ATM, KAP1 and the Epstein-Barr virus polymerase processivity factor direct traffic at the intersection of transcription and replication.

The timing of transcription and replication must be carefully regulated for heavily-transcribed genomes of double-stranded DNA viruses: transcription of immediate early/early genes must decline as replication ramps up from the same genome-ensuring efficient and timely replication of viral genomes followed by their packaging by structural proteins. To understand how the prototypic DNA virus Epstein-Barr virus tackles the logistical challenge of switching from transcription to DNA replication, we examined the proteome at viral replication forks. Specifically, to transition from transcription, the viral DNA polymerase-processivity factor EA-D is SUMOylated by the epigenetic regulator and E3 SUMO-ligase KAP1/TRIM28. KAP1's SUMO2-ligase function is triggered by phosphorylation via the PI3K-related kinase ATM and the RNA polymerase II-associated helicase RECQ5 at the transcription machinery. SUMO2-EA-D then recruits the histone loader CAF1 and the methyltransferase SETDB1 to silence the parental genome via H3K9 methylation, prioritizing replication. Thus, a key viral protein and host DNA repair, epigenetic and transcription-replication interference pathways orchestrate the handover from transcription-to-replication, a fundamental feature of DNA viruses.

Beta human papillomavirus 8E6 promotes alternative end joining.

Double strand breaks (DSBs) are one of the most lethal DNA lesions in cells. The E6 protein of beta-human papillomavirus (HPV8 E6) impairs two critical DSB repair pathways: homologous recombination (HR) and non-homologous end joining (NHEJ). However, HPV8 E6 only delays DSB repair. How DSBs are repaired in cells with HPV8 E6 remains to be studied. We hypothesize that HPV8 E6 promotes a less commonly used DSB repair pathway, alternative end joining (Alt-EJ). Using CAS9-based Alt-EJ reporters, we show that HPV8 E6 promotes Alt-EJ. Further, using small molecule inhibitors, CRISPR/CAS9 gene knockout, and HPV8 E6 mutant, we find that HPV8 E6 promotes Alt-EJ by binding p300, an acetyltransferase that facilitates DSB repair by HR and NHEJ. At least some of this repair occurs through a subset of Alt-EJ known as polymerase theta dependent end joining. Finally, whole genome sequencing analysis showed HPV8 E6 caused an increased frequency of deletions bearing the microhomology signatures of Alt-EJ. This study fills the knowledge gap of how DSB is repaired in cells with HPV8 E6 and the mutagenic consequences of HPV8 E6 mediated p300 destabilization. Broadly, this study supports the hypothesis that beta-HPV promotes cancer formation by increasing genomic instability.

IFI16 Partners with KAP1 to Maintain Epstein-Barr Virus Latency.

Herpesviruses establish latency to ensure permanent residence in their hosts. Upon entry into a cell, these viruses are rapidly silenced by the host, thereby limiting the destructive viral lytic phase while allowing the virus to hide from the immune system. Notably, although the establishment of latency by the oncogenic herpesvirus Epstein-Barr virus (EBV) requires the expression of viral latency genes, latency can be maintained with a negligible expression of viral genes. Indeed, in several herpesviruses, the host DNA sensor IFI16 facilitated latency via H3K9me3 heterochromatinization. This silencing mark is typically imposed by the constitutive heterochromatin machinery (HCM). The HCM, in an antiviral role, also silences the lytic phase of EBV and other herpes viruses. We investigated if IFI16 restricted EBV lytic activation by partnering with the HCM and found that IFI16 interacted with core components of the HCM, including the KRAB-associated protein 1 (KAP1) and the site-specific DNA binding KRAB-ZFP SZF1. This partnership silenced the EBV lytic switch protein ZEBRA, encoded by the BZLF1 gene, thereby favoring viral latency. Indeed, IFI16 contributed to H3K9 trimethylation at lytic genes of all kinetic classes. In defining topology, we found that IFI16 coenriched with KAP1 at the BZLF1 promoter, and while IFI16 and SZF1 were each adjacent to KAP1 in latent cells, IFI16 and SZF1 were not. Importantly, we also found that disruption of latency involved rapid downregulation of IFI16 transcription. These findings revealed a previously unknown partnership between IFI16 and the core HCM that supports EBV latency via antiviral heterochromatic silencing. IMPORTANCE The interferon-gamma inducible protein 16 (IFI16) is a nuclear DNA sensor that mediates antiviral responses by activating the inflammasome, triggering an interferon response, and silencing lytic genes of herpesviruses. The last, which helps maintain latency of the oncoherpesvirus Epstein-Barr virus (EBV), is accomplished via H3K9me3 heterochromatinization through unknown mechanisms. Here, we report that IFI16 physically partners with the core constitutive heterochromatin machinery to silence the key EBV lytic switch protein, thereby ensuring continued viral latency in B lymphocytes. We also find that disruption of latency involves rapid transcriptional downregulation of IFI16. These findings point to hitherto unknown physical and functional partnerships between a well-known antiviral mechanism and the core components of the constitutive heterochromatin machinery.

