Polar Mutations Research Articles

Predicting the changes in neutralizing antibody interaction with G protein derived from Bangladesh isolates of Nipah virus: molecular dynamics based approach

The infectious Nipah virus (NiV) is categorized into NiV-M (Malaysia) and NiV-B (Bangladesh) groups based on its genome comparison, pathogenicity, and mortality rate. The development of therapeutic molecules has used NiV-M-derived data in multiple studies than NiV-B. In continuation with this, the protein level investigation is also less explored to understand the interaction with therapeutic neutralizing antibodies for NiV-B. So, this study focuses on understanding the impact of NiV-B-specific mutations on the interaction of therapeutic neutralizing antibodies with the G protein. The population-based comparative analysis of NiV-B G protein sequences with NiV-M sequence identified twenty-six mutations. These predominantly polar mutations were then used to model the mutant protein (G_MT). In a comparative study, the G protein G_MT and reference protein G_WT (Malaysian origin) were subjected to a protein docking with neutralizing human monoclonal antibody HENV26. The binding affinity and the free binding energy of the glycoprotein in complex with G-WT and G_MT were calculated using PRODIGY and MM/PBSA tools respectively. Based on the PRODIGY report, G-WT showed stronger binding (-13.8 kcal/mol) compared to that of the G_MT (-9.0 kcal/mol) with the HENV26 antibody. The stability of the complexes was evaluated using MM/PBSA which showed higher binding energy with HENV26 for G_WT (-75.11 kcal/mol) in contrast to G_MT (-41.66 kcal/mol). The results indicate that the mutant G protein has a reduced ability to bind to neutralizing antibodies, resulting in a decreased effectiveness against strains carrying these mutations. Communicated by Ramaswamy H. Sarma

Structural implications of SARS-CoV-2 Surface Glycoprotein N501Y mutation within receptor-binding domain [499-505] – computational analysis of the most frequent Asn501 polar uncharged amino acid mutations

The aim of this study was to evaluate the impact of the most frequent Asn501 polar uncharged amino acid mutations upon important structural properties of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) Surface Glycoprotein RBD – hACE2 (human angiotensin-converting enzyme 2) heterodimer. Mutations N501Y, N501T and N501S were considered and their impact upon complex solubility, secondary motifs formation and intermolecular hydrogen bonding interface was analyzed. Results and findings are reported based on 50 ns run in Gromacs molecular dynamics simulation software. Special attention is paid on the biomechanical shifts in the receptor-binding domain (RBD) [499-505]: ProThrAsn(Tyr)GlyValGlyTyr, having substituted Asparagine to Tyrosine at position 501. The main findings indicate that the N501S mutation increases SARS-CoV-2 S-protein RBD – hACE2 solubility over N501T, N501 (wild type): , . The N501Y mutation shifts -helix S-protein RBD [366-370]: SerValLeuTyrAsn into π-helix for t > 38.5 ns. An S-protein RBD [503-505]: ValGlyTyr shift from -helix into a turn is observed due to the N501Y mutation in t > 33 ns. An empirical proof for the presence of a Y501-binding pocket, based on RBD [499-505]: PTYGVGY ’s RMSF peak formation is presented. There is enhanced electrostatic interaction between Tyr505 (RBD) phenolic -OH group and Glu37 (hACE2) side chain oxygen atoms due to the N501Y mutation. The N501Y mutation shifts the hydrogen bond into permanent polar contact; ; .

Mutational analysis of a conserved positive charge in the c-ring of E. coli ATP synthase

F1Fo ATP synthase is a ubiquitous molecular motor that utilizes a rotary mechanism to synthesize adenosine triphosphate (ATP), the fundamental energy currency of life. The membrane-embedded Fo motor converts the electrochemical gradient of protons into rotation, which is then used to drive the conformational changes in the soluble F1 motor that catalyze ATP synthesis. In E. coli, the Fo motor is composed of a c10 ring (rotor) alongside subunit a (stator), which together provide two aqueous half channels that facilitate proton translocation. Previous work has suggested that Arg50 and Thr51 on the cytoplasmic side of each subunit c are involved in the proton translocation process, and positive charge is conserved in this region of subunit c. To further investigate the role of these residues and the chemical requirements for activity at these positions, we generated 13 substitution mutants and assayed their in vitro ATP synthesis, H+ pumping, and passive H+ permeability activities, as well as the ability of mutants to carry out oxidative phosphorylation in vivo. While polar and hydrophobic mutations were generally tolerated in either position, introduction of negative charge or removal of polarity caused a substantial defect. We discuss the possible effects of altered electrostatics on the interaction between the rotor and stator, water structure in the aqueous channel, and interaction of the rotor with cardiolipin.

