene expression in a species-specific manner. Of note, independently evolved ERVs are associated with the expression of human-specific germline genes, demonstrating the prevalence of ERV-driven mechanisms in mammals. Together, we propose that ERVs fine-tune species-specific transcriptomes in the mammalian germline.

Differential SP1 interactions in SV40 chromatin from virions and minichromosomes.

SP1 binding in SV40 chromatin in vitro and in vivo was characterized in order to better understand its role during the initiation of early transcription. We observed that chromatin from disrupted virions, but not minichromosomes, was efficiently bound by HIS-tagged SP1 in vitro, while the opposite was true for the presence of endogenous SP1 introduced in vivo. Using ChIP-Seq to compare the location of SP1 to nucleosomes carrying modified histones, we found that SP1 could occupy its whole binding site in virion chromatin but only the early side of its binding site in most of the minichromosomes carrying modified histones due to the presence of overlapping nucleosomes. The results suggest that during the initiation of an SV40 infection, SP1 binds to an open region in SV40 virion chromatin but quickly triggers chromatin reorganization and its own removal.

Epigenetic and epitranscriptomic regulation of viral replication.

Eukaryotic gene expression is regulated not only by genomic enhancers and promoters, but also by covalent modifications added to both chromatin and RNAs. Whereas cellular gene expression may be either enhanced or inhibited by specific epigenetic modifications deposited on histones (in particular, histone H3), these epigenetic modifications can also repress viral gene expression, potentially functioning as a potent antiviral innate immune response in DNA virus-infected cells. However, viruses have evolved countermeasures that prevent the epigenetic silencing of their genes during lytic replication, and they can also take advantage of epigenetic silencing to establish latent infections. By contrast, the various covalent modifications added to RNAs, termed epitranscriptomic modifications, can positively regulate mRNA translation and/or stability, and both DNA and RNA viruses have evolved to utilize epitranscriptomic modifications as a means to maximize viral gene expression. As a consequence, both chromatin and RNA modifications could serve as novel targets for the development of antivirals. In this Review, we discuss how host epigenetic and epitranscriptomic processes regulate viral gene expression at the levels of chromatin and RNA function, respectively, and explore how viruses modify, avoid or utilize these processes in order to regulate viral gene expression.

A viral toolkit for recording transcription factor-DNA interactions in live mouse tissues.

Transcription factors (TFs) enact precise regulation of gene expression through site-specific, genome-wide binding. Common methods for TF-occupancy profiling, such as chromatin immunoprecipitation, are limited by requirement of TF-specific antibodies and provide only end-point snapshots of TF binding. Alternatively, TF-tagging techniques, in which a TF is fused to a DNA-modifying enzyme that marks TF-binding events across the genome as they occur, do not require TF-specific antibodies and offer the potential for unique applications, such as recording of TF occupancy over time and cell type specificity through conditional expression of the TF-enzyme fusion. Here, we create a viral toolkit for one such method, calling cards, and demonstrate that these reagents can be delivered to the live mouse brain and used to report TF occupancy. Further, we establish a Cre-dependent calling cards system and, in proof-of-principle experiments, show utility in defining cell type-specific TF profiles and recording and integrating TF-binding events across time. This versatile approach will enable unique studies of TF-mediated gene regulation in live animal models.

Chromatin maturation of the HIV-1 provirus in primary resting CD4+ T cells.

Human immunodeficiency virus type 1 (HIV-1) infection is a chronic condition, where viral DNA integrates into the genome. Latently infected cells form a persistent, heterogeneous reservoir that at any time can reactivate the integrated HIV-1. Here we confirmed that latently infected cells from HIV-1 positive study participants exhibited active HIV-1 transcription but without production of mature spliced mRNAs. To elucidate the mechanisms behind this we employed primary HIV-1 latency models to study latency establishment and maintenance. We characterized proviral transcription and chromatin development in cultures of resting primary CD4+ T-cells for four months after ex vivo HIV-1 infection. As heterochromatin (marked with H3K9me3 or H3K27me3) gradually stabilized, the provirus became less accessible with reduced activation potential. In a subset of infected cells, active marks (e.g. H3K27ac) and elongating RNAPII remained detectable at the latent provirus, despite prolonged proviral silencing. In many aspects, latent HIV-1 resembled an active enhancer in a subset of resting cells. The enhancer chromatin actively promoted latency and the enhancer-specific CBP/P300-inhibitor GNE049 was identified as a new latency reversal agent. The division of the latent reservoir according to distinct chromatin compositions with different reactivation potential enforces the notion that even though a relatively large set of cells contains the HIV-1 provirus, only a discrete subset is readily able to reactivate the provirus and spread the infection.