SARS-CoV-2 viroporin encoded by ORF3a triggers the NLRP3 inflammatory pathway.

Heightened inflammatory response is a prominent feature of severe COVID-19 disease. We report that the SARS-CoV-2 ORF3a viroporin activates the NLRP3 inflammasome, the most promiscuous of known inflammasomes. Ectopically expressed ORF3a triggers IL-1ß expression via NFκB, thus priming the inflammasome. ORF3a also activates the NLRP3 inflammasome but not NLRP1 or NLRC4, resulting in maturation of IL-1ß and cleavage/activation of Gasdermin. Notably, ORF3a activates the NLRP3 inflammasome via both ASC-dependent and -independent modes. This inflammasome activation requires efflux of potassium ions and oligomerization between the kinase NEK7 and NLRP3. Importantly, infection of epithelial cells with SARS-CoV-2 similarly activates the NLRP3 inflammasome. With the NLRP3 inhibitor MCC950 and select FDA-approved oral drugs able to block ORF3a-mediated inflammasome activation, as well as key ORF3a amino acid residues needed for virus release and inflammasome activation conserved in the new variants of SARS-CoV-2 isolates across continents, ORF3a and NLRP3 present prime targets for intervention.

The danger molecule HMGB1 cooperates with the NLRP3 inflammasome to sustain expression of the EBV lytic switch protein in Burkitt lymphoma cells.

High mobility group box 1 (HMGB1) is an important chromatin protein and a pro-inflammatory molecule. Though shown to enhance target DNA binding by the Epstein-Barr virus (EBV) lytic switch protein ZEBRA, whether HMGB1 actually contributes to gammaherpesvirus biology is not known. In investigating the contribution of HMGB1 to the lytic phase of EBV, important for development of EBV-mediated diseases, we find that compared to latently-infected cells, lytic phase Burkitt lymphoma-derived cells and peripheral blood lytic cells during primary EBV infection express high levels of HMGB1. Our experiments place HMGB1 upstream of ZEBRA and reveal that HMGB1, through the NLRP3 inflammasome, sustains the expression of ZEBRA. These findings indicate that in addition to the NLRP3 inflammasome's recently discovered role in turning the EBV lytic switch on, NLRP3 cooperates with the danger molecule HMGB1 to also maintain ZEBRA expression, thereby sustaining the lytic signal.

An Ancestral Retrovirus Envelope Protein Regulates Persistent Gammaherpesvirus Lifecycles.

