Polymerase theta is a synthetic lethal target for killing Epstein-Barr virus lymphomas.

Treatment options for Epstein-Barr virus (EBV)-cancers are limited, underscoring the need for new therapeutic approaches. We have previously shown that EBV-transformed cells and cancers lack homologous recombination (HR) repair, a prominent error-free pathway that repairs double-stranded DNA breaks; instead, EBV-transformed cells demonstrate genome-wide scars of the error-prone microhomology-mediated end joining (MMEJ) repair pathway. This suggests that EBV-cancers are vulnerable to synthetic lethal therapeutic approaches that target MMEJ repair. Indeed, we have previously found that targeting PARP, an enzyme that contributes to MMEJ, results in the death of EBV-lymphoma cells. With the emergence of clinical resistance to PARP inhibitors and the recent discovery of inhibitors of Polymerase theta (POLθ), the polymerase essential for MMEJ, we investigated the role of POLθ in EBV-lymphoma cells. We report that EBV-transformed cell lines, EBV-lymphoma cell lines, and EBV-lymphomas in AIDS patients demonstrate greater abundance of POLθ, driven by the EBV protein EBNA1, compared to EBV-uninfected primary lymphocytes and EBV-negative lymphomas from AIDS patients (a group that also abundantly expresses POLθ). We also find POLθ enriched at cellular DNA replication forks and exposure to the POLθ inhibitor Novobiocin impedes replication fork progress, impairs MMEJ-mediated repair of DNA double-stranded breaks, and kills EBV-lymphoma cells. Notably, cell killing is not due to Novobiocin-induced activation of the lytic/replicative phase of EBV. These findings support a role for POLθ not just in DNA repair but also DNA replication and as a therapeutic target in EBV-lymphomas and potentially other EBV-cancers as EBNA1 is expressed in all EBV-cancers.IMPORTANCEEpstein-Barr virus (EBV) contributes to ~2% of the global cancer burden. With a recent estimate of >200,000 deaths a year, identifying molecular vulnerabilities will be key to the management of these frequently aggressive and treatment-resistant cancers. Building on our earlier work demonstrating reliance of EBV-cancers on microhomology-mediated end-joining repair, we now report that EBV lymphomas and transformed B cell lines abundantly express the MMEJ enzyme POLθ that likely protects cellular replication forks and repairs replication-related cellular DNA breaks. Importantly also, we show that a newly identified POLθ inhibitor kills EBV-cancer cells, revealing a novel strategy to block DNA replication and repair of these aggressive cancers.

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.

Development of Porcine Monoclonal Antibodies with In Vitro Neutralizing Activity against Classical Swine Fever Virus from C-Strain E2-Specific Single B Cells.

Neutralizing antibodies (nAbs) can be used before or after infection to prevent or treat viral diseases. However, there are few efficacious nAbs against classical swine fever virus (CSFV) that have been produced, especially the porcine-originated nAbs. In this study, we generated three porcine monoclonal antibodies (mAbs) with in vitro neutralizing activity against CSFV, aiming to facilitate the development of passive antibody vaccines or antiviral drugs against CSFV that offer the advantages of stability and low immunogenicity. Pigs were immunized with the C-strain E2 (CE2) subunit vaccine, KNB-E2. At 42 days post vaccination (DPV), CE2-specific single B cells were isolated via fluorescent-activated cell sorting (FACS) baited by Alexa Fluor™ 647-labeled CE2 (positive), goat anti-porcine IgG (H + L)-FITC antibody (positive), PE mouse anti-pig CD3ε (negative) and PE mouse anti-pig CD8a (negative). The full coding region of IgG heavy (H) chains and light (L) chains was amplified by reverse transcription-polymerase chain reaction (RT-PCR). Overall, we obtained 3 IgG H chains, 9 kappa L chains and 36 lambda L chains, which include three paired chains (two H + κ and one H + λ). CE2-specific mAbs were successfully expressed in 293T cells with the three paired chains. The mAbs exhibit potent neutralizing activity against CSFVs. They can protect ST cells from infections in vitro with potent IC50 values from 14.43 µg/mL to 25.98 µg/mL for the CSFV C-strain, and 27.66 µg/mL to 42.61 µg/mL for the CSFV Alfort strain. This study is the first report to describe the amplification of whole-porcine IgG genes from single B cells of KNB-E2-vaccinated pig. The method is versatile, sensitive, and reliable. The generated natural porcine nAbs can be used to develop long-acting and low-immunogenicity passive antibody vaccine or anti-CSFV agents for CSF control and prevention.