Application of CRISPR/Cas9 Gene Editing Technology in Studies of Intestinal Anaerobic Bacteria

Human intestinal bacteria play important roles in the nutrition, development, immunity and even psychological diseases of the host, researches about such microorganisms is prevalent in recent years. New omics technologies are of great help for us to explore new functions and composition of intestinal microbiome, while studies about the molecular mechanisms of the symbiosis and reciprocity between these commensals and host are significant and too, and an important approach in the field is to conduct targeted gene editing for intestinal anaerobes and verify the function of genes by knocking out, replacing or inserting genes. Although the traditional bacterial gene editing technologies show high accuracy for DNA modification, they have low recombination efficiency and a polar mutation effect. This hinders the functional researches of intestinal bacteria. CRISPR/Cas9 technology, which emerged about 10 years ago, has advantages of wide universality, simple design and operation, and has been widely used in gene manipulation of different organisms. This paper briefly introduces the technical principle of CRISPR/Cas9 and summarizes the current application of this technology in researches of intestinal anaerobic bacteria, a few aspects we are concerning and the trends for future development are analyzed as well.

Fast-forwarding evolution-Accelerated adaptation in a proofreading-deficient hypermutator herpesvirus.

Evolution relies on the availability of genetic diversity for fitness-based selection. However, most deoxyribonucleic acid (DNA) viruses employ DNA polymerases (Pol) capable of exonucleolytic proofreading to limit mutation rates during DNA replication. The relative genetic stability produced by high-fidelity genome replication can make studying DNA virus adaptation and evolution an intensive endeavor, especially in slowly replicating viruses. Here, we present a proofreading-impaired Pol mutant (Y547S) of Marek's disease virus that exhibits a hypermutator phenotype while maintaining unimpaired growth in vitro and wild-type (WT)-like pathogenicity in vivo. At the same time, mutation frequencies observed in Y547S virus populations are 2-5-fold higher compared to the parental WT virus. We find that Y547S adapts faster to growth in originally non-permissive cells, evades pressure conferred by antiviral inhibitors more efficiently, and is more easily attenuated by serial passage in cultured cells compared to WT. Our results suggest that hypermutator viruses can serve as a tool to accelerate evolutionary processes and help identify key genetic changes required for adaptation to novel host cells and resistance to antiviral therapy. Similarly, the rapid attenuation achieved through adaptation of hypermutators to growth in cell culture enables identification of genetic changes underlying attenuation and virulence, knowledge that could practically exploited, e.g.in the rational design of vaccines.

Genotoxic hazard and oxidative stress induced by wastewater irrigated soil with special reference to pesticides and heavy metal pollution

Due to enhancement of industrial growth and urbanization, soil contamination is increasing prominently. Therefore, it is important to examine possible adverse effects of industrial waste. Soil samples were might to be polluted with several heavy-metals and pesticides. Gas chromatographic results showed occurrence of high-level of organochlorine and organophosphate pesticides in studied soil samples. Genotoxicity of soil extracts was assessed using environmental-risk assessment models. Soil samples were extracted in hexane and dichloromethane solvents and were evaluated for genotoxic potential by prokaryotic (Ames test, plasmid nicking assay and E. coli K-12 DNA repair defective mutants) and eukaryotic (Allium cepa root chromosomal aberration and Vigna radiata seed-germination test) bioassays. Strain TA98 was found the most susceptible among soil extracts. The mutagenicity of hexane soil extract from wastewater irrigation was found to be higher than that of DCM samples in terms of mutagenic index, mutagenic potential, and induction factor for Ames strains. The damage in DNA repair defective mutants of hexane extracts were found higher compared to DCM extracts at dose of 20 μl/ml of culture. Survival in polA, lexA and recA mutants were 39%, 47% and 55% while treated with hexane extract. Allium cepa test, mitotic index was decreased in dose-dependent way and various kinds of chromosomal aberrations were found. Vigna radiata seeds germination and other parameters were also affected when treated with wastewater irrigated (WWI) soil. Oxidative stress in V. radiata roots were also showed under CLS microscope. Genotoxicity of WWI soil extract was also confirmed by plasmid nicking test. Our study provides possible explanation for the assessment of potential health and environmental hazards of the industrial region.

