SARS-CoV-2 Nsp3 unique domain SUD interacts with guanine quadruplexes and G4-ligands inhibit this interaction

The multidomain non-structural protein 3 (Nsp3) is the largest protein encoded by coronavirus (CoV) genomes and several regions of this protein are essential for viral replication. Of note, SARS-CoV Nsp3 contains a SARS-Unique Domain (SUD), which can bind Guanine-rich non-canonical nucleic acid structures called G-quadruplexes (G4) and is essential for SARS-CoV replication. We show herein that the SARS-CoV-2 Nsp3 protein also contains a SUD domain that interacts with G4s. Indeed, interactions between SUD proteins and both DNA and RNA G4s were evidenced by G4 pull-down, Surface Plasmon Resonance and Homogenous Time Resolved Fluorescence. These interactions can be disrupted by mutations that prevent oligonucleotides from folding into G4 structures and, interestingly, by molecules known as specific ligands of these G4s. Structural models for these interactions are proposed and reveal significant differences with the crystallographic and modeled 3D structures of the SARS-CoV SUD-NM/G4 interaction. Altogether, our results pave the way for further studies on the role of SUD/G4 interactions during SARS-CoV-2 replication and the use of inhibitors of these interactions as potential antiviral compounds.     

SCB1, a BURP-domain protein gene,from developing soybean seed coats

We describe a gene, SCB1 (Seed Coat BURP-domain protein 1), that is expressed specifically within the soybean (Glycine max [L.] Merrill) seed coat early in its development. Northern blot analysis and mRNA in situ hybridization revealed novel patterns of gene expression during seed development. SCB1 mRNA accumulated first within the developing thick-walled parenchyma cells of the inner integument and later in the thick- and thin-walled parenchyma cells of the outer integument. This occurred prior to the period of seed coat maturation and seed filling and before either of the layers started to degrade. SCB1 may therefore play a role in the differentiation of the seed coat parenchyma cells. In addition, the protein product appears to be located within cell walls. The SCB1 gene codes for a new member of a class of modular proteins that possess a carboxy-terminal BURP domain and a variety of different repeated sequences. The sequence of the genomic clone revealed the insertion of a Tgm transposable element in the upstream promoter region but it is not certain whether it contributes to the tissue-specific pattern of SCB1 expression.     

TAXONOMIC REVISION OF THE DINOFLAGELLATE AMPHIDOMA CAUDATA: TRANSFER TO THE GENUS AZADINIUM (DINOPHYCEAE) AND PROPOSAL OF TWO VARIETIES,BASED ON MORPHOLOGICAL AND MOLECULAR PHYLOGENETIC ANALYSES1

The systematic position of Amphidoma caudata Halldal within the genus Amphidoma has remained uncertain as a result of its plate formula and the absence of molecular phylogenetic data. Also, this thecate dinoflagellate taxon has been used to designate two distinct morphotypes. The present study aims to clarify the generic affiliation of Amphidoma caudata and the taxonomic value of two different morphotypes M1 and M2. The new examination of the plate formula using SEM showed that it was the same for both morphotypes and that it corresponded to the tabulation of the recent erected genus Azadinium Elbrächter et Tillmann. Morphometric analysis, using cell size, length of apical projection in conjunction with the cell length, and the ratio of horn and spine showed that M1 and M2 formed two distinct groups. These results were supported by a molecular approach, revealing notable differences in the sequences of LSU rDNA and ITS region between these two morphotypes. Phylogenetic analyses inferred either from LSU and combined SSU, ITS region and COI data positioned M1 and M2 in a sister cluster of Azadinium species while Amphidoma languida Tillmann, Salas et Elbrächter, the only species of Amphidoma for which sequence data were available, was situated in a basal position of the Azadinium clade. Thus, we propose the transfer of Amphidoma caudata to the genus Azadinium and, consequently, the rehabilitation of the original tabulation of the genus Amphidoma Stein. To discriminate the two morphotypes, we propose a rank of variety with the following designations: Azadinium caudatum var. caudatum and Azadinium caudatum var. margalefii.     

Synthesis and biological evaluation of novel heterocyclic derivatives of combretastatin A-4

A novel series of combretastatin A-4 heterocyclic analogues was prepared by replacement of the B ring with indole, benzofurane or benzothiophene, attached at the C2 position. These compounds were evaluated for their abilities to inhibit tubulin assembly: derivative cis 3b, having a benzothiophene, showed an activity similar to those of colchicine or deoxypodophyllotoxine. The antiproliferative and antimitotic properties of cis 3b against keratinocyte cancer cell lines were also evaluated and the intracellular organization of microtubules in the cells after treatment with both stereoisomers of 3b was also determined, using confocal microscopy.     

Mutations in the mitochondrial split gene COXI are preferentially located in exons: a mapping study of 170 mutants

We have analysed the precise location of a large number (170) of mutations affecting the structural gene for subunit 1 of the cytochrome c oxidase complex. This gene, COXI, is 12.9 kb long and the major part of the sequence (i.e. 11.3 kb) is composed of introns. Several conclusions can be drawn from this study: (1) A significant proportion (84/170) of the mutations cannot be assigned to a single position within the gene by deletion mapping, in spite of clearly being located in it. These mutations are probably large deletions or multiple mutations. (2) Four mutants carry distant double mutations, which have been individually localized. (3) Eighty-two mutants have lesions that are restricted to very short regions of the gene and we therefore conclude that they are most probably due to single hits; amongst these single mutations, 41 are unambiguously located in exons and 28 in introns. This result implies that, at least in this particular split gene, the probability of selection of a mutant phenotype in an exon is, on the average, 13.3 times greater than in an intron, in spite of the existence, within most of these introns, of open reading frames specifying intronic proteins. The evolutionary significance and biological implications of these results are discussed.     