Increased expression of BCL11B and its recruited chromatin remodeling factors during highly active antiretroviral therapy synergistically represses the transcription of human immunodeficiency virus type 1 and is associated with residual immune activation.

Persistence of human immunodeficiency virus 1 (HIV-1) latency and residual immune activation remain major barriers to treatment in patients receiving highly active antiretroviral therapy (HAART). In the present study, we investigated the molecular mechanisms of persistent HIV infection and residual immune activation in HAART-treated patients. We showed that the expression level of B-cell CLL/lymphoma 11B (BCL11B) was significantly increased in CD4+T cells from HIV-infected patients undergoing HAART, and this was accompanied by increased expression of BCL11B-associated chromatin modifiers and inflammatory factors in comparison to healthy controls and untreated patients with HIV. In vitro assays showed that BCL11B significantly inhibited HIV-1 long terminal repeat (LTR)-mediated transcription. Knockdown of BCL11B resulted in the activation of HIV latent cells, and dissociation of BCL11B and its related chromatin remodeling factors from the HIV LTR. Our findings suggested that increased expression of BCL11B and its related chromatin modifiers contribute to HIV-1 transcriptional silencing, and alteration of BCL11B levels might lead to abnormal transcription and inflammation.

Epigenetics and the dynamics of chromatin during adenovirus infections.

The DNA genome of eukaryotic cells is compacted by histone proteins within the nucleus to form chromatin. Nuclear-replicating viruses such as adenovirus have evolved mechanisms of chromatin manipulation to promote infection and subvert host defenses. Epigenetic factors may also regulate persistent adenovirus infection and reactivation in lymphoid tissues. In this review, we discuss the viral proteins E1A and protein VII that interact with and alter host chromatin, as well as E4orf3, which separates host chromatin from sites of viral replication. We also highlight recent advances in chromatin technologies that offer new insights into virus-directed chromatin manipulation. Beyond the role of chromatin in the viral replication cycle, we discuss the nature of persistent viral genomes in lymphoid tissue and cell lines, and the potential contribution of epigenetic signals in maintaining adenovirus in a quiescent state. By understanding the mechanisms through which adenovirus manipulates host chromatin, we will understand new aspects of this ubiquitous virus and shed light on previously unknown aspects of chromatin biology.

Quantitative Microscopy Reveals Stepwise Alteration of Chromatin Structure during Herpesvirus Infection.

During lytic herpes simplex virus 1 (HSV-1) infection, the expansion of the viral replication compartments leads to an enrichment of the host chromatin in the peripheral nucleoplasm. We have shown previously that HSV-1 infection induces the formation of channels through the compacted peripheral chromatin. Here, we used three-dimensional confocal and expansion microscopy, soft X-ray tomography, electron microscopy, and random walk simulations to analyze the kinetics of host chromatin redistribution and capsid localization relative to their egress site at the nuclear envelope. Our data demonstrated a gradual increase in chromatin marginalization, and the kinetics of chromatin smoothening around the viral replication compartments correlated with their expansion. We also observed a gradual transfer of capsids to the nuclear envelope. Later in the infection, random walk modeling indicated a gradually faster transport of capsids to the nuclear envelope that correlated with an increase in the interchromatin channels in the nuclear periphery. Our study reveals a stepwise and time-dependent mechanism of herpesvirus nuclear egress, in which progeny viral capsids approach the egress sites at the nuclear envelope via interchromatin spaces.

