Viral Packaging Research Articles

N terminus of SARS-CoV-2 nonstructural protein 3 interrupts RNA-driven phase separation of N protein by displacing RNA

The connection between SARS-CoV-2 replication-transcription complexes (RTCs) and nucleocapsid (N) protein is critical for regulating genomic RNA replication and virion packaging over the viral life cycle. However, the mechanism that dynamically regulates genomic RNA packaging and replication remains elusive. Here, we demonstrate that the N-terminal domain (NTD) of SARS-CoV-2 nonstructural protein 3 (Nsp3), a core component of viral RTCs, binds N protein and displaces RNA in a concentration-dependent manner. This interaction disrupts liquid-liquid phase separation of N protein driven by N protein-RNA interactions which is crucial for virion packaging and viral replication. We also report a high-resolution crystal structure of the Nsp3 ubiquitin-like domain 1 (Ubl1) at 1.49 Å, which reveals abundant negative charges on the protein surface. Sequence and structural analyses identify several conserved motifs at the Ubl1-N protein interface and a previously unexplored highly negative groove, providing insights into the molecular mechanism of Ubl1-mediated modulation of N protein-RNA binding. Our findings elucidate the mechanism of dynamic regulation of SARS-CoV-2 genomic RNA replication and packaging over the viral life cycle. Targeting the conserved Ubl1-N protein interaction hotspots also promises to aid in the development of broad-spectrum antivirals against pathogenic coronaviruses.

Phase separation and viral factories: unveiling the physical processes supporting RNA packaging in dsRNA viruses.

Understanding of the physicochemical properties and functions of biomolecular condensates has rapidly advanced over the past decade. More recently, many RNA viruses have been shown to form cytoplasmic replication factories, or viroplasms, via phase separation of their components, akin to numerous cellular membraneless organelles. Notably, diverse viruses from the Reoviridae family containing 10-12 segmented double-stranded RNA genomes induce the formation of viroplasms in infected cells. Little is known about the inner workings of these membraneless cytoplasmic inclusions and how they may support stoichiometric RNA assembly in viruses with segmented RNA genomes, raising questions about the roles of phase separation in coordinating viral genome packaging. Here, we discuss how the molecular composition of viroplasms determines their properties, highlighting the interplay between RNA structure, RNA remodelling, and condensate self-organisation. Advancements in RNA structural probing and theoretical modelling of condensates can reveal the mechanisms through which these ribonucleoprotein complexes support the selective enrichment and stoichiometric assembly of distinct viral RNAs.

Crystal structure and nucleic acid binding mode of CPV NSP9: implications for viroplasm in Reovirales.

Cytoplasmic polyhedrosis viruses (CPVs), like other members of the order Reovirales, produce viroplasms, hubs of viral assembly that shield them from host immunity. Our study investigates the potential role of NSP9, a nucleic acid-binding non-structural protein encoded by CPVs, in viroplasm biogenesis. We determined the crystal structure of the NSP9 core (NSP9ΔC), which shows a dimeric organization topologically similar to the P9-1 homodimers of plant reoviruses. The disordered C-terminal region of NSP9 facilitates oligomerization but is dispensable for nucleic acid binding. NSP9 robustly binds to single- and double-stranded nucleic acids, regardless of RNA or DNA origin. Mutagenesis studies further confirmed that the dimeric form of NSP9 is critical for nucleic acid binding due to positively charged residues that form a tunnel during homodimerization. Gel migration assays reveal a unique nucleic acid binding pattern, with the sequential appearance of two distinct complexes dependent on protein concentration. The similar gel migration pattern shared by NSP9 and rotavirus NSP3, coupled with its structural resemblance to P9-1, hints at a potential role in translational regulation or viral genome packaging, which may be linked to viroplasm. This study advances our understanding of viroplasm biogenesis and Reovirales replication, providing insights into potential antiviral drug targets.

