Growth Transformation of B Cells by Epstein-Barr Virus Requires IMPDH2 Induction and Nucleolar Hypertrophy.

The in vitro growth transformation of primary B cells by Epstein-Barr virus (EBV) is the initial step in the development of posttransplant lymphoproliferative disorder (PTLD). We performed electron microscopic analysis and immunostaining of primary B cells infected with wild-type EBV. Interestingly, the nucleolar size was increased by two days after infection. A recent study found that nucleolar hypertrophy, which is caused by the induction of the IMPDH2 gene, is required for the efficient promotion of growth in cancers. In the present study, RNA-seq revealed that the IMPDH2 gene was significantly induced by EBV and that its level peaked at day 2. Even without EBV infection, the activation of primary B cells by the CD40 ligand and interleukin-4 increased IMPDH2 expression and nucleolar hypertrophy. Using EBNA2 or LMP1 knockout viruses, we found that EBNA2 and MYC, but not LMP1, induced the IMPDH2 gene during primary infections. IMPDH2 inhibition by mycophenolic acid (MPA) blocked the growth transformation of primary B cells by EBV, leading to smaller nucleoli, nuclei, and cells. Mycophenolate mofetil (MMF), which is a prodrug of MPA that is approved for use as an immunosuppressant, was tested in a mouse xenograft model. Oral MMF significantly improved the survival of mice and reduced splenomegaly. Taken together, these results indicate that EBV induces IMPDH2 expression through EBNA2-dependent and MYC-dependent mechanisms, leading to the hypertrophy of the nucleoli, nuclei, and cells as well as efficient cell proliferation. Our results provide basic evidence that IMPDH2 induction and nucleolar enlargement are crucial for B cell transformation by EBV. In addition, the use of MMF suppresses PTLD. IMPORTANCE EBV infections cause nucleolar enlargement via the induction of IMPDH2, which are essential for B cell growth transformation by EBV. Although the significance of IMPDH2 induction and nuclear hypertrophy in the tumorigenesis of glioblastoma has been reported, EBV infection brings about the change quickly by using its transcriptional cofactor, EBNA2, and MYC. Moreover, we present here, for the novel, basic evidence that an IMPDH2 inhibitor, namely, MPA or MMF, can be used for EBV-positive posttransplant lymphoproliferative disorder (PTLD).

Cellular APOBEC3A deaminase drives mutations in the SARS-CoV-2 genome.

The number of genetic variations in the SARS-CoV-2 genome has been increasing primarily due to continuous viral mutations. Here, we report that the human APOBEC3A (A3A) cytidine deaminase plays a critical role in the induction of C-to-U substitutions in the SARS-CoV-2 genome. Bioinformatic analysis of the chronological genetic changes in a sequence database indicated that the largest UC-to-UU mutation signature, consistent with APOBEC-recognized nucleotide motifs, was predominant in single-stranded RNA regions of the viral genome. In SARS-CoV-2-infected cells, exogenous expression of A3A but not expression of other APOBEC proteins induced UC-to-UU mutations in viral RNA (vRNA). Additionally, the mutated C bases were often located at the tips in bulge or loop regions in the vRNA secondary structure. Interestingly, A3A mRNA expression was drastically increased by interferons (IFNs) and tumour necrosis factor-α (TNF-α) in epithelial cells derived from the respiratory system, a site of efficient SARS-CoV-2 replication. Moreover, the UC-to-UU mutation rate was increased in SARS-CoV-2 produced from lung epithelial cells treated with IFN-ß and TNF-α, but not from CRISPR/Cas9-based A3A knockout cells. Collectively, these findings demonstrate that A3A is a primary host factor that drives mutations in the SARS-CoV-2 RNA genome via RNA editing.

The Effect of Maternal Coagulation Parameters on Fetal Acidemia in Placental Abruption.

This study aimed to identify factors predicting the probability of serious fetal acidemia at delivery in placental abruption. We identified 5769 women who delivered at >22 weeks' gestation at two institutions in a tertiary referral unit specializing in neonatal infant care between January 2007 and December 2011. Ninety-one abruption cases were identified based on clinical and histological diagnoses. Serious fetal acidemia was defined as a pH < 7.0 in the umbilical arterial blood at delivery. Using a linear discriminant function, we calculated the score to determine the probability of serious fetal acidemia. Serious fetal acidemia was observed in 34 patients (37.4%). A logistic regression model showed that abnormal fetal heart rate patterns (bradycardia and late decelerations), uterine spasm, and maternal plasma concentration of fibrinogen less than 288 ng/dL were significantly associated with the occurrence of serious fetal acidemia. We suggest that the implementation of maternal fibrinogen in patients with placental abruption is a prognostic factor for serious fetal acidemia at delivery.

