Cip1-interacting zinc-finger protein 1 (CIZ1) interacts with Xist lncRNA to form large RNA-protein assemblies at the inactive X-chromosome (Xi) in female mammalian nuclei, plus smaller assemblies in both sexes. CIZ1 assemblies influence underlying chromatin, and their disruption alters the expression of autosomal and X-linked gene clusters. Here, we explore the regulated dissolution of CIZ1-Xi assemblies during mitosis and show that, like Xist, CIZ1 is released in prometaphase under the regulation of Aurora Kinase B (AURKB). The part of human/mouse CIZ1 comprising 179/181 C-terminal amino acids encodes a matrin-3 domain that facilitates dimerization to form a compact folded core with disordered C-terminal extensions. Mass spectrometry revealed 56 high-confidence interacting partners of the C-terminal fragment, predominantly chromatin, nuclear matrix, and RNA-binding proteins. Phosphomimetic mutation of three conserved AURKB sites in the C-terminal extensions released CIZ1 from its nuclear anchor points, but did not affect its interaction with chromatin or nuclear matrix proteins. In contrast, the same mutations, or deletion of the C-terminal extensions, abolished interaction with RNAs, including Xist. Together, the data suggest CIZ1 is a regulatable component of the protein-RNA assemblies that preserve epigenetic stability across the nucleus, and that AURKB drives their dissolution in mitosis via dissociation of CIZ1 from RNA.

Gene transfer agents (GTAs) are phage-derived elements that have evolved repeatedly across diverse prokaryotes, where they drive high-frequency horizontal gene transfer (HGT). Here, we demonstrate that the Rhodobacter capsulatus GTA (RcGTA) tailspike protein, TspA, is a potent biofilm-degrading enzyme. Purified TspA is effective at both preventing initial biofilm formation and clearing established, mature biofilms. Crucially, TspA enhances RcGTA-mediated gene transfer, suggesting that this enzyme facilitates GTA navigation through the extracellular matrix. Unlike the permanently anchored tailspikes of canonical phages, TspA possesses a unique β-sandwich N-terminal domain that enables its dissociation from mature particles and engages in biofilm polysaccharide recognition. Our findings indicate that TspA is an evolutionary adaptation used by GTAs to optimize HGT within complex, densely packed microbial biofilm communities.

ABSTRACT Bacteriophage Exclusion (BREX) systems are a diverse family of bacterial and archaeal defense mechanisms defined by two conserved genes encoding a putative alkaline phosphatase BrxZ (PglZ) and an ATPase BrxC (PglY). Here, we characterize a Type V BREX system from the archaeon Haloarcula hispanica . Similar to bacterial Type I BREX systems, host–virus discrimination relies on methylation of specific non-palindromic DNA motifs. Notably, the H. hispanica BREX locus encodes two methyltransferases, BrxX1 and BrxX2, which independently target distinct motifs (GTAYCCG and GACCCC). The system protects H. hispanica against three of seven tested archaeal viruses. However, two related viruses, SH1 and HHIV-2, despite encoding multiple BREX target sites, escape BREX-mediated defense. We demonstrate that a large virion protein, VP1, encoded by these viruses inhibits the BREX system, as mutant viruses carrying partial deletions of VP1 lose resistance to the host defense. Together, these findings provide the first characterization of a Type V BREX system and demonstrate that archaeal viruses can counteract BREX through virion-associated anti-defense proteins.

