Structural basis for DNA recognition by a viral genome-packaging machine.

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.

Gene transfer agents: The ambiguous role of selfless viruses in genetic exchange and bacterial evolution.

Gene transfer agents (GTAs) are genetic elements derived from ancestral bacteriophages that have become domesticated by the host. GTAs are present in diverse prokaryotic organisms, where they can facilitate horizontal gene transfer under certain conditions. Unlike typical bacteriophages, GTAs do not exhibit any preference for the replication or transfer of the genes encoding them; instead, they exhibit a remarkable capacity to package chromosomal, and sometimes extrachromosomal, DNA into virus-like capsids and disseminate it to neighboring cells. Because GTAs resemble defective prophages, identification of novel GTAs is not trivial. The detection of candidates relies on the genetic similarity to known GTAs, which has been fruitful in α-proteobacterial lineages but challenging in more distant bacteria. Here we consider several fundamental questions: What is the true prevalence of GTAs in prokaryote genomes? Given there are high costs for GTA production, what advantage do GTAs provide to the bacterial host to justify their maintenance? How is the bacterial chromosome recognized and processed for inclusion in GTA particles? This article highlights the challenges in comprehensively understanding GTAs' prevalence, function and DNA packaging method. Going forward, broad study of atypical GTAs and use of ecologically relevant conditions are required to uncover their true impact on bacterial chromosome evolution.

DNA Packaging Specificity in the λ-Like Phages: Gifsy-1.

DNA viruses recognize viral DNA and package it into virions. Specific recognition is needed to distinguish viral DNA from host cell DNA. The λ-like Escherichia coli phages are interesting and good models to examine genome packaging by large DNA viruses. Gifsy-1 is a λ-like Salmonella phage. Gifsy-1's DNA packaging specificity was compared with those of closely related phages λ, 21, and N15. In vivo packaging studies showed that a Gifsy-1-specific phage packaged λ DNA at ca. 50% efficiency and λ packages Gifsy-1-specific DNA at ~30% efficiency. The results indicate that Gifsy-1 and λ share the same DNA packaging specificity. N15 is also shown to package Gifsy-1 DNA. Phage 21 fails to package λ, N15, and Gifsy-1-specific DNAs; the efficiencies are 0.01%, 0.01%, and 1%, respectively. A known incompatibility between the 21 helix-turn-helix motif and cosBλ is proposed to account for the inability of 21 to package Gifsy-1 DNA. A model is proposed to explain the 100-fold difference in packaging efficiency between λ and Gifsy-1-specific DNAs by phage 21. Database sequences of enteric prophages indicate that phages with Gifsy-1's DNA packaging determinants are confined to Salmonella species. Similarly, prophages with λ DNA packaging specificity are rarely found in Salmonella. It is proposed that λ and Gifsy-1 have diverged from a common ancestor phage, and that the differences may reflect adaptation of their packaging systems to host cell differences.

Disenfranchised DNA: biochemical analysis of mutant øX174 DNA-binding proteins may further elucidate the evolutionary significance of the unessential packaging protein A.

Most icosahedral DNA viruses package and condense their genomes into pre-formed, volumetrically constrained capsids. However, concurrent genome biosynthesis and packaging are specific to single-stranded (ss) DNA micro- and parvoviruses. Before packaging, ~120 copies of the øX174 DNA-binding protein J interact with double-stranded DNA. 60 J proteins enter the procapsid with the ssDNA genome, guiding it between 60 icosahedrally ordered DNA-binding pockets formed by the capsid proteins. Although J proteins are small, 28-37 residues in length, they have two domains. The basic, positively charged N-terminus guides the genome between binding pockets, whereas the C-terminus acts as an anchor to the capsid's inner surface. Three C-terminal aromatic residues, W30, Y31, and F37, interact most extensively with the coat protein. Their corresponding codons were mutated, and the resulting strains were biochemically and genetically characterized. Depending on the mutation, the substitutions produced unstable packaging complexes, unstable virions, infectious progeny, or particles packaged with smaller genomes, the latter being a novel phenomenon. The smaller genomes contained internal deletions. The juncture sequences suggest that the unessential A* (A star) protein mediates deletion formation.IMPORTANCEUnessential but strongly conserved gene products are understudied, especially when mutations do not confer discernable phenotypes or the protein's contribution to fitness is too small to reliably determine in laboratory-based assays. Consequently, their functions and evolutionary impact remain obscure. The data presented herein suggest that microvirus A* proteins, discovered over 40 years ago, may hasten the termination of non-productive packaging events. Thus, performing a salvage function by liberating the reusable components of the failed packaging complexes, such as DNA templates and replication enzymes.