Older Patients and Patients with Severe Arteriosclerosis Are Less Likely to Develop Keloids and Hypertrophic Scars after Thoracic Midline Incision: A Survey-Based Analysis of 328 Cases.

BACKGROUND: Surgery is a well-known trigger of keloid and hypertrophic scarring. Sternotomy scars are subject to high skin tension, which is known to promote pathologic scarring. This suggests that sternotomies in adults are associated with high pathologic scarring rates, which aligns with the authors' anecdotal experience. However, this notion has never been examined formally. Therefore, the authors conducted a survey-based cohort study of patients who had undergone a sternotomy. METHODS: All consecutive Japanese adults (18 years of age or older) who underwent cardiovascular surgery with sternotomy in 2014 to 2017 were identified in 2019 by chart review and sent a questionnaire. Respondents formed the study cohort. The questionnaire presented randomly ordered photographs of representative mature, keloid, and hypertrophic scars and asked the patients to choose the image that best resembled their midline scar when it was particularly noticeable. The incidence of self-reported pathologic scarring (keloids and hypertrophic scars were grouped together) and the patient demographic (age and sex) and clinical characteristics (intima-media thickness of the left and right common and internal carotid arteries) that were associated with pathologic scarring were determined. RESULTS: Of the 548 patients who underwent sternotomy, 328 responded for a 60 percent response rate. The mean patient age was 67 years, and 68.0 percent were male. Of these patients, 195 (59.5 percent) reported they had a pathologic scar. Compared with patients who had a mature scar, patients who had a pathologic scar had younger mean age (65 versus 69 years; p = 0.0002) and lower intima-media thickness (0.92 versus 1.05 mm; p = 0.028). CONCLUSIONS: Sternotomy was associated with a high rate of pathologic scarring. Older age and arteriosclerosis were associated with less pathologic scarring. CLINICAL QUESTION/LEVEL OF EVIDENCE: Risk, III.

Nanopore Sequencing for Characterization of HIV-1 Recombinant Forms.

High genetic diversity, including the emergence of recombinant forms (RFs), is one of the most prominent features of human immunodeficiency virus type 1 (HIV-1). Conventional detection of HIV-1 RFs requires pretreatments, i.e., cloning or single-genome amplification, to distinguish them from dual- or multiple-infection variants. However, these processes are time-consuming and labor-intensive. Here, we constructed a new nanopore sequencing-based platform that enables us to obtain distinctive genetic information for intersubtype RFs and dual-infection HIV-1 variants by using amplicons of HIV-1 near-full-length genomes or two overlapping half-length genome fragments. Repeated benchmark tests of HIV-1 proviral DNA revealed consensus sequence inference with a reduced error rate, allowing us to obtain sufficiently accurate sequence data. In addition, we applied the platform for sequence analyses of 9 clinical samples with suspected HIV-1 RF infection or dual infection according to Sanger sequencing-based genotyping tests for HIV-1 drug resistance. For each RF infection case, replicated analyses involving our nanopore sequencing-based platform consistently produced long consecutive analogous consensus sequences with mosaic genomic structures consisting of two different subtypes. In contrast, we detected multiple heterologous sequences in each dual-infection case. These results demonstrate that our new nanopore sequencing platform is applicable to identify the full-length HIV-1 genome structure of intersubtype RFs as well as dual-infection heterologous HIV-1. Since the genetic diversity of HIV-1 continues to gradually increase, this system will help accelerate full-length genome analysis and molecular epidemiological surveillance for HIV-1. IMPORTANCE HIV-1 is characterized by large genetic differences, including HIV-1 recombinant forms (RFs). Conventional genetic analyses require time-consuming pretreatments, i.e., cloning or single-genome amplification, to distinguish RFs from dual- or multiple-infection cases. In this study, we developed a new analytical system for HIV-1 sequence data obtained by nanopore sequencing. The error rate of this method was reduced to ~0.06%. We applied this system for sequence analyses of 9 clinical samples with suspected HIV-1 RF infection or dual infection, which were extracted from 373 cases of HIV patients based on our retrospective analysis of HIV-1 drug resistance genotyping test results. We found that our new nanopore sequencing platform is applicable to identify the full-length HIV-1 genome structure of intersubtype RFs as well as dual-infection heterologous HIV-1. Our protocol will be useful for epidemiological surveillance to examine HIV-1 transmission as well as for genotypic tests of HIV-1 drug resistance in clinical settings.