Spatially clustered loci with multiple enhancers are frequent targets of HIV-1 integration.

HIV-1 recurrently targets active genes and integrates in the proximity of the nuclear pore compartment in CD4+ T cells. However, the genomic features of these genes and the relevance of their transcriptional activity for HIV-1 integration have so far remained unclear. Here we show that recurrently targeted genes are proximal to super-enhancer genomic elements and that they cluster in specific spatial compartments of the T cell nucleus. We further show that these gene clusters acquire their location during the activation of T cells. The clustering of these genes along with their transcriptional activity are the major determinants of HIV-1 integration in T cells. Our results provide evidence of the relevance of the spatial compartmentalization of the genome for HIV-1 integration, thus further strengthening the role of nuclear architecture in viral infection.

KSHV lytic proteins K-RTA and K8 bind to cellular and viral chromatin to modulate gene expression.

The oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV) has two distinct life cycles with lifelong latent/non-productive and a sporadic lytic-reactivating/productive phases in the infected immune compromised human hosts. The virus reactivates from latency in response to various chemical or environmental stimuli, which triggers the lytic cascade and leads to the expression of immediate early gene, i.e. Replication and Transcription Activator (K-RTA). K-RTA, the latent-to-lytic switch protein, activates the expression of early (E) and late (L) lytic genes by transactivating multiple viral promoters. Expression of K-RTA is shown to be sufficient and essential to switch the latent virus to enter into the lytic phase of infection. Similarly, the virus-encoded bZIP family of protein, K8 also plays an important role in viral lytic DNA replication. Although, both K-RTA and K8 are found to be the ori-Lyt binding proteins and are required for lytic DNA replication, the detailed DNA-binding profile of these proteins in the KSHV and host genomes remains uncharacterized. In this study, using chromatin immunoprecipitation combined with high-throughput sequencing (ChIP-seq) assay, we performed a comprehensive analysis of K-RTA and K8 binding sites in the KSHV and human genomes in order to identify specific DNA binding sequences/motifs. We identified two novel K-RTA binding motifs, (i.e. AGAGAGAGGA/motif RB and AGAAAAATTC/motif RV) and one K8 binding motif (i.e. AAAATGAAAA/motif KB), respectively. The binding of K-RTA/K8 proteins with these motifs and resulting transcriptional modulation of downstream genes was further confirmed by DNA electrophoretic gel mobility shift assay (EMSA), reporter promoter assay, Chromatin Immunoprecipitation (ChIP) assay and mRNA quantitation assay. Our data conclusively shows that K-RTA/K8 proteins specifically bind to these motifs on the host/viral genomes to modulate transcription of host/viral genes during KSHV lytic reactivation.

Adenovirus 5 E1A Interacts with E4orf3 To Regulate Viral Chromatin Organization.

Human adenovirus expresses several early proteins that control various aspects of the viral replication program, including an orchestrated expression of viral genes. Two of the earliest viral transcriptional units activated after viral genome entry into the host cell nucleus are the E1 and E4 units, which each express a variety of proteins. Chief among these are the E1A proteins that function to reprogram the host cell and activate transcription of all other viral genes. The E4 gene encodes multiple proteins, including E4orf3, which functions to disrupt cellular antiviral defenses, including the DNA damage response pathway and activation of antiviral genes. Here we report that E1A directly interacts with E4orf3 via the conserved N terminus of E1A to regulate the expression of viral genes. We show that E4orf3 indiscriminately drives high nucleosomal density of viral genomes, which is restrictive to viral gene expression and which E1A overcomes via a direct interaction with E4orf3. We also show that during infection E1A colocalizes with E4orf3 to nuclear tracks that are associated with heterochromatin formation. The inability of E1A to interact with E4orf3 has a significant negative impact on overall viral replication, the ability of the virus to reprogram the host cell, and the levels of viral gene expression. Together these results show that E1A and E4orf3 work together to fine-tune the viral replication program during the course of infection and highlight a novel mechanism that regulates viral gene expression.IMPORTANCE To successfully replicate, human adenovirus needs to carry out a rapid yet ordered transcriptional program that executes and drives viral replication. Early in infection, the viral E1A proteins are the key activators and regulators of viral transcription. Here we report, for the first time, that E1A works together with E4orf3 to perfect the viral transcriptional program and identify a novel mechanism by which the virus can adjust viral gene expression by modifying its genome's nucleosomal organization via cooperation between E1A and E4orf3.