Viral packaging ATPases utilize a glutamate switch to couple ATPase activity and DNA translocation.

Many viruses utilize ringed packaging ATPases to translocate double-stranded DNA into procapsids during replication. A critical step in the mechanochemical cycle of such ATPases is ATP binding, which causes a subunit within the motor to grip DNA tightly. Here, we probe the underlying molecular mechanism by which ATP binding is coupled to DNA gripping and show that a glutamate-switch residue found in AAA+ enzymes is central to this coupling in viral packaging ATPases. Using free-energy landscapes computed through molecular dynamics simulations, we determined the stable conformational state of the ATPase active site in ATP- and ADP-bound states. Our results show that the catalytic glutamate residue transitions from an active to an inactive pose upon ATP hydrolysis and that a residue assigned as the glutamate switch is necessary for regulating this transition. Furthermore, we identified via mutual information analyses the intramolecular signaling pathway mediated by the glutamate switch that is responsible for coupling ATP binding to conformational transitions of DNA-gripping motifs. We corroborated these predictions with both structural and functional experimental measurements. Specifically, we showed that the crystal structure of the ADP-bound P74-26 packaging ATPase is consistent with the structural coupling predicted from simulations, and we further showed that disrupting the predicted signaling pathway indeed decouples ATPase activity from DNA translocation activity in the φ29 DNA packaging motor. Our work thus establishes a signaling pathway that couples chemical and mechanical events in viral DNA packaging motors.

Structure and dynamics of the mitochondrial DNA-compaction factor Abf2 from S. cerevisiae.

Mitochondria are essential organelles that produce most of the energy via the oxidative phosphorylation (OXPHOS) system in all eukaryotic cells. Several essential subunits of the OXPHOS system are encoded by the mitochondrial genome (mtDNA) despite its small size. Defects in mtDNA maintenance and expression can lead to severe OXPHOS deficiencies and are amongst the most common causes of mitochondrial disease. The mtDNA is packaged as nucleoprotein structures, referred to as nucleoids. The conserved mitochondrial proteins, ARS-binding factor 2 (Abf2) in the budding yeast Saccharomyces cerevisiae (S. cerevisiae) and mitochondrial transcription factor A (TFAM) in mammals, are nucleoid associated proteins (NAPs) acting as condensing factors needed for packaging and maintenance of the mtDNA. Interestingly, gene knockout of Abf2 leads, in yeast, to the loss of mtDNA and respiratory growth, providing evidence for its crucial role. On a structural level, the condensing factors usually contain two DNA binding domains called high-mobility group boxes (HMG boxes). The co-operating mechanical activities of these domains are crucial in establishing the nucleoid architecture by bending the DNA strands. Here we used advanced solution NMR spectroscopy methods to characterize the dynamical properties of Abf2 together with its interaction with DNA. We find that the two HMG-domains react notably different to external cues like temperature and salt, indicating distinct functional properties. Biophysical characterizations show the pronounced difference of these domains upon DNA-binding, suggesting a refined picture of the Abf2 functional cycle.

Frozen-hydrated chromatin from metaphase chromosomes has an interdigitated multilayer structure.

