Active Replication Research Articles

A Computational Study on the Interaction of NSP10 and NSP14: Unraveling the RNA Synthesis Proofreading Mechanism in SARS-CoV-2, SARS-CoV, and MERS-CoV.

The interaction of exoribonuclease (ExoN) nonstructural protein (NSP14) with NSP10 co-factors is crucial for high-fidelity proofreading activity of coronavirus replication and transcription. Proofreading function is critical for maintaining the large genomes to ensure replication proficiency; therefore, while maintaining the viral replication fitness, quick resistance has been reported to the nucleotide analogue (NA) drugs. Therefore, targeting the NSP14 and NSP10 interacting interface with small molecules or peptides could be a better strategy to obstruct replication processes of coronaviruses (CoVs). A comparative study on the binding mechanism of NSP10 with the NSP14 ExoN domain of SARS-CoV-2, SARS-CoV, MERS-CoV, and four SARS-CoV-2 NSP14mutant complexes has been carried out. Protein–protein interaction (PPI) dynamics, per-residue binding free energy (BFE) analyses, and the identification of interface hotspot residues have been studied using molecular dynamics simulations and various computational tools. The BFE of the SARS-CoV NSP14–NSP10 complex was higher when compared to novel SARS-CoV-2 and MERS. However, SARS-CoV-2 NSP14mutant systems display a higher BFE as compared to the wild type (WT) but lower than SARS-CoV and MERS. Despite the high BFE, the SARS-CoV NSP14–NSP10 complex appears to be structurally more flexible in many regions especially the catalytic site, which is not seen in SARS-CoV-2 and its mutant or MERS complexes. The significantly high residue energy contribution of key interface residues and hotspots reveals that the high binding energy between NSP14 and NSP10 may enhance the functional activity of the proofreading complex, as the NSP10–NSP14 interaction is essential in maintaining the stability of the ExoN domain for the replicative fitness of CoVs. The factors discussed for SARS-CoV-2 complexes may be responsible for NSP14 ExoN having a high replication proficiency, significantly leading to the evolution of new variants of SARS-CoV-2. The NSP14 residues V66, T69, D126, and I201and eight residues of NSP10 (L16, F19, V21, V42, M44, H80, K93, and F96) are identified as common hotspots. Overall, the interface area, hotspot locations, bonded/nonbonded contacts, and energies between NSP14 and NSP10 may pave a way in designing potential inhibitors to disrupt NSP14–NSP10 interactions of CoVs especially SARS-CoV-2.

High Predictive Value of the Soluble ZEBRA Antigen (Epstein-Barr Virus Trans-Activator Zta) in Transplant Patients with PTLD.

The ZEBRA (Z EBV replication activator) protein is the major transcription factor of EBV, expressed upon EBV lytic cycle activation. An increasing body of studies have highlighted the critical role of EBV lytic infection as a risk factor for lymphoproliferative disorders, such as post-transplant lymphoproliferative disease (PTLD). We studied 108 transplanted patients (17 PTLD and 91 controls), retrospectively selected from different hospitals in France and in the Netherlands. The majority of PTLD were EBV-positive diffuse large B-cell lymphomas, five patients experienced atypical PTLD forms (EBV-negative lymphomas, Hodgkin’s lymphomas, and T-cell lymphomas). Fourteen patients among the seventeen who developed a pathologically confirmed PTLD were sZEBRA positive (soluble ZEBRA, plasma level above 20 ng/mL, measured by an ELISA test). The specificity and positive predictive value (PPV) of the sZEBRA detection in plasma were 98% and 85%, respectively. Considering a positivity threshold of 20 ng/mL, the sensitivity of the sZEBRA was 82.35% and the specificity was 94.51%. The mean of the sZEBRA values in the PTLD cases were significantly higher than in the controls (p < 0.0001). The relevance of the lytic cycle and, particularly, the role of ZEBRA in lymphomagenesis is a new paradigm pertaining to the prevention and treatment strategies for PTLD. Given the high-specificity and the predictive values of this test, it now appears relevant to investigate the lytic EBV infection in transplanted patients as a prognostic biomarker.