Human H4 tail stimulates HIV-1 integration through binding to the carboxy-terminal domain of integrase.

The integration of the retroviral genome into the chromatin of the infected cell is catalysed by the integrase (IN)•viral DNA complex (intasome). This process requires functional association between the integration complex and the nucleosomes. Direct intasome/histone contacts have been reported to modulate the interaction between the integration complex and the target DNA (tDNA). Both prototype foamy virus (PFV) and HIV-1 integrases can directly bind histone amino-terminal tails. We have further investigated this final association by studying the effect of isolated histone tails on HIV-1 integration. We show here that the binding of HIV-1 IN to a peptide derived from the H4 tail strongly stimulates integration catalysis in vitro. This stimulation was not observed with peptide tails from other variants or with alpha-retroviral (RAV) and spuma-retroviral PFV integrases. Biochemical analyses show that the peptide tail induces both an increase in the IN oligomerization state and affinity for the target DNA, which are associated with substantial structural rearrangements in the IN carboxy-terminal domain (CTD) observed by NMR. Our data indicate that the H4 peptide tail promotes the formation of active strand transfer complexes (STCs) and support an activation step of the incoming intasome at the contact of the histone tail.

Directed Nucleosome Sliding during the Formation of the Simian Virus 40 Particle Exposes DNA Sequences Required for Early Transcription.

Simian virus 40 (SV40) exists as chromatin throughout its life cycle and undergoes typical epigenetic regulation mediated by changes in nucleosome location and associated histone modifications. In order to investigate the role of epigenetic regulation during the encapsidation of late-stage minichromosomes into virions, we mapped the locations of nucleosomes containing acetylated or methylated lysines in the histone tails of H3 and H4 present in the chromatin from 48-h-postinfection minichromosomes and disrupted virions. In minichromosomes obtained late in infection, nucleosomes were found carrying various histone modifications primarily in the regulatory region, with a major nucleosome located within the enhancer and other nucleosomes at the early and late transcriptional start sites. The nucleosome found in the enhancer would be expected to repress early transcription by blocking access to part of the SP1 binding sites and the left side of the enhancer in late-stage minichromosomes while also allowing late transcription. In chromatin from virions, the principal nucleosome located in the enhancer was shifted ∼70 bases in the late direction from what was found in minichromosomes, and the level of modified histones was increased throughout the genome. The shifting of the enhancer-associated nucleosome to the late side would effectively serve as a switch to relieve the repression of early transcription found in late minichromosomes while likely also repressing late transcription by blocking access to necessary regulatory sequences. This epigenetic switch appeared to occur during the final stage of virion formation.IMPORTANCE For a virus to complete infection, it must produce a new virus particle in which the genome is able to support a new infection. This is particularly important for viruses like simian virus 40 (SV40), which exist as chromatin throughout their life cycles, since chromatin structure plays a major role in the regulation of the life cycle. In order to determine the role of SV40 chromatin structure late in infection, we mapped the locations of nucleosomes and their histone tail modifications in SV40 minichromosomes and in the SV40 chromatin found in virions using chromatin immunoprecipitation-DNA sequencing (ChIP-Seq). We have identified a novel viral transcriptional control mechanism in which a nucleosome found in the regulatory region of the SV40 minichromosome is directed to slide during the formation of the virus particle, exposing transcription factor binding sites required for early transcription that were previously blocked by the presence of the nucleosome.

Differential viral accessibility (DIVA) identifies alterations in chromatin architecture through large-scale mapping of lentiviral integration sites.