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.

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.

Inflammasome, the Constitutive Heterochromatin Machinery, and Replication of an Oncogenic Herpesvirus.

The success of long-term host-virus partnerships is predicated on the ability of the host to limit the destructive potential of the virus and the virus's skill in manipulating its host to persist undetected yet replicate efficiently when needed. By mastering such skills, herpesviruses persist silently in their hosts, though perturbations in this host-virus equilibrium can result in disease. The heterochromatin machinery that tightly regulates endogenous retroviral elements and pericentromeric repeats also silences invading genomes of alpha-, beta-, and gammaherpesviruses. That said, how these viruses disrupt this constitutive heterochromatin machinery to replicate and spread, particularly in response to disparate lytic triggers, is unclear. Here, we review how the cancer-causing gammaherpesvirus Epstein-Barr virus (EBV) uses the inflammasome as a security system to alert itself of threats to its cellular home as well as to flip the virus-encoded lytic switch, allowing it to replicate and escape in response to a variety of lytic triggers. EBV provides the first example of an infectious agent able to actively exploit the inflammasome to spark its replication. Revealing an unexpected link between the inflammasome and the epigenome, this further brings insights into how the heterochromatin machinery uses differential strategies to maintain the integrity of the cellular genome whilst guarding against invading pathogens. These recent insights into EBV biology and host-viral epigenetic regulation ultimately point to the NLRP3 inflammasome as an attractive target to thwart herpesvirus reactivation.

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.

STAT3 imparts BRCAness by impairing homologous recombination repair in Epstein-Barr virus-transformed B lymphocytes.

Epstein-Barr virus (EBV) causes lymphomas and epithelial cell cancers. Though generally silent in B lymphocytes, this widely prevalent virus can cause endemic Burkitt lymphoma and post-transplant lymphoproliferative disorders/lymphomas in immunocompromised hosts. By learning how EBV breaches barriers to cell proliferation, we hope to undermine those strategies to treat EBV lymphomas and potentially other cancers. We had previously found that EBV, through activation of cellular STAT3 prevents phosphorylation of Chk1, and thereby, suppresses activation of the intra-S phase cell-cycle checkpoint, a potent barrier to oncogene-driven proliferation. This observation prompted us to examine the consequences on DNA repair since homologous recombination repair, the most error-free form, requires phosphoChk1. We now report that the defect in Chk1 phosphorylation also curtails RAD51 nucleation, and thereby, homologous recombination repair of DNA double strand breaks. The resulting reliance on error-prone microhomology-mediated end-joining (MMEJ) repair makes EBV-transformed cells susceptible to PARP inhibition and simultaneous accrual of genome-wide deletions and insertions resulting from synthesis-dependent MMEJ. Analysis of transcriptomic and drug susceptibility data from hundreds of cancer lines reveals a STAT3-dependent gene-set predictive of susceptibility of cancers to synthetic lethal PARP inhibition. These findings i) demonstrate how the tumor virus EBV re-shapes cellular DNA repair, ii) provide the first genome-wide evidence for insertions resulting from MMEJ in human cells, and iii) expand the range of cancers (EBV-related and -unrelated) that are likely to respond to synthetic lethal inhibitors given the high prevalence of cancers with constitutively active STAT3.

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.

Novel replisome-associated proteins at cellular replication forks in EBV-transformed B lymphocytes.