Replication Compartments-The Great Survival Strategy for Epstein-Barr Virus Lytic Replication.

During Epstein-Barr virus (EBV) lytic replication, viral DNA synthesis is carried out in viral replication factories called replication compartments (RCs), which are located at discrete sites in the nucleus. Viral proteins constituting the viral replication machinery are accumulated in the RCs to amplify viral genomes. Newly synthesized viral DNA is stored in a subdomain of the RC termed the BMRF1-core, matured by host factors, and finally packed into assembled viral capsids. Late (L) genes are transcribed from DNA stored in the BMRF1-core through a process that is mainly dependent on the viral pre-initiation complex (vPIC). RC formation is a well-regulated system and strongly advantageous for EBV survival because of the following aspects: (1) RCs enable the spatial separation of newly synthesized viral DNA from the cellular chromosome for protection and maturation of viral DNA; (2) EBV-coded proteins and their interaction partners are recruited to RCs, which enhances the interactions among viral proteins, cellular proteins, and viral DNA; (3) the formation of RCs benefits continuous replication, leading to L gene transcription; and (4) DNA storage and maturation leads to efficient progeny viral production. Here, we review the state of knowledge of this important viral structure and discuss its roles in EBV survival.

Molecular epidemiological features of SARS-CoV-2 in Japan, 2020-1.

There were five epidemic waves of coronavirus disease 2019 in Japan between 2020 and 2021. It remains unclear how the domestic waves arose and abated. To better understand this, we analyzed the pangenomic sequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and characterized the molecular epidemiological features of the five epidemic waves in Japan. In this study, we performed deep sequencing to determine the pangenomic SARS-CoV-2 sequences of 1,286 samples collected in two cities far from each other, Tokyo Metropolis and Nagoya. Then, the spatiotemporal genetic changes of the obtained sequences were compared with the sequences available in the Global Initiative on Sharing All Influenza Data (GISAID) database. A total of 873 genotypes carrying different sets of mutations were identified in the five epidemic waves. Phylogenetic analysis demonstrated that sharp displacements of lineages and genotypes occurred between consecutive waves over the 2 years. In addition, a wide variety of genotypes were observed in the early half of each wave, whereas a few genotypes were detected across Japan during an entire wave. Phylogenetically, putative descendant genotypes observed late in each wave displayed regional clustering and evolution in Japan. The genetic diversity of SARS-CoV-2 displayed uneven dynamics during each epidemic wave in Japan. Our findings provide an important molecular epidemiological basis to aid in controlling future SARS-CoV-2 epidemics.

SARS-CoV-2 accessory protein ORF8 is secreted extracellularly as a glycoprotein homodimer.

ORF8 is an accessory protein encoded by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Consensus regarding the biological functions of ORF8 is lacking, largely because the fundamental characteristics of this protein in cells have not been determined. To clarify these features, we herein established an ORF8 expression system in 293T cells. Using this system, approximately 41% of the ORF8 expressed in 293T cells were secreted extracellularly as a glycoprotein homodimer with inter/intramolecular disulfide bonds. Intracellular ORF8 was sensitive to the glycosidase Endo H, whereas the secreted portion was Endo-H-resistant, suggesting that secretion occurs via a conventional pathway. Additionally, immunoblotting analysis showed that the total amounts of the major histocompatibility complex class Ι (MHC-I), angiotensin-converting enzyme 2 (ACE2), and SARS-CoV-2 spike (CoV-2 S) proteins coexpressed in cells were not changed by the increased ORF8 expression, although FACS analysis revealed that the expression of the cell surface MHC-I protein, but not that of ACE2 and CoV-2 S proteins, was reduced by ORF8 expression. Finally, we demonstrate by RNA-seq analysis that ORF8 had no significant stimulatory effects in human primary monocyte-derived macrophages (MDMs). Taken together, our results provide fundamental evidence that the ORF8 glycoprotein acts as a secreted homodimer, and its functions are likely associated with the intracellular transport and/or extracellular signaling in SARS-CoV-2 infection.

