Albumin influences leucocyte FcRn expression in the early days of kidney transplantation.

FcRn, a receptor originally known for its involvement in IgG and albumin transcytosis and recycling, is also important in the establishment of the innate and adaptive immune response. Dysregulation of the immune response has been associated with variations in FcRn expression, as observed in cancer. Recently, a link between autophagy and FcRn expression has been demonstrated. Knowing that autophagy is strongly involved in the development of reperfusion injury in kidney transplantation and that albuminemia is transiently decreased in the first 2 weeks after transplantation, we investigated variations in FcRn expression after kidney transplantation. We monitored FcRn levels by flow cytometry in leukocytes from 25 renal transplant patients and considered parameters such as albumin concentrations, estimated glomerular filtration rate, serum creatinine, serum IgG levels, and ischaemia/reperfusion time. Two groups of patients could be distinguished according to their increased or non-increased FcRn expression levels between days 2 and 6 (d2-d6) post-transplantation. Leukocyte FcRn expression at d2-d6 was correlated with albumin concentrations at d0-d2. These results suggest that albumin concentrations at d0-d2 influence FcRn expression at d2-d6, raising new questions about the mechanisms underlying these original observations.

Detection of the alternative lengthening of telomeres pathway in malignant gliomas for improved molecular diagnosis.

Human malignant gliomas exhibit acquisition of either one of two telomere maintenance mechanisms, resulting from either reactivation of telomerase expression or activation of an alternative lengthening of telomeres (ALT) mechanism. In the present study, we analyzed 63 human malignant gliomas for the presence of ALT-specific extrachromosomal circles of telomeric DNA (C-circles) and measured telomerase expression, telomeric DNA content (Telo/Alu method), and telomeric repeat-containing RNAs (TERRA) levels. We also assessed histomolecular markers routinely used in clinical practice. The presence of C-circles significantly correlated with IDH1/2 mutation, MGMT exon 1 methylation, low Ki-67 immunostaining, increased telomeric DNA content, absence of functional ATRX protein and level of HTERT gene expression. In multivariate analysis, we observed a trend to a correlation between elevated TERRA levels and increased survival. Interestingly, the C-circles assay allowed to detect ALT activation in glioblastomas exhibiting wild-type IDH1/2 and ATRX expression. These results suggest that, after the correlations uncovered here have been confirmed on larger numbers of tumors, telomeric markers might be useful in improving diagnosis. They also point out to the utility of using the specific, sensitive and quantitative C-circle and Telo/Alu assays that can work with as few as 30 ng of tumor DNA.

Measurement of Telomere Length in Colorectal Cancers for Improved Molecular Diagnosis.

All tumors have in common to reactivate a telomere maintenance mechanism to allow for unlimited proliferation. On the other hand, genetic instability found in some tumors can result from the loss of telomeres. Here, we measured telomere length in colorectal cancers (CRCs) using TRF (Telomere Restriction Fragment) analysis. Telomeric DNA content was also quantified as the ratio of total telomeric (TTAGGG) sequences over that of the invariable Alu sequences. In most of the 125 CRCs analyzed, there was a significant diminution in telomere length compared with that in control healthy tissue. Only 34 tumors exhibited no telomere erosion and, in some cases, a slight telomere lengthening. Telomere length did not correlate with age, gender, tumor stage, tumor localization or stage of tumor differentiation. In addition, while telomere length did not correlate with the presence of a mutation in BRAF (V-raf murine sarcoma viral oncogene homolog B), PIK3CA (phosphatidylinositol 3-kinase catalytic subunit), or MSI status, it was significantly associated with the occurrence of a mutation in KRAS. Interestingly, we found that the shorter the telomeres in healthy tissue of a patient, the larger an increase in telomere length in the tumor. Our study points to the existence of two types of CRCs based on telomere length and reveals that telomere length in healthy tissue might influence telomere maintenance mechanisms in the tumor.