Cryo-electron tomography and small-angle X-ray scattering were used to investigate the chromatin folding in metaphase chromosomes. The tomographic 3D reconstructions show that frozen-hydrated chromatin emanated from chromosomes is planar and forms multilayered plates. The layer thickness was measured accounting for the contrast transfer function fringes at the plate edges, yielding a width of ~ 7.5 nm, which is compatible with the dimensions of a monolayer of nucleosomes slightly tilted with respect to the layer surface. Individual nucleosomes are visible decorating distorted plates, but typical plates are very dense and nucleosomes are not identifiable as individual units, indicating that they are tightly packed. Two layers in contact are ~ 13 nm thick, which is thinner than the sum of two independent layers, suggesting that nucleosomes in the layers interdigitate. X-ray scattering of whole chromosomes shows a main scattering peak at ~ 6 nm, which can be correlated with the distance between layers and between interdigitating nucleosomes interacting through their faces. These observations support a model where compact chromosomes are composed of many chromatin layers stacked along the chromosome axis.

Low-Temperature Adaptation Targets Genome Packing Reactions in an Icosahedral Single-Stranded DNA Virus.

øX174, G4, and α3 represent the three sister genera of a Microviridae subfamily. α3-like genomes are considerably larger than their sister genera genomes, yet they are packaged into capsids of similar internal volumes. They also contain multiple A* genes, which are nested within the larger A gene reading frame. Although unessential under most conditions, A* proteins mediate the fidelity of packaging reactions. Larger genomes and multiple A* genes may indicate that genome packaging is more problematic for α3-like viruses, especially at lower temperatures, where DNA persistence lengths would be longer. Unlike members of the other genera, which reliably form plaques at 20°C, α3-like phages are naturally cold sensitive below 28°C. To determine whether there was a connection between the uniquely α3-like genome characteristics and the cold-sensitive phenotype, the α3 assembly pathway was characterized at low temperature. Although virions were not detected, particles consistent with off-pathway packaging complexes were observed. In a complementary evolutionary approach, α3 was experimentally evolved to grow at progressively lower temperatures. The two major responses to cold adaptation were genome reduction and elevated A* gene expression. IMPORTANCE The production of enzymes, transcription factors, and viral receptors directly influences the niches viruses can inhabit. Some prokaryotic hosts can thrive in widely differing environments; thus, physical parameters, such as temperature, should also be considered. These variables may directly alter host physiology, preventing viral replication. Alternatively, they could negatively inhibit infection processes in a host-independent manner. The members of three sister Microviridae genera (canonical species øX174, G4 and α3) infect the same host, but α3-like viruses are naturally cold sensitive, which could effectively exclude them from low-temperature environments (<28°C). Exclusion appeared to be independent of host cell physiology. Instead, it could be largely attributed to low-temperature packaging defects. The results presented here demonstrate how physical parameters, such as temperature, can directly influence viral diversification and niche determination in a host-independent manner.

Nipple fluid for breast cancer diagnosis using the nanopore of Phi29 DNA-packaging motor.

Detection of cancer in its early stage is a challenging task for oncologists. Inflammatory breast cancer has symptoms that are similar to mastitis and can be mistaken for microbial infection. Currently, the differential diagnosis between mastitis and Inflammatory breast cancer via nipple aspirate fluid (NAF) is difficult. Here, we report a label-free and amplification-free detection platform using an engineered nanopore of the phi29 DNA-packaging motor with biomarker Galectin3 (GAL3), Thomsen-Friedenreich (TF) binding peptide as the probe fused at its C-terminus. The binding of the biomarker in NAF samples from breast cancer patients to the probe results in the connector's conformational change with a current blockage of 32 %. Utilization of dwell time, blockage ratio, and peak signature enable us to detect basal levels of biomarkers from patient NAF samples at the single-molecule level. This platform will allow for breast cancers to be resolved at an early stage with accuracy and thoroughness.

The Effects of Packaged, but Misguided, Single-Stranded DNA Genomes Are Transmitted to the Outer Surface of the ϕX174 Capsid.

