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PubMed2026

Maintenance of nucleosome organization through replication and transcription counteracts aberrant coalescence of active chromatin.

Nucleosomes with their associated modifications organize and regulate the genome. It is unclear how this is integrated with the requirement of replication and transcription to access the DNA template without jeopardizing chromatin function. Here, we reveal a unified requirement for the histone chaperone FACT in mediating nucleosome disruption and reassembly during mammalian replication and transcription. Upon acute FACT depletion, replisome and RNA polymerase progression is halted genome wide, and chromatin structure in their wake collapses, with reduced nucleosome occupancy, irregular spacing, and intermediate assemblies. Chromatin states deteriorate as modified histones are lost due to a lack of histone recycling. Chromatin fiber disorder further manifests in the 3D genome, triggering active genes to coalesce in aberrant microcompartments. Similarly, aberrant compartments form in cells failing to maintain chromatin fiber structure through replication. Nucleosome organization therefore dynamically regulates genome architecture, guarding against spurious chromatin aggregation.

Nucleosomes↗
PubMed2026

Halorubrum marinum sp. nov., Halorubrum rarum sp. nov., Halorubrum wangae sp. nov., Halorubrum shenae sp. nov., and Halorubrum zhoui sp. nov., halophilic archaea from coastal tidal flats, a saline lake, and a marine solar saltern.

Five novel halophilic archaeal strains, designated DTA46T, DTA98T, HHNYT27T, N11T, and SY-15T, were isolated from diverse saline environments across various regions of China. Amplicon and metagenome analyses revealed that three amplicon reads were affiliated with strains DTA46T, HHNYT27T, and N11T while two MAGs related to strains N11T and SY-15T. The sequence similarities among these five strains and current species of the genus Halorubrum were 93.1%-99.1% and 86.0%-95.9% judged by 16S rRNA and rpoB' genes, respectively. Phylogenomic and comparative genomic analyses revealed their close affiliation with Halorubrum. The average nucleotide identity (ANI), digital DNA-DNA hybridization (dDDH), and average amino acid identity (AAI) values between these strains and existing Halorubrum species ranged from 74.9%-93.6%, 22.3%-58.3%, and 68.3%-93.7%, respectively. All are below the recommended thresholds for species delineation, which supports their classification as novel taxa. The growth characteristics of strains DTA46T, DTA98T, HHNYT27T, N11T, and SY-15T were determined as follows: temperature range 20-60 °C (optima: 35, 37-42, 37, 35, and 42 °C), NaCl concentration 1.4-5.5 M (optima: 2.6, 3.1, 3.1, 3.1, and 5.1 M), and pH range 5.5-9.5 (optima: 8.0, 8.0, 7.0, 7.5, and 7.0). Based on the polyphasic characterization integrating phenotypic, chemotaxonomic, phylogenetic, and phylogenomic evidence, strains DTA46T, DTA98T, HHNYT27T, N11T, and SY-15T are proposed to represent five novel species of the genus Halorubrum, for which the names Halorubrum marinum sp. nov., Halorubrum rarum sp. nov., Halorubrum wangae sp. nov., Halorubrum shenae sp. nov., and Halorubrum zhoui sp. nov. are designated, respectively.

Phylogeny↗
PubMed2026

Cold-adapted RNA polymerase from Pseudomonas phage Njord improves synthesis of therapeutic mRNA.

An RNA polymerase identified in the genome of Pseudomonas phage Njord offers a promising tool for the synthesis of mRNA and other therapeutic nucleic acids. Originating from a marine microbial ecosystem, Njord RNAP transcribes RNA at high yield even under low temperature conditions. Key properties of the enzyme relevant to mRNA synthesis are presented including transcriptional fidelity, promoter specificity, incorporation of modified nucleotides, and the impurity profile of the RNA. Specific attention is given to the formation of contaminating double-stranded RNA (dsRNA) species. Analysis of transcription reactions shows that DNA-templated promoter-independent transcription is a major source of detectable dsRNA impurities and that Njord RNAP displays a minimal level of this activity. Consistent with the known inflammatory role of dsRNA in synthetic mRNA, transcriptomic analysis of cell culture and a live animal study demonstrates that mRNA synthesized with Njord RNAP elicits only a minimal immune response. This natural enzyme enables efficient mRNA synthesis at ambient temperature and produces transcripts essentially free of dsRNA, offering significant potential to streamline mRNA manufacturing processes.

DNA-Directed RNA Polymerases↗
PubMed2026

OsCLSY4 modulates epigenomic patterns and grain size in rice.