DNA contamination within recombinant adeno-associated virus preparations correlates with decreased CD34+ cell clonogenic potential

Recombinant adeno-associated viruses (rAAV) are promising for applications in many genome editing techniques through their effectiveness as carriers of DNA homologous donors into primary hematopoietic stem and progenitor cells (HSPC) but have many outstanding concerns. Specifically, their biomanufacturing and the variety of factors that influence the quality and consistency of rAAV preps are in question. During the process of rAAV packaging, a cell line is transfected with several DNA plasmids that collectively encode all the necessary information to allow for viral packaging. Ideally, this process results in packaging of complete viral particles only containing rAAV genomes; however, this is not the case. Through this study, we were able to leverage Single-Stranded Virus (SSV)-seq, an NGS-based method to quantify all DNA species present within rAAV preps. From this, it was determined that much of the DNA within some rAAV preps is not vector-genome derived, and there is wide variability in the contamination by DNA across various preps. Furthermore, we demonstrate that transducing CD34+ hematopoietic stem and progenitor cells (HSPC) with preps with higher contaminating DNA resulted in decreased clonogenic potential, altered transcriptomic profiles, and decreased genomic editing. Collectively, this study characterized the effects of DNA contamination within rAAV preps on CD34+ HSPC cellular potential.

Visualizing Viral RNA Packaging Signals in Action

Here we confirm, using genome-scale RNA fragments in assembly competition assays, that multiple sub-sites (Packaging Signals, PSs) across the 5′ two-thirds of the gRNA of Satellite Tobacco Necrosis Virus-1 make sequence-specific contacts to the viral CPs helping to nucleate formation of its T = 1 virus-like particle (VLP). These contacts explain why natural virions only package their positive-sense genomes. Asymmetric cryo-EM reconstructions of these VLPs suggest that interactions occur between amino acid residues in the N-terminal ends of the CP subunits and the gRNA PS loop sequences. The base-paired stems of PSs also act non-sequence-specifically by electrostatically promoting the assembly of CP trimers. Importantly, alterations in PS-CP affinity result in an asymmetric distribution of bound PSs inside VLPs, with fuller occupation of the higher affinity 5′ PS RNAs around one vertex, decreasing to an RNA-free opposite vertex within the VLP shell. This distribution suggests that gRNA folding regulates cytoplasmic genome extrusion so that the weakly bound 3′ end of the gRNA, containing the RNA polymerase binding site, extrudes first. This probably occurs after cation-loss induced swelling of the CP-shell, weakening contacts between CP subunits. These data reveal for the first time in any virus how differential PS folding propensity and CP affinities support the multiple roles genomes play in virion assembly and infection. The high degree of conservation between the CP fold of STNV-1 and those of the CPs of many other viruses suggests that these aspects of genome function will be widely shared.

Nuclear reorganization by NPM1-mediated phase separation triggered by adenovirus core protein VII.

In this study, we explored how adenoviruses utilize a process known as liquid-liquid phase separation (LLPS) to enhance their replication. We focused on a cellular chromatin remodeling protein, NPM1, which plays a crucial role in nucleolar formation through LLPS. NPM1 facilitates LLPS by interacting with adenovirus protein VII, effectively accumulating protein VII into membrane-less compartments called virus-induced post-replication bodies. NPM1 functions as a molecular chaperone of protein VII to assemble viral chromatin by transferring protein VII to viral DNA. Remarkably, when NPM1 was depleted, this process was disrupted, decreasing viral genome packaging. These findings shed light on a critical aspect of virus-host interactions, illustrating how adenovirus utilizes NPM1-mediated LLPS activity. Our findings provide valuable insights into the dynamic interplay between viruses and the host nucleus.

Development and Evaluation of a Shrimp Virus (IHHNV)-Mediated Gene Transfer and Expression System for Shrimps.