Polar mutagenesis of polycistronic bacterial transcriptional units using Cas12a

BackgroundFunctionally related genes in bacteria are often organized and transcribed as polycistronic transcriptional units. Examples are the fim operon, which codes for biogenesis of type 1 fimbriae in Escherichia coli, and the atp operon, which codes for the FoF1 ATP synthase. We tested the hypothesis that markerless polar mutations could be efficiently engineered using CRISPR/Cas12a in these loci.ResultsCas12a-mediated engineering of a terminator sequence inside the fimA gene occurred with efficiencies between 10 and 80% and depended on the terminator’s sequence, whilst other types of mutations, such as a 97 bp deletion, occurred with 100% efficiency. Polar mutations using a terminator sequence were also engineered in the atp locus, which induced its transcriptional shutdown and produced identical phenotypes as a deletion of the whole atp locus (ΔatpIBEFHAGDC). Measuring the expression levels in the fim and atp loci showed that many supposedly non-polar mutants induced a significant polar effect on downstream genes. Finally, we also showed that transcriptional shutdown or deletion of the atp locus induces elevated levels of intracellular ATP during the exponential growth phase.ConclusionsWe conclude that Cas12a-mediated mutagenesis is an efficient simple system to generate polar mutants in E. coli. Different mutations were induced with varying degrees of efficiency, and we confirmed that all these mutations abolished the functions encoded in the fim and atp loci. We also conclude that it is difficult to predict which mutagenesis strategy will induce a polar effect in genes downstream of the mutation site. Furthermore the strategies described here can be used to manipulate the metabolism of E. coli as showcased by the increase in intracellular ATP in the markerless ΔatpIBEFHAGDC mutant.

Regulation of Endothelial Cell function by Integration of Gαi and β‐arrestin signaling at Atypical Chemokine Receptor 3

Atypical Chemokine receptor 3 (ACKR3), also known as C‐X‐C chemokine receptor type 7 (CXCR7), is a G protein‐coupled receptor (GPCR) implicated in several physiological processes including leukocyte trafficking, cancer, and angiogenesis. Commonly co‐expressed with C‐X‐C chemokine receptor type 4 (CXCR4), ACKR3 is classified as an β‐arrestin‐biased “scavenger” receptor due to its lack of G‐protein signaling and its ability to efficiently internalize and degrade its ligands, CXCL11 and CXCL12. However, much is still unknown regarding ACKR3 signaling mechanisms and its implications on cellular physiology. Here we describe ongoing studies to characterize the mechanisms that underlie ACKR3 signaling and endothelial cell function. We demonstrated two ACKR3 specific agonists, WW36 and WW38, promote proliferation and migration of human umbilical vein endothelial cells (HUVECs). Surprisingly, we found that ACKR3‐promoted proliferation and migration was sensitive to pertussis toxin (PTX), suggesting the involvement of Gαi/o proteins at a receptor which does not canonically signal via G proteins. We also observed similar sensitivity to PTX on ERK activation in HUVECs. We hypothesized that these results may in part be explained by the recently described coordination of Gαi and β‐arrestin (βarr) to promote Gαi:βarr complex‐specific downstream signaling. In support of this hypothesis, we demonstrated the formation of a Gαi:βarr complex upon ACKR3 activation in a dose‐dependent manner. To characterize the necessary components in Gαi and βarr, we first developed active proximal polar core mutant and proximal finger loop βarr mutant and tested these at the prototypical class B Vasopressin 2 Receptor. Both mutants lead to considerable decrease in complex formation with the finger loop mutation nearly abolishing our split signal. Within the Gαi subfamily, we observed considerable differences in the ability to form complex, with Gαi2 and Gαi3 promoting less complex formation than Gαi1 and Gαo. We developed several Gαi mutants, hypothesizing that residues that differ between Gαi isoforms may impact Gαi:βarr formation. Using NanoBRET we demonstrated that these mutants impair complex formation, suggesting that they may be involved either via direct contacts with βarr or impacting Gαi conformation such that complex formation is no longer as favorable. These studies reveal a novel role for Gαi:βarr complexes in mediating ACKR3 function that contribute to endothelial cell biology. Additionally, these findings support an emerging paradigm in which G proteins and βarr coordinate their signaling downstream of GPCRs.