Characterization of oviduct epithelial spheroids for the study of embryo-maternal communication in cattle.

Most in vitro models of oviduct epithelial cells (OEC) used thus far to gain insights into embryo-maternal communication induce cell dedifferentiation or are technically challenging. Moreover, although the presence of developing embryos has been shown to alter gene expression in OEC, the effect of embryos on OEC physiology remains largely unknown. Here, we propose a model based on bovine oviduct epithelial spheroids (OES) with specific shape and diameter (100-200 µm) criteria. The aims of this study were to i) determine the appropriate culture conditions of bovine OES cultured in suspension by evaluating their morphology, total cell number, viability, and activity of ciliated cells; ii) monitor gene expression in OES at the time of their formation (day 0) and over the 10 days of culture; and iii) test whether the vicinity of developing embryos affects OES quality criteria. On day 10, the proportions of vesicle-shaped OES (V-OES) were higher in M199/500 (500 µl of HEPES-buffered TCM-199) and synthetic oviduct fluid (SOF)/25 (25-µL droplet of SOF medium under mineral oil) than in M199/25 (25-µL droplet of M199 under mineral oil). The proportion of viable cells in V-OES was not affected by culture conditions and remained high (>80%) through day 10. The total number of cells per V-OES decreased over time except in SOF/25, while the proportions of ciliated cells increased over time in M199/500 but decreased in M199/25 and SOF/25. The movement amplitude of OES in suspension decreased over time under all culture conditions. Moreover, the gene expression of ANXA1, ESR1, HSPA8, and HSPA1A in OES remained stable during culture, while that of PGR and OVGP1 decreased from day 0 to day 10. Last, the co-culture of developing embryos with OES in SOF/25 increased the rates of blastocysts on days 7 and 8 compared to embryos cultured alone, and increased the proportion of V-OES compared to OES cultured alone. In conclusion, M199/500 and SOF/25 provided the optimal conditions for the long-time culture of OES. The supporting effect of OES on embryo development and of developing embryos on OES morphology was evidenced for the first time. Altogether, these results point OES as an easy-to-use, standardizable, and physiological model to study embryo-maternal interactions in cattle.

The neonatal Fc receptor expression during macrophage differentiation is related to autophagy.

The neonatal Fc receptor (FcRn) plays a central role in recycling and biodistributing immunoglobulin G. FcRn is also involved in many physiological immune functions as well as pathological immune responses in cancer or autoimmune diseases. Low levels of FcRn in tumor cells and the microenvironment is associated with poor prognosis in non-small cell lung cancers. Among cells that are present in the tumor microenvironment, macrophages express high levels of FcRn. Macrophages are involved in these pathophysiological contexts by their dual differentiation states of pro- or anti-inflammatory macrophages. However, variations in FcRn protein expression have not been described in macrophage subtypes. In this work, we studied FcRn expression in an in vitro model of pro- and anti-inflammatory macrophage differentiation. We demonstrated an inverse relation between FcRn protein and mRNA expression in macrophage populations. Autophagy, which is involved in protein degradation and acquisition of phagocytic function in macrophages, participated in regulating FcRn levels. Intravenous immunoglobulin protected FcRn against autophagosome degradation in anti-inflammatory macrophages. Our data demonstrate that autophagy participates in regulating FcRn expression in pro- and anti-inflammatory macrophages. This finding raises new questions concerning the regulation of FcRn in immune functions.

In vitro recombination and inverted terminal repeat binding activities of the Mcmar1 transposase.

