Five families of diverse DNA viruses comprehensively restructure the nucleus.

Many viruses have evolved ways to restructure their host cell's nucleus profoundly and unexpectedly upon infection. In particular, DNA viruses that need to commandeer their host's cellular synthetic functions to produce their progeny can induce the condensation and margination of host chromatin during productive infection, a phenomenon known as virus-induced reorganization of cellular chromatin (ROCC). These ROCC-inducing DNA viruses belong to 5 families (herpesviruses, baculoviruses, adenoviruses, parvoviruses, and geminiviruses) that infect a wide range of hosts and are important for human and ecosystem health, as well as for biotechnology. Although the study of virus-induced ROCC is in its infancy, investigations are already raising important questions, such as why only some DNA viruses that replicate their genomes in the nucleus elicit ROCC. Studying the shared and distinct properties of ROCC-inducing viruses will provide valuable insights into viral reorganization of host chromatin that could have implications for future therapies that target the viral life cycle.

Four-dimensional analyses show that replication compartments are clonal factories in which Epstein-Barr viral DNA amplification is coordinated.

Herpesviruses must amplify their DNA to load viral particles and they do so in replication compartments. The development and functions of replication compartments during DNA amplification are poorly understood, though. Here we examine 2 functionally distinct replicons in the same cells to dissect DNA amplification within replication compartments. Using a combination of single-cell assays, computational modeling, and population approaches, we show that compartments initially were seeded by single genomes of Epstein-Barr virus (EBV). Their amplification subsequently took 13 to 14 h in individual cells during which their compartments occupied up to 30% of the nucleus and the nuclear volume grew by 50%. The compartmental volumes increased in proportion to the amount of DNA and viral replication proteins they contained. Each compartment synthesized similar levels of DNA, indicating that the total number of compartments determined the total levels of DNA amplification. Further, the amplification, which depended on the number of origins, was regulated differently early and late during the lytic phase; early during the lytic phase, the templates limited DNA synthesis, while later the templates were in excess, coinciding with a decline in levels of the viral replication protein, BMRF1, in the replication compartments. These findings show that replication compartments are factories in which EBV DNA amplification is both clonal and coordinated.

How Kaposi's sarcoma-associated herpesvirus stably transforms peripheral B cells towards lymphomagenesis.

Primary effusion lymphomas (PELs) are causally associated with Kaposi's sarcoma-associated herpesvirus (KSHV) and 86% of PELs are coinfected with Epstein-Barr virus (EBV). Understanding how PELs develop has been impaired by the difficulty of infecting B cells with KSHV in vitro, and the inability of KSHV to transform them. We show that EBV supports an optimal coinfection of 2.5% of peripheral B cells by KSHV. This coinfection requires 1 or more transforming genes of EBV but not entry into KSHV's lytic cycle. We demonstrate that dually infected B cells are stably transformed in vitro and show that while both viruses can be maintained, different cells exhibit distinct, transformed properties. Transformed cells that grow to predominate in a culture express increased levels of most KSHV genes and differentially express a subset of cellular genes, as do bona fide PEL cells. These dually infected peripheral B cells are thus both stably transformed and allow in vitro molecular dissection of early steps in the progression to lymphomagenesis.

Long-distance communication: Looping of human papillomavirus genomes regulates expression of viral oncogenes.

High-risk human papillomaviruses (HPVs) are a major cause of cancers. HPVs infect epithelial cells, and viral oncogenes disrupt several cellular processes, including cell division, differentiation, and apoptosis. Expression of these oncogenes is relatively low in undifferentiated epithelial cells but increases in differentiating cells by unknown mechanisms. In a new study, Parish and colleagues unveil how two cellular proteins, CCCTC-binding factor (CTCF) and Yin Yang 1 (YY1), mediate looping of the HPV18 genome, which regulates expression of viral oncogenes in both dividing and differentiating epithelial cells.

An Epigenetic Journey: Epstein-Barr Virus Transcribes Chromatinized and Subsequently Unchromatinized Templates during Its Lytic Cycle.