Role of a Polar c‐Ring Residue in the Fo Motor of E. coli ATP Synthase

ATP Synthase is a molecular motor that utilizes a rotary mechanism to synthesize adenosine triphosphate (ATP), the fundamental energy currency of life. The torque for this mechanism is generated in the membrane‐embedded FO motor by harnessing the proton motive force, where protons flow down the electrochemical gradient through two half channels in the motor. The exit half channel on the cytoplasmic side is found at the interface of subunit a (stator) and the subunit c ring (rotor). Previous work on the E. coli ATP synthase has suggested that Thr51 in subunit c is involved in the proton translocation process. To investigate the role of this residue and chemical requirements at this position, we generated six substitution mutants and assayed their in vitro ATP synthesis, H+ pumping, and H+ permeability activities as well as the ability of mutants to carry out oxidative phosphorylation in vivo. Polar and hydrophobic mutations were generally tolerated, except for a notable decrease in function when a negative charge is introduced. Unusually, substitution with Cys completely abolished in vitro functions but only mildly inhibited growth on succinate. Overall, our results suggest that cThr51 may play a supporting role in the interaction between the c‐ring and subunit a in the region of the proton exit channel.

The Pbo Cluster from Pseudomonas syringae pv. Phaseolicola NPS3121 Is Thermoregulated and Required for Phaseolotoxin Biosynthesis

The bean (Phaseolus vulgaris) pathogen Pseudomonas syringae pv. phaseolicola NPS3121 synthesizes phaseolotoxin in a thermoregulated way, with optimum production at 18 °C. Gene PSPPH_4550 was previously shown to be thermoregulated and required for phaseolotoxin biosynthesis. Here, we established that PSPPH_4550 is part of a cluster of 16 genes, the Pbo cluster, included in a genomic island with a limited distribution in P. syringae and unrelated to the possession of the phaseolotoxin biosynthesis cluster. We identified typical non-ribosomal peptide synthetase, and polyketide synthetase domains in several of the pbo deduced products. RT-PCR and the analysis of polar mutants showed that the Pbo cluster is organized in four transcriptional units, including one monocistronic and three polycistronic. Operons pboA and pboO are both essential for phaseolotoxin biosynthesis, while pboK and pboJ only influence the amount of toxin produced. The three polycistronic units were transcribed at high levels at 18 °C but not at 28 °C, whereas gene pboJ was constitutively expressed. Together, our data suggest that the Pbo cluster synthesizes secondary metabolite(s), which could participate in the regulation of phaseolotoxin biosynthesis.

Approaches to Understanding Mechanisms of Dentilisin Protease Complex Expression in Treponema denticola.