Human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) persist as life-long infections alternating between latency and lytic replication. Human endogenous retroviruses (HERVs), via integration into the host genome, represent genetic remnants of ancient retroviral infections. Both show similar epigenetic silencing while dormant, but can reactivate in response to cell signaling cues or triggers that, for gammaherpesviruses, result in productive lytic replication. Given their co-existence with humans and shared epigenetic silencing, we asked if HERV expression might be linked to lytic activation of human gammaherpesviruses. We found ERVW-1 mRNA, encoding the functional HERV-W envelope protein Syncytin-1, along with other repeat class elements, to be elevated upon lytic activation of EBV. Knockdown/knockout of ERVW-1 reduced lytic activation of EBV and KSHV in response to various lytic cycle triggers. In this regard, reduced expression of immediate early proteins ZEBRA and RTA for EBV and KSHV, respectively, places Syncytin-1's influence on lytic activation mechanistically upstream of the latent-to-lytic switch. Conversely, overexpression of Syncytin-1 enhanced lytic activation of EBV and KSHV in response to lytic triggers, though this was not sufficient to induce lytic activation in the absence of such triggers. Syncytin-1 is expressed in replicating B cell blasts and lymphoma-derived B cell lines where it appears to contribute to cell cycle progression. Together, human gammaherpesviruses and B cells appear to have adapted a dependency on Syncytin-1 that facilitates the ability of EBV and KSHV to activate lytic replication from latency, while promoting viral persistence during latency by contributing to B cell proliferation.

A heterochromatin inducing protein differentially recognizes self versus foreign genomes.

Krüppel-associated box-domain zinc finger protein (KRAB-ZFP) transcriptional repressors recruit TRIM28/KAP1 to heterochromatinize the mammalian genome while also guarding the host by silencing invading foreign genomes. However, how a KRAB-ZFP recognizes target sequences in the natural context of its own or foreign genomes is unclear. Our studies on B-lymphocytes permanently harboring the cancer-causing Epstein-Barr virus (EBV) have shown that SZF1, a KRAB-ZFP, binds to several lytic/replicative phase genes to silence them, thereby promoting the latent/quiescent phase of the virus. As a result, unless SZF1 and its binding partners are displaced from target regions on the viral genome, EBV remains dormant, i.e. refractory to lytic phase-inducing triggers. As SZF1 also heterochromatinizes the cellular genome, we performed in situ footprint mapping on both viral and host genomes in physically separated B-lymphocytes bearing latent or replicative/active EBV genomes. By analyzing footprints, we learned that SZF1 recognizes the host genome through a repeat sequence-bearing motif near centromeres. Remarkably, SZF1 does not use this motif to recognize the EBV genome. Instead, it uses distinct binding sites that lack obvious similarities to each other or the above motif, to silence the viral genome. Virus mutagenesis studies show that these distinct binding sites are not only key to maintaining the established latent phase but also silencing the lytic phase in newly-infected cells, thus enabling the virus to establish latency and transform cells. Notably, these binding sites on the viral genome, when also present on the human genome, are not used by SZF1 to silence host genes during latency. This differential approach towards target site recognition may reflect a strategy by which the host silences and regulates genomes of persistent invaders without jeopardizing its own homeostasis.

A Mechanism-Based Targeted Screen To Identify Epstein-Barr Virus-Directed Antiviral Agents.

Epstein-Barr virus (EBV) is one of nine human herpesviruses that persist latently to establish permanent residence in their hosts. Periodic activation into the lytic/replicative phase allows such viruses to propagate and spread, but can also cause disease in the host. This lytic phase is also essential for EBV to cause infectious mononucleosis and cancers, including B lymphocyte-derived Burkitt lymphoma and immunocompromise-associated lymphoproliferative diseases/lymphomas as well as epithelial cell-derived nasopharyngeal cell carcinoma. In the absence of anti-EBV agents, however, therapeutic options for EBV-related diseases are limited. In earlier work, we discovered that through the activities of the viral protein kinase conserved across herpesviruses and two cellular proteins, ATM and KAP1, a lytic cycle amplification loop is established, and disruption of this loop disables the EBV lytic cascade. We therefore devised a high-throughput screening assay, screened a small-molecule-compound library, and identified 17 candidates that impair the release of lytically replicated EBV. The identified compounds will (i) serve as lead compounds or may be modified to inhibit EBV and potentially other herpesviruses, and (ii) be developed into anticancer agents, as functions of KAP1 and ATM are tightly linked to cancer. Importantly, our screening strategy may also be used to screen additional compound libraries for antiherpesviral and anticancer drugs.IMPORTANCE Epstein-Barr virus, which is nearly ubiquitous in humans, is causal to infectious mononucleosis, chronic active EBV infection, and lymphoid and epithelial cancers. However, EBV-specific antiviral agents are not yet available. To aid in the identification of compounds that may be developed as antivirals, we pursued a mechanism-based approach. Since many of these diseases rely on EBV's lytic phase, we developed a high-throughput assay that is able to measure a key step that is essential for successful completion of EBV's lytic cascade. We used this assay to screen a library of small-molecule compounds and identified inhibitors that may be pursued for their anti-EBV and possibly even antiherpesviral potential, as this key mechanism appears to be common to several human herpesviruses. Given the prominent role of this mechanism in both herpesvirus biology and cancer, our screening assay may be used as a platform to identify both antiherpesviral and anticancer drugs.