An efficient gene transfer and expression tool is lacking for shrimps and shrimp cells. To solve this, this study has developed a shrimp DNA virus-mediated gene transfer and expression system, consisting of insect Sf9 cells for viral packaging, the shrimp viral vector of pUC19-IHHNV-PH-GUS and the baculoviral vector of Bacmid or Bacmid-VP28 encoding the shrimp WSSV envelope protein VP28. The pUC19-IHHNV-PH-GUS vector was constructed by assembling the genomic DNA of shrimp infectious hypodermal and hematopoietic necrosis virus (IHHNV), which has shortened inverted terminal repeats, into a pUC19 backbone, and then an expression cassette of baculoviral polyhedron (PH) promoter-driven GUS (β-glucuronidase) reporter gene was inserted immediately downstream of IHHNV for proof-of-concept. It was found that the viral vector of pUC19-IHHNV-PH-GUS could be successfully packaged into IHHNV-like infective virions in the Sf9 cells, and the gene transfer efficiency of this system was evaluated and verified in three systems of Sf9 cells, shrimp hemolymph cells and tissues of infected shrimps, but the GUS expression could only be detected in cases where the viral vector was co-transfected or co-infected with a baculovirus of Bacmid or Bacmid-VP28 due to the Bacmid-dependence of the PH promoter. Moreover, the packaging and infection efficiencies could be significantly improved when Bacmid-VP28 was used instead of Bacmid.

A phage satellite manipulates the viral DNA packaging motor to inhibit phage and promote satellite spread.

ICP1, a lytic bacteriophage of Vibrio cholerae, is parasitized by phage satellites, PLEs, which hijack ICP1 proteins for their own horizontal spread. PLEs' dependence on ICP1's DNA replication machineryand virion components results in inhibition of ICP1's lifecycle. PLEsare expected to depend on ICP1 factors for genome packaging, but the mechanism(s) PLEs use to inhibit ICP1 genome packaging is currently unknown. Here, we identify and characterize Gpi, PLE's indiscriminate genome packaging inhibitor. Gpi binds to ICP1's large terminase (TerL), the packaging motor, and blocks genome packaging. To overcome Gpi's negative effect on TerL, a component PLE also requires, PLE uses two genome packaging specifiers, GpsA and GpsB, that specifically allow packaging of PLE genomes. Surprisingly, PLE also uses mimicry of ICP1's pac site as a backup strategy to ensure genome packaging. PLE's pac site mimicry, however, is only sufficient if PLE can inhibit ICP1 at other stages of its lifecycle, suggesting an advantage to maintaining Gpi, GpsAand GpsB. Collectively, these results provide mechanistic insights into another stage of ICP1's lifecycle that is inhibited by PLE, which is currently the most inhibitory of the documented phage satellites. More broadly, Gpi represents the first satellite-encoded inhibitor of a phage TerL.

Role of RNA structural plasticity in modulating HIV-1 genome packaging and translation

HIV-1 transcript function is controlled in part by twinned transcriptional start site usage, where 5' capped RNAs beginning with a single guanosine (1G) are preferentially packaged into progeny virions as genomic RNA (gRNA) whereas those beginning with three sequential guanosines (3G) are retained in cells as mRNAs. In 3G transcripts, one of the additional guanosines base pairs with a cytosine located within a conserved 5' polyA element, resulting in formation of an extended 5' polyA structure as opposed to the hairpin structure formed in 1G RNAs. To understand how this remodeling influences overall transcript function, we applied in vitro biophysical studies with in-cell genome packaging and competitive translation assays to native and 5' polyA mutant transcripts generated with promoters that differentially produce 1G or 3G RNAs. We identified mutations that stabilize the 5' polyA hairpin structure in 3G RNAs, which promote RNA dimerization and Gag binding without sequestering the 5' cap. None of these 3G transcripts were competitively packaged, confirming that cap exposure is a dominant negative determinant of viral genome packaging. For all RNAs examined, conformations that favored 5' cap exposure were both poorly packaged and more efficiently translated than those that favored 5' cap sequestration. We propose that structural plasticity of 5' polyA and other conserved RNA elements place the 5' leader on a thermodynamic tipping point for low-energetic (~3 kcal/mol) control of global transcript structure and function.