The Mcmar1 mariner element (MLE) presents some intriguing features with two large, perfectly conserved, 355 bp inverted terminal repeats (ITRs) containing two 28 bp direct repeats (DRs). The presence of a complete ORF in Mcmar1 makes it possible to explore the transposition of this unusual MLE. Mcmar1 transposase (MCMAR1) was purified, and in vitro transposition assays showed that it is able to promote ITR-dependent DNA cleavages and recombination events, which correspond to plasmid fusions and transpositions with imprecise ends. Further analyses indicated that MCMAR1 is able to interact with the 355 bp ITR through two DRs: the EDR (external DR) is a high-affinity binding site for MCMAR1, whereas the IDR (internal DR) is a low-affinity binding site. The main complex detected within the EDR contained a transposase dimer and only one DNA molecule. We hypothesize that the inability of MCMAR1 to promote precise in vitro transposition events could be due to mutations in its ORF sequence or to the specific features of transposase binding to the ITR. Indeed, the ITR region spanning from EDR to IDR resembles a MITE and could be bent by specific host factors. This suggests that the assembly of the transposition complex is more complex than that of those involved in the mobility of the Mos1 and Himar1 mariner elements.

Site-directed integration of transgenes: transposons revisited using DNA-binding-domain technologies.

In the last 20 years, tools derived from DNA transposons have made major contributions to genetic studies from gene delivery to gene discovery. Various complementary and fairly ubiquitous DNA vehicles have been developed. Although many transposons are efficient DNA vehicles, they appear to have limited ability to target specific sequences, since all that is required at the integration locus is the presence of a short 2- to 4-bp sequence. Consequently, insertions mediated by transposon-based vectors occur somewhat randomly. In the past 5 years, strategies have emerged to enhance the site-specificity of transposon-based vectors, and to avoid random integrations. The first proposes that new target site specificity could be grafted onto a transposase by adding a new DNA-binding domain. Alternative strategies consist of indirectly targeting either the transposase or the transposon to a chosen genomic locus. The most important information available about each strategy are presented, and limitations and future prospects are discussed.

Target site selection by the mariner-like element, Mos1.

The eukaryotic transposon Mos1 is a class-II transposable element that moves using a "cut-and-paste" mechanism in which the transposase is the only protein factor required. The formation of the excision complex is well documented, but the integration step has so far received less investigation. Like all mariner-like elements, Mos1 was thought to integrate into a TA dinucleotide without displaying any other target selection preferences. We set out to synthesize what is currently known about Mos1 insertion sites, and to define the characteristics of Mos1 insertion sequences in vitro and in vivo. Statistical analysis can be used to identify the TA dinucleotides that are non-randomly targeted for transposon integration. In vitro, no specific feature determining target choice other than the requirement for a TA dinucleotide has been identified. In vivo, data were obtained from two previously reported integration hotspots: the bacterial cat gene and the Caenorhabditis elegans rDNA locus. Analysis of these insertion sites revealed a preference for TA dinucleotides that are included in TATA or TA x TA motifs, or located within AT-rich regions. Analysis of the physical properties of sequences obtained in vitro and in vivo do not help to explain Mos1 integration preferences, suggesting that other characteristics must be involved in Mos1 target choice.

The mariner Mos1 transposase produced in tobacco is active in vitro.

The mariner-like transposon Mos1 is used for insertional mutagenesis and transgenesis in different animals (insects, nematodes), but has never been used in plants. In this paper, the transposition activity of Mos1 was tested in Nicotiana tabacum, but no transposition event was detected. In an attempt to understand the absence of in planta transposition, Mos1 transposase (MOS1) was produced and purified from transgenic tobacco (HMNtMOS1). HMNtMOS1 was able to perform all transposition reaction steps in vitro: binding to ITR, excision and integration of the same pseudo-transposon used in in planta transposition assays. The in vitro transposition reaction was not inhibited by tobacco nuclear proteins, and did not depend on the temperature used for plant growth. Several hypotheses are proposed that could explain the inhibition of HMNtMOS1 activity in planta.

Lack of FcRn Impairs Natural Killer Cell Development and Functions in the Tumor Microenvironment.