Nascent Transcriptomics Reveal Cellular Prolytic Factors Upregulated Upstream of the Latent-to-Lytic Switch Protein of Epstein-Barr Virus.

Lytic activation from latency is a key transition point in the life cycle of herpesviruses. Epstein-Barr virus (EBV) is a human herpesvirus that can cause lymphomas, epithelial cancers, and other diseases, most of which require the lytic cycle. While the lytic cycle of EBV can be triggered by chemicals and immunologic ligands, the lytic cascade is activated only when expression of the EBV latent-to-lytic switch protein ZEBRA is turned on. ZEBRA then transcriptionally activates other EBV genes and, together with some of those gene products, ensures completion of the lytic cycle. However, not every latently infected cell exposed to a lytic trigger turns on the expression of ZEBRA, resulting in responsive and refractory subpopulations. What governs this dichotomy? By examining the nascent transcriptome following exposure to a lytic trigger, we find that several cellular genes are transcriptionally upregulated temporally upstream of ZEBRA. These genes regulate lytic susceptibility to various degrees in latently infected cells that respond to mechanistically distinct lytic triggers. While increased expression of these cellular genes defines a prolytic state, such upregulation also runs counter to the well-known mechanism of viral-nuclease-mediated host shutoff that is activated downstream of ZEBRA. Furthermore, a subset of upregulated cellular genes is transcriptionally repressed temporally downstream of ZEBRA, indicating an additional mode of virus-mediated host shutoff through transcriptional repression. Thus, increased transcription of a set of host genes contributes to a prolytic state that allows a subpopulation of cells to support the EBV lytic cycle.IMPORTANCE Transition from latency to the lytic phase is necessary for herpesvirus-mediated pathology as well as viral spread and persistence in the population at large. Yet, viral genomes in only some cells in a population of latently infected cells respond to lytic triggers, resulting in subpopulations of responsive/lytic and refractory cells. Our investigations into this partially permissive phenotype of the herpesvirus Epstein-Barr virus (EBV) indicate that upon exposure to lytic triggers, certain cellular genes are transcriptionally upregulated, while viral latency genes are downregulated ahead of expression of the viral latent-to-lytic switch protein. These cellular genes contribute to lytic susceptibility to various degrees. Apart from indicating that there may be a cellular "prolytic" state, our findings indicate that (i) early transcriptional upregulation of cellular genes counters the well-known viral-nuclease-mediated host shutoff and (ii) subsequent transcriptional downregulation of a subset of early upregulated cellular genes is a previously undescribed mode of host shutoff.

Characterization and comparative analysis of microRNAs in the rice pest Sogatella furcifera.

MicroRNAs (miRNAs) are a class of endogenous regulatory RNA molecules 21-24 nucleotides in length that act as functional regulators of post-transcriptional repression of messenger RNA. We report the identification and characterization of a conserved miRNA and 171 novel miRNAs in the migratory rice pest Sogatella furcifera by deep sequencing, which were observed to be biased towards female adults of the insect, modulating the functionality and targets of the miRNAs in sex differentiation. A switch in arm usage was also observed in 9 miRNA when compared to the insect ancestor during insect evolution. The miRNA loci showed high 5' fidelity in both miRNA and star species and about 93.4% of WBPH miRNAs conserved within non-planthopper species were homologous with planthopper species. The novel miRNAs identified in this study provide a better understanding of the sRNA and the regulatory role of miRNA in sexual dimorphism and alteration in the expression or function of miRNAs in the rice pest.

Genome characterization of the newly identified maize-associated totivirus Anhui.