Microviruses are single-stranded DNA viruses infecting bacteria, characterized by T = 1 shells made of single jelly-roll capsid proteins. To understand how microviruses infect their host cells, we have isolated and studied an unusually large microvirus, Ebor. Ebor belongs to the proposed "Tainavirinae" subfamily of Microviridae and infects the model Alphaproteobacterium Rhodobacter capsulatus. Using cryogenic electron microscopy, we show that the enlarged capsid of Ebor is the result of an extended C-terminus of the major capsid protein. The extra packaging space accommodates genes encoding a lytic enzyme and putative methylase, both absent in microviruses with shorter genomes. The capsid is decorated with protrusions at its 3-fold axes, which we show to recognize lipopolysaccharides on the host surface. Cryogenic electron tomography shows that during infection, Ebor attaches to the host cell via five such protrusions. This attachment brings a single pentameric capsomer into close contact with the cell membrane, creating a special vertex through which the genome is ejected. Both subtomogram averaging and single particle analysis identified two intermediates of capsid opening, showing that the interacting penton opens from its center via the separation of individual capsomer subunits. Structural comparison with the model Bullavirinae phage phiX174 suggests that this genome delivery mechanism may be widely present across Microviridae. IMPORTANCE:Tailless Microviridae bacteriophages are major components of the global virosphere. Notably, microviruses are prominent members of the mammalian gut virome, and certain compositions have been linked to serious health disorders; however, a molecular understanding of how they initiate infection of their host remains poorly characterized. We demonstrate that trimeric protrusions located at the corners of a single microvirus capsomer mediate host cell attachment. This interaction triggers opening of the capsomer, driven by separation of subunits from its center, much like flower petals open during blooming. This extensive opening explains how the genome translocation apparatus, along with the genome itself, is able to exit the capsid. "Penton blooming" likely represents a conserved mechanism shared by diverse viruses possessing similar capsid architectures.

PIF1 is a conserved helicase and G4 DNA binding and unwinding enzyme, with roles in genome stability. Human PIF1 (hPIF1) is poorly understood, but its functions can become critical for tumour cell survival during oncogene-driven replication stress. Here we report the discovery, via an X-ray crystallographic fragment screen (XChem), of hPIF1 DNA binding and unwinding inhibitors. A structure was obtained with a 4-phenylthiazol-2-amine fragment bound in a pocket between helicase domains 2A and 2B, with additional contacts to Valine 258 from domain 1A. The compound makes specific interactions, notably through Leucine 548 and Alanine 551, that constrain conformational adjustments between domains 2A and 2B, previously linked to ATP hydrolysis and DNA unwinding. We next synthesized a range of related compounds and characterized their effects on hPIF1 DNA-binding and helicase activity in vitro, expanding the structure activity relationship (SAR) around the initial hit. A systematic analysis of clinical cancer databases is also presented here, supporting the notion that hPIF1 upregulation may represent a specific cancer cell vulnerability. The research demonstrates that hPIF1 is a tractable target through 4-phenylthiazol-2-amine derivatives as inhibitors of its helicase action, setting a foundation for creation of a novel class of anti-cancer therapeutics.

Tailed double-stranded DNA bacteriophage employs a protein terminase motor to package their genome into a preformed protein shell-a system shared with eukaryotic dsDNA viruses such as herpesviruses. DNA packaging motor proteins represent excellent targets for antiviral therapy, with Letermovir, which binds Cytomegalovirus terminase, already licensed as an effective prophylaxis. In the realm of bacterial viruses, these DNA packaging motors comprise three protein constituents: the portal protein, small terminase and large terminase. The portal protein guards the passage of DNA into the preformed protein shell and acts as a protein interaction hub throughout viral assembly. Small terminase recognises the viral DNA and recruits large terminase, which in turn pumps DNA in an ATP-dependent manner. Large terminase also cleaves DNA at the termination of packaging. Multiple high-resolution structures of each component have been resolved for different phages, but it is only more recently that the field has moved towards cryo-EM reconstructions of protein complexes. In conjunction with highly informative single-particle studies of packaging kinetics, these structures have begun to inspire models for the packaging process and its place among other DNA machines.