Control of occult hepatitis B virus infection.

The diagnosis of hepatitis B virus (HBV) infection requires HBV DNA testing and serologic testing for detection of the surface antigen (HBsAg) and the hepatitis B core antibody (anti-HBc). There is a population of patients with occult HBV infection (OBI), which is not detected by HBsAg or HBV DNA quantification in blood, despite the presence of active replication in the liver. This document provides a definition of OBI and describes the diagnostic techniques currently used. It also addresses the detection of patients with risk factors and the need for screening for OBI in these patients. Correct diagnosis of OBI prevents HBV reactivation and transmission. Diagnosis of OBI is based on the detection of HBV DNA in patients with undetectable HBsAg in blood. A high number of patients with OBI may remain undiagnosed; therefore, screening for OBI in patients with factor risks is essential. For a correct diagnosis of OBI, it is necessary that new markers such as ultrasensitive HBsAg are incorporated, and a more comprehensive marker study is performed by including markers such as cccDNA.

Identification of a non-canonical G3BP-binding sequence in a Mayaro virus nsP3 hypervariable domain.

Ras-GTPase-activating SH3 domain-binding-proteins 1 (G3BP1) and 2 (G3BP2) are multifunctional RNA-binding proteins involved in stress granule nucleation, previously identified as essential cofactors of Old World alphaviruses. They are recruited to viral replication complexes formed by the Chikungunya virus (CHIKV), Semliki Forest virus (SFV), and Sindbis virus (SINV) via an interaction with a duplicated FGxF motif conserved in the hypervariable domain (HVD) of virus-encoded nsP3. According to mutagenesis studies, this FGxF duplication is strictly required for G3BP binding and optimal viral growth. Contrasting with this scenario, nsP3 encoded by Mayaro virus (MAYV), an arthritogenic virus grouped with Old World alphaviruses, contains a single canonical FGxF sequence. In light of this unusual feature, we questioned MAYV nsP3/G3BPs relationships. We report that G3BP1 and G3BP2 are both required for MAYV growth in human cells and bind nsP3 protein. In infected cells, they are recruited to nsP3-containing cytosolic foci and active replication complexes. Unexpectedly, deletion of the single FGxF sequence in MAYV nsP3 did not abolish these phenotypes. Using mutagenesis and in silico modeling, we identify an upstream FGAP amino acid sequence as an additional MAYV nsP3/G3BP interaction motif required for optimal viral infectivity. These results, therefore, highlight a non-conventional G3BP binding sequence in MAYV nsP3.

Global burden of occult hepatitis B virus infection: where we are and what to do

Occult hepatitis B virus (HBV) infection is characterised by the persistence of replication-competent HBV DNA in the liver (with or without HBV DNA in the blood) of individuals testing negative for HBsAg, using currently available assays.1 Occult HBV infection represents a phase of the natural history of HBV infection,2 in which HBV DNA in the form of a covalently closed circular DNA minichromosome has suppressed replicative activity and gene expression with extremely low rates of virion production.

Development and Use of a Kinetical and Real-Time Monitoring System to Analyze the Replication of Hepatitis C Virus.

In microbiological research, it is important to understand the time course of each step in a pathogen’s lifecycle and changes in the host cell environment induced by infection. This study is the first to develop a real-time monitoring system that kinetically detects luminescence reporter activity over time without sampling cells or culture supernatants for analyzing the virus replication. Subgenomic replicon experiments with hepatitis C virus (HCV) showed that transient translation and genome replication can be detected separately, with the first peak of translation observed at 3–4 h and replication beginning around 20 h after viral RNA introduction into cells. From the bioluminescence data set measured every 30 min (48 measurements per day), the initial rates of translation and replication were calculated, and their capacity levels were expressed as the sums of the measured signals in each process, which correspond to the areas on the kinetics graphs. The comparison of various HuH-7-derived cell lines showed that the bioluminescence profile differs among cell lines, suggesting that both translation and replication capacities potentially influence differences in HCV susceptibility. The effects of RNA mutations within the 5′ UTR of the replicon on viral translation and replication were further analyzed in the system developed, confirming that mutations to the miR-122 binding sites primarily reduce replication activity rather than translation. The newly developed real-time monitoring system should be applied to the studies of various viruses and contribute to the analysis of transitions and progression of each process of their life cycle.