The oral spirochete Treponema denticola is a keystone periodontal pathogen that, in association with members of a complex polymicrobial oral biofilm, contributes to tissue damage and alveolar bone loss in periodontal disease. Virulence-associated behaviors attributed to T. denticola include disruption of the host cell extracellular matrix, tissue penetration and disruption of host cell membranes accompanied by dysregulation of host immunoregulatory factors. T. denticola dentilisin is associated with several of these behaviors. Dentilisin is an outer membrane-associated complex of acylated subtilisin-family PrtP protease and two other lipoproteins, PrcB and PrcA, that are unique to oral spirochetes. Dentilisin is encoded in a single operon consisting of prcB-prcA-prtP. We employ multiple approaches to study mechanisms of dentilisin assembly and PrtP protease activity. To determine the role of each protein in the protease complex, we have made targeted mutations throughout the protease locus, including polar and nonpolar mutations in each gene (prcB, prcA, prtP) and deletions of specific PrtP domains, including single base mutagenesis of key PrtP residues. These will facilitate distinguishing between host cell responses to dentilisin protease activity and its acyl groups. The boundaries of the divergent promoter region and the relationship between dentilisin and the adjacent iron transport operon are being resolved by incremental deletions in the sequence immediately 5’ to the protease locus. Comparison of the predicted three-dimensional structure of PrtP to that of other subtilisin-like proteases shows a unique PrtP C-terminal domain of approximately 250 residues. A survey of global gene expression in the presence or absence of protease gene expression reveals potential links between dentilisin and iron uptake and homeostasis in T. denticola. Understanding the mechanisms of dentilisin transport, assembly and activity of this unique protease complex may lead to more effective prophylactic or therapeutic treatments for periodontal disease.

Model-driven analysis of mutant fitness experiments improves genome-scale metabolic models of Zymomonas mobilis ZM4.

Genome-scale metabolic models have been utilized extensively in the study and engineering of the organisms they describe. Here we present the analysis of a published dataset from pooled transposon mutant fitness experiments as an approach for improving the accuracy and gene-reaction associations of a metabolic model for Zymomonas mobilis ZM4, an industrially relevant ethanologenic organism with extremely high glycolytic flux and low biomass yield. Gene essentiality predictions made by the draft model were compared to data from individual pooled mutant experiments to identify areas of the model requiring deeper validation. Subsequent experiments showed that some of the discrepancies between the model and dataset were caused by polar effects, mis-mapped barcodes, or mutants carrying both wild-type and transposon disrupted gene copies—highlighting potential limitations inherent to data from individual mutants in these high-throughput datasets. Therefore, we analyzed correlations in fitness scores across all 492 experiments in the dataset in the context of functionally related metabolic reaction modules identified within the model via flux coupling analysis. These correlations were used to identify candidate genes for a reaction in histidine biosynthesis lacking an annotated gene and highlight metabolic modules with poorly correlated gene fitness scores. Additional genes for reactions involved in biotin, ubiquinone, and pyridoxine biosynthesis in Z. mobilis were identified and confirmed using mutant complementation experiments. These discovered genes, were incorporated into the final model, iZM4_478, which contains 747 metabolic and transport reactions (of which 612 have gene-protein-reaction associations), 478 genes, and 616 unique metabolites, making it one of the most complete models of Z. mobilis ZM4 to date. The methods of analysis that we applied here with the Z. mobilis transposon mutant dataset, could easily be utilized to improve future genome-scale metabolic reconstructions for organisms where these, or similar, high-throughput datasets are available.

Mechanism of tumor-suppressive cell competition in flies.

Oncogenic mutations often trigger antitumor cellular response such as induction of apoptosis or cellular senescence. Studies in the last decade have identified the presence of the third guardian against mutation‐induced tumorigenesis, namely “cell competition.” Cell competition is a context‐dependent cell elimination whereby cells with higher fitness eliminate neighboring cells with lower fitness by inducing cell death. While oncogene‐induced apoptosis or oncogene‐induced senescence acts as a cell‐autonomous tumor suppressor, cell competition protects the tissue from tumorigenesis via cell‐cell communication. For instance, in Drosophila epithelium, oncogenic cells with cell polarity mutations overproliferate and develop into tumors on their own but are eliminated from the tissue when surrounded by wild‐type cells. Genetic studies in flies have unraveled that such tumor‐suppressive cell competition is regulated by at least three mechanisms: direct cell‐cell interaction between polarity‐deficient cells and wild‐type cells, secreted factors from epithelial cells, and systemic factors from distant organs. Molecular manipulation of tumor‐suppressive cell competition could provide a novel therapeutic strategy against human cancers.