Aquatic viruses act as key players in shaping microbial communities. In polar environments, they face significant challenges like limited host availability and harsh conditions. However, due to restricted ecosystem accessibility, our understanding of viral diversity, abundance, adaptations, and host interactions remains limited. To fill this knowledge gap, we studied viruses from atmosphere-close aquatic ecosystems in the Central Arctic and Northern Greenland. Aquatic samples for virus-host analysis were collected from ~60 cm depth and the submillimeter surface microlayer (SML) during the Synoptic Arctic Survey 2021 on icebreaker Oden in Arctic summer. Water was sampled from a melt pond and open water before undergoing size-fractioned filtration and followed by genome-resolved metagenomic and cultivation investigations. The prokaryotic diversity in the melt pond was considerably lower compared to open water. The melt pond was dominated by a Flavobacterium sp. and Aquiluna sp., the latter having a relatively small genome size of 1.2 Mb and the metabolic potential to generate ATP using the phosphate acetyltransferase-acetate kinase pathway. Viral diversity on the host fraction (0.2 – 5 µm) of the melt pond was strikingly limited compared to open water. From 1154 dereplicated viral operational taxonomic units (vOTUs), of which two-thirds were predicted bacteriophages, 17.2% encoded for auxiliary metabolic genes (AMGs) with metabolic functions. Some AMGs like glycerol-3-phosphate cytidylyltransferase and ice-binding like proteins might serve cryoprotection of the host. Prophages were often associated with SML genomes, and two active prophages of a new viral genera from the Arctic SML strain Leeuwenhoekiella aequorea Arc30 were induced. We found evidence that vOTU abundance in the SML compared to ~60 cm depth was more positively correlated to the distribution of a vOTU across five different Arctic stations. The results indicate that viruses employ elaborated strategies to endure in extreme and host-limited environments. Moreover, our observations suggest that the immediate air-sea interface serves as a platform for viral distribution in the Central Arctic. ### Competing Interest Statement The authors have declared no competing interest. * AMG : Auxiliary metabolic gene CRISPR : Clustered Regularly Interspaced Short Palindromic Repeat EPS : Extracellular polymeric substances IBP : Ice-binding protein iRep : Index of replication KEGG : Kyoto Encyclopedia of Genes and Genomes MAG : Metagenome-assembled genomes SML : Sea-surface microlayer SSW : Subsurface water TEM : Transmission electron microscopy vOTU : Viral operational taxonomic unit

Aquatic viruses act as key players in shaping microbial communities. In polar environments, they face significant challenges such as limited host availability and harsh conditions. However, due to the restricted accessibility of these ecosystems, our understanding of viral diversity, abundance, adaptations, and host interactions remains limited. To fill this knowledge gap, we studied viruses from atmosphere-close aquatic ecosystems in the Central Arctic and Northern Greenland. Aquatic samples for virus-host analysis were collected from 60 cm depth and the submillimeter surface microlayer (SML) during the Synoptic Arctic Survey 2021 on icebreaker Oden in the Arctic summer. Water was sampled from a melt pond and open water before undergoing size-fractioned filtration, followed by genome-resolved metagenomic and cultivation investigations. The prokaryotic diversity in the melt pond was considerably lower compared to that of open water. The melt pond was dominated by a Flavobacterium sp. and Aquiluna sp., the latter having a relatively small genome size of 1.2 Mb and the metabolic potential to generate ATP using the phosphate acetyltransferase-acetate kinase pathway. Viral diversity on the host fraction (0.2–5 µm) of the melt pond was strikingly limited compared to that of open water. From the 1154 viral operational taxonomic units (vOTUs), of which two-thirds were predicted bacteriophages, 17.2

DNA recognition is critical for assembly of double-stranded DNA viruses, particularly for the initiation of packaging the viral genome into the capsid. The key component that recognizes viral DNA is the small terminase protein. Despite prior studies, the molecular mechanism for DNA recognition remained elusive. Here, we address this question by identifying the minimal site in the bacteriophage HK97 genome specifically recognized by the small terminase and determining the structure of this complex by cryoEM. The circular small terminase employs an entirely unexpected mechanism in which DNA transits through the central tunnel, and sequence-specific recognition takes place as it emerges. This recognition stems from a substructure formed by the N- and C-terminal segments of two adjacent protomers which are unstructured when DNA is absent. Such interaction ensures continuous engagement of the small terminase with DNA, enabling it to slide along the DNA while simultaneously monitoring its sequence. This mechanism allows locating and instigating packaging initiation and termination precisely at the specific cos sequence.