Chronic unilateral anterior nodular scleritis with local inflammation of the ciliary body associated with the varicella-zoster virus

AIM: To analyze the etiopathogenesis, clinical features, and treatment algorithm for chronic unilateral anterior nodular scleritis with local inflammation of the ciliary body to increase medical alertness to the herpetic etiology of the disease in the absence of extraocular manifestations of herpes infection, reduce the disease duration, and increase the effectiveness of treatment.&#x0D; RESULTS: Features of etiopathogenesis were analyzed. The characteristic clinical symptoms of chronic nodular scleritis, local anterior cyclitis, and pars planitis caused by the varicella-zoster virus (VZV) were described. The etiological role of VZV has been established based on high levels of VZV-IgG antibodies, presence of VZV-gE-IgG antibodies (markers of active virus replication), and effectiveness of antiherpetic therapy.&#x0D; DISCUSSION: The surgical removal of a melanocytic skin nevus with skin autotransplantation in the paraorbital region of the left eye, in the zone of innervation of the first branch of the trigeminal nerve, contributed to the reactivation of the ophthalmic herpes and the development of anterior nodular scleritis of the left eye. An intensive long-term ineffective therapy with corticosteroids and antibacterial drugs in the absence of etiotropic treatment caused a chronic course of anterior nodular scleritis, spread of the inflammatory process to the ciliary body, and development of local anterior cyclitis and pars planitis of herpetic etiology in a 17-year-old child.&#x0D; CONCLUSION: Maximum medical alertness and early and accurate clinical differential diagnosis between scleritis associated with immunoinflammatory rheumatic diseases and herpesvirus infections are necessary since the expansion of the range and number of anti-inflammatory drugs used in the absence of positive dynamics from their use leads to a chronic disease course, damage not only to deep layers of the sclera but also the spread of inflammation to the deeper layers of the eyeball, a decrease in visual acuity, undesirable effects of local glucocorticoid therapy, and an increase in intraocular pressure and development of cataracts. With any scleritis resistant to conventional treatment, the likelihood of a herpetic etiology of the inflammatory process and laboratory diagnosis of ophthalmic herpes should be considered. In the absence of a specialized laboratory, for etiological diagnosis, the possibility of ex juvantibus antiviral therapy should be considered. The described clinical symptoms of chronic nodular scleritis with local lesions of the ciliary body contribute to the early diagnosis of ophthalmoherpes, which allows the timely initiation of antiviral therapy with an antiherpetic effect, prevents the development of a chronic disease course, occurrence of complications, and preservation and/or restoration of visual acuity.

Coronavirus Infections in Animals: Risks of Direct and Reverse Zoonoses.

The publications on animal coronavirus infections that have the greatest emerging potential, as well as official data from the World Organization for Animal Health (OIE) on cases of animal infection with COVID-19, are analyzed. Like most infectious diseases common to humans, coronavirus infections were first discovered in animals. Due to the increased rate of replication and recombination activity compared to other viruses, mutations occur more often in the genome of coronaviruses, which contribute to the acquisition of new qualities in order to consolidate in the host organism. Examples of cross-species transmission are not only SARS-CoV, MERS-CoV, and SARS-CoV-2, which are dangerous to humans, but also coronaviruses of agricultural and domestic animals, between which there is a genetic relationship. There are several known cases of zoo, wild, domestic, and farm animals displaying symptoms characteristic of COVID-19 and identification of the genome of the SARS-CoV-2 virus in them. The issue of cross-species transmission of coronavirus infections, in particular the reverse zoonosis of SARS-CoV-2 from animals to humans, is widely discussed. According to the conclusions of many researchers, including OIE experts, there is no direct evidence base for infection of humans with COVID-19 from animals. However, people with suspected COVID-19 and with a confirmed diagnosis are still advised to isolate not only from people but also from animals. A number of methods for specific prevention, diagnosis, and immunization against a wide range of coronavirus infections are being developed at the All-Russia Research Institute for Animal Protection.