Most icosahedral viruses condense their genomes into volumetrically constrained capsids. However, concurrent genome biosynthesis and packaging are specific to single-stranded DNA (ssDNA) viruses. ssDNA genome packaging combines elements found in both double-stranded DNA (dsDNA) and ssRNA systems. Similar to dsDNA viruses, the genome is packaged into a preformed capsid. Like ssRNA viruses, there are numerous capsid-genome associations. In ssDNA microviruses, the DNA-binding protein J guides the genome between 60 icosahedrally ordered DNA binding pockets. It also partially neutralizes the DNA's negative phosphate backbone. ϕX174-related microviruses, such as G4 and α3, have J proteins that differ in length and charge organization. This suggests that interchanging J proteins could alter the path used to guide DNA in the capsid. Previously, a ϕXG4J chimera, in which the ϕX174 J gene was replaced with the G4 gene, was characterized. It displayed lethal packaging defects, which resulted in procapsids being removed from productive assembly. Here, we report the characterization of another inviable chimera, ϕXα3J. Unlike ϕXG4J, ϕXα3J efficiently packaged DNA but produced noninfectious particles. These particles displayed a reduced ability to attach to host cells, suggesting that internal DNA organization could distort the capsid's outer surface. Mutations that restored viability altered J-coat protein contact sites. These results provide evidence that the organization of ssDNA can affect both packaging and postpackaging phenomena. IMPORTANCE ssDNA viruses utilize icosahedrally ordered protein-nucleic acids interactions to guide and organize their genomes into preformed shells. As previously demonstrated, chaotic genome-capsid associations can inhibit ϕX174 packaging by destabilizing packaging complexes. However, the consequences of poorly organized genomes may extend beyond the packaging reaction. As demonstrated herein, it can lead to uninfectious packaged particles. Thus, ssDNA genomes should be considered an integral and structural virion component, affecting the properties of the entire particle, which includes the capsid's outer surface.

Whole genome sequence analysis of bacteriophage P1 that infects the Lactobacillus plantarum.

Phage P1 was isolated from the abnormal fermented liquid using Lactobacillus plantarum (L. plantarum) IMAU10120. To date, genetic knowledge regarding L. plantarum phage diversity is still limited, and further in-depth sequencing analysis of isolated L. plantarum phages can fill this gap. Here, we investigated the whole genome sequence of L. plantarum phage P1, sequenced by Illumina HiSeq platform, to decipher its genomic characteristics and putative DNA packaging mechanism. It was revealed that phage P1 was 73,787 bp in length, which was composed of linear double-stranded DNA (dsDNA), and the GC content was 39.17%. Its genome contained 86 coding sequences for various functions, such as adsorption, injection, replication, assembly, and release. Moreover, it was observed that L. plantarum phage P1 utilized the 'cohesive ends' DNA packaging mechanism. This study furthered the genomic knowledge of L. plantarum phages and provided some basis for the control of L. plantarum phages in the dairy fermentation industry.

Cryo-EM structure and in vitro DNA packaging of a thermophilic virus with supersized T=7 capsids.

Double-stranded DNA viruses, including bacteriophages and herpesviruses, package their genomes into preformed capsids, using ATP-driven motors. Seeking to advance structural and mechanistic understanding, we established in vitro packaging for a thermostable bacteriophage, P23-45 of Thermus thermophilus Both the unexpanded procapsid and the expanded mature capsid can package DNA in the presence of packaging ATPase over the 20 °C to 70 °C temperature range, with optimum activity at 50 °C to 65 °C. Cryo-EM reconstructions for the mature and immature capsids at 3.7-Å and 4.4-Å resolution, respectively, reveal conformational changes during capsid expansion. Capsomer interactions in the expanded capsid are reinforced by formation of intersubunit ß-sheets with N-terminal segments of auxiliary protein trimers. Unexpectedly, the capsid has T=7 quasi-symmetry, despite the P23-45 genome being twice as large as those of known T=7 phages, in which the DNA is compacted to near-crystalline density. Our data explain this anomaly, showing how the canonical HK97 fold has adapted to double the volume of the capsid, while maintaining its structural integrity. Reconstructions of the procapsid and the expanded capsid defined the structure of the single vertex containing the portal protein. Together with a 1.95-Å resolution crystal structure of the portal protein and DNA packaging assays, these reconstructions indicate that capsid expansion affects the conformation of the portal protein, while still allowing DNA to be packaged. These observations suggest a mechanism by which structural events inside the capsid can be communicated to the outside.