Soil propagule banks of ectomycorrhizal fungi associated with Larix cajanderi above the treeline in the Siberian Arctic.

Microbial symbionts are essential for plant niche expansion into novel habitats. Dormant propagules of ectomycorrhizal (EM) fungi are thought to play an important role in seedling establishment in invasion fronts; however, propagule bank communities above the treeline are poorly understood in the Eurasian Arctic, where treelines are expected to advance under rapid climate change. To investigate the availability of EM fungal propagules, we collected 100 soil samples from Arctic tundra sites and applied bioassay experiments using Larix cajanderi as bait seedlings. We detected 11 EM fungal operational taxonomic units (OTUs) by obtaining entire ITS regions. Suillus clintonianus was the most frequently observed OTU, followed by Cenococcum geophilum and Sebacinales OTU1. Three Suillus and one Rhizopogon species were detected in the bioassay seedlings, indicating the availability of Larix-specific suilloid spores at least 30 km from the contemporary treeline. Spores of S. clintonianus and S. spectabilis remained infective after preservation for 14 mo and heat treatment at 60 °C, implying the durability of the spores. Long-distance dispersal capability and spore resistance to adverse conditions may represent ecological strategies employed by suilloid fungi to quickly associate with emerging seedlings of compatible hosts in treeless habitats.

Squamous Cell Carcinoma of Unknown Primary that Metastasized to an Inguinal Lymph Node: A Case Report.

Carcinoma of unknown primary (CUP) is a malignant tumor with histological characteristics indicating metastasis in a patient with an unidentified primary lesion after whole-body evaluation at the time of examination. CUP incidence is similar in men and women, and average age at diagnosis is 60 years. Reports of overall incidence vary but CUP is believed to account for 1-5% of all cancers. We encountered a case of apparently metastatic squamous cell carcinoma of the inguinal region in a patient without a detectable primary lesion. We report this case and review the literature on CUP, to increase awareness of this rare lesion.

Molecular Basis of Epstein-Barr Virus Latency Establishment and Lytic Reactivation.

Epstein-Barr virus (EBV) is a causative agent of infectious mononucleosis and several types of cancer. Like other herpesviruses, it establishes an asymptomatic, life-long latent infection, with occasional reactivation and shedding of progeny viruses. During latency, EBV expresses a small number of viral genes, and exists as an episome in the host-cell nucleus. Expression patterns of latency genes are dependent on the cell type, time after infection, and milieu of the cell (e.g., germinal center or peripheral blood). Upon lytic induction, expression of the viral immediate-early genes, BZLF1 and BRLF1, are induced, followed by early gene expression, viral DNA replication, late gene expression, and maturation and egress of progeny virions. Furthermore, EBV reactivation involves more than just progeny production. The EBV life cycle is regulated by signal transduction, transcription factors, promoter sequences, epigenetics, and the 3D structure of the genome. In this article, the molecular basis of EBV latency establishment and reactivation is summarized.

Infantile Hemangiomas Cleared by Combined Therapy With Pulsed Dye Laser and Propranolol.

BACKGROUND: Oral propranolol is the first-line therapy for infantile hemangioma. Combining it with pulse dye laser (PDL) (595nm-long PDL) could reduce treatment duration and sequelae incidence and severity. OBJECTIVE: To determine the effect of PDL-propranolol treatment on duration to cure and sequelae. METHODS: All consecutive patients with infantile hemangioma who were cured by PDL-propranolol treatment were identified. RESULTS: In the 27 cases, average age at treatment start was 4.3 ± 3.8 months, mean tumor diameter was 11.1 ± 14.0 cm2, and tumor-type was most common (72.4% of lesions). The patients received 9.8 ± 10.5 PDL sessions. After ensuring patients had no physical contraindications, including heart disease, oral propranolol was started at 1 mg/kg/d, increased up to 3 mg/kg/d as a maintenance dose. Mean propranolol treatment duration was 11.1 ± 4.9 months. Total treatment duration was 15.3 ± 10.8 months. CONCLUSION: Our data in the context of recent literature suggest combining propranolol with PDL may reduce propranolol duration without increasing harms.

The FAT10 post-translational modification is involved in the lytic replication of Kaposi's sarcoma-associated herpesvirus.

During Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication, host cell functions including protein expression and post-translational modification pathways are dysregulated by KSHV to promote virus production. Here, we attempted to identify key proteins for KSHV lytic replication by profiling protein expression in the latent and lytic phases using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Proteomic analysis, immunoblotting, and quantitative PCR demonstrated that antigen-F (HLA-F) adjacent transcript 10 (FAT10) and UBE1L2 (also known as ubiquitin-like modifier-activating enzyme 6, UBA6) were upregulated during lytic replication. FAT10 is a ubiquitin-like protein (UBL). UBE1L2 is the FAT10-activating enzyme (E1), which is essential for FAT10 modification (FAT10ylation). FAT10ylated proteins were immediately expressed after lytic induction and increased over time during lytic replication. Knockout of UBE1L2 suppressed KSHV production but not KSHV DNA synthesis. In order to isolate FAT10ylated proteins during KSHV lytic replication, we conducted immunoprecipitations using anti-FAT10 antibody and Ni-NTA chromatography of exogenously expressed His-tagged FAT10 from cells undergoing latent or lytic replication. LC-MS/MS was performed to identify FAT10ylated proteins. We identified KSHV ORF59 and ORF61 as FAT10ylation substrates. Our study revealed that the UBE1L2-FAT10 system is upregulated during KSHV lytic replication, and it contributes to viral propagation.ImportanceUbiquitin and UBL post-translational modifications, including FAT10, are utilized and dysregulated by viruses for achievement of effective infection and virion production. The UBE1L2-FAT10 system catalyzes FAT10ylation, where one or more FAT10 molecules are covalently linked to a substrate. FAT10ylation is catalyzed by the sequential actions of E1 (activation enzyme), E2 (conjugation enzyme), and E3 (ligase) enzymes. The E1 enzyme for FAT10ylation is UBE1L2, which activates FAT10 and transfers it to E2/USE1. FAT10ylation regulates the cell cycle, IFN signaling, and protein degradation; however, its primary biological function remains unknown. Here, we revealed that KSHV lytic replication induces UBE1L2 expression and production of FAT10ylated proteins including KSHV lytic proteins. Moreover, UBE1L2 knockout suppressed virus production during the lytic cycle. This is the first report demonstrating the contribution of the UBE1L2-FAT10 system to KSHV lytic replication. Our findings provide insight into the physiological function(s) of novel post-translational modifications in KSHV lytic replication.

MG132 exerts anti-viral activity against HSV-1 by overcoming virus-mediated suppression of the ERK signaling pathway.

Herpes simplex virus 1 (HSV-1) causes a number of clinical manifestations including cold sores, keratitis, meningitis and encephalitis. Although current drugs are available to treat HSV-1 infection, they can cause side effects such as nephrotoxicity. Moreover, owing to the emergence of drug-resistant HSV-1 strains, new anti-HSV-1 compounds are needed. Because many viruses exploit cellular host proteases and encode their own viral proteases for survival, we investigated the inhibitory effects of a panel of protease inhibitors (TLCK, TPCK, E64, bortezomib, or MG132) on HSV-1 replication and several host cell signaling pathways. We found that HSV-1 infection suppressed c-Raf-MEK1/2-ERK1/2-p90RSK signaling in host cells, which facilitated viral replication. The mechanism by which HSV-1 inhibited ERK signaling was mediated through the polyubiquitination and proteasomal degradation of Ras-guanine nucleotide-releasing factor 2 (Ras-GRF2). Importantly, the proteasome inhibitor MG132 inhibited HSV-1 replication by reversing ERK suppression in infected cells, inhibiting lytic genes (ICP5, ICP27 and UL42) expression, and overcoming the downregulation of Ras-GRF2. These results indicate that the suppression of ERK signaling via proteasomal degradation of Ras-GRF2 is necessary for HSV-1 infection and replication. Given that ERK activation by MG132 exhibits anti-HSV-1 activity, these results suggest that the proteasome inhibitor could serve as a novel therapeutic agent against HSV-1 infection.

Excessive positive response of model-simulated land net primary production to climate changes over circumboreal forests.