De novo DNA methylation, orchestrated by the RNA-directed DNA methylation (RdDM) pathway, is essential for gene regulation and transposon silencing. While CLASSY (CLSY) proteins facilitate RNA POLYMERASE IV (Pol IV) recruitment to initiate the RdDM pathway in plants, their roles in crops are incompletely explored. Here, we report OsCLSY4 as the dominant regulator within the OsCLSY family, driving Pol IV-mediated epigenomic patterns and influencing diverse agricultural traits. Epigenomics analyses reveal that OsCLSY4 controls over 95% of Pol IV-dependent 24-nucleotide small interfering RNA (24-nt siRNA) clusters and more than 70% of Pol IV-dependent hypomethylated CHH differentially methylated regions (DMRs), predominantly at miniature inverted-repeat transposable elements (MITEs). Loss of OsCLSY4 leads to dysregulation of MADS22 and GA20ox1 in a DNA methylation-dependent manner. SunTag-mediated targeted demethylation confirms that reduced DNA methylation in promoter regions leads to MADS22 activation and GA20ox1 repression to influence grain size, linking epigenetic changes to phenotypic outcomes of osclsy4. Moreover, OsCLSY4 governs tissue-specific methylation patterns in panicle and seedling. Mechanistically, OsCLSY4 is the predominantly expressed OsCLSY family member and interacts with Pol IV. Collectively, our findings position OsCLSY4 as a central hub for Pol IV-mediated epigenomic regulation in rice and suggest its potential utility in epigenetic breeding strategies.

Oryza↗
PubMed1976

Preparations and properties of ribonucleic acid polymerase from Acinetobacter calcoaceticus.

Deoxyribonucleic acid (DNA)-dependent ribonucleic acid (RNA) polymerase (EC 2.7.7.6) from Acinetobacter calcoaceticus was purified to apparent homogeneity and its properties were compared with those of the Escherichia coli B enzyme. The molecular weights of the two native active enzymes as well as their alpha and beta subunits appeared to be similar. No subunit corresponding to that of sigma from E. coli was found, and furthermore no separation between the beta subunits could be detected by gel electrophoresis. A number of different DNAs were transcribed by the enzyme from A. calcoaceticus. Maximal RNA synthesis occurred at pH 8.7, 10 mM Mg2+, or 0.3 mM Mn2+ and at a total ionic strength of 0.1. Higher ionic strengths led to increasing inhibition of transcription and at mu = 0.4 complete inhibition was observed. The mechanism of inhibition of salt was not related to the initiation event as observed with T4 core RNA polymerase (R.Kleppe, 1975). In an attempt to understand the mechanism of inhibition by salt, the effect of ionic strength on the sedimentation properties of the enzyme was investigated. At low ionic strength, enzyme species with sedimentation coefficients, s20,w, of 5.8S, 12.4S, and 19.3S were present. In buffers with higher ionic strengths the relative amounts of the 12.4S species decreased. It is suggested, therefore, that the inhibition of activity at higher salt concentrations is caused by a decrease in concentration of the active enzyme species.

Acinetobacter↗
PubMed2026

Genomic insights into low-level rifampicin resistance mediated by borderline rpoB mutations in Mycobacterium tuberculosis: prevalence and phylogeny in Northeast China.

The emergence of low-level rifampicin (RIF) resistance in Mycobacterium tuberculosis poses a challenge to tuberculosis (TB) control, as it often leads to discordance between genotypic resistance detected by molecular assays (e.g., Xpert MTB/RIF) and phenotypic susceptibility in conventional drug susceptibility testing (DST). In this study, we performed whole-genome sequencing (WGS) on 17 clinical isolates from Changchun, Northeast China, which exhibited such discordance. All isolates harbored functional borderline mutations in the rpoB RRDR region, predominantly Leu452Pro and Leu430Pro (29% each), followed by His445Asn (18%). RIF minimum inhibitory concentration (MIC) values ranged from ≤0.25 to 1.0 mg/L, confirming low-level resistance. Notably, 53% (9/17) of the isolates were co-resistant to fluoroquinolones and 24% (4/17) to isoniazid (INH). According to WHO classification, 59% (10/17) were pre-extensively drug-resistant TB (Pre-XDR-TB) or multidrug-resistant TB (MDR-TB). Phylogenetic analysis revealed that 94% (16/17) belonged to the East Asian Beijing lineage (Lineage 2.2.1), with no evidence of recent local transmission. These findings underscore the complexity of low-level RIF resistance and its frequent association with broader drug resistance in a dominant lineage, highlighting the need for integrating MIC and WGS into diagnostic algorithms to guide appropriate treatment and surveillance.IMPORTANCEThe accurate detection of RIF resistance is critical for the management of TB, yet standard phenotypic methods often fail to identify strains with low-level resistance conferred by borderline rpoB mutations. This study provides the first genomic characterization of such discordant isolates in Northeast China, revealing a high prevalence of co-resistance to other key drugs and a strong association with the locally dominant Beijing lineage. The findings emphasize that reliance on phenotypic DST alone may lead to underestimation of drug resistance and inappropriate treatment, potentially contributing to the emergence and spread of Pre-XDR-TB and MDR-TB. Incorporating MIC determination and WGS into routine diagnostics could enhance detection, inform tailored therapy, and improve surveillance of these clinically significant strains.

