Mechanical Capsid Maturation Facilitates the Resolution of Conflicting Requirements for Herpesvirus Assembly

Most viruses undergo a maturation process from a weakly self-assembled, noninfectious particle to a stable, infectious virion. For herpesviruses, this maturation process resolves several conflicting requirements: i) assembly must be driven by weak, reversible interactions between viral particle subunits to reduce errors and minimize energy of self-assembly; ii) the viral particle must be stable enough to withstand tens of atmospheres of DNA pressure resulting from its strong confinement in the capsid. With herpes simplex virus type 1 (HSV-1) as a prototype of human herpesviruses, we demonstrate that this mechanical capsid maturation is mainly facilitated through capsid-binding auxiliary protein UL25, orthologs of which are present in all herpesviruses. Through genetic manipulation of UL25 mutants of HSV-1 combined with interrogation of capsid mechanics with atomic force microscopy nano-indentation, we suggest the mechanism of stepwise binding of distinct UL25 domains correlated with capsid maturation and DNA packaging. These findings demonstrate another paradigm of viruses as elegantly programmed nano-machines, where an intimate relationship between mechanical and genetic information is preserved in UL25 architecture. IMPORTANCE Minor capsid protein UL25 plays a critical role in mechanical maturation of HSV-1 capsid during virus assembly, required for stable DNA packaging. We modulate UL25-capsid interactions by genetically deleting different UL25 regions and quantify the effect on mechanical capsid stability using an atomic force microscopy (AFM) nano-indentation approach. This approach reveals how UL25 regions reinforce the herpesvirus capsid in order to stably package and retain pressurized DNA. Our data suggests a mechanism of stepwise binding of two main UL25 domains timed with DNA packaging.

Helical inchworming: a novel translocation mechanism for a ring ATPase

Ring ATPases perform a variety of tasks in the cell. Their function involves complex communication and coordination among the often identical subunits. Translocases in this group are of particular interest as they involve both chemical and mechanical actions in their operation. We study the DNA packaging motor of bacteriophage φ29, and using single-molecule optical tweezers and single-particle cryo-electron microscopy, have discovered a novel translocation mechanism for a molecular motor.

A viral genome packaging ring-ATPase is a flexibly coordinated pentamer

Multi-subunit ring-ATPases carry out a myriad of biological functions, including genome packaging in viruses. Though the basic structures and functions of these motors have been well-established, the mechanisms of ATPase firing and motor coordination are poorly understood. Here, using single-molecule fluorescence, we determine that the active bacteriophage T4 DNA packaging motor consists of five subunits of gp17. By systematically doping motors with an ATPase-defective subunit and selecting single motors containing a precise number of active or inactive subunits, we find that the packaging motor can tolerate an inactive subunit. However, motors containing one or more inactive subunits exhibit fewer DNA engagements, a higher failure rate in encapsidation, reduced packaging velocity, and increased pausing. These findings suggest a DNA packaging model in which the motor, by re-adjusting its grip on DNA, can skip an inactive subunit and resume DNA translocation, suggesting that strict coordination amongst motor subunits of packaging motors is not crucial for function.

Experimental evolution of extremophile levels of radiation resistance in Escherichia coli

Recent human development of high-level sources of ionizing radiation (IR) prompts a corresponding need to understand the effects of IR on living systems. One approach has focused on the capacity of some organisms to survive astonishing levels of IR exposure. Using experimental evolution, we have generated populations of Escherichia coli with IR resistance comparable to the extremophile Deinococcus radiodurans. Every aspect of cell physiology is affected. Cellular isolates exhibit approximately 1,000 base pair changes plus major genomic and proteomic alterations. The IR resistance phenotype is stable without selection for at least 100 generations. Defined and probable contributions include alterations in cellular systems involved in DNA repair, amelioration of reactive oxygen species, Fe metabolism and repair of iron-sulfur centers, DNA packaging, and intermediary metabolism. A path to new mechanistic discoveries, exemplified by an exploration of rssB function, is evident. Most important, there is no single molecular mechanism underlying extreme IR resistance.

Controlling the topology of mammalian mitochondrial DNA

The genome of mitochondria, called mtDNA, is a small circular DNA molecule present at thousands of copies per human cell. MtDNA is packaged into nucleoprotein complexes called nucleoids, and the density of mtDNA packaging affects mitochondrial gene expression. Genetic processes such as transcription, DNA replication and DNA packaging alter DNA topology, and these topological problems are solved by a family of enzymes called topoisomerases. Within mitochondria, topoisomerases are involved firstly in the regulation of mtDNA supercoiling and secondly in disentangling interlinked mtDNA molecules following mtDNA replication. The loss of mitochondrial topoisomerase activity leads to defects in mitochondrial function, and variants in the dual-localized type IA topoisomerase TOP3A have also been reported to cause human mitochondrial disease. We review the current knowledge on processes that alter mtDNA topology, how mtDNA topology is modulated by the action of topoisomerases, and the consequences of altered mtDNA topology for mitochondrial function and human health.