Maize-associated totivirus Anhui (MATV-Ah) is a novel totivirus with a 5536-nt genome and two large ORFs that encode a putative coat protein (CP) and an RNA-dependent RNA polymerase (RdRP). The two ORFs share amino acid identities of 32 and 56% when compared to other plant-associated totiviruses, respectively. Based on genome sequence similarity and phylogenetic analysis, MATV-Ah is proposed to be a member of the family Totiviridae genus Totivirus.

Genome sequence of a rice pest, the white-backed planthopper (Sogatella furcifera).

Background: Sogatella furcifera is an important phloem sap-sucking and plant virus-transmitting migratory insect of rice. Because of its high reproductive potential, dispersal capability and transmission of plant viral diseases, S. furcifera causes considerable damage to rice grain production and has great economical and agricultural impacts. Comprehensive studies into ecological aspects and virus-host interactions of S. furcifera have been limited because of the lack of a well-assembled genome sequence. Findings: A total of 241.3 Gb of raw reads from the whole genome of S. furcifera were generated by Illumina sequencing using different combinations of mate-pair and paired-end libraries from 17 insert libraries ranging between 180 bp and 40 kbp. The final genome assembly (0.72 Gb), with average N50 contig size of 70.7 kb and scaffold N50 of 1.18 Mb, covers 98.6 % of the estimated genome size of S. furcifera . Genome annotation, assisted by eight different developmental stages (embryos, 1 st -5 th instar nymphs, 5-day-old adults and 10-day-old adults), generated 21 254 protein-coding genes, which captured 99.59 % (247/248) of core CEGMA genes and 91.7 % (2453/2675) of BUSCO genes. Conclusions: We report the first assembled and annotated whole genome sequence and transcriptome of S. furcifera . The assembled draft genome of S. furcifera will be a valuable resource for ecological and virus-host interaction studies of this pest.

Understanding the immune system architecture and transcriptome responses to southern rice black-streaked dwarf virus in Sogatella furcifera.

Sogatella furcifera, the white-backed planthopper (WBPH), has become one of the most destructive pests in rice production owing to its plant sap-sucking behavior and efficient transmission of Southern rice black-streaked dwarf virus (SRBSDV) in a circulative, propagative and persistent manner. The dynamic and complex SRBSDV-WBPH-rice plant interaction is still poorly understood. In this study, based on a homology-based genome-wide analysis, 348 immune-related genes belonging to 28 families were identified in WBPH. A transcriptome analysis of non-viruliferous (NVF) and viruliferous groups with high viral titers (HVT) and median viral titers (MVT) revealed that feeding on SRBSDV-infected rice plants has a significant impact on gene expression, regardless of viral titers in insects. We identified 278 up-regulated and 406 down-regulated genes shared among the NVF, MVT, and HVT groups and detected significant down-regulation of primary metabolism-related genes and oxidoreductase. In viruliferous WBPH with viral titer-specific transcriptome changes, 1,906 and 1,467 genes exhibited strict monotonically increasing and decreasing expression, respectively. The RNAi pathway was the major antiviral response to increasing viral titers among diverse immune responses. These results clarify the responses of immune genes and the transcriptome of WBPH to SRBSDV and improve our knowledge of the functional relationship between pathogen, vector, and host.

Ecogenomic survey of plant viruses infecting Tobacco by Next generation sequencing.