Enhanced receptor binding of SARS-CoV-2 through networks of hydrogen-bonding and hydrophobic interactions

Molecular dynamics and free energy simulations have been carried out to elucidate the structural origin of differential protein-protein interactions between the common receptor protein angiotensin converting enzyme 2 (ACE2) and the receptor binding domains of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [A. E. Gorbalenya et al., Nat. Microbiol. 5, 536-544 (2020)] that causes coronavirus disease 2019 (COVID-19) [P. Zhou et al., Nature 579, 270-273 (2020)] and the SARS coronavirus in the 2002-2003 (SARS-CoV) [T. Kuiken et al., Lancet 362, 263-270 (2003)] outbreak. Analysis of the dynamic trajectories reveals that the binding interface consists of a primarily hydrophobic region and a delicate hydrogen-bonding network in the 2019 novel coronavirus. A key mutation from a hydrophobic residue in the SARS-CoV sequence to Lys417 in SARS-CoV-2 creates a salt bridge across the central hydrophobic contact region, which along with polar residue mutations results in greater electrostatic complementarity than that of the SARS-CoV complex. Furthermore, both electrostatic effects and enhanced hydrophobic packing due to removal of four out of five proline residues in a short 12-residue loop lead to conformation shift toward a more tilted binding groove in the complex in comparison with the SARS-CoV complex. On the other hand, hydrophobic contacts in the complex of the SARS-CoV-neutralizing antibody 80R are disrupted in the SARS-CoV-2 homology complex model, which is attributed to failure of recognition of SARS-CoV-2 by 80R.

A Critical Effect of Pol Escape Mutations Associated with a Detrimental Allele HLA-C*15:05 on Clinical Outcome in HIV-1 Subtype A/E Infection

Background: The mechanism explaining the role of detrimental HLA alleles in HIV-1 infections has not been studied. We recently demonstrated that HLA-A*29:01-B*07:05-C*15:05 is a detrimental haplotype and that Pol mutations associated with this haplotype significantly correlated with poor clinical outcome in HIV-1 subtype A/E-infected individuals. To clarify the mechanism underlying this detrimental haplotype, we here investigated the effect of these Pol mutations on the ability of HIV-1-specific CD8+ T-cells to recognize HIV-1 and on the clinical outcome. Methods: To identify a T-cell epitope including Pol653/657 where this haplotype-associated mutations were found, we analyzed T-cell responses to overlapping or truncated peptides. The recognition of epitope-specific CD8+ T-cells were analyzed for target cells infected with the mutant virus. We further analyzed the existence of T-cells specific for this epitope and its mutant ones and their role in suppression of HIV-1 replication in the subtype A/E-infected HLA-C*15:05+ Vietnamese. Findings: We identified HLA-C*15:05-restricted T-cells specific for the Pol SL9 epitope and found that 3 mutants (Pol S653A/T/L) had escaped from the T-cells. HLA-C*15:05+ individuals infected with these mutants showed significantly lower CD4+ T-cell counts than those with the wild-type virus or Pol S653K/Q mutants, which are not associated with HLA-C*15:05. Interpretation: These findings indicate that the accumulation of Pol S653A/T/L mutants critically and negatively affected control of HIV-1 by SL9-specific T-cells in the subtype A/E-infected individuals having the HLA-C*15:05 detrimental allele. Funding Statement: This research was supported by a grant-in-aid (15fk0410019) for AIDS Research from AMED, a JSPS KAKENHI grant-in-aid for scientific research (A) (grant No. 15H02658), and by a Joint Research Grant with the Institute of Tropical Medicine, Nagasaki University. Declaration of Interests: The authors have no financial conflicts of interest. Ethics Approval Statement: This study was approved by the Ethical Committee of NHTD and Kumamoto University. Informed consent was obtained from all individuals according to the Declaration of Helsinki.