SV40 miR-S1 and Cellular miR-1266 Sequester Each Other from Their Targets, Enhancing Telomerase Activity and Viral Replication.

Virus-encoded microRNAs (miRNAs) target viral and host mRNAs to repress protein production from viral and host genes, and regulate viral persistence, cell transformation, and evasion of the immune system. The present study demonstrated that simian virus 40 (SV40)-encoded miRNA miR-S1 targets a cellular miRNA miR-1266 to derepress their respective target proteins, namely, T antigens (Tags) and telomerase reverse transcriptase (TERT). An in silico search for cellular miRNAs to interact with viral miR-S1 yielded nine potential miRNAs, five of which, including miR-1266, were found to interact with miR-S1 in dual-luciferase tests employing reporter plasmids containing the miRNA sequences with miR-S1. Intracellular bindings of miR-1266 to miR-S1 were also verified by the pull-down assay. These miRNAs were recruited into the Ago2-associated RNA-induced silencing complex. Intracellular coexpression of miR-S1 with miR-1266 abrogated the downregulation of TERT and decrease in telomerase activity induced by miR-1266. These effects of miR-S1 were also observed in miR-1266-expressing A549 cells infected with SV40. Moreover, the infected cells contained more Tag, replicated more viral DNA, and released more viral particles than control A549 cells infected with SV40, indicating that miR-S1-induced Tag downregulation was antagonized by miR-1266. Collectively, the present results revealed an interplay of viral and cellular miRNAs to sequester each other from their respective targets. This is a novel mechanism for viruses to manipulate the expression of viral and cellular proteins, contributing to not only viral lytic and latent replication but also cell transformation observed in viral infectious diseases including oncogenesis.

Early Emergence of 5' Terminally Deleted Coxsackievirus-B3 RNA Forms Is Associated with Acute and Persistent Infections in Mouse Target Tissues.

Major EV-B populations characterized by 5′ terminal deletions (5′TD) have been shown to be associated with the development of myocarditis and type 1 diabetes in mice or humans. To date, the dynamics of EV-B 5′TD-RNA forms’ emergence during the course of infection and their impact on cellular functions remain unclear. Using a RACE-PCR approach in CVB3/28-infected mouse organs, we showed an early (3 days post infection, DPI) emergence of major 5′TD populations associated with minor full-length RNA forms. Viral replication activities with infectious particle production were associated with heart, liver, and pancreas acute inflammatory lesions, whereas clearance of viral RNA without organ lesions was observed in the brain, lung, intestines, and muscles from 3 to 7 DPI. At 28 DPI, low viral RNA levels, +/-RNA ratios < 5 associated with viral protein 1 expression revealed a persistent infection in the heart and pancreas. This persistent infection was characterized by molecular detection of only 5′TD RNA forms that were associated with dystrophin cleavage in the heart and insulin production impairment in beta-pancreatic cells. These results demonstrated that major EV-B 5′TD RNA forms can be early selected during systemic infection and that their maintenance may drive EV-induced acute and persistent infections with target cell dysfunctions.

Design, synthesis and biological evaluation of novel dihydrobenzodioxine derivatives as HBV capsid protein inhibitors

Capsid assembly modulators (CAMs) have recently been revealed to be effective in blocking HBV replication. HBV capsid protein inhibitors reduce and ultimately eliminate HBV by inhibiting virus replication and blocking hepatocyte infection. Sulfonamides are synthetic functional groups in development of different kinds of drugs. Sulfonyl benzamide clinical drugs NVR 3-778 and BA-38017 are lead compounds in discovery of antiviral compounds with increased activity and reduced cytotoxicity by drug design strategies including pharmacophore hybrid, bioisosterism and scaffold hopping. In current study, three series of target compounds were synthesized, and their anti-HBV activity was evaluated against HepAD38 cells. Compound 5a (EC50 = 0.50 ± 0.07 μM, CC50 = 48.16 ± 9.15 μM) showed better anti-HBV DNA replication activity than the lead compound BA-38017, and showed good inhibitory effect on the assembly of HBV capsid protein compared with the clinical drug NVR 3-778. In addition, preliminary structure–activity relationship (SAR) and molecular docking studies were conducted to explore potential interactions and binding modes between compounds and target proteins, which may help researchers to find more effective anti-HBV drugs.