The Epstein-Barr virus (EBV) lytic phase, like those of all herpesviruses, proceeds via an orderly cascade that integrates DNA replication and gene expression. EBV early genes are expressed independently of viral DNA amplification, and several early gene products facilitate DNA amplification. On the other hand, EBV late genes are defined by their dependence on viral DNA replication for expression. Recently, a set of orthologous genes found in beta- and gammaherpesviruses have been determined to encode a viral preinitiation complex (vPIC) that mediates late gene expression. The EBV vPIC requires an origin of lytic replication in cis, implying that the vPIC mediates transcription from newly replicated DNA. In agreement with this implication, EBV late gene mRNAs localize to replication factories. Notably, these factories exclude canonical histones. In this review, we compare and contrast the mechanisms and epigenetics of EBV early and late gene expression. We summarize recent findings, propose a model explaining the dependence of EBV late gene expression on lytic DNA amplification, and suggest some directions for future study.

Plasmid Partitioning by Human Tumor Viruses.

The human tumor viruses that replicate as plasmids (we use the term plasmid to avoid any confusion in the term episome, which was coined to mean DNA elements that occur both extrachromosomally and as integrated forms during their life cycles, as does phage lambda) share many features in their DNA synthesis. We know less about their mechanisms of maintenance in proliferating cells, but these mechanisms must underlie their partitioning to daughter cells. One amazing implication of how these viruses are thought to maintain themselves is that while host chromosomes commit themselves to partitioning in mitosis, these tumor viruses would commit themselves to partitioning before mitosis and probably in S phase shortly after their synthesis.

Epstein-Barr virus microRNAs reduce immune surveillance by virus-specific CD8+ T cells.

Infection with Epstein-Barr virus (EBV) affects most humans worldwide and persists life-long in the presence of robust virus-specific T-cell responses. In both immunocompromised and some immunocompetent people, EBV causes several cancers and lymphoproliferative diseases. EBV transforms B cells in vitro and encodes at least 44 microRNAs (miRNAs), most of which are expressed in EBV-transformed B cells, but their functions are largely unknown. Recently, we showed that EBV miRNAs inhibit CD4+ T-cell responses to infected B cells by targeting IL-12, MHC class II, and lysosomal proteases. Here we investigated whether EBV miRNAs also counteract surveillance by CD8+ T cells. We have found that EBV miRNAs strongly inhibit recognition and killing of infected B cells by EBV-specific CD8+ T cells through multiple mechanisms. EBV miRNAs directly target the peptide transporter subunit TAP2 and reduce levels of the TAP1 subunit, MHC class I molecules, and EBNA1, a protein expressed in most forms of EBV latency and a target of EBV-specific CD8+ T cells. Moreover, miRNA-mediated down-regulation of the cytokine IL-12 decreases the recognition of infected cells by EBV-specific CD8+ T cells. Thus, EBV miRNAs use multiple, distinct pathways, allowing the virus to evade surveillance not only by CD4+ but also by antiviral CD8+ T cells.

Identification of properties of the Kaposi's sarcoma-associated herpesvirus latent origin of replication that are essential for the efficient establishment and maintenance of intact plasmids.

UNLABELLED: The maintenance of latent Kaposi's sarcoma-associated herpesvirus (KSHV) genomes is mediated in cis by their terminal repeats (TR). A KSHV genome can have 16 to 50 copies of the 801-bp TR, each of which harbors a 71-bp-long minimal replicator element (MRE). A single MRE can support replication in transient assays, and the presence of as few as two TRs appears to support establishment of KSHV-derived plasmids. Why then does KSHV have such redundancy and heterogeneity in the number of TRs? By determining the abilities of KSHV-derived plasmids containing various numbers of the TRs and MREs to be established and maintained in the long term, we have found that plasmids with fewer than 16 TRs or those with tandem repeats of the MREs are maintained inefficiently, as shown by both their decreased abilities to support formation of colonies and their instability, resulting in frequent rearrangements yielding larger plasmids during and after establishment. These defects often can be overcome by adding the Epstein-Barr virus (EBV) partitioning element, FR (i.e., family of repeats), in cis to these plasmids. In addition we have found that the spacing between MREs is important for their functions, too. Thus, two properties of KSHV's origin of latent replication essential for the efficient establishment and maintenance of viral plasmids stably are (i) the presence of approximately 16 copies of the TR, which are needed for efficient partitioning, and (ii) the presence of at least 2 MRE units separated by 801 bp of center-to-center spacing, which are required for efficient synthesis. IMPORTANCE: KSHV is a human tumor virus that maintains its genome as a plasmid in lymphoid tumor cells. Each plasmid DNA molecule encodes many origins of synthesis. Here we show that these many origins provide an essential advantage to KSHV, allowing the DNAs to be maintained without rearrangement. We find also that the correct spacing between KSHV's origins of DNA synthesis is required for them to support synthesis efficiently. The identification of these properties illuminates plasmid replication in mammalian cells and should lead to the development of rational means to inhibit these tumorigenic replicons.