High density of unrepaired genomic ribonucleotides leads to Topoisomerase 1-mediated severe growth defects in absence of ribonucleotide reductase

Cellular levels of ribonucleoside triphosphates (rNTPs) are much higher than those of deoxyribonucleoside triphosphates (dNTPs), thereby influencing the frequency of incorporation of ribonucleoside monophosphates (rNMPs) by DNA polymerases (Pol) into DNA. RNase H2-initiated ribonucleotide excision repair (RER) efficiently removes single rNMPs in genomic DNA. However, processing of rNMPs by Topoisomerase 1 (Top1) in absence of RER induces mutations and genome instability. Here, we greatly increased the abundance of genomic rNMPs in Saccharomyces cerevisiae by depleting Rnr1, the major subunit of ribonucleotide reductase, which converts ribonucleotides to deoxyribonucleotides. We found that in strains that are depleted of Rnr1, RER-deficient, and harbor an rNTP-permissive replicative Pol mutant, excessive accumulation of single genomic rNMPs severely compromised growth, but this was reversed in absence of Top1. Thus, under Rnr1 depletion, limited dNTP pools slow DNA synthesis by replicative Pols and provoke the incorporation of high levels of rNMPs in genomic DNA. If a threshold of single genomic rNMPs is exceeded in absence of RER and presence of limited dNTP pools, Top1-mediated genome instability leads to severe growth defects. Finally, we provide evidence showing that accumulation of RNA/DNA hybrids in absence of RNase H1 and RNase H2 leads to cell lethality under Rnr1 depletion.

Analysis of unusual and signature APOBEC-mutations in HIV-1 pol next-generation sequences.

At low mutation-detection thresholds, next generation sequencing (NGS) for HIV-1 genotypic resistance testing is susceptible to artifactual detection of mutations arising from PCR error and APOBEC-mediated G-to-A hypermutation. We analyzed published HIV-1 pol Illumina NGS data to characterize the distribution of mutations at eight NGS mutation detection thresholds: 20%, 10%, 5%, 2%, 1%, 0.5%, 0.2%, and 0.1%. At each threshold, we determined proportions of amino acid mutations that were unusual (defined as having a prevalence <0.01% in HIV-1 group M sequences) or signature APOBEC mutations. Eight studies, containing 855 samples, in the NCBI Sequence Read Archive were analyzed. As detection thresholds were lowered, there was a progressive increase in the proportion of positions with usual and unusual mutations and in the proportion of all mutations that were unusual. The median proportion of positions with an unusual mutation increased gradually from 0% at the 20% threshold to 0.3% at the 1% threshold and then exponentially to 1.3% (0.5% threshold), 6.9% (0.2% threshold), and 23.2% (0.1% threshold). In two of three studies with available plasma HIV-1 RNA levels, the proportion of positions with unusual mutations was negatively associated with virus levels. Although the complete set of signature APOBEC mutations was much smaller than that of unusual mutations, the former outnumbered the latter in one-sixth of samples at the 0.5%, 1%, and 2% thresholds. The marked increase in the proportion of positions with unusual mutations at thresholds below 1% and in samples with lower virus loads suggests that, at low thresholds, many unusual mutations are artifactual, reflecting PCR error or G-to-A hypermutation. Profiling the numbers of unusual and signature APOBEC pol mutations at different NGS mutation detection thresholds may be useful to avoid selecting a threshold that is too low and poses an unacceptable risk of identifying artifactual mutations.

Fertilization-Coupled Sperm Nuclear Fusion Is Required for Normal Endosperm Nuclear Proliferation.

Angiosperms exhibit double fertilization, a process in which one of the sperm cells released from the pollen tube fertilizes the egg, while the other sperm cell fertilizes the central cell, giving rise to the embryo and endosperm, respectively. We have previously reported two polar nuclear fusion-defective double knockout mutants of Arabidopsis thaliana immunoglobulin binding protein (BiP), a molecular chaperone of the heat shock protein 70 (Hsp70) localized in the endoplasmic reticulum (ER), (bip1 bip2) and its partner ER-resident J-proteins, ERdj3A and P58IPK (erdj3a p58ipk). These mutants are defective in the fusion of outer nuclear membrane and exhibit characteristic seed developmental defects after fertilization with wild-type pollen, which are accompanied by aberrant endosperm nuclear proliferation. In this study, we used time-lapse live-cell imaging analysis to determine the cause of aberrant endosperm nuclear division in these mutant seeds. We found that the central cell of bip1 bip2 or erdj3a p58ipk double mutant female gametophytes was also defective in sperm nuclear fusion at fertilization. Sperm nuclear fusion was achieved after the onset of the first endosperm nuclear division. However, division of the condensed sperm nucleus resulted in aberrant endosperm nuclear divisions and delayed expression of paternally derived genes. By contrast, the other double knockout mutant, erdj3b p58ipk, which is defective in the fusion of inner membrane of polar nuclei but does not show aberrant endosperm nuclear proliferation, was not defective in sperm nuclear fusion at fertilization. We thus propose that premitotic sperm nuclear fusion in the central cell is critical for normal endosperm nuclear proliferation.