Targeting Replication Stress Response Pathways to Enhance Genotoxic Chemo- and Radiotherapy.

Proliferating cells regularly experience replication stress caused by spontaneous DNA damage that results from endogenous reactive oxygen species (ROS), DNA sequences that can assume secondary and tertiary structures, and collisions between opposing transcription and replication machineries. Cancer cells face additional replication stress, including oncogenic stress that results from the dysregulation of fork progression and origin firing, and from DNA damage induced by radiotherapy and most cancer chemotherapeutic agents. Cells respond to such stress by activating a complex network of sensor, signaling and effector pathways that protect genome integrity. These responses include slowing or stopping active replication forks, protecting stalled replication forks from collapse, preventing late origin replication firing, stimulating DNA repair pathways that promote the repair and restart of stalled or collapsed replication forks, and activating dormant origins to rescue adjacent stressed forks. Currently, most cancer patients are treated with genotoxic chemotherapeutics and/or ionizing radiation, and cancer cells can gain resistance to the resulting replication stress by activating pro-survival replication stress pathways. Thus, there has been substantial effort to develop small molecule inhibitors of key replication stress proteins to enhance tumor cell killing by these agents. Replication stress targets include ATR, the master kinase that regulates both normal replication and replication stress responses; the downstream signaling kinase Chk1; nucleases that process stressed replication forks (MUS81, EEPD1, Metnase); the homologous recombination catalyst RAD51; and other factors including ATM, DNA-PKcs, and PARP1. This review provides an overview of replication stress response pathways and discusses recent pre-clinical studies and clinical trials aimed at improving cancer therapy by targeting replication stress response factors.

An anti-picornaviral strategy based on the crystal structure of foot-and-mouth disease virus 2C protein.

The foot-and-mouth disease virus (FMDV) 2C protein shares conserved motifs with enterovirus 2Cs despite low sequence identity. Here, we determine the crystal structure of an FMDV 2C fragment to 1.83Å resolution, which comprises an ATPase domain, a region equivalent to the enterovirus 2C zinc-finger (ZFER), and a C-terminal domain harboring a loop (PBL) that occupies a hydrophobic cleft (Pocket) in an adjacent 2C molecule. Mutations at ZFER, PBL, and Pocket affect FMDV 2C ATPase activity and are lethal to FMDV infectious clones. Because the PBL-Pocket interaction between FMDV 2C molecules is essential for its functions, we design an anti-FMDV peptide derived from PBL (PBL-peptide). PBL-peptide inhibits FMDV 2C ATPase activity, binds FMDV 2C with nanomolar affinity, and disrupts FMDV 2C oligomerization. FMDV 2C targets lipid droplets (LDs) and induces LD clustering in cells, and PBL-peptide disrupts FMDV 2C-induced LD clustering. Finally, we demonstrate that PBL-peptide exhibits anti-FMDV activity in cells.

Replication stress triggered by nucleotide pool imbalance drives DNA damage and cGAS-STING pathway activation in NAFLD.

Non-alcoholic steatotic liver disease (NAFLD) is the most common cause of chronic liver disease worldwide. NAFLD has a major effect on the intrinsic proliferative properties of hepatocytes. Here, we investigated the mechanisms underlying the activation of DNA damage response during NAFLD. Proliferating mouse NAFLD hepatocytes harbor replication stress (RS) with an alteration of the replication fork's speed and activation of ATR pathway, which is sufficient to cause DNA breaks. Nucleotide pool imbalance occurring during NAFLD is the key driver of RS. Remarkably, DNA lesions drive cGAS/STING pathway activation, a major component of cells' intrinsic immune response. The translational significance of this study was reiterated by showing that lipid overload in proliferating HepaRG was sufficient to induce RS and nucleotide pool imbalance. Moreover, livers from NAFLD patients displayed nucleotide pathway deregulation and cGAS/STING gene alteration. Altogether, our findings shed light on the mechanisms by which damaged NAFLD hepatocytes might promote disease progression.