The latent membrane protein 1 (LMP1) oncogene of Epstein-Barr virus can simultaneously induce and inhibit apoptosis in B cells.

The latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) regulates its own expression and the expression of human genes via its two functional moieties; the transmembrane domains of LMP1 are required to regulate its expression via the unfolded protein response (UPR) and autophagy in B cells, and the carboxy-terminal domain of LMP1 activates cellular signaling pathways that affect cellular proliferation and survival. An apparent anomaly in the complex regulation of the UPR and autophagy by LMP1 is that the induction of either pathway can lead to cellular death, yet neither EBV-infected B cells nor B cells expressing only LMP1 die. Thus, we sought to understand how B cells that express LMP1 survive. The transmembrane domains of LMP1 activated apoptosis in B cells, the apoptosis required the UPR, and the carboxy-terminal domain of LMP1 blocked this apoptosis. The expression of the mRNA of Bcl2a1, encoding an antiapoptotic homolog of BCL2, correlated directly with the expression of LMP1 in EBV-positive B-cell strains, and its expression inhibited the apoptosis induced by the transmembrane domains of LMP1. These findings illustrate how the carboxy-terminal domain of LMP1 supports survival of B cells in the presence of the deleterious effects of the complex regulation of this viral oncogene.

Characterization and intracellular trafficking of Epstein-Barr virus BBLF1, a protein involved in virion maturation.

Epstein-Barr virus (EBV) BBLF1 shares 13 to 15% amino acid sequence identities with the herpes simplex virus 1 UL11 and cytomegalovirus UL99 tegument proteins, which are involved in the final envelopment during viral maturation. This study demonstrates that BBLF1 is a myristoylated and palmitoylated protein, as are UL11 and UL99. Myristoylation of BBLF1 both facilitates its membrane anchoring and stabilizes it. BBLF1 is shown to localize to the trans-Golgi network (TGN) along with gp350/220, a site where final envelopment of EBV particles takes place. The localization of BBLF1 at the TGN requires myristoylation and two acidic clusters, which interact with PACS-1, a cytosolic protein, to mediate retrograde transport from the endosomes to the TGN. Knockdown of the expression of BBLF1 during EBV lytic replication reduces the production of virus particles, demonstrating the requirement of BBLF1 to achieve optimal production of virus particles. BBLF1 is hypothesized to facilitate the budding of tegumented capsid into glycoprotein-embedded membrane during viral maturation.

Lymphomas differ in their dependence on Epstein-Barr virus.

Epstein-Barr virus (EBV) encodes oncogenic information and, oftentimes concomitant with host immunosuppression, gives rise to malignancies in all major categories of lymphoma defined by the World Health Organization. Here, we conditionally evicted the viral extrachromosomal genome from tumor cells in vitro to examine the role of EBV in different lymphomas, including Burkitt lymphoma (BL) and posttransplant lymphoproliferative disorder. Cells derived from 2 canonical BLs were found to have the least dependence on the virus; some required EBV to prevent the inefficient induction of apoptosis. In contrast, cells derived from a subset of BL, Wp-restricted BL, required EBV to block a robust apoptotic program that involves the up-regulation of the proapoptotic protein Bim. Wp-restricted BL cells also relied on the virus to promote efficient proliferation, a distinction that highlights the multiple contributions EBV makes to affect proliferation of its host cells. Like Wp-BL cells, posttransplant lymphoproliferative disorder cells depended on the virus to inhibit apoptosis. They furthermore required the virus to drive them out of G(1)/G(0). Together, these results reveal a graded dependence on EBV among tumor cells that directly correlates with the number of viral genes expressed in the tumor cell.

How Epstein-Barr Virus Induces the Reorganization of Cellular Chromatin.