UL25 capsid binding facilitates mechanical maturation of the Herpesvirus capsid and allows retention of pressurized DNA

The maturation process that occurs in most viruses is evolutionarily driven as it resolves several conflicting virion assembly requirements. During herpesvirus assembly in a host cell nucleus, micron-long double-stranded herpes DNA is packaged into a nanometer-sized procapsid. This leads to strong confinement of the viral genome with resulting tens of atmospheres of intra-capsid DNA pressure. Yet, the procapsid is unstable due to weak, reversible interactions between its protein subunits, which ensures free energy minimization and reduces assembly errors. In this work we show that herpesviruses resolve these contradictory capsid requirements through a mechanical capsid maturation process facilitated by multi-functional auxiliary protein UL25. Through mechanical interrogation of herpes simplex virus type 1 (HSV-1) capsid with atomic force microscopy nano-indentation, we show that UL25 binding at capsid vertices post-assembly provides the critical capsid reinforcement required for stable DNA encapsidation; the absence of UL25 binding leads to capsid rupture. Furthermore, we demonstrate that gradual capsid reinforcement is a feasible maturation mechanism facilitated by progressive UL25 capsid binding, which is likely correlated with DNA packaging progression. This work provides insight into elegantly programmed viral assembly machinery where targeting of capsid assembly mechanics presents a new antiviral strategy that is resilient to development of drug resistance. Importance: Most viruses undergo a maturation process from a weakly assembled particle to a stable virion. Herpesvirus capsid undergoes mechanical maturation to withstand tens of atmospheres of DNA pressure. We demonstrate that this mechanical capsid maturation is mainly facilitated through binding of auxiliary protein UL25 in HSV-1 capsid vertices. We show that UL25 binding provides the critical capsid reinforcement required for stable DNA encapsidation. Our data also suggests that gradual capsid reinforcement by progressive UL25 binding is a feasible capsid maturation mechanism, correlated with DNA packaging progression.

Detection and Molecular Characterization of Two Gammaherpesviruses from Pantesco Breed Donkeys during an Outbreak of Mild Respiratory Disease

Equid and asinine gammaherpesviruses (GHVs; genus Percavirus) are members of the Herpesviridae family. Though GHVs have been reported in horse populations, less studies are available on gammaherpesviral infections in donkeys. This study reports the co-infection with two GHVs in Pantesco breed donkeys, an endangered Italian donkey breed. Samples (n = 124) were collected on a breeding farm in Southern Italy from 40 donkeys, some of which were healthy or presented erosive tongue lesions and/or mild respiratory signs. Samples were analysed by using a set of nested PCRs targeting the DNA polymerase, glycoprotein B, and DNA-packaging protein genes, and sequence and phylogenetic analyses were performed. Twenty-nine donkeys (72.5%) tested positive, and the presence of Equid gammaherpesvirus 7 and asinine herpesvirus 5 was evidenced. In 11 animals, we found evidence for co-infection with viruses from the two species. Virions with herpesvirus-like morphology were observed by electron microscopic examination, and viruses were successfully isolated in RK-13-KY cell monolayers. The histological evaluation of tongue lesions revealed moderate lympho-granulocytic infiltrates and rare eosinophilic inclusions. The detection of GHVs in this endangered asinine breed suggests the need long-life monitoring within conservation programs and reinforces the need for further investigations of GHV’s pathogenetic role in asinine species.

Intravirion DNA Can Access the Space Occupied by the Bacteriophage P22 Ejection Proteins

Tailed double-stranded DNA bacteriophages inject some proteins with their dsDNA during infection. Phage P22 injects about 12, 12, and 30 molecules of the proteins encoded by genes 7, 16 and 20, respectively. After their ejection from the virion, they assemble into a trans-periplasmic conduit through which the DNA passes to enter the cytoplasm. The location of these proteins in the virion before injection is not well understood, although we recently showed they reside near the portal protein barrel in DNA-filled heads. In this report we show that when these proteins are missing from the virion, a longer than normal DNA molecule is encapsidated by the P22 headful DNA packaging machinery. Thus, the ejection proteins occupy positions within the virion that can be occupied by packaged DNA when they are absent.