Stalled replication fork protection limits cGAS-STING and P-body-dependent innate immune signalling.

Protection of stalled replication forks is crucial for cells to respond to replication stress and maintain genome stability. Genome instability and replication stress have been linked to immune activation. Here we show that Abro1 and FANCD2 protect replication forks, which is linked with the restriction of innate immune responses. We reveal that stalled replication fork degradation induced by Abro1 or FANCD2 deficiency leads to accumulation of cytosolic single-stranded DNA and activation of a cGAS-STING-dependent innate immune response that is dependent on DNA2 nuclease. We further show that the increased cytosolic single-stranded DNA contains ribosomal DNA that can bind to cGAS. In addition, Abro1 and FANCD2 limit the formation of replication stress-induced P-bodies, and P-bodies are capable of modulating activation of the innate immune response after prolonged replication stress. Our study demonstrates a connection between replication stress and activation of the innate immune response that may be targeted for therapeutic purpose.

THU310 - RIPK1 plays a survival role in the liver of mice with active HDV replication

THU310 - RIPK1 plays a survival role in the liver of mice with active HDV replication

BK Polyomavirus bkv-miR-B1-5p: A Stable Micro-RNA to Monitor Active Viral Replication after Kidney Transplantation.

Background: Bkv-miR-B1-5p is a viral micro-RNA (miRNA) specifically produced during BK polyomavirus (BKPyV) replication. Recent studies have suggested using bkv-miR-B1-5p as a biomarker to monitor viral infection and predict complications in kidney transplant patients. To identify the technical limitations of this miRNA quantification in biological samples, knowledge of its stability and distribution in the extracellular compartment is necessary. Moreover, a proof of concept for using bkv-miR-B1-5p as a biomarker of active replication in chronic infection is still missing in the published literature. Methods: The stability of bkv-miR-B1-5p was evaluated in samples derived from cell cultures and in urine from BKPyV-infected kidney transplant recipients. The miRNA was quantified in different fractions of the extracellular compartment, including exosomes, and protein binding was evaluated. Finally, we developed an in vitro model for chronic culture of BKPyV clinical isolates to observe changes in the bkv-miR-B1-5p level during persistent infections. Results: Bkv-miR-B1-5p is a stable biomarker in samples from humans and in vitro experiments. Marginally associated with the exosomes, most of the circulating bkv-miR-B1-5p is bound to proteins, especially Ago2, so the miRNA quantification does not require specific exosome isolation. The bkv-miR-B1-5p level is predictable of viral infectivity, which makes it a potential specific biomarker of active BKPyV replication after kidney transplantation.

Plasma Virome Reveals Blooms and Transmission of Anellovirus in Intravenous Drug Users with HIV-1, HCV, and/or HBV Infections.