Alterations in chromatin structure play a major role in the epigenetic regulation of gene expression. Here, we describe a step-by-step protocol for differential viral accessibility (DIVA), a method for identifying changes in chromatin accessibility genome-wide. Commonly used methods for mapping accessible genomic loci have strong preferences toward detecting 'open' chromatin found at regulatory regions but are not well suited to studying chromatin accessibility in gene bodies and intergenic regions. DIVA overcomes this limitation, enabling a broader range of sites to be interrogated. Conceptually, DIVA is similar to ATAC-seq in that it relies on the integration of exogenous DNA into the genome to map accessible chromatin, except that chromatin architecture is probed through mapping integration sites of exogenous lentiviruses. An isogenic pair of cell lines are transduced with a lentiviral vector, followed by PCR amplification and Illumina sequencing of virus-genome junctions; the resulting sequences define a set of unique lentiviral integration sites, which are compared to determine whether genomic loci exhibit significantly altered accessibility between experimental and control cells. Experienced researchers will take 6 d to generate lentiviral stocks and transduce the target cells, a further 5 d to prepare the Illumina sequencing libraries and a few hours to perform the bioinformatic analysis.

In Vivo Labelling of Adenovirus DNA Identifies Chromatin Anchoring and Biphasic Genome Replication.

Adenoviruses are DNA viruses with a lytic infection cycle. Following the fate of incoming as well as recently replicated genomes during infections is a challenge. In this study, we used the ANCHOR3 technology based on a bacterial partitioning system to establish a versatile in vivo imaging system for adenoviral genomes. The system allows the visualization of both individual incoming and newly replicated genomes in real time in living cells. We demonstrate that incoming adenoviral genomes are attached to condensed cellular chromatin during mitosis, facilitating the equal distribution of viral genomes in daughter cells after cell division. We show that the formation of replication centers occurs in conjunction with in vivo genome replication and determine replication rates. Visualization of adenoviral DNA revealed that adenoviruses exhibit two kinetically distinct phases of genome replication. Low-level replication occurred during early replication, while high-level replication was associated with late replication phases. The transition between these phases occurred concomitantly with morphological changes of viral replication compartments and with the appearance of virus-induced postreplication (ViPR) bodies, identified by the nucleolar protein Mybbp1A. Taken together, our real-time genome imaging system revealed hitherto uncharacterized features of adenoviral genomes in vivo The system is able to identify novel spatiotemporal aspects of the adenovirus life cycle and is potentially transferable to other viral systems with a double-stranded DNA phase.IMPORTANCE Viruses must deliver their genomes to host cells to ensure replication and propagation. Characterizing the fate of viral genomes is crucial to understand the viral life cycle and the fate of virus-derived vector tools. Here, we integrated the ANCHOR3 system, an in vivo DNA-tagging technology, into the adenoviral genome for real-time genome detection. ANCHOR3 tagging permitted the in vivo visualization of incoming genomes at the onset of infection and of replicated genomes at late phases of infection. Using this system, we show viral genome attachment to condensed host chromosomes during mitosis, identifying this mechanism as a mode of cell-to-cell transfer. We characterize the spatiotemporal organization of adenovirus replication and identify two kinetically distinct phases of viral genome replication. The ANCHOR3 system is the first technique that allows the continuous visualization of adenoviral genomes during the entire virus life cycle, opening the way for further in-depth study.

The human leukemia virus HTLV-1 alters the structure and transcription of host chromatin in cis.

Chromatin looping controls gene expression by regulating promoter-enhancer contacts, the spread of epigenetic modifications, and the segregation of the genome into transcriptionally active and inactive compartments. We studied the impact on the structure and expression of host chromatin by the human retrovirus HTLV-1. We show that HTLV-1 disrupts host chromatin structure by forming loops between the provirus and the host genome; certain loops depend on the critical chromatin architectural protein CTCF, which we recently discovered binds to the HTLV-1 provirus. We show that the provirus causes two distinct patterns of abnormal transcription of the host genome in cis: bidirectional transcription in the host genome immediately flanking the provirus, and clone-specific transcription in cis at non-contiguous loci up to >300 kb from the integration site. We conclude that HTLV-1 causes insertional mutagenesis up to the megabase range in the host genome in >104 persistently-maintained HTLV-1+ T-cell clones in vivo.