A thermophilic phage uses a small terminase protein with a fixed helix-turn-helix geometry.

Tailed bacteriophages use a DNA-packaging motor to encapsulate their genome during viral particle assembly. The small terminase (TerS) component of this DNA-packaging machinery acts as a molecular matchmaker that recognizes both the viral genome and the main motor component, the large terminase (TerL). However, how TerS binds DNA and the TerL protein remains unclear. Here we identified gp83 of the thermophilic bacteriophage P74-26 as the TerS protein. We found that TerSP76-26 oligomerizes into a nonamer that binds DNA, stimulates TerL ATPase activity, and inhibits TerL nuclease activity. A cryo-EM structure of TerSP76-26 revealed that it forms a ring with a wide central pore and radially arrayed helix-turn-helix domains. The structure further showed that these helix-turn-helix domains, which are thought to bind DNA by wrapping the double helix around the ring, are rigidly held in an orientation distinct from that seen in other TerS proteins. This rigid arrangement of the putative DNA-binding domain imposed strong constraints on how TerSP76-26 can bind DNA. Finally, the TerSP76-26 structure lacked the conserved C-terminal ß-barrel domain used by other TerS proteins for binding TerL. This suggests that a well-ordered C-terminal ß-barrel domain is not required for TerSP76-26 to carry out its matchmaking function. Our work highlights a thermophilic system for studying the role of small terminase proteins in viral maturation and presents the structure of TerSP76-26, revealing key differences between this thermophilic phage and its mesophilic counterparts.

Finally, a Role Befitting Astar: Strongly Conserved, Unessential Microvirus A* Proteins Ensure the Product Fidelity of Packaging Reactions.

In microviruses, 60 copies of the positively charged DNA binding protein J guide the single-stranded DNA genome into the icosahedral capsid. Consequently, ∼12% of the genome is icosahedrally ordered within virions. Although the internal volume of the ϕX174, G4, and α3 capsids are nearly identical, their genome lengths vary widely from 5,386 (ϕX174) to 6,067 (α3) nucleotides. As the genome size increases, the J protein's length and charge decreases. The ϕX174 J protein is 37 amino acids long and has a charge of +12, whereas the 23-residue G4 and α3 proteins have respective +6 and +8 charges. While the large ϕX174 J protein can substitute for the smaller ones, the converse is not true. Thus, the smallest genome, ϕX174, requires the more stringent J protein packaging guide. To investigate this further, a chimeric virus (ϕXG4J) was generated by replacing the indigenous ϕX174 J gene with that of G4. The resulting mutant, ϕXG4J, was not viable on the level of plaque formation without ϕX174 J gene complementation. During uncomplemented infections, capsids dissociated during packaging or quickly thereafter. Those that survived were significantly less stable and infectious than the wild type. Complementation-independent ϕXG4J variants were isolated. They contained duplications that increased genome size by as much as 3.8%. Each duplication started at nucleotide 991, creating an additional DNA substrate for the unessential but highly conserved A* protein. Accordingly, ϕXG4J viability and infectivity was also restored by the exogenous expression of a cloned A* gene.IMPORTANCE Double-stranded DNA viruses typically package their genomes into a preformed capsid. In contrast, single-stranded RNA viruses assemble their coat proteins around their genomes via extensive nucleotide-protein interactions. Single-stranded DNA (ssDNA) viruses appear to blend both strategies, using nucleotide-protein interactions to organize their genomes into preformed shells, likely by a controlled process. Chaotic genome-capsid associations could inhibit packaging or genome release during the subsequent infection. This process appears to be partially controlled by the unessential A* protein, a shorter version of the essential A protein that mediates rolling-circle DNA replication. Protein A* may elevate fitness by ensuring the product fidelity of packaging reactions. This phenomenon may be widespread in ssDNA viruses that simultaneously synthesize and package DNA with rolling circle and rolling circle-like DNA replication proteins. Many of these viruses encode smaller, unessential, and/or functionally undefined in-frame versions of A/A*-like proteins.