Land carbon cycle components in an Earth system model (ESM) play a crucial role in the projections of forest ecosystem responses to climate/environmental changes. Evaluating models from the viewpoint of observations is essential for an improved understanding of model performance and for identifying uncertainties in their outputs. Herein, we evaluated the land net primary production (NPP) for circumboreal forests simulated with 10 ESMs in Phase 5 of the Coupled Model Intercomparison Project by comparisons with observation-based indexes for forest productivity, namely, the composite version 3G of the normalized difference vegetation index (NDVI3g) and tree-ring width index (RWI). These indexes show similar patterns in response to past climate change over the forests, i.e., a one-year time lag response and smaller positive responses to past climate changes in comparison with the land NPP simulated by the ESMs. The latter showed overly positive responses to past temperature and/or precipitation changes in comparison with the NDVI3g and RWI. These results indicate that ESMs may overestimate the future forest NPP of circumboreal forests (particularly for inland dry regions, such as inner Alaska and Canada, and eastern Siberia, and for hotter, southern regions, such as central Europe) under the expected increases in both average global temperature and precipitation, which are common to all current ESMs.

Isotopic compositions of ground ice in near-surface permafrost in relation to vegetation and microtopography at the Taiga-Tundra boundary in the Indigirka River lowlands, northeastern Siberia.

The warming trend in the Arctic region is expected to cause drastic changes including permafrost degradation and vegetation shifts. We investigated the spatial distribution of ice content and stable isotopic compositions of water in near-surface permafrost down to a depth of 1 m in the Indigirka River lowlands of northeastern Siberia to examine how the permafrost conditions control vegetation and microtopography in the Taiga-Tundra boundary ecosystem. The gravimetric water content (GWC) in the frozen soil layer was significantly higher at microtopographically high elevations with growing larch trees (i.e., tree mounds) than at low elevations with wetland vegetation (i.e., wet areas). The observed ground ice (ice-rich layer) with a high GWC in the tree mounds suggests that the relatively elevated microtopography of the land surface, which was formed by frost heave, strongly affects the survival of larch trees. The isotopic composition of the ground ice indicated that equilibrium isotopic fractionation occurred during ice segregation at the tree mounds, which implies that the ice formed with sufficient time for the migration of unfrozen soil water to the freezing front. In contrast, the isotopic data for the wet areas indicated that rapid freezing occurred under relatively non-equilibrium conditions, implying that there was insufficient time for ice segregation to occur. The freezing rate of the tree mounds was slower than that of the wet areas due to the difference of such as soil moisture and snow cover depends on vegetation and microtopography. These results indicate that future changes in snow cover, soil moisture, and organic layer, which control underground thermal conductivity, will have significant impacts on the freezing environment of the ground ice at the Taiga-Tundra boundary in northeastern Siberia. Such changes in the freezing environment will then affect vegetation due to changes in the microtopography of the ground surface.

First Days in the Life of Naive Human B Lymphocytes Infected with Epstein-Barr Virus.

Epstein-Barr virus (EBV) infects and activates resting human B lymphocytes, reprograms them, induces their proliferation, and establishes a latent infection in them. In established EBV-infected cell lines, many viral latent genes are expressed. Their roles in supporting the continuous proliferation of EBV-infected B cells in vitro are known, but their functions in the early, prelatent phase of infection have not been investigated systematically. In studies during the first 8 days of infection using derivatives of EBV with mutations in single genes of EBVs, we found only Epstein-Barr nuclear antigen 2 (EBNA2) to be essential for activating naive human B lymphocytes, inducing their growth in cell volume, driving them into rapid cell divisions, and preventing cell death in a subset of infected cells. EBNA-LP, latent membrane protein 2A (LMP2A), and the viral microRNAs have supportive, auxiliary functions, but mutants of LMP1, EBNA3A, EBNA3C, and the noncoding Epstein-Barr virus with small RNA (EBERs) had no discernible phenotype compared with wild-type EBV. B cells infected with a double mutant of EBNA3A and 3C had an unexpected proliferative advantage and did not regulate the DNA damage response (DDR) of the infected host cell in the prelatent phase. Even EBNA1, which has very critical long-term functions in maintaining and replicating the viral genomic DNA in established cell lines, was dispensable for the early activation of infected cells. Our findings document that the virus dose is a decisive parameter and indicate that EBNA2 governs the infected cells initially and implements a strictly controlled temporal program independent of other viral latent genes. It thus appears that EBNA2 is sufficient to control all requirements for clonal cellular expansion and to reprogram human B lymphocytes from energetically quiescent to activated cells.IMPORTANCE The preferred target of Epstein-Barr virus (EBV) is human resting B lymphocytes. We found that their infection induces a well-coordinated, time-driven program that starts with a substantial increase in cell volume, followed by cellular DNA synthesis after 3 days and subsequent rapid rounds of cell divisions on the next day accompanied by some DNA replication stress (DRS). Two to 3 days later, the cells decelerate and turn into stably proliferating lymphoblast cell lines. With the aid of 16 different recombinant EBV strains, we investigated the individual contributions of EBV's multiple latent genes during early B-cell infection and found that many do not exert a detectable phenotype or contribute little to EBV's prelatent phase. The exception is EBNA2 that is essential in governing all aspects of B-cell reprogramming. EBV relies on EBNA2 to turn the infected B lymphocytes into proliferating lymphoblasts preparing the infected host cell for the ensuing stable, latent phase of viral infection. In the early steps of B-cell reprogramming, viral latent genes other than EBNA2 are dispensable, but some, EBNA-LP, for example, support the viral program and presumably stabilize the infected cells once viral latency is established.