The copper-inducible copAB operon in Xanthomonas citri subsp. citri is regulated at transcriptional and translational levels.

Upstream open reading frames (ORFs) are frequently found in the 5'-flanking regions of genes and may have a regulatory role in gene expression. A small ORF (named cohL here) was identified upstream from the copAB copper operon in Xanthomonascitri subsp. citri (Xac). We previously demonstrated that copAB expression was induced by copper and that gene inactivation produced a mutant strain that was unable to grow in the presence of copper. Here, we address the role of cohL in copAB expression control. We demonstrate that cohL expression is induced by copper in a copAB-independent manner. Although cohL is transcribed, the CohL protein is either not expressed in vivo or is synthesized at undetectable levels. Inactivation of cohL (X. citri cohL polar mutant strain) leads to an inability to synthesize cohL and copAB transcripts and consequently the inability to grow in the presence of copper. Bioinformatic tools predicted a stem-loop structure for the cohL-copAB intergenic region and revealed that this region may arrange itself in a secondary structure. Using in vitro gene expression, we found out that the structured 5'-UTR mRNA of copAB is responsible for sequestering the ribosome-binding site that drives the translation of copA. However, copper alone was not able to release the sequence. Based on the results, we speculate that cohL plays a role as a regulatory RNA rather than as a protein-coding gene.

Genetic diversity of the human adenovirus species C DNA polymerase

BackgroundHuman Adenovirus (HAdV) are responsible for severe infections in hematopoietic stem cells transplant (HSCT) recipient, species C viruses being the most commonly observed in this population. There is no approved antiviral treatment yet. Cidofovir (CDV), a cytidine analog, is the most frequently used and its lipophilic conjugate, brincidofovir (BCV), is under clinical development. These drugs target the viral DNA polymerase (DNA pol). Little is known about the natural polymorphism of HAdV DNA pol in clinical strains. MethodsWe assessed the inter- and intra-species variability of the whole gene coding for HAdV DNA pol of HAdV clinical strains of species C. The study included 60 species C HAdV (21 C1, 27 C2 and 12 C5) strains isolated from patients with symptomatic infections who had never experienced CDV or BCV treatments and 20 reference strains. We also evaluated the emergence of mutations in thrirteen patients with persistent HAdV infection despite antiviral treatment. ResultsWe identified 356 polymorphic nucleotide positions (9.9% of the whole gene), including 102 positions with nonsynonymous mutations (28.0%) representing 8.7% of all amino acids. The mean numbers of nucleotide and amino acid mutations per strain were 23.1 (±6.2) and 5.2 (±2.4) respectively. Most of amino acid substitutions (60.6%) were observed in one instance only. A minority (13.8%) were observed in more than 10% of all strains. The most variable region was the NH2 terminal domain (44.2% of amino acid mutations). Mutations in the exonuclease domain accounted for 27.8%. The binding domains for the terminal protein (TPR), TPR1 and TPR2, presented a limited number of mutations, which were nonetheless frequently observed (62.5% and 58.8% of strains for TPR1 and TPR2, respectively). None of the mutations associated with CDV or BCV resistance were detected. In patients receieving antiviral drugs with persistent HAdV replication, we identified a new mutation in the NH2 terminal region. ConclusionsOur study shows a high diversity in HAdV DNA pol sequences in clinical species C HAdV and provides a comprehensive mapping of its natural polymorphism. These data will contribute to the interpretation of HAdV DNA pol mutations selected in patients receiving antiviral treatments.