Epstein-Barr virus (EBV) is an oncogenic herpesvirus and WHO class 1 carcinogen that resides in B lymphocytes of nearly all humans. While silent in most, EBV can cause endemic Burkitt lymphoma in children and post-transplant lymphoproliferative disorders/lymphomas in immunocompromised hosts. The pathogenesis of such lymphomas is multifactorial but to a large extent depends on EBV's ability to aggressively drive cellular DNA replication and B cell proliferation despite cell-intrinsic barriers to replication. One such barrier is oncogenic replication stress which hinders the progression of DNA replication forks. To understand how EBV successfully overcomes replication stress, we examined cellular replication forks in EBV-transformed B cells using iPOND (isolation of Proteins on Nascent DNA)-mass spectrometry and identified several cellular proteins that had not previously been linked to DNA replication. Of eight candidate replisome-associated proteins that we validated at forks in EBV-transformed cells and Burkitt lymphoma-derived cells, three zinc finger proteins (ZFPs) were upregulated early in B cells newly-infected with EBV in culture as well as expressed at high levels in EBV-infected B blasts in the blood of immunocompromised transplant recipients. Expressed highly in S- and G2-phase cells, knockdown of each ZFP resulted in stalling of proliferating cells in the S-phase, cleavage of caspase 3, and cell death. These proteins, newly-identified at replication forks of EBV-transformed and Burkitt lymphoma cells therefore contribute to cell survival and cell cycle progression, and represent novel targets for intervention of EBV-lymphomas while simultaneously offering a window into how the replication machinery may be similarly modified in other cancers.

Pure Hemozoin is inflammatory in vivo and activates the NALP3 inflammasome via release of uric acid.

The role of proinflammatory cytokine production in the pathogenesis of malaria is well established, but the identification of the parasite products that initiate inflammation is not complete. Hemozoin is a crystalline metabolite of hemoglobin digestion that is released during malaria infection. In the present study, we characterized the immunostimulatory activity of pure synthetic hemozoin (sHz) in vitro and in vivo. Stimulation of naive murine macrophages with sHz results in the MyD88-independent activation of NF-kappaB and ERK, as well as the release of the chemokine MCP-1; these responses are augmented by IFN-gamma. In macrophages prestimulated with IFN-gamma, sHz also results in a MyD88-dependent release of TNF-alpha. Endothelial cells, which encounter hemozoin after schizont rupture, respond to sHz by releasing IL-6 and the chemokines MCP-1 and IL-8. In vivo, the introduction of sHz into the peritoneal cavity produces an inflammatory response characterized by neutrophil recruitment and the production of MCP-1, KC, IL-6, IL-1alpha, and IL-1beta. MCP-1 and KC are produced independently of MyD88, TLR2/4 and TLR9, and components of the inflammasome; however, neutrophil recruitment, the localized production of IL-1beta, and the increase in circulating IL-6 require MyD88 signaling, the IL-1R pathway, and the inflammasome components ICE (IL-1beta-converting enzyme), ASC (apoptosis-associated, speck-like protein containing CARD), and NALP3. Of note, inflammasome activation by sHz is reduced by allopurinol, which is an inhibitor of uric acid synthesis. These data suggest that uric acid is released during malaria infection and may serve to augment the initial host response to hemozoin via activation of the NALP3 inflammasome.

Discovery of swine as a host for the Reston ebolavirus.

Since the discovery of the Marburg and Ebola species of filovirus, seemingly random, sporadic fatal outbreaks of disease in humans and nonhuman primates have given impetus to identification of host tropisms and potential reservoirs. Domestic swine in the Philippines, experiencing unusually severe outbreaks of porcine reproductive and respiratory disease syndrome, have now been discovered to host Reston ebolavirus (REBOV). Although REBOV is the only member of Filoviridae that has not been associated with disease in humans, its emergence in the human food chain is of concern. REBOV isolates were found to be more divergent from each other than from the original virus isolated in 1989, indicating polyphyletic origins and that REBOV has been circulating since, and possibly before, the initial discovery of REBOV in monkeys.

A pandemic strain of calicivirus threatens rabbit industries in the Americas.