The neonatal Fc receptor (FcRn) is responsible for the recycling and transcytosis of IgG and albumin. FcRn level was found altered in cancer tissues and implicated in tumor immunosurveillance and neoplastic cell growth. However, the consequences of FcRn down-regulation in the anti-tumor immune response are not fully elucidated. By using the B16F10 experimental lung metastasis model in an FcRn-deficient microenvironment (FcRn-/- mice), we found lung metastasis associated with an abnormal natural killer (NK) cell phenotype. In FcRn-/- mice, NK cells were immature, as shown by their surface marker profile and their decreased ability to degranulate and synthesize interferon γ after chemical and IL-2 or IL-12, IL-15 and IL-18 activation. These new findings support the critical role of FcRn downregulation in the tumor microenvironment in anti-tumor immunity, via NK cell maturation and activation.

[Zinc finger proteins: tools for site-specific correction or modification of the genome]. / Les domaines à doigts de zinc: vers la modification de la structure et de l'activité des génomes.

The ability to achieve site-specific correction or modification of the genome has widespread implications for basic and applied research. Individual zinc finger (ZF) domain recognizes DNA triplets with high specificity and affinity. They are used to create zinc finger protein (ZFP), like the ZF-nucleases, which could be designed to be specific for nearly any site in the genome. These domains can be tandemly linked to recognize DNA sequences of different lengths, with high fidelity. Different methods have been developed to design ZF specifically targeted to any triplet. This modular design offers a large number of combinatorial possibilities for the specific recognition of DNA. By fusing ZF to repression or activation domains, genes can be selectively targeted and switched off and on. Zinc-finger proteins (ZFPs) that recognize novel DNA sequences are the basis of a powerful technology platform with many uses in therapeutics. The ZF have been used as the DNA-binding domains of novel transcription factors (ZFP TFs) which are used to inhibit or activate genes involved in different diseases. ZF-nucleases are developed to modify genes implicated in different diseases. Many clinical trials using ZFPs are currently under investigation.

Alteration Analysis of Bone Marrow Mesenchymal Stromal Cells from De Novo Acute Myeloid Leukemia Patients at Diagnosis.

Bone marrow (BM)-derived mesenchymal stromal cells (MSCs) frequently display alterations in several hematologic disorders, such as acute lymphoid leukemia, acute myeloid leukemia (AML), and myelodysplastic syndromes. In acute leukemias, it is not clear whether MSC alterations contribute to the development of the malignant clone or whether they are simply the effect of tumor expansion on the microenvironment. We extensively investigated the characteristics of MSCs isolated from the BM of patients with de novo AML at diagnosis (L-MSCs) in terms of phenotype (gene and protein expression, apoptosis and senescence levels, DNA double-strand break formation) and functions (proliferation and clonogenic potentials, normal and leukemic hematopoiesis-supporting activity). We found that L-MSCs show reduced proliferation capacity and increased apoptosis levels compared with MSCs from healthy controls. Longer population doubling time in L-MSCs was not related to the AML characteristics at diagnosis (French-American-British type, cytogenetics, or tumor burden), but was related to patient age and independently associated with poorer patient outcome, as was cytogenetic prognostic feature. Analyzing, among others, the expression of 93 genes, we found that proliferative deficiency of L-MSCs was associated with a perivascular feature at the expense of the osteo-chondroblastic lineage with lower expression of several niche factors, such as KITLG, THPO, and ANGPT1 genes, the cell adhesion molecule VCAM1, and the developmental/embryonic genes, BMI1 and DICER1. L-MSC proliferative capacity was correlated positively with CXCL12, THPO, and ANGPT1 expression and negatively with JAG1 expression. Anyway, these changes did not affect their in vitro capacity to support normal hematopoiesis and to modify leukemic cell behavior (protection from apoptosis and quiescence induction). Our findings indicate that BM-derived MSCs from patients with newly diagnosed AML display phenotypic and functional alterations such as proliferative deficiency that could be attributed to tumor progression, but does not seem to play a special role in the leukemic process.

Genetic Inactivation of ATRX Leads to a Decrease in the Amount of Telomeric Cohesin and Level of Telomere Transcription in Human Glioma Cells.