Mycobacterium tuberculosis↗
PubMed2026

Genetic diversity and drug resistance profiles of Mycobacterium tuberculosis among Ethiopian children as determined by whole-genome sequencing.

UNLABELLED: Ethiopia ranks 30th among the tuberculosis (TB) burden countries, with children representing a significant yet understudied population group. This study aims to investigate the genetic diversity and drug-resistant profile among Ethiopian children. We included children under 15 years of age diagnosed with culture-confirmed pulmonary TB/drug-resistant TB between January 2017 and June 2023. Phenotypic drug susceptibility testing and whole-genome sequencing were conducted for 85 Mycobacterium tuberculosis (MTB) isolates. Demographic data were combined with genomic information. Lineage 4 was the most dominant (77.6%), while lineage 2 was less common (1%). Within lineage 4, several sub-lineages were identified, with lineage 4.2.2.2 being notably the most predominant (48%). Most of these cases were from Oromia (58%), including the hotspot areas for lineage 4 that were identified at a 99% confidence level. Among 17 MDR/pre-XDR-TB isolates, lineages 3 and 4.2.2.2 were the dominantly observed lineages/sub-lineages, with proportions of 29% and 65%, respectively. Of the 85 cases, 30.5% were drug-resistant TB to at least one of the five first-line anti-TB drugs tested by phenotypic drug susceptibility testing. Of these 26 drug-resistant TB cases, 23 were concordant with whole-genome sequencing characterization. The most frequent resistance mutations to rifampicin were found in the rpoB gene, specifically p.Ser450Leu (88%), followed by isoniazid in the katG gene, p.Ser315Thr (86%). Multidrug-resistant TB was strongly associated with MTB lineages (P = 0.007). This study identified high genetic diversity of M. tuberculosis and related drug-resistance mutations, with a strong concordance between whole-genome sequencing-based predictions and phenotypic drug susceptibility testing. IMPORTANCE: Our findings revealed a high genetic diversity of Mycobacterium tuberculosis among Ethiopian children, with the most common lineage being lineage 4, specifically lineage 4.2.2.2, in which a higher frequency of multidrug-resistant tuberculosis (TB) was observed. Additionally, we identified regional hotspots, suggesting ongoing community transmission. Moreover, whole-genome sequencing demonstrated high concordance with phenotypic drug susceptibility testing and identified mutation genes associated with first- and second-line anti-TB drugs, highlighting its usefulness in providing comprehensive results for resistance detection in children. Thus, it is essential for integrating genomic surveillance into childhood TB and drug resistance control.

Humans↗
PubMed2026

Pathogenic POLRMT variants in mice impair mtDNA transcription and affect perinatal survival.

Mitochondrial gene expression is essential for oxidative phosphorylation that generates the bulk of the cellular ATP, and mitochondrial dysfunction is a common cause of human metabolic diseases. Recently, the first pathogenic variants in the only known mitochondrial RNA polymerase (POLRMT) were described in patients presenting with a wide variety of clinical manifestations, including hypotonia, short stature, and developmental delay. Here, we modeled two human pathogenic POLRMT variants by creating the corresponding substitutions in mice: the dominant S582F and the recessive R984C variant. Mice homozygous for the R984C variant showed perinatal lethality without apparent embryonic developmental defects, a finding consistent with a failure to adapt to the metabolic transition to oxidative metabolism at birth. Mice carrying the S582F variant were viable and exhibited decreased mitochondrial transcript levels due to impaired de novo transcription. However, mtDNA levels and in organello mtDNA replication remained normal, which recapitulates the molecular phenotypes observed in patients. Altogether, our findings indicate that the conserved arginine near the active site is essential for POLRMT function, while the serine in the intercalating hairpin of the N-terminal domain is required for near-genome length transcription but not primase activity. This study highlights genotype-phenotype differences and provides new insights into POLRMT function.