We have discovered how Epstein-Barr virus (EBV) induces the reorganization of cellular chromatin (ROCC), in which host chromatin is compacted and marginated within the nucleus, with viral DNA replication occurring in the chromatin-free regions. Five families of DNA viruses induce ROCC: herpesviruses, adenoviruses, parvoviruses, baculoviruses, and geminiviruses. These families infect a variety of hosts, including vertebrates, insects, and plants. They also share several characteristics: they replicate and encapsidate their genomes in the host nucleus and package their genomes unbound by histones. We have identified the viral genes and processes required for EBV's ROCC. Each of EBV's seven core DNA synthesis genes and its origin of lytic replication (oriLyt), in trans, are required, while its protein kinase, BGLF4, and its true late genes are not. Following these findings, we tested the role of EBV lytic DNA amplification in driving ROCC. Surprisingly, the inhibition of EBV's lytic DNA synthesis still supports chromatin compaction but blocks its margination. We propose a two-step model for ROCC. First, the initiation of viral lytic DNA synthesis induces a cellular response that results in global chromatin compaction. Second, the histone-free, productive viral DNA synthesis leads to the margination of compacted chromatin to the nuclear periphery. We have tested this model by asking if the histone-associated simian virus 40 (SV40) DNA synthesis could substitute for oriLyt-mediated synthesis and found that EBV's ROCC is incompatible with SV40 DNA replication. Elucidating EBV's induction of ROCC both illuminates how other viruses can do so and indicates how this spatial control of cellular chromatin benefits them. IMPORTANCE Five families of viruses support the reorganization of cellular chromatin (ROCC), the compaction and margination of host chromatin, upon their productive infection. That they all share this phenotype implies the importance of ROCC in viral life cycles. With Epstein-Barr virus (EBV), a herpesvirus, we show that the viral replication complex and origin of lytic replication (oriLyt) are essential for ROCC. In contrast, its protein kinase and true late genes are not. We show that, unexpectedly, the viral lytic amplification is not required for chromatin compaction but is required for its margination. We propose a two-step model for ROCC: first, global chromatin compaction occurs as a cellular response to the initiation of viral DNA synthesis; then, the accumulation of newly synthesized, histone-free viral DNA leads to cellular chromatin margination. Taken together, our findings provide insights into a process contributing to the productive phase of five families of viruses.

Optimal LentiCRISPR-Based System for Sequential CRISPR/Cas9 Screens.

The advent of genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screening has advanced the understanding of molecular systems within cells. Here, we demonstrate the utility of sequentially performed CRISPR knockout screens that use an existing library to explore a biological question across the human genome, and then the remaining cells are used to examine each gene candidate against one common gene of interest. We call this approach "Many vs One" CRISPR screening, made possible by a modified 7SK promoter in place of the U6 promoter to drive expression of a single guide RNA. Inserting this novel 7SK promoter into the ubiquitously used lentiCRISPRv2 backbone is crucial, because it overcomes the need for a substantial increase in CRISPR library coverage during screening, sample processing, and next generation sequencing. This new 7SK vector equals the original lentiCRISPRv2 in lentiviral titer, knockout efficiency, and ease of use.

Coupled transcriptome and proteome analysis of human lymphotropic tumor viruses: insights on the detection and discovery of viral genes.

BACKGROUND: Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) are related human tumor viruses that cause primary effusion lymphomas (PEL) and Burkitt's lymphomas (BL), respectively. Viral genes expressed in naturally-infected cancer cells contribute to disease pathogenesis; knowing which viral genes are expressed is critical in understanding how these viruses cause cancer. To evaluate the expression of viral genes, we used high-resolution separation and mass spectrometry coupled with custom tiling arrays to align the viral proteomes and transcriptomes of three PEL and two BL cell lines under latent and lytic culture conditions. RESULTS: The majority of viral genes were efficiently detected at the transcript and/or protein level on manipulating the viral life cycle. Overall the correlation of expressed viral proteins and transcripts was highly complementary in both validating and providing orthogonal data with latent/lytic viral gene expression. Our approach also identified novel viral genes in both KSHV and EBV, and extends viral genome annotation. Several previously uncharacterized genes were validated at both transcript and protein levels. CONCLUSIONS: This systems biology approach coupling proteome and transcriptome measurements provides a comprehensive view of viral gene expression that could not have been attained using each methodology independently. Detection of viral proteins in combination with viral transcripts is a potentially powerful method for establishing virus-disease relationships.

Identifying a property of origins of DNA synthesis required to support plasmids stably in human cells.