Accessibility and Structural Role of Histone Domains in Chromatin. Biophysical and Immunochemical Studies of Progressive Digestion with Immobilized Proteases

Abstract

The accessibility and role of histone regions in chromatin fibres were investigated using limited proteolysis with enzymes covalently bound to collagen membranes. The changes in chromatin conformation and condensation monitored by various biophysical methods, were correlated to the degradation of the histone proteins revealed by antibodies specific for histones and histone peptides.

Upon digestion with trypsin and subtilisin, chromatin undergoes successive structural transitions. The cleavage of the C-terminal domains of Hl, H2A and H2B, and of the N-terminal tail of H3 led to a decondensation of chromatin fibres, indicated by increases in electric birefringence and orientational relaxation times. It corresponds to a 15% increase in linear dimensions. The degradation of the other terminal regions of histones H3, H2A and H2B resulted in the appearance of hinge points between nucleosomes without alteration of the overall orientation of polynucleosome chains. Despite the loss of all the basic domains of HI, H3, H2A and H2B, no significant change in DNA-protein interactions occurred, suggesting that most of these protease-accessible regions interact weakly, if at all, with DNA in chromatin. Further proteolysis led to H4 degradation and other additional cleavages of Hl, H2B and H3. This caused the relaxation of no more than 8% of the total DNA but resulted in changes in the ability of chromatin to condense at high ionic strength. More extensive digestion resulted in a total unravelling of nucleosomal chains which acquired properties similar to those of Hl- depleted chromatin, although the globular part of HI was still present.

The data suggest that histone-histone interactions between HI and core histone domains play a central role in stabilizing the chromatin fibres, and cuts in H3, H2A and H2B as well as HI, seem necessary for chromatin expansion. On the contrary, H4 might be involved in the stabilization of nucleosomes only.     

NK cell responses to Plasmodium infection and control of intrahepatic parasite development

Various components of innate and adaptive immunity contribute to host defenses against Plasmodium infection. We investigated the contribution of NK cells to the immune response to primary infection with Plasmodium yoelii sporozoites in C57BL/6 mice. We found that hepatic and splenic NK cells were activated during infection and displayed different phenotypic and functional properties. The number of hepatic NK cells increased whereas the number of splenic NK cells decreased. Expression of the Ly49 repertoire was modified in the spleen but not in the liver. Splenic and hepatic NK cells have a different inflammatory cytokines profile production. In addition, liver NK cells were cytotoxic to YAC-1 cells and P. yoelii liver stages in vitro but not to erythrocytic stages. No such activity was observed with splenic NK cells from infected mice. These in vitro results were confirmed by the in vivo observation that Rag2(-/-) mice were more resistant to sporozoite infection than Rag2(-/-) gamma c(-/-) mice, whereas survival rates were similar for the two strains following blood-stage infection. Thus, NK cells are involved in early immune mechanisms controlling Plasmodium infection, mostly at the pre-erythrocytic stage.     

IgG Autoantibody to Brain Beta Tubulin III Associated with Cytokine Cluster-II Discriminate Cerebral Malaria in Central India

Background

The main processes in the pathogenesis of cerebral malaria caused by Plasmodium falciparum involved sequestration of parasitized red blood cells and immunopathological responses. Among immune factors, IgG autoantibodies to brain antigens are increased in P. falciparum infected patients and correlate with disease severity in African children. Nevertheless, their role in the pathophysiology of cerebral malaria (CM) is not fully defined. We extended our analysis to an Indian population with genetic backgrounds and endemic and environmental status different from Africa to determine if these autoantibodies could be either a biomarker or a risk factor of developing CM.

Methods/Principal Findings

We investigated the significance of these self-reactive antibodies in clinically well-defined groups of P. falciparum infected patients manifesting mild malaria (MM), severe non-cerebral malaria (SM), or cerebral malaria (CM) and in control subjects from Gondia, a malaria epidemic site in central India using quantitative immunoprinting and multivariate statistical analyses. A two-fold complete-linkage hierarchical clustering allows classifying the different patient groups and to distinguish the CM from the others on the basis of their profile of IgG reactivity to brain proteins defined by PANAMA Blot. We identified beta tubulin III (TBB3) as a novel discriminant brain antigen in the prevalence of CM. In addition, circulating IgG from CM patients highly react with recombinant TBB3. Overall, correspondence analyses based on singular value decomposition show a strong correlation between IgG anti-TBB3 and elevated concentration of cluster-II cytokine (IFNγ, IL1β, TNFα, TGFβ) previously demonstrated to be a predictor of CM in the same population.

Conclusions/Significance

Collectively, these findings validate the relationship between antibody response to brain induced by P. falciparum infection and plasma cytokine patterns with clinical outcome of malaria. They also provide significant insight into the immune mechanisms associated to CM by the identification of TBB3 as a new disease-specific marker and potential therapeutic target.