Mutations in ATRX (alpha thalassemia/mental retardation syndrome X-linked), a chromatin-remodeling protein, are associated with the telomerase-independent ALT (alternative lengthening of telomeres) pathway of telomere maintenance in several types of cancer, including human gliomas. In telomerase-positive glioma cells, we found by immunofluorescence that ATRX localized not far from the chromosome ends but not exactly at the telomere termini. Chromatin immunoprecipitation (ChIP) experiments confirmed a subtelomeric localization for ATRX, yet short hairpin RNA (shRNA)-mediated genetic inactivation of ATRX failed to trigger the ALT pathway. Cohesin has been recently shown to be part of telomeric chromatin. Here, using ChIP, we showed that genetic inactivation of ATRX provoked diminution in the amount of cohesin in subtelomeric regions of telomerase-positive glioma cells. Inactivation of ATRX also led to diminution in the amount of TERRAs, noncoding RNAs resulting from transcription of telomeric DNA, as well as to a decrease in RNA polymerase II (RNAP II) levels at the telomeres. Our data suggest that ATRX might establish functional interactions with cohesin on telomeric chromatin in order to control TERRA levels and that one or the other or both of these events might be relevant to the triggering of the ALT pathway in cancer cells that exhibit genetic inactivation of ATRX.

Nuclear importation of Mariner transposases among eukaryotes: motif requirements and homo-protein interactions.

Mariner-like elements (MLEs) are widespread transposable elements in animal genomes. They have been divided into at least five sub-families with differing host ranges. We investigated whether the ability of transposases encoded by Mos1, Himar1 and Mcmar1 to be actively imported into nuclei varies between host belonging to different eukaryotic taxa. Our findings demonstrate that nuclear importation could restrict the host range of some MLEs in certain eukaryotic lineages, depending on their expression level. We then focused on the nuclear localization signal (NLS) in these proteins, and showed that the first 175 N-terminal residues in the three transposases were required for nuclear importation. We found that two components are involved in the nuclear importation of the Mos1 transposase: an SV40 NLS-like motif (position: aa 168 to 174), and a dimerization sub-domain located within the first 80 residues. Sequence analyses revealed that the dimerization moiety is conserved among MLE transposases, but the Himar1 and Mcmar1 transposases do not contain any conserved NLS motif. This suggests that other NLS-like motifs must intervene in these proteins. Finally, we showed that the over-expression of the Mos1 transposase prevents its nuclear importation in HeLa cells, due to the assembly of transposase aggregates in the cytoplasm.

Symbiotic virus at the evolutionary intersection of three types of large DNA viruses; iridoviruses, ascoviruses, and ichnoviruses.

BACKGROUND: The ascovirus, DpAV4a (family Ascoviridae), is a symbiotic virus that markedly increases the fitness of its vector, the parasitic ichneumonid wasp, Diadromus puchellus, by increasing survival of wasp eggs and larvae in their lepidopteran host, Acrolepiopsis assectella. Previous phylogenetic studies have indicated that DpAV4a is related to the pathogenic ascoviruses, such as the Spodoptera frugiperda ascovirus 1a (SfAV1a) and the lepidopteran iridovirus (family Iridoviridae), Chilo iridescent virus (CIV), and is also likely related to the ancestral source of certain ichnoviruses (family Polydnaviridae). METHODOLOGY/PRINCIPAL FINDINGS: To clarify the evolutionary relationships of these large double-stranded DNA viruses, we sequenced the genome of DpAV4a and undertook phylogenetic analyses of the above viruses and others, including iridoviruses pathogenic to vertebrates. The DpAV4a genome consisted of 119,343 bp and contained at least 119 open reading frames (ORFs), the analysis of which confirmed the relatedness of this virus to iridoviruses and other ascoviruses. CONCLUSIONS: Analyses of core DpAV4a genes confirmed that ascoviruses and iridoviruses are evolutionary related. Nevertheless, our results suggested that the symbiotic DpAV4a had a separate origin in the iridoviruses from the pathogenic ascoviruses, and that these two types shared parallel evolutionary paths, which converged with respect to virion structure (icosahedral to bacilliform), genome configuration (linear to circular), and cytopathology (plasmalemma blebbing to virion-containing vesicles). Our analyses also revealed that DpAV4a shared more core genes with CIV than with other ascoviruses and iridoviruses, providing additional evidence that DpAV4a represents a separate lineage. Given the differences in the biology of the various iridoviruses and ascoviruses studied, these results provide an interesting model for how viruses of different families evolved from one another.