ABSTRACTIntravenous drug users (IDUs) are a high-risk group for HIV-1, hepatitis C virus (HCV), and hepatitis B virus (HBV) infections, which are the leading causes of death in IDUs. However, the plasma virome of IDUs and how it is influenced by above viral infections remain unclear. Using viral metagenomics, we determined the plasma virome of IDUs and its association with HIV-1, HCV, and/or HBV infections. Compared with healthy individuals, IDUs especially those with major viral infections had higher viral abundance and diversity. Anelloviridae dominated plasma virome. Coinfections of multiple anelloviruses were common, and anelloviruses from the same genus tended to coexist together. In this study, 4,487 anellovirus ORF1 sequences were identified, including 1,620 (36.1%) with less than 69% identity to any known sequences, which tripled the current number. Compared with healthy controls (HC), more anellovirus sequences were observed in neg-IDUs, and HIV-1, HCV, and/or HBV infections further expanded the sequence number in IDUs, which was characterized by the emergence of novel divergent taxons and blooms of resident anelloviruses. Pegivirus was mainly identified in infected IDUs. Five main pegivirus transmission clusters (TCs) were identified by phylogenetic analysis, suggesting a transmission link. Similar anellovirus profiles were observed in IDUs within the same TC, suggesting transmission of anellome among IDUs. Our data suggested that IDUs suffered higher plasma viral burden especially anelloviruses, which was associated with HIV-1, HCV, and/or HBV infections. Blooms in abundance and unprecedented diversity of anellovirus highlighted active evolution and replication of this virus in blood circulation, and an uncharacterized role it may engage with the host.IMPORTANCE Virome is associated with immune status and determines or influences disease progression through both pathogenic and resident viruses. Increased viral burden in IDUs especially those with major viral infections indicated the suboptimal immune status and high infection risks of these population. Blooms in abundance and unprecedented diversity of anellovirus highlighted its active evolution and replication in the blood circulation, and sensitive response to other viral infections. In addition, transmission cluster analysis revealed the transmission link of pegivirus among IDUs, and the individuals with transmission links shared similar anellome profiles. In-depth monitoring of the plasma virome in high-risk populations is not only needed for surveillance for emerging viruses and transmission networks of major and neglected bloodborne viruses, but also important for a better understanding of commensal viruses and their role it may engage with immune system.

Mitochondrial transport and metabolism of the major methyl donor and versatile cofactor S-adenosylmethionine, and related diseases: A review†.

S-adenosyl-L-methionine (SAM) is a coenzyme and the most commonly used methyl-group donor for the modification of metabolites, DNA, RNA and proteins. SAM biosynthesis and SAM regeneration from the methylation reaction product S-adenosyl-L-homocysteine (SAH) take place in the cytoplasm. Therefore, the intramitochondrial SAM-dependent methyltransferases require the import of SAM and export of SAH for recycling. Orthologous mitochondrial transporters belonging to the mitochondrial carrier family have been identified to catalyze this antiport transport step: Sam5p in yeast, SLC25A26 (SAMC) in humans, and SAMC1-2 in plants. In mitochondria SAM is used by a vast number of enzymes implicated in the following processes: the regulation of replication, transcription, translation, and enzymatic activities; the maturation and assembly of mitochondrial tRNAs, ribosomes and protein complexes; and the biosynthesis of cofactors, such as ubiquinone, lipoate, and molybdopterin. Mutations in SLC25A26 and mitochondrial SAM-dependent enzymes have been found to cause human diseases, which emphasizes the physiological importance of these proteins.

DNA replication dynamics during erythrocytic schizogony in the malaria parasites Plasmodium falciparum and Plasmodium knowlesi.

Malaria parasites are unusual, early-diverging protozoans with non-canonical cell cycles. They do not undergo binary fission, but divide primarily by schizogony. This involves the asynchronous production of multiple nuclei within the same cytoplasm, culminating in a single mass cytokinesis event. The rate and efficiency of parasite reproduction is fundamentally important to malarial disease, which tends to be severe in hosts with high parasite loads. Here, we have studied for the first time the dynamics of schizogony in two human malaria parasite species, Plasmodium falciparum and Plasmodium knowlesi. These differ in their cell-cycle length, the number of progeny produced and the genome composition, among other factors. Comparing them could therefore yield new information about the parameters and limitations of schizogony. We report that the dynamics of schizogony differ significantly between these two species, most strikingly in the gap phases between successive nuclear multiplications, which are longer in P. falciparum and shorter, but more heterogenous, in P. knowlesi. In both species, gaps become longer as schizogony progresses, whereas each period of active DNA replication grows shorter. In both species there is also extreme variability between individual cells, with some schizonts producing many more nuclei than others, and some individual nuclei arresting their DNA replication for many hours while adjacent nuclei continue to replicate. The efficiency of schizogony is probably influenced by a complex set of factors in both the parasite and its host cell.