The plasmid origin of replication, oriP, of Epstein-Barr Virus (EBV) was identified in an assay to detect autonomously replicating sequences (ARSs) in human cells. Raji ori, a second origin in EBV, functions in vivo but fails in long-term ARS assays. We examined the initiating element, DS, within oriP and Raji ori to resolve this paradox. DS, but not Raji ori, binds EBNA1; whereas both act as ARSs in short-term assays, with DS being more efficient, only DS can act as an ARS in long-term assays. Surprisingly, we found that DS supported the establishment of a plasmid with Raji ori in cis and that after deletion of DS, Raji ori could now act as an ARS in the long term. This finding explains the frequent failure of ARS assays in mammalian cells. More origins can initially act as ARSs than can be established. We identified one requirement for ARSs to be established: They must function efficiently enough initially to generate a wide distribution of numbers of plasmids per cell. Only the cells that have more than a threshold number of plasmids can survive selections imposed on the cells to retain these replicons.

MicroRNA 29c is down-regulated in nasopharyngeal carcinomas, up-regulating mRNAs encoding extracellular matrix proteins.

Using highly sensitive microarray-based procedures, we identified eight microRNAs (miRNAs) showing robust differential expression between 31 laser-capture-microdissected nasopharyngeal carcinomas (NPCs) and 10 normal healthy nasopharyngeal epithelial samples. In particular, miRNA mir-29c was expressed at one-fifth the levels in tumors as in normal epithelium. In NPC tumors, the lower mir-29c levels correlated with higher levels of multiple mRNAs whose 3' UTRs can bind mir-29c at target sequences conserved across many vertebrates. In cultured cells, introduction of mir-29c down-regulated these genes at the level of mRNA and inhibited expression of luciferase encoded by vectors having the 3' UTRs of these genes. Moreover, for each of several genes tested, mutating the mir-29c target sites in the 3' UTR abrogated mir-29c-induced inhibition of luciferase expression. Most of the mir-29c-targeted genes identified encode extracellular matrix proteins, including multiple collagens and laminin gamma1, that are associated with tumor cell invasiveness and metastatic potential, prominent characteristics of NPC. Thus, we identify eight miRNAs differentially expressed in NPC and demonstrate the involvement of one in regulating genes involved in metastasis.

Proof for EBV's sustaining role in Burkitt's lymphomas.

We have found that not all Epstein-Barr viral (EBV) plasmids are duplicated each cell cycle. This inefficiency is intrinsic to EBV's mechanism of DNA synthesis in latently infected cells and necessarily leads to a loss of EBV plasmids from proliferating cells. If EBV provides its host cells advantages that allow those cells that retain EBV to outgrow those that lose it, then such proliferating populations will be EBV-positive. EBV-associated human tumors are EBV-positive. Thus, the presence of EBV plasmids in most cells of a tumor demonstrates that EBV sustains these tumors in vivo. The virus can provide multiple selective advantages to tumor cells, including promoting cell proliferation and inhibiting cell death. In the case of Burkitt's lymphomas (BL), most current evidence indicates that the tumor requires the virus minimally to block apoptosis.

Epstein-Barr Virus Limits the Accumulation of IPO7, an Essential Gene Product.

Epstein-Barr virus (EBV) encodes more than 40 miRNAs that target cellular mRNAs to aid its infection, replication, and maintenance in individual cells and in its human host. Importin-7 (IPO7), also termed Imp7 or RanBPM7, is a nucleocytoplasmic transport protein that has been frequently identified as a target for two of these viral miRNAs. How the viral life cycle might benefit from regulating IPO7 has been unclear, though. We demonstrate with CRISPR-Cas9 mutagenesis that IPO7 is essential in at least three cells lines and that increasing its levels of expression inhibits growth of infected cells. EBV thus regulates the level of IPO7 to limit its accumulation consistent with its being required for survival of its host cell.

How Epstein-Barr Virus and Kaposi's Sarcoma-Associated Herpesvirus Are Maintained Together to Transform the Same B-Cell.

Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) independently cause human cancers, and both are maintained as plasmids in tumor cells. They differ, however, in their mechanisms of segregation; EBV partitions its genomes quasi-faithfully, while KSHV often clusters its genomes and partitions them randomly. Both viruses can infect the same B-cell to transform it in vitro and to cause primary effusion lymphomas (PELs) in vivo. We have developed simulations based on our measurements of these replicons in B-cells transformed in vitro to elucidate the synthesis and partitioning of these two viral genomes when in the same cell. These simulations successfully capture the biology of EBV and KSHV in PELs. They have revealed that EBV and KSHV replicate and partition independently, that they both contribute selective advantages to their host cell, and that KSHV pays a penalty to cluster its genomes.