Single-stranded DNA bacteriophages of the Microviridae family are major components of the global virosphere. Microviruses are highly abundant in aquatic ecosystems and are prominent members of the mammalian gut microbiome, where their diversity has been linked to various chronic health disorders. Despite the clear importance of microviruses, little is known about the molecular mechanism of host infection. Here, we have characterized an exceptionally large microvirus, Ebor, and provide crucial insights into long-standing mechanistic questions. Cryogenic electron microscopy of Ebor revealed a capsid with trimeric protrusions that recognise lipopolysaccharides on the host surface. Cryogenic electron tomography of the host cell colonized with virus particles demonstrated that the virus initially attaches to the cell via five such protrusions, located at the corners of a single pentamer. This interaction triggers a stargate mechanism of capsid opening along the 5-fold symmetry axis, enabling delivery of the virus genome. Despite variations in specific virus-host interactions among different Microviridae family viruses, structural data indicate that the stargate mechanism of infection is universally employed by all members of the family. Startlingly, our data reveal a mechanistic link for the opening of relatively small capsids made out of a single jelly-roll fold with the structurally unrelated giant viruses.

DNA recognition is critical for assembly of double-stranded DNA viruses, in particular for the initiation of packaging the viral genome into the capsid. DNA packaging has been extensively studied for three archetypal bacteriophage systems: cos, pac and phi29. We identified the minimal site within the cos region of bacteriophage HK97 specifically recognised by the small terminase and determined a cryoEM structure for the small terminase:DNA complex. This nonameric circular protein utilizes a previously unknown mechanism of DNA binding. While DNA threads through the central tunnel, unexpectedly, DNA-recognition is generated at its exit by a substructure formed by the N- and C-terminal segments of two adjacent protomers of the terminase which are unstructured in the absence of DNA. Such interaction ensures continuous engagement of the small terminase with DNA, allowing sliding along DNA while simultaneously checking the DNA sequence. This mechanism allows locating and instigating packaging initiation and termination precisely at the cos site.

Double-stranded DNA viruses utilise machinery, made of terminase proteins, to package viral DNA into the capsid. For cos bacteriophage, a defined signal, recognised by small terminase, flanks each genome unit. Here we present the first structural data for a cos virus DNA packaging motor, assembled from the bacteriophage HK97 terminase proteins, procapsids encompassing the portal protein, and DNA containing a cos site. The cryo-EM structure is consistent with the packaging termination state adopted after DNA cleavage, with DNA density within the large terminase assembly ending abruptly at the portal protein entrance. Retention of the large terminase complex after cleavage of the short DNA substrate suggests that motor dissociation from the capsid requires headful pressure, in common with pac viruses. Interestingly, the clip domain of the 12-subunit portal protein does not adhere to C-12 symmetry, indicating asymmetry induced by binding of the large terminase/DNA. The motor assembly is also highly asymmetric, showing a ring of 5 large terminase monomers, tilted against the portal. Variable degrees of extension between N- and C-terminal domains of individual subunits suggest a mechanism of DNA translocation driven by inter-domain contraction and relaxation.

Although membrane-containing dsDNA bacterial viruses are some of the most prevalent predators in aquatic environments, we know little about how they function due to their intractability in the laboratory.Here, we have identified and thoroughly characterized a new type of membrane-containing bacteriophage, Jorvik, that infects the freshwater mixotrophic model bacterium Rhodobacter capsulatus.Jorvik is extremely virulent, can persist in the host integrated into the RuBisCo operon and encodes two experimentally verified cell wall hydrolases.Jorvik-like prophages are abundant in the genomes of alpha-proteobacteria, are distantly related to known viruses of the class Tectiliviricetes, and we propose they should be classified as a new family.Crucially, we demonstrate how widely used phage manipulation methods should be adjusted to prevent loss of virus infectivity.Our thorough characterization of environmental phage Jorvik provides important experimental insights about phage diversity and interactions in microbial communities that are often unexplored in common metagenomic analyses.