Epstein-Barr virus genome packaging factors accumulate in BMRF1-cores within viral replication compartments.

Productive replication of Epstein-Barr virus (EBV) during the lytic cycle occurs in discrete sites within nuclei, termed replication compartments. We previously proposed that replication compartments consist of two subnuclear domains: "ongoing replication foci" and "BMRF1-cores". Viral genome replication takes place in ongoing replication foci, which are enriched with viral replication proteins, such as BALF5 and BALF2. Amplified DNA and BMRF1 protein accumulate in BMRF1-cores, which are surrounded by ongoing replication foci. We here determined the locations of procapsid and genome-packaging proteins of EBV via three-dimensional (3D) surface reconstruction and correlative fluorescence microscopy-electron microscopy (FM-EM). The results revealed that viral factors required for DNA packaging, such as BGLF1, BVRF1, and BFLF1 proteins, are located in the innermost subdomains of the BMRF1-cores. In contrast, capsid structural proteins, such as BBRF1, BORF1, BDLF1, and BVRF2, were found both outside and inside the BMRF1-cores. Based on these observations, we propose a model in which viral procapsids are assembled outside the BMRF1-cores and subsequently migrate therein, where viral DNA encapsidation occurs. To our knowledge, this is the first report describing capsid assembly sites in relation to EBV replication compartments.

Effect of floods on the δ13C values in plant leaves: a study of willows in Northeastern Siberia.

Although stable carbon isotopic composition (δ13C) of plants has been widely used to indicate different water regimes in terrestrial ecosystems over the past four decades, the changes in the plant δ13C value under waterlogging have not been sufficiently clarified. With the enhanced global warming in recent years, the increasing frequency and severity of river floods in Arctic regions lead to more waterlogging on willows that are widely distributed in river lowland. To investigate the δ13C changes in plants under different water conditions (including waterlogging), we measured the δ13C values in the leaves of willows with three species, Salix boganidensis, S. glauca, and S. pulchra, and also monitored changes in plant physiology, under several major flooding conditions in Northeastern Siberia. The foliar δ13C values of willows varied, ranging from -31.6 to -25.7‰ under the different hydrological status, which can be explained by: (i) under normal conditions, the foliar δ13C values decrease from dry (far from a river) to wet (along a river bank) areas; (ii) the δ13C values increase in frequently waterlogged areas owing to stomatal closure; and (iii) after prolonged flooding periods, the δ13C values again decrease, probably owing to the effects of not only the closure of stomata but also the reduction of foliar photosynthetic ability under long period of waterlogging. Based on these results, we predict that plant δ13C values are strongly influenced by plant physiological responses to diverse hydrological conditions, particularly the long periods of flooding, as occurs in Arctic regions.

Signal Transduction Pathways Associated with KSHV-Related Tumors.

Signal transduction pathways play a key role in the regulation of cell growth, cell differentiation, cell survival, apoptosis, and immune responses. Bacterial and viral pathogens utilize the cell signal pathways by encoding their own proteins or noncoding RNAs to serve their survival and replication in infected cells. Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV-8), is classified as a rhadinovirus in the γ-herpesvirus subfamily and was the eighth human herpesvirus to be discovered from Kaposi's sarcoma specimens. KSHV is closely associated with an endothelial cell malignancy, Kaposi's sarcoma, and B-cell malignancies, primary effusion lymphoma, and multicentric Castleman's disease. Recent studies have revealed that KSHV manipulates the cellular signaling pathways to achieve persistent infection, viral replication, cell proliferation, anti-apoptosis, and evasion of immune surveillance in infected cells. This chapter summarizes recent developments in our understanding of the molecular mechanisms used by KSHV to interact with the cell signaling machinery.