Autism and nonsyndromic mental retardation associated with a de novo mutation in the NLGN4X gene promoter causing an increased expression level.

BACKGROUND: Pathogenic mutations in the X-linked Neuroligin 4 gene (NLGN4X) in autism spectrum disorders (ASDs) and/or mental retardation (MR) are rare. However, nothing is known regarding a possible altered expression level of NLGN4X that would be caused by mutations in regulatory sequences. We investigated this issue by analyzing these regions in patients with ASDs and no mutation in the NLGN4X coding sequence. METHODS: We studied 96 patients who met all DSM-IV criteria for autism. The entire coding sequence and the regulatory sequences of the NLGN4X gene were analyzed by polymerase chain reaction and direct sequencing. RESULTS: We identified a de novo 1 base pair (-335G>A) substitution located in the promoter region in a patient with autism and nonsyndromic profound MR. Interestingly, this variation is associated with an increased level of the NLGN4X transcript in the patient compared with male control subjects as well as his father. Further in vitro luciferase reporter and electrophoretic mobility shift assays confirmed, respectively, that this mutation increases gene expression and is probably caused by altered binding of transcription factors in the mutated promoter sequence. CONCLUSIONS: This result brings further insight about the phenotypic spectrum of NLGN4X mutations and suggests that the analysis of the expression level of NLGN4X might detect new cases.

Metal influence on metallothionein synthesis in the hydrothermal vent mussel Bathymodiolus thermophilus.

The present study reports on the metallothionein expression in the hydrothermal vent mussel Bathymodiolus thermophilus. Metallothioneins (MT) are proteins involved in intracellular metal regulation and conserved throughout the animal kingdom. The hydrothermal vent environment presents peculiarities (high levels of sulfides and metals, low pH, anoxia) that may have driven associated species to develop original evolutionary ways to face these extreme living conditions. Mussels were exposed to different metal solutions at the atmospheric pressure. The MT mRNA levels and MT contents were measured in gills and mantles of each exposed mussel. The intracellular metal distribution was estimated in fractions obtained after the centrifugation of tissue homogenates. A few of the tested metals (Ag, Cu, Cd, Hg and Zn) were able to significantly induce MT mRNA levels. Silver was the only one that produced a significant increase of the MT protein level in both mantle and gills. The gills always presented higher MT protein levels than the mantle did, while their MT mRNA levels were similar. Our data show that MT mRNA and MT protein levels do not follow a clear relationship in the gills and mantle of B. thermophilus and we assume that a posttranscriptional control occurs in these mussels.

Genomic sequence of Spodoptera frugiperda Ascovirus 1a, an enveloped, double-stranded DNA insect virus that manipulates apoptosis for viral reproduction.