Rabbit Hemorrhagic Disease (RHD) is a severe acute viral disease specifically affecting the European rabbit Oryctolagus cuniculus. As the European rabbit is the predominant species of domestic rabbit throughout the world, RHD contributes towards significant losses to rabbit farming industries and endangers wild populations of rabbits in Europe and other predatory animals in Europe that depend upon rabbits as a food source. Rabbit Hemorrhagic Disease virus (RHDV) - a Lagovirus belonging to the family Caliciviridae is the etiological agent of RHD. Typically, RHD presents with sudden death in 70% to 95% of infected animals. There have been four separate incursions of RHDV in the USA, the most recent of which occurred in the state of Indiana in June of 2005. Animal inoculation studies confirmed the pathogenicity of the Indiana 2005 isolate, which caused acute death and pathological changes characterized by acute diffuse severe liver necrosis and pulmonary hemorrhages. Complete viral genome sequences of all USA outbreak isolates were determined and comparative genomics revealed that each outbreak was the result of a separate introduction of virus rather than from a single virus lineage. All of the USA isolates clustered with RHDV genomes from China, and phylogenetic analysis of the major capsid protein (VP60) revealed that they were related to a pandemic antigenic variant strain known as RHDVa. Rapid spread of the RHDVa pandemic suggests a selective advantage for this new subtype. Given its rapid spread, pathogenic nature, and potential to further evolve, possibly broadening its host range to include other genera native to the Americas, RHDVa should be regarded as a threat.

Traffic to the malaria parasite food vacuole: a novel pathway involving a phosphatidylinositol 3-phosphate-binding protein.

Phosphatidylinositol 3-phosphate (PI3P) is a key ligand for recruitment of endosomal regulatory proteins in higher eukaryotes. Subsets of these endosomal proteins possess a highly selective PI3P binding zinc finger motif belonging to the FYVE domain family. We have identified a single FYVE domain-containing protein in Plasmodium falciparum which we term FCP. Expression and mutagenesis studies demonstrate that key residues are involved in specific binding to PI3P. In contrast to FYVE proteins in other organisms, endogenous FCP localizes to a lysosomal compartment, the malaria parasite food vacuole (FV), rather than to cytoplasmic endocytic organelles. Transfections of deletion mutants further indicate that FCP is essential for trophozoite and FV maturation and that it traffics to the FV via a novel constitutive cytoplasmic to vacuole targeting pathway. This newly discovered pathway excludes the secretory pathway and is directed by a C-terminal 44-amino acid peptide domain. We conclude that an FYVE protein that might be expected to participate in vesicle targeting in the parasite cytosol instead has a vital and functional role in the malaria parasite FV.

Vacuolar type H+ pumping pyrophosphatases of parasitic protozoa.

Trans-membrane proton pumping is responsible for a myriad of physiological processes including the generation of proton motive force that drives bioenergetics. Among the various proton pumping enzymes, vacuolar pyrophosphatases (V-PPases) form a distinct class of proton pumps, which are characterised by their ability to translocate protons across a membrane by using the potential energy released by hydrolysis of the phosphoanhydride bond of inorganic pyrophosphate. Until recently, V-PPases were known to be the purview of only plant vacuoles and plasma membranes of phototrophic bacteria. Recent discoveries of V-PPases in kinetoplastid and apicomplexan parasites, however, have expanded our view of the evolutionary reach of these enzymes. The lack of V-PPases in the vertebrate hosts of these parasites makes them potentially excellent targets for developing broad-spectrum antiparasitic agents. This review surveys the current understanding of V-PPases in parasitic protozoa with an emphasis on malaria parasites. Topological predictions suggest remarkable similarity of the parasite enzymes to their plant homologues with 15-16 membrane spanning domains and conserved sequences shown to constitute critical catalytic residues. Remarkably, malaria parasites have been shown to possess two V-PPase genes, one is an apparent orthologue of the canonical plant enzyme, whereas the other is a more distantly related paralogue with homology to a recently identified new class of K+-insensitive plant V-PPases. V-PPases appear to localise both to the plasma membrane and cytoplasmic organelles believed to be acidocalcisomes or polyphosphate bodies. Gene transfer experiments suggest that one of the malarial V-PPases is predominantly localised to the surface of intraerythrocytic parasites. We suggest a model in which V-PPase localised to the malaria parasite plasma membrane may serve as an electrogenic pump utilising pyrophosphate as an energy source, thus sparing the more precious ATP. Searching for V-PPase inhibitors could prove fruitful as a novel means of antiparasitic chemotherapy.