Selection and characterization of human scFvs targeting the SARS-CoV-2 nucleocapsid protein isolated from antibody libraries of COVID-19 patients

In 2019, the novel SARS-CoV-2 coronavirus emerged in China, causing the pneumonia named COVID-19. At the beginning, all research efforts were focused on the spike (S) glycoprotein. However, it became evident that the nucleocapsid (N) protein is pivotal in viral replication, genome packaging and evasion of the immune system, is highly immunogenic, which makes it another compelling target for antibody development alongside the spike protein. This study focused on the construction of single chain fragments variable (scFvs) libraries from SARS-CoV-2-infected patients to establish a valuable, immortalized and extensive antibodies source. We used the Intracellular Antibody Capture Technology to select a panel of scFvs against the SARS-CoV-2 N protein. The whole panel of scFv was expressed and characterized both as intrabodies and recombinant proteins. ScFvs were then divided into 2 subgroups: those that exhibited high binding activity to N protein when expressed in yeast or in mammalian cells as intrabodies, and those purified as recombinant proteins, displaying affinity for recombinant N protein in the nanomolar range. This panel of scFvs against the N protein represents a novel platform for research and potential diagnostic applications.

Reprogramming Glioblastoma Cells into Non-Cancerous Neuronal Cells as a Novel Anti-Cancer Strategy.

Glioblastoma Multiforme (GBM) is an aggressive brain tumor with a high mortality rate. Direct reprogramming of glial cells to different cell lineages, such as induced neural stem cells (iNSCs) and induced neurons (iNeurons), provides genetic tools to manipulate a cell's fate as a potential therapy for neurological diseases. NeuroD1 (ND1) is a master transcriptional factor for neurogenesis and it promotes neuronal differentiation. In the present study, we tested the hypothesis that the expression of ND1 in GBM cells can force them to differentiate toward post-mitotic neurons and halt GBM tumor progression. In cultured human GBM cell lines, including LN229, U87, and U373 as temozolomide (TMZ)-sensitive and T98G as TMZ-resistant cells, the neuronal lineage conversion was induced by an adeno-associated virus (AAV) package carrying ND1. Twenty-one days after AAV-ND1 transduction, ND1-expressing cells displayed neuronal markers MAP2, TUJ1, and NeuN. The ND1-induced transdifferentiation was regulated by Wnt signaling and markedly enhanced under a hypoxic condition (2% O2 vs. 21% O2). ND1-expressing GBM cultures had fewer BrdU-positive proliferating cells compared to vector control cultures. Increased cell death was visualized by TUNEL staining, and reduced migrative activity was demonstrated in the wound-healing test after ND1 reprogramming in both TMZ-sensitive and -resistant GBM cells. In a striking contrast to cancer cells, converted cells expressed the anti-tumor gene p53. In an orthotopical GBM mouse model, AAV-ND1-reprogrammed U373 cells were transplanted into the fornix of the cyclosporine-immunocompromised C57BL/6 mouse brain. Compared to control GBM cell-formed tumors, cells from ND1-reprogrammed cultures formed smaller tumors and expressed neuronal markers such as TUJ1 in the brain. Thus, reprogramming using a single-factor ND1 overcame drug resistance, converting malignant cells of heterogeneous GBM cells to normal neuron-like cells in vitro and in vivo. These novel observations warrant further research using patient-derived GBM cells and patient-derived xenograft (PDX) models as a potentially effective treatment for a deadly brain cancer and likely other astrocytoma tumors.

Advances in Understanding the Envelope Protein in Coronavirus Infection

The COVID-19 pandemic continues to impart devastating effects on human health, healthcare systems, and the economy. Vaccination, monoclonal antibodies, and antiviral therapies prevent and limit early infection. Unfortunately, few strategies exist to mitigate the disease burden in the vast number of individuals who seek medical attention with established infection and severe disease. While we have a limited understanding of the mechanistic basis by which SARS-CoV-2 causes critical illness, increasing evidence suggests that host-pathogen interactions shape immune responses that drive the pathogenesis of COVID-19. Therefore, it is imperative to understand the roles of the viral proteins and how they shape the course of infection. One interesting protein is the envelope (E) protein of SARS-CoV-2; this tiny structural protein has been implicated in many phases of the viral life cycle. Importantly, the E protein facilitates viral packaging and replication, and its deletion reduces viral pathogenicity. The E protein also possesses ion channel functions, interacts with host proteins, and has the potential to have various structural topologies. This review aims to establish an updated understanding by highlighting recent developments in the investigation of the SARS-CoV-2 E protein, particularly in comparison to the envelope protein of SARS-CoV. thorough knowledge of this protein will enable targeted studies in hopes of tailored efficacious treatments.