CIZ1 is a nuclear matrix protein that is part of the large RNA-dependent supramolecular assembly complexes (SMACs) that form at the inactive X chromosome (Xi) in female cells, and smaller assemblies throughout the nucleus in males and females. It plays a role in maintenance of epigenetic state and gene expression in differentiated cells, via stabilisation of histone post-translational modifications H2AK119ub1 and H3K27me3, added by polycomb repressive complexes (PRC) 1 and 2. Here, we show that expression of the N-terminal replication domain (RD) and C-terminal anchor domain (AD) of human CIZ1 transcript is uncoupled, with consistently elevated AD in early stage breast cancers, and sporadically elevated AD in other common solid tumours. At the protein level CIZ1-Xi SMACs are corrupted in female breast cancers cells, and this is accompanied by elevated AD-encoding transcripts. We modelled the effect of AD fragments in primary murine embryonic fibroblasts and observed dominant-negative interference with CIZ1 SMACs during their assembly in early G1 phase. Mutagenesis identified the matrin 3 homology domain as essential for self-interaction to form stable homodimers in vitro , and as a determinant of its dominant-negative effect in cells, implicating the dimerization interface in CIZ1 SMAC integrity. SMAC disruption was coincident with depletion of PRC1-dependent H2AK119ub1 from Xi chromatin, in a manner abrogated by the PR-deubiquitinase inhibitor PR619, suggesting that CIZ1 SMACs normally stabilise H2AK119ub1 by shielding Xi chromatin from attack by deubiquitinases. Moreover, SMAC disruption was accompanied by changes in gene expression within days. Together, the data suggest that inappropriate expression of CIZ1 AD fragments could drive epigenetic instability in early stage breast cancers by destabilizing the CIZ1 SMACs that normally protect repressed chromatin. ![Figure][1] ### Competing Interest Statement DC and JA are founders and shareholders in Cizzle Biotechnology Ltd, which part funded the research. Other authors disclosed no potential conflicts of interest. [1]: pending:yes

Visible surface films, termed slicks, can extensively cover freshwater and marine ecosystems, with coastal regions being particularly susceptible to their presence. The sea-surface microlayer (SML), the upper 1-mm at the air-water interface in slicks (herein slick SML) harbors a distinctive bacterial community, but generally little is known about SML viruses. Using flow cytometry, metagenomics, and cultivation, we characterized viruses and bacteria in a brackish slick SML in comparison to non-slick SML as well as seawater below slick and non-slick areas (subsurface water = SSW). Size-fractionated filtration of all samples distinguished viral attachment to hosts and particles. The slick SML contained higher abundances of virus-like particles, prokaryotic cells, and dissolved organic carbon compared to non-slick SML and SSW. The community of 428 viral operational taxonomic units (vOTUs), 426 predicted as lytic, distinctly differed across all size fractions in the slick SML compared to non-slick SML and SSW. Specific metabolic profiles of bacterial metagenome-assembled genomes and isolates in the slick SML included a prevalence of genes encoding motility and carbohydrate-active enzymes (CAZymes). Several vOTUs were enriched in slick SML, and many virus variants were associated with particles. Nine vOTUs were only found in slick SML, six of them being targeted by slick SML-specific clustered-regularly interspaced short palindromic repeats (CRISPR) spacers likely originating from Gammaproteobacteria. Moreover, isolation of three previously unknown lytic phages for Alishewanella sp. and Pseudoalteromonas tunicata, abundant and actively replicating slick SML bacteria, suggests that viral activity in slicks contributes to biogeochemical cycling in coastal ecosystems.

Phosphorylation of SARS-CoV-2 nucleoprotein recruits human cytosolic 14-3-3 proteins playing a well-recognized role in replication of many viruses. Here we use genetic code expansion to demonstrate that 14-3-3 binding is triggered by phosphorylation of SARS-CoV-2 nucleoprotein at either of two pseudo-repeats centered at Ser197 and Thr205. According to fluorescence anisotropy measurements, the pT205-motif,presentin SARS-CoV-2 but not in SARS-CoV, is preferred over the pS197-motif by all seven human 14-3-3 isoforms, which collectively display an unforeseen pT205/pS197 peptide binding selectivity hierarchy. Crystal structures demonstrate that pS197 and pT205 are mutually exclusive 14-3-3-binding sites, whereas SAXS and biochemical data obtained on the full protein-protein complex indicate that 14-3-3 binding occludes the Ser/Arg-rich region of the nucleoprotein, inhibiting its dephosphorylation. This Ser/Arg-rich region is highly prone to mutations, as exemplified by the Omicron and Delta variants, with our data suggesting that the strength of 14-3-3/nucleoprotein interaction can be linked with the replicative fitness of the virus.