Ascoviruses (family Ascoviridae) are double-stranded DNA viruses with circular genomes that attack lepidopterans, where they produce large, enveloped virions, 150 by 400 nm, and cause a chronic, fatal disease with a cytopathology resembling that of apoptosis. After infection, host cell DNA is degraded, the nucleus fragments, and the cell then cleaves into large virion-containing vesicles. These vesicles and virions circulate in the hemolymph, where they are acquired by parasitic wasps during oviposition and subsequently transmitted to new hosts. To develop a better understanding of ascovirus biology, we sequenced the genome of the type species Spodoptera frugiperda ascovirus 1a (SfAV-1a). The genome consisted of 156,922 bp, with a G+C ratio of 49.2%, and contained 123 putative open reading frames coding for a variety of enzymes and virion structural proteins, of which tentative functions were assigned to 44. Among the most interesting enzymes, due to their potential role in apoptosis and viral vesicle formation, were a caspase, a cathepsin B, several kinases, E3 ubiquitin ligases, and especially several enzymes involved in lipid metabolism, including a fatty acid elongase, a sphingomyelinase, a phosphate acyltransferase, and a patatin-like phospholipase. Comparison of SfAV-1a proteins with those of other viruses showed that 10% were orthologs of Chilo iridescent virus proteins, the highest correspondence with any virus, providing further evidence that ascoviruses evolved from a lepidopteran iridovirus. The SfAV-1a genome sequence will facilitate the determination of how ascoviruses manipulate apoptosis to generate the novel virion-containing vesicles characteristic of these viruses and enable study of their origin and evolution.

Evolution of full-length and deleted forms of the mariner-like element, Botmar1, in the Genome of the bumble bee, Bombus terrestris (Hymenoptera: Apidae).

Mariner-like elements (MLE) are Class II transposable elements that are very widespread among eukaryotic genomes. One MLE belonging to the mauritiana subfamily, named Botmar1, has been identified in the genome of the bumble bee, Bombus terrestris. gDNA hybridization with the Botmar1 transposase ORF revealed that about 230 elements are present in each haploid genome of B. terrestris that consist entirely of 1.3- and 0.85-kbp elements. The analysis of their sequences revealed that there are two Botmar1 subfamilies of similar ages in the Bombus terrestris genome: one is composed entirely of 1.3-kpb elements, whereas the second comprises both completed and deleted elements. Our previous data indicated that the internally deleted form, which correspond to the 0.85-kbp Botmar1-related elements occur in other distantly related hymenopteran genomes. Because the presence of similar 1.3- and 0.85-kbp Botmar1-related elements in some distantly related hymenopteran species cannot be explained by horizontal transfers, the nucleic acid sequence properties of these elements were further investigated. We found that certain structural properties in their nucleic acid sequence might explain the occurrence of 0.85-kbp Botmar1-related elements presenting similarly located internal deletions in hymenopteran genomes.

Evidence for the evolution of ascoviruses from iridoviruses.

Ascoviruses (family Ascoviridae) are large, enveloped, double-stranded (ds)DNA viruses that attack lepidopteran larvae and pupae, and are unusual in that they are transmitted by parasitic wasps during oviposition. Previous comparisons of DNA polymerase sequences from vertebrate and invertebrate viruses suggested that ascoviruses are closely related to iridoviruses. This relationship was unexpected because these viruses differ markedly in virion symmetry, genome configuration and cellular pathology. Here we present evidence based on sequence comparisons and phylogenetic analyses of a greater range of ascovirus proteins and their homologues in other large dsDNA viruses that ascoviruses evolved from iridoviruses. Consensus trees for the major capsid protein, DNA polymerase, thymidine kinase and ATPase III from representative ascoviruses, algal viruses (family Phycodnaviridae), vertebrate and invertebrate iridoviruses (family Iridoviridae) and African swine fever virus (ASFV; family Asfarviridae) showed that ascovirus proteins clustered most closely with those of the lepidopteran iridovirus Chilo iridescent virus (CIV) (Invertebrate iridescent virus 6). Moreover, analysis of the presence or absence of homologues of an additional 50 proteins encoded in the genome of Spodoptera frugiperda ascovirus (SfAV-1a) showed that about 40 % occurred in CIV, with lower percentages encoded by the genomes of, respectively, vertebrate iridoviruses, phycodnaviruses and ASFV. The occurrence of three of these genes in SfAV-1a but not CIV was indicative of the evolutionary differentiation of ascoviruses from invertebrate iridoviruses.