Comparative Analysis of the Capsid Structures of AAVrh.10, AAVrh.39, and AAV8.

Adeno-associated viruses (AAVs) from clade E are often used as vectors in gene delivery applications. This clade includes rhesus isolate 10 (AAVrh.10) and 39 (AAVrh.39) which, unlike representative AAV8, are capable of crossing the blood-brain barrier (BBB), thereby enabling the delivery of therapeutic genes to the central nervous system. Here, the capsid structures of AAV8, AAVrh.10 and AAVrh.39 have been determined by cryo-electron microscopy and three-dimensional image reconstruction to 3.08-, 2.75-, and 3.39-Šresolution, respectively, to enable a direct structural comparison. AAVrh.10 and AAVrh.39 are 98% identical in amino acid sequence but only ∼93.5% identical to AAV8. However, the capsid structures of all three viruses are similar, with only minor differences observed in the previously described surface variable regions, suggesting that specific residues S269 and N472, absent in AAV8, may confer the ability to cross the BBB in AAVrh.10 and AAVrh.39. Head-to-head comparison of empty and genome-containing particles showed DNA ordered in the previously described nucleotide-binding pocket, supporting the suggested role of this pocket in DNA packaging for the Dependoparvovirus The structural characterization of these viruses provides a platform for future vector engineering efforts toward improved gene delivery success with respect to specific tissue targeting, transduction efficiency, antigenicity, or receptor retargeting.IMPORTANCE Recombinant adeno-associated virus vectors (rAAVs), based on AAV8 and AAVrh.10, have been utilized in multiple clinical trials to treat different monogenetic diseases. The closely related AAVrh.39 has also shown promise in vivo As recently attained for other AAV biologics, e.g., Luxturna and Zolgensma, based on AAV2 and AAV9, respectively, the vectors in this study will likely gain U.S. Food and Drug Administration approval for commercialization in the near future. This study characterized the capsid structures of these clinical vectors at atomic resolution using cryo-electron microscopy and image reconstruction for comparative analysis. The analysis suggested two key residues, S269 and N472, as determinants of BBB crossing for AAVrh.10 and AAVrh.39, a feature utilized for central nervous system delivery of therapeutic genes. The structure information thus provides a platform for engineering to improve receptor retargeting or tissue specificity. These are important challenges in the field that need attention. Capsid structure information also provides knowledge potentially applicable for regulatory product approval.

VP39 of Spodoptera litura multicapsid nucleopolyhedrovirus cannot efficiently rescue the nucleocapsid assembly of vp39-null Autographa californica multiple nucleopolyhedrovirus.