Animals↗
PubMed2026

Development and optimization of T-ARMS PCR assays for detection of lethal haplotypes of TADA2A, UR1B, and PORL1B in pigs in Vietnam.

Marker-assisted selection has increasingly relied on single-nucleotide polymorphisms (SNPs) as robust genetic markers, particularly in livestock breeding programs. In pig farming, embryonic mortality significantly affects litter size, and SNPs in reference genes have been implicated as potential causal factors. We developed and optimized a tetra-primer amplification refractory mutation system (T-ARMS) PCR assay for rapid, cost-effective detection of SNPs in 3 candidate genes-TADA2A, PORL1B, URB1-that are associated with embryonic lethality and reproductive performance. Primer sets were designed based on known mutation sites and validated using synthetic gene constructs and porcine genomic DNA from pigs of Duroc and Landrace breeds. Optimization of annealing temperatures and primer concentration ratios yielded distinct and reproducible allele-specific amplicon patterns that were corroborated by PCR-RFLP and Sanger sequencing. Our T-ARMS PCR protocol, which requires minimal equipment and reduces processing time to <3&#x2009;h, had high specificity and efficiency in differentiating wild-type, heterozygous, and homozygous mutant genotypes in 20 Duroc and 20 Landrace pigs. Our Tetra-ARMS PCR assay is a robust and economically viable tool for SNP genotyping in pig breeding programs, potentially contributing to the reduction of embryonic lethality and the improvement of overall reproductive outcomes.

Sus scrofa↗
PubMed2021

Mechanism, cellular functions and cancer roles of polymerase-theta-mediated DNA end joining.

Cellular pathways that repair chromosomal double-strand breaks (DSBs) have pivotal roles in cell growth, development and cancer. These DSB repair pathways have been the target of intensive investigation, but one pathway - alternative end joining (a-EJ) - has long resisted elucidation. In this Review, we highlight recent progress in our understanding of a-EJ, especially the assignment of DNA polymerase theta (Pol&#x3b8;) as the predominant mediator of a-EJ in most eukaryotes, and discuss a potential molecular mechanism by which Pol&#x3b8;-mediated end joining (TMEJ) occurs. We address possible cellular functions of TMEJ in resolving DSBs that are refractory to repair by non-homologous end joining (NHEJ), DSBs generated following replication fork collapse and DSBs present owing to stalling of repair by homologous recombination. We also discuss how these context-dependent cellular roles explain how TMEJ can both protect against and cause genome instability, and the emerging potential of Pol&#x3b8; as a therapeutic target in cancer.

Animals↗
PubMed2023

Pol&#x3b3; coordinates DNA synthesis and proofreading to ensure mitochondrial genome integrity.

Accurate replication of mitochondrial DNA (mtDNA) by DNA polymerase &#x3b3; (Pol&#x3b3;) is essential for maintaining cellular energy supplies, metabolism, and cell cycle control. To illustrate the structural mechanism for Pol&#x3b3; coordinating polymerase (pol) and exonuclease (exo) activities to ensure rapid and accurate DNA synthesis, we determined four cryo-EM structures of Pol&#x3b3; captured after accurate or erroneous incorporation to a resolution of 2.4-3.0&#x2009;&#xc5;. The structures show that Pol&#x3b3; employs a dual-checkpoint mechanism to sense nucleotide misincorporation and initiate proofreading. The transition from replication to error editing is accompanied by increased dynamics in both DNA and enzyme, in which the polymerase relaxes its processivity and the primer-template DNA unwinds, rotates, and backtracks to shuttle the mismatch-containing primer terminus 32&#x2009;&#xc5; to the exo site for editing. Our structural and functional studies also provide a foundation for analyses of Pol&#x3b3; mutation-induced human diseases and aging.

Humans↗
PubMed2024

N6-methyladenosine modification of a parvovirus-encoded small noncoding RNA facilitates viral DNA replication through recruiting Y-family DNA polymerases.