BACKGROUND: The invasion of plant by viruses cause major damage to plants and reduces crop yield and integrity. Devastating plant virus infection has been experienced at different times all over the world, which are attributed to different events of mutation, re-assortment and recombination occurring in the viruses. Strategies for proper virus management has been mostly limited to eradicating the vectors that spreads the plant viruses. However, development of prompt and effective diagnostic methods are required to monitor emerging and re-emerging diseases that may be symptomatic or asymptomatic in the plant as well as the genetic variation and evolution in the plant viruses. A survey of plant viruses infecting field-grown Tobacco crop was conducted in Anhui Province of China by the deep sequencing of sRNAs. METHODS: Survey of plant viruses infecting Tobacco was carried based on 104 samples collected across the province. Nine different sRNA libraries was prepared and custom-made bioinformatics pipeline coupled with molecular techniques was developed to sequence, assemble and analyze the siRNAs for plant virus discovery. We also carried out phylogenetic and recombination analysis of the identified viruses. RESULTS: Twenty two isolates from eight different virus species including Cucumber mosaic virus, Potato virus Y, Tobacco mosaic virus, Tobacco vein banding Mosaic virus, Pepper mottle virus, Brassica yellow virus, Chilli venial mottle virus, Broad bean wilt virus 2 were identified in tobacco across the survey area. The near-complete genome sequence of the 22 new isolates were determined and analyzed. The isolates were grouped together with known strains in the phylogenetic tree. Molecular variation in the isolates indicated the conserved coding regions have majorly a nucleotide sequence identity of 80-94 % with previously identified isolates. Various events of recombination were discovered among some of the isolates indicating that two or more viruses or different isolates of one virus infect the same host cell. CONCLUSION: This study describes the discovery of a consortium of plant viruses infecting Tobacco that are broadly distributed in Anhui province of China. It also demonstrates the effectiveness of NGS in identifying plant viruses without a prior knowledge of the virus and the genetic diversity that enhanced mixed infection.

Identification and characterization of microRNAs in the white-backed planthopper, Sogatella furcifera.

MicroRNAs (miRNAs) are a novel class of small, non-coding endogenous RNAs that play critical regulatory roles in many metabolic activities in eukaryotes. Reports of the identification of miRNAs in Sogatella furcifera (white-backed planthopper), the insect that acts as the only confirmed vector of the southern rice black-streaked dwarf virus (SRBSDV), are limited. In this study, a total of 382 miRNAs were identified in S. furcifera, including 106 conserved and 276 novel miRNAs, using high-throughput sequencing based on two small RNA libraries from viruliferous and non-viruliferous S. furcifera, and these miRNAs belonged to 52 conserved miRNA families and 58 S. furcifera-specific families, respectively. Comparison with miRNAs from 26 insect species and five other species in miRBase showed that more than half of the conserved miRNA families are highly conserved in Hexapoda, while other miRNAs are only conserved in non-dipterans. Furthermore, 4 117 target genes predicted for the 382 identified miRNAs could be categorized into 45 functional groups annotated by Gene Ontology. Compared with non-viruliferous cells, eight up-regulated miRNAs and four down-regulated miRNAs were identified in cells inoculated with SRBSDV, among which miR-14 and miR-n98a may be involved in the immune response to SRBSDV infection. Analyses of the identified miRNAs will provide insights into the roles of these miRNAs in the regulation and expression of genes involved in the metabolism, development and viral infection of S. furcifera.

Complete genome sequence of tobacco virus 1, a closterovirus from Nicotiana tabacum.

The complete genome sequence of a novel virus, provisionally named tobacco virus 1 (TV1), was determined, and this virus was identified in leaves of tobacco (Nicotiana tabacum) exhibiting leaf mosaic and yellowing symptoms in Anhui Province, China. The genome sequence of TV1 consists of 15,395 nucleotides with 61.6 % nucleotide sequence identity to mint virus 1 (MV1). Its genome organization is similar to that of MV1, containing nine open reading frames (ORFs) that potentially encode proteins with putative functions in virion assembly, cell-to-cell movement and suppression of RNA silencing. Phylogenetic analysis of the heat shock protein 70 homolog (HSP70h) placed TV1 alongside members of the genus Closterovirus in the family Closteroviridae. To our knowledge, this study is the first report of the complete genome sequence of a closterovirus identified in tobacco.

Complete genome sequence of a novel velarivirus infecting areca palm in China.

The complete genome of a novel virus, provisionally named areca palm velarivirus 1 (APV1), was identified in areca palm exhibiting leaf yellowing symptoms in Hainan province, China. The genome of APV1 consists of 16,080 nucleotides and possesses 11 open reading frames (ORFs), sharing 56.4% nucleotide sequence identity with little cherry virus 1 (NC_001836.1). The genome organization of APV1 is highly similar to that of members of the genus Velarivirus (family Closteroviridae). Phylogenetic analysis placed APV1 together with members of the genus Velarivirus.