CrAssphage and related viruses of the order Crassvirales (hereafter referred to as crassviruses) were originally discovered by cross-assembly of metagenomic sequences. They are the most abundant viruses in the human gut, are found in the majority of individual gut viromes, and account for up to 95% of the viral sequences in some individuals 1–4 . Crassviruses are likely to have major roles in shaping the composition and functionality of the human microbiome, but the structures and roles of most of the virally encoded proteins are unknown, with only generic predictions resulting from bioinformatic analyses 4,5 . Here we present a cryo-electron microscopy reconstruction of Bacteroides intestinalis virus ΦcrAss001 6 , providing the structural basis for the functional assignment of most of its virion proteins. The muzzle protein forms an assembly about 1 MDa in size at the end of the tail and exhibits a previously unknown fold that we designate the ‘crass fold’, that is likely to serve as a gatekeeper that controls the ejection of cargos. In addition to packing the approximately 103 kb of virus DNA, the ΦcrAss001 virion has extensive storage space for virally encoded cargo proteins in the capsid and, unusually, within the tail. One of the cargo proteins is present in both the capsid and the tail, suggesting a general mechanism for protein ejection, which involves partial unfolding of proteins during their extrusion through the tail. These findings provide a structural basis for understanding the mechanisms of assembly and infection of these highly abundant crassviruses.

In recent years, the third-generation nanopore sequencing technology (nanopore sequencing) has stood out due to its long read lengths, simple operation, portability, and real-time sequence readout, and has also shown good prospects in biomolecular detection. Mainstream nanopore architectures implement transmembrane proteins that insert into an isolative membrane, where the signal resolution of the nanopore is dominated by the nature of the pore protein. Current choices focus on naturally bio-membrane dwelling proteins, whereas a broad range of other proteins may also provide pores suitable for nanopore sensing, although their implementation has been limited by a poor ability to insert into organic membranes. Here, we describe a strategy for inserting viral portal proteins with flexible inner pores into lipid membranes: We expressed different mutants of G20C portal protein to 1) enable chemical modifications of hydrophobic anchors to the portal's exterior structure and 2) substitute hydrophobic amino acid residues into the portal's base in order to facilitate membrane insertion. In this work, we engineered and purified different portal mutants, and verified their structural integrity with electron microscopy. Building on our recent demonstration of DNA sensing with G20C mutants, we are in the process of characterizing the sensing capabilities of these mutants through ionic current recordings of DNA and peptide translocation through the portal mutant. We expect to apply this approach to a wide range of non-membrane, ring-shaped proteins and facilitate the development of new biological nanopores that can provide improved signal resolution and data quality.

The application of nanopores as label-free, single-molecule biosensors for electrical or optical probing of structural features in biomolecules has been widely explored. While biological nanopores (membrane proteins and bacteriophage portal proteins) and solid-state nanopores (thin films and two-dimensional materials) have been extensively employed, the third class of nanopores known as hybrid nanopores, where an artificial membrane substitutes the organic support membrane of proteins, has been only sparsely studied due to challenges in implementation. G20c portal protein contains a natural DNA pore that is used by viruses for filling their capsid with viral genomic DNA. We have previously developed a lipid-free hybrid nanopore by "corking" the G20c portal protein into a SiNx nanopore. Herein, we demonstrate that through chemical functionalization of the synthetic nanopore, covalent linkage between the solid-state pore and the G20c portal protein considerably improves the hybrid pore stability, lifetime, and voltage resilience. Moreover, we demonstrate electric-field-driven and motor protein-mediated transport of DNA molecules through this hybrid nanopore. Our integrated protein/solid-state device can serve as a robust and durable framework for sensing and sequencing at high voltages, potentially providing higher resolution, higher signal-to-noise ratio, and higher throughput compared to the more conventional membrane-embedded protein platforms.