Human bocavirus 1 (HBoV1) is a human parvovirus that causes lower respiratory tract infections in young children. It contains a single-stranded (ss) DNA genome of ~5.5 kb that encodes a small noncoding RNA of 140 nucleotides known as bocavirus-encoded small RNA (BocaSR), in addition to viral proteins. Here, we determined the secondary structure of BocaSR in vivo by using DMS-MaPseq. Our findings reveal that BocaSR undergoes N6-methyladenosine (m6A) modification at multiple sites, which is critical for viral DNA replication in both dividing HEK293 cells and nondividing cells of the human airway epithelium. Mechanistically, we found that m6A-modified BocaSR serves as a mediator for recruiting Y-family DNA repair DNA polymerase (Pol) &#x3b7; and Pol &#x3ba; likely through a direct interaction between BocaSR and the viral DNA replication origin at the right terminus of the viral genome. Thus, this report represents direct involvement of a viral small noncoding RNA in viral DNA replication through m6A modification.

Humans↗
PubMed2024

DNA polymerase delta governs parental histone transfer to DNA replication lagging strand.

Chromatin replication is intricately intertwined with the recycling of parental histones to the newly duplicated DNA strands for faithful genetic and epigenetic inheritance. The transfer of parental histones occurs through two distinct pathways: leading strand deposition, mediated by the DNA polymerase &#x3b5; subunits Dpb3/Dpb4, and lagging strand deposition, facilitated by the MCM helicase subunit Mcm2. However, the mechanism of the facilitation of Mcm2 transferring parental histones to the lagging strand while moving along the leading strand remains unclear. Here, we show that the deletion of Pol32, a nonessential subunit of major lagging-strand DNA polymerase &#x3b4;, results in a predominant transfer of parental histone H3-H4 to the leading strand during replication. Biochemical analyses further demonstrate that Pol32 can bind histone H3-H4 both in vivo and in vitro. The interaction of Pol32 with parental histone H3-H4 is disrupted through the mutation of the histone H3-H4 binding domain within Mcm2. Our findings identify the DNA polymerase &#x3b4; subunit Pol32 as a critical histone chaperone downstream of Mcm2, mediating the transfer of parental histones to the lagging strand during DNA replication.

DNA Polymerase III↗
PubMed2024

Natural history of eukaryotic DNA viruses with double jelly-roll major capsid proteins.

The phylum Preplasmiviricota (kingdom Bamfordvirae, realm Varidnaviria) is a broad assemblage of diverse viruses with comparatively short double-stranded DNA genomes (<50 kbp) that produce icosahedral capsids built from double jelly-roll major capsid proteins. Preplasmiviricots infect hosts from all cellular domains, testifying to their ancient origin, and, in particular, are associated with six of the seven supergroups of eukaryotes. Preplasmiviricots comprise four major groups of viruses, namely, polintons, polinton-like viruses (PLVs), virophages, and adenovirids. We used protein structure modeling and analysis to show that protein-primed DNA polymerases (pPolBs) of polintons, virophages, and cytoplasmic linear plasmids encompass an N-terminal domain homologous to the terminal proteins (TPs) of prokaryotic PRD1-like tectivirids and eukaryotic adenovirids that are involved in protein-primed replication initiation, followed by a viral ovarian tumor-like cysteine deubiquitinylase (vOTU) domain. The vOTU domain is likely responsible for the cleavage of the TP from the large pPolB polypeptide and is inactivated in adenovirids, in which TP is a separate protein. Many PLVs and transpovirons encode a distinct derivative of polinton-like pPolB that retains the TP, vOTU, and pPolB polymerization palm domains but lacks the exonuclease domain and instead contains a superfamily 1 helicase domain. Analysis of the presence/absence and inactivation of the vOTU domains and replacement of pPolB with other DNA polymerases in eukaryotic preplasmiviricots enabled us to outline a complete scenario for their origin and evolution.

Capsid Proteins↗
PubMed2025

Identification of EppR, a Second Repressor of Error-Prone DNA Polymerase Genes in Acinetobacter baumannii.

Acinetobacter baumannii is an opportunistic pathogen causing several infections that are increasingly difficult to treat due to its ability to rapidly gain antibiotic resistances. These resistances can arise due to mutations through the activity of error-prone DNA polymerases, such as DNA polymerase V (DNA Pol V) in response to DNA damage. The regulation of the DNA damage response (DDR) in A. baumannii is not completely understood; the regulation of genes encoding multiple copies of DNA Pol V is not fully characterized. Through genome-wide mutagenesis, we have identified a novel TetR-like family regulator of the umuDC and umuC genes, which we have named Error-prone polymerase regulator (EppR). We have found that EppR represses the expression of the genes encoding DNA Pol V and itself through direct binding to an EppR motif in their promoters. Lastly, we show that EppR also regulates UmuDAb, previously identified as a regulator of genes encoding DNA Pol V. These two gene products are functionally required to ensure regulation of the expression of the two umuDC, the two umuC genes as well as the regulators umuDAb and eppR genes. With these results, we propose a model in which multiple transcription factors regulate the expression of all these genes.