BACKGROUND: Autographa californica multiple nucleopolyhedrovirus (AcMNPV) vp39 is conserved in all sequenced baculovirus genomes. In previous studies, VP39 has been identified as the major capsid structure protein of baculoviruses and found to be essential for nucleocapsid assembly. The nucleocapsid composition and structure of Group I and II NPVs of the Alphabaculovirus genus are very similar. It is not clear whether the major capsid structure protein VP39 of Group I NPVs is functionally identical to or substitutable with the Group II NPV VP39. In this study, the function of Group II Spodoptera litura MNPV (SpltMNPV) VP39 in Group I AcMNPV was characterized. METHODS: Sequence alignment of AcMNPV VP39 and SpltMNPV VP39 was performed using Clustal X and edited with GeneDoc. To determine whether VP39 of Group I NPVs can be functionally substituted by Group II NPV VP39, a vp39-null AcMNPV (vAcvp39KO) and a vp39-pseudotyped AcMNPV (vAcSpltvp39:FLAG), in which the Group I AcMNPV vp39 coding sequence was replaced with that of SpltMNPV from Group II NPVs, were constructed via homologous recombination in Escherichia coli. Using an anti-FLAG monoclonal antibody, immunoblot analysis was performed to examine SpltMNPV VP39 expression. Fluorescence and light microscopy were used to monitor viral replication and infection. Viral growth curve analysis was performed using a fifty percent tissue culture infective dose (TCID50) endpoint dilution assay. Viral morphogenesis was detected using an electron microscope. RESULTS: Sequence alignment indicated that the N-termini of AcMNPV VP39 and SpltMNPV VP39 are relatively conserved, whereas the C-terminus of SpltMNPV VP39 lacks the domain of amino acid residues 306-334 homologous to AcMNPV VP39. Immunoblot analysis showed that SpltMNPV VP39 was expressed in vAcSpltvp39:FLAG. Fluorescence and light microscopy showed that vAcSpltvp39:FLAG did not spread by infection. Viral growth curve analysis confirmed a defect in infectious budded virion production. Electron microscopy revealed that although masses of abnormally elongated empty capsid structures existed inside the nuclei of Sf9 cells transfected with vAcSpltvp39:FLAG, no nucleocapsids were observed. CONCLUSION: Altogether, our results demonstrated that VP39 from SpltMNPV cannot efficiently substitute AcMNPV VP39 during nucleocapsid assembly in AcMNPV.

UL28 and UL33 homologs of Marek's disease virus terminase complex involved in the regulation of cleavage and packaging of viral DNA are indispensable for replication in cultured cells.

Processing and packaging of herpesvirus genomic DNA is regulated by a packaging-associated terminase complex comprising of viral proteins pUL15, pUL28 and pUL33. Marek's disease virus (MDV) homologs UL28 and UL33 showed conserved functional features with high sequence identity with the corresponding Herpes simplex virus 1 (HSV-1) homologs. As part of the investigations into the role of the UL28 and UL33 homologs of oncogenic MDV for DNA packaging and replication in cultured cells, we generated MDV mutant clones deficient in UL28 or UL33 of full-length MDV genomes. Transfection of UL28- or UL33-deleted BAC DNA into chicken embryo fibroblast (CEF) did not result either in the production of visible virus plaques, or detectable single cell infection after passaging onto fresh CEF cells. However, typical MDV plaques were detectable in CEF transfected with the DNA of revertant mutants where the deleted genes were precisely reinserted. Moreover, the replication defect of the UL28-deficient mutant was completely restored when fragment encoding the full UL28 gene was co-transfected into CEF cells. Viruses recovered from the revertant construct, as well as by the UL28 co-transfection, showed replication ability comparable with parental virus. Furthermore, the transmission electron microscopy study indicated that immature capsids were assembled without the UL28 expression, but with the loss of infectivity. Importantly, predicted three-dimensional structures of UL28 between MDV and HSV-1 suggests conserved function in virus replication. For the first time, these results revealed that both UL28 and UL33 are essential for MDV replication through regulating DNA cleavage and packaging.

Phage Mu Gam protein promotes NHEJ in concert with Escherichia coli ligase.