Enhancing prime editor flexibility with coiled-coil heterodimers

BackgroundPrime editing enables precise base substitutions, insertions, and deletions at targeted sites without the involvement of double-strand DNA breaks or exogenous donor DNA templates. However, the large size of prime editors (PEs) hampers their delivery in vivo via adeno-associated virus (AAV) due to the viral packaging limit. Previously reported split PE versions provide a size reduction, but they require intricate engineering and potentially compromise editing efficiency.ResultsHerein, we present a simplified split PE named as CC-PE, created through non-covalent recruitment of reverse transcriptase to the Cas9 nickase via coiled-coil heterodimers, which are widely used in protein design due to their modularity and well-understood sequence-structure relationship. We demonstrate that the CC-PE maintains or even surpasses the efficiency of unsplit PE in installing intended edits, with no increase in the levels of undesired byproducts within tested loci amongst a variety of cell types (HEK293T, A549, HCT116, and U2OS). Furthermore, coiled-coil heterodimers are used to engineer SpCas9-NG-PE and SpRY-PE, two Cas9 variants with more flexible editing scope. Similarly, the resulting NG-CC-PE and SpRY-CC-PE also achieve equivalent or enhanced efficiency of precise editing compared to the intact PE. When the dual AAV vectors carrying CC-PE are delivered into mice to target the Pcsk9 gene in the liver, CC-PE enables highly efficient precise editing, resulting in a significant reduction of plasma low-density lipoprotein cholesterol and total cholesterol.ConclusionsOur innovative, modular system enhances flexibility, thus potentially facilitating the in vivo applicability of prime editing.

The Role of the Host Cytoskeleton in the Formation and Dynamics of Rotavirus Viroplasms.

Rotavirus (RV) replicates within viroplasms, membraneless electron-dense globular cytosolic inclusions with liquid-liquid phase properties. In these structures occur the virus transcription, replication, and packaging of the virus genome in newly assembled double-layered particles. The viroplasms are composed of virus proteins (NSP2, NSP5, NSP4, VP1, VP2, VP3, and VP6), single- and double-stranded virus RNAs, and host components such as microtubules, perilipin-1, and chaperonins. The formation, coalescence, maintenance, and perinuclear localization of viroplasms rely on their association with the cytoskeleton. A stabilized microtubule network involving microtubules and kinesin Eg5 and dynein molecular motors is associated with NSP5, NSP2, and VP2, facilitating dynamic processes such as viroplasm coalescence and perinuclear localization. Key post-translation modifications, particularly phosphorylation events of RV proteins NSP5 and NSP2, play pivotal roles in orchestrating these interactions. Actin filaments also contribute, triggering the formation of the viroplasms through the association of soluble cytosolic VP4 with actin and the molecular motor myosin. This review explores the evolving understanding of RV replication, emphasizing the host requirements essential for viroplasm formation and highlighting their dynamic interplay within the host cell.

Picornavirus security proteins promote the release of extracellular vesicle enclosed viruses via the modulation of host kinases.