Acinetobacter baumannii↗
PubMed2025

Enzymatic and Structural Roles of Candida albicans Rev1 in DNA Damage Response and Disseminated Candidiasis.

Translesion DNA synthesis (TLS) is a fundamental biological process that enables DNA replication through various lesions to ensure genome stability and to prevent cell death due to replication fork collapse. Rev1, a member of Y-family DNA polymerase (Pol), functions in concert with a B-family enzyme Pol&#x3b6; in promoting TLS through various lesions. Interestingly, for such a function, the catalytic activity of Rev1 seems to be dispensable in Saccharomyces cerevisiae. Unlike Pol&#x3b6;, which possesses robust DNA polymerase activity, biochemical assays suggest that Rev1 predominantly incorporates a "C" opposite any templating residues, but the biological relevance of this activity of Rev1 remains elusive. Here we characterized Rev1 from Candida albicans, an opportunistic fungal pathogen responsible for maximum casualties due to systemic candidiasis in immunosuppressed individuals. Concerted genetic analyses of several Rev1 mutants in various DNA-damaging conditions suggested that in most lesion bypasses except 4-NQO-induced DNA lesions, the catalytic role of Rev1 is not important. However, simultaneous interactions of BRCT and the C-terminal domain of Rev1 with PCNA and Pol&#x3b6;, respectively, enable Rev1 to be essential during TLS. DNA damage recovery and mutagenesis assays further confirmed the lesion-specific roles of various domains of Rev1. Contrary to ex&#xa0;vivo data, animal studies suggested that CaRev1 is dispensable for systemic candidiasis development. We discuss the possible involvement of other TLS DNA polymerases in DNA damage response while C. albicans replicates and establishes itself in the host.

Candida albicans↗
PubMed2026

Refining a giant virus lineage: a novel order unifying Mamonoviridae and "Manesviridae," unveiled by the discovery of furtivovirus.

UNLABELLED: The evolutionary origins and taxonomic framework of giant viruses related to the family Mamonoviridae and its relative group, including clandestinovirus, remain unclassified due to gaps in genome size and host range between these two groups. This study aimed to address this gap by integrating our newly isolated virus with publicly available metagenome-assembled genomes (MAGs) to construct a more robust phylogenetic framework. Here, we report the isolation and characterization of a new giant virus, furtivovirus, using the unicellular amoeba Vermamoeba vermiformis as a host. Furtivovirus has a genome of approximately 560 kbp and shares key features with its closest relative, clandestinovirus. Ultrastructural analysis revealed a unique host-nucleus-dependent replication strategy characterized by the breakdown of the nuclear membrane and the packaging of nascent virions directly within the nucleoplasm, distinguishing it from canonical cytoplasmic virion factories. Comprehensive phylogenetic and comparative genomic analyses of shared orthologous groups and nucleocytovirus marker proteins revealed that furtivovirus, clandestinovirus, ushikuvirus, and usurpativirus form a distinct monophyletic clade, for which we propose a new family, "Manesviridae." Further analysis using amino acid-based similarity metrics of Nucleocytoviricota viral genomes, including established MAGs, demonstrated that this new family is robustly placed as a sister group to the family Mamonoviridae. This study elucidated the evolutionary relationships between viruses with large and small genomes that possess similar virion sizes within this lineage. Based on this cumulative evidence, we propose the establishment of a new order to unify these two families, thereby expanding their diversity and clarifying the evolutionary history of this branch within Nucleocytoviricota. IMPORTANCE: Giant viruses challenge our traditional understanding of viral evolution, raising the question of how a single related group can diverge to infect different hosts while evolving into vastly different genome sizes and replication strategies. The family Mamonoviridae and its relatives epitomize this evolutionary divergence: one group possesses massive genomes, whereas the other has genomes that are less than half their size. The discovery of furtivovirus and its unique nucleoplasm-dependent replication cycle provides a critical biological context for this genomic disparity. Through deep comparative genomic analysis, we demonstrated that these seemingly disparate lineages share a cohesive evolutionary origin that is distinct from other established orders. This finding highlights the complexity of genome evolution, demonstrating that giant viruses can expand their overall genome size to adapt to uncertain environments while reducing their core essential genes, thereby providing new insights into the evolutionary pressures that shape the diversity of the virosphere.

Giant Viruses↗