The Gam protein of transposable phage Mu is an ortholog of eukaryotic and bacterial Ku proteins, which carry out nonhomologous DNA end joining (NHEJ) with the help of dedicated ATP-dependent ligases. Many bacteria carry Gam homologs associated with either complete or defective Mu-like prophages, but the role of Gam in the life cycle of Mu or in bacteria is unknown. Here, we show that MuGam is part of a two-component bacterial NHEJ DNA repair system. Ensemble and single-molecule experiments reveal that MuGam binds to DNA ends, slows the progress of RecBCD exonuclease, promotes binding of NAD+-dependent Escherichia coli ligase A, and stimulates ligation. In vivo, Gam equally promotes both precise and imprecise joining of restriction enzyme-digested linear plasmid DNA, as well as of a double-strand break (DSB) at an engineered I-SceI site in the chromosome. Cell survival after the induced DSB is specific to the stationary phase. In long-term growth competition experiments, particularly upon treatment with a clastogen, the presence of gam in a Mu lysogen confers a distinct fitness advantage. We also show that the role of Gam in the life of phage Mu is related not to transposition but to protection of genomic Mu copies from RecBCD when viral DNA packaging begins. Taken together, our data show that MuGam provides bacteria with an NHEJ system and suggest that the resulting fitness advantage is a reason that bacteria continue to retain the gam gene in the absence of an intact prophage.

Molecular switch-like regulation enables global subunit coordination in a viral ring ATPase.

Subunits in multimeric ring-shaped motors must coordinate their activities to ensure correct and efficient performance of their mechanical tasks. Here, we study WT and arginine finger mutants of the pentameric bacteriophage φ29 DNA packaging motor. Our results reveal the molecular interactions necessary for the coordination of ADP-ATP exchange and ATP hydrolysis of the motor's biphasic mechanochemical cycle. We show that two distinct regulatory mechanisms determine this coordination. In the first mechanism, the DNA up-regulates a single subunit's catalytic activity, transforming it into a global regulator that initiates the nucleotide exchange phase and the hydrolysis phase. In the second, an arginine finger in each subunit promotes ADP-ATP exchange and ATP hydrolysis of its neighbor. Accordingly, we suggest that the subunits perform the roles described for GDP exchange factors and GTPase-activating proteins observed in small GTPases. We propose that these mechanisms are fundamental to intersubunit coordination and are likely present in other ring ATPases.

DNA Packaging and Genomics of the Salmonella 9NA-Like Phages.

We present the genome sequences of Salmonella enterica tailed phages Sasha, Sergei, and Solent. These phages, along with Salmonella phages 9NA, FSL_SP-062, and FSL_SP-069 and the more distantly related Proteus phage PmiS-Isfahan, have similarly sized genomes of between 52 and 57 kbp in length that are largely syntenic. Their genomes also show substantial genome mosaicism relative to one another, which is common within tailed phage clusters. Their gene content ranges from 80 to 99 predicted genes, of which 40 are common to all seven and form the core genome, which includes all identifiable virion assembly and DNA replication genes. The total number of gene types (pangenome) in the seven phages is 176, and 59 of these are unique to individual phages. Their core genomes are much more closely related to one another than to the genome of any other known phage, and they comprise a well-defined cluster within the family Siphoviridae To begin to characterize this group of phages in more experimental detail, we identified the genes that encode the major virion proteins and examined the DNA packaging of the prototypic member, phage 9NA. We show that it uses a pac site-directed headful packaging mechanism that results in virion chromosomes that are circularly permuted and about 13% terminally redundant. We also show that its packaging series initiates with double-stranded DNA cleavages that are scattered across a 170-bp region and that its headful measuring device has a precision of ±1.8%.IMPORTANCE The 9NA-like phages are clearly highly related to each other but are not closely related to any other known phage type. This work describes the genomes of three new 9NA-like phages and the results of experimental analysis of the proteome of the 9NA virion and DNA packaging into the 9NA phage head. There is increasing interest in the biology of phages because of their potential for use as antibacterial agents and for their ecological roles in bacterial communities. 9NA-like phages that infect two bacterial genera have been identified to date, and related phages infecting additional Gram-negative bacterial hosts are likely to be found in the future. This work provides a foundation for the study of these phages, which will facilitate their study and potential use.