The discovery that extracellular vesicles (EVs) serve as carriers of virus particles calls for a reevaluation of the release strategies of non-enveloped viruses. Little is currently known about the molecular mechanisms that determine the release and composition of EVs produced by virus-infected cells, as well as conservation of these mechanisms among viruses. We previously described an important role for the Leader protein of the picornavirus encephalomyocarditis virus (EMCV) in the induction of virus-carrying EV subsets with distinct molecular and physical properties. EMCV L acts as a 'viral security protein' by suppressing host antiviral stress and type-I interferon (IFN) responses. Here, we tested the ability of functionally related picornavirus proteins of Theilers murine encephalitis virus (TMEV L), Saffold virus (SAFV L), and coxsackievirus B3 (CVB3 2Apro), to rescue EV and EV-enclosed virus release when introduced in Leader-deficient EMCV. We show that all viral security proteins tested were able to promote virus packaging in EVs, but that only the expression of EMCV L and CVB3 2Apro increased overall EV production. We provide evidence that one of the main antiviral pathways counteracted by this class of picornaviral proteins, i.e. the inhibition of PKR-mediated stress responses, affected EV and EV-enclosed virus release during infection. Moreover, we show that the enhanced capacity of the viral proteins EMCV L and CVB3 2Apro to promote EV-enclosed virus release is linked to their ability to simultaneously promote the activation of the stress kinase P38 MAPK. Taken together, we demonstrate that cellular stress pathways involving the kinases PKR and P38 are modulated by the activity of non-structural viral proteins to increase the release EV-enclosed viruses during picornavirus infections. These data shed new light on the molecular regulation of EV production in response to virus infection.

E-M349E and NS2A/2B–P1(T) are compensatory mutations of rDTMUV-NS2AB-P1P1’(AA), which regain virus proliferation by enhancing the virus package and restoring NS2A/2B cleavage

Duck Tembusu virus (DTMUV) belongs to the Flaviviridae family and mainly infects ducks. The genome of DTMUV is translated into a polyprotein, which is further cleaved into several protein by viral NS2B3 protease and host proteases. Crucially, the cleavage of the NS2A/2B precursor during this process is essential for the formation of replication complexes and viral packaging. Previous research has demonstrated that alanine mutations in NS2A/2B (P1P1’ (AA)) result in an attenuated strain (rDTMUV-NS2A/2B–P1P1’ (AA)) by disrupting NS2A/2B cleavage. In this study, we investigate the effects of the P1P1’ (AA) mutation on the viral life cycle and explore compensatory mutations in rDTMUV-NS2A/2B–P1P1’ (AA). Infected ducklings exhibit similar body weight gain and viral tissue loads to DTMUV-WT. Compensatory mutations E-M349E and P1(T) emerge, restoring proliferation levels to those of rDTMUV-WT. Specifically, E-M349E enhances viral packaging, while P1(T) reinstates NS2A/2B proteolysis in vitro. Thus, our findings reveal novel compensatory sites capable of restoring the attenuated DTMUV during polyprotein cleavage and packaging.

RNA genome packaging and capsid assembly of bluetongue virus visualized in host cells

Unlike those of double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and ssRNA viruses, the mechanism of genome packaging of dsRNA viruses is poorly understood. Here, we combined the techniques of high-resolution cryoelectron microscopy (cryo-EM), cellular cryoelectron tomography (cryo-ET), and structure-guided mutagenesis to investigate genome packaging and capsid assembly of bluetongue virus (BTV), a member of the Reoviridae family of dsRNA viruses. A total of eleven assembly states of BTV capsid were captured, with resolutions up to 2.8Å, with most visualized in the host cytoplasm. ATPase VP6 was found underneath the vertices of capsid shell protein VP3 as an RNA-harboring pentamer, facilitating RNA packaging. RNA packaging expands the VP3 shell, which then engages middle- and outer-layer proteins to generate infectious virions. These revealed "duality" characteristics of the BTV assembly mechanism reconcile previous contradictory co-assembly and core-filling models and provide insights into the mysterious RNA packaging and capsid assembly of Reoviridae members and beyond.

Mutagenesis and functional analysis of the varicella-zoster virus portal protein.

Herpesviruses replicate by cleaving concatemeric dsDNA into single genomic units that are packaged through an oligomeric portal present in preformed procapsids. In contrast to what is known about phage portal proteins, details concerning herpesvirus portal structure and function are not as well understood. A panel of 65 Varicella-Zoster virus (VZV) recombinant portal proteins with five amino acid in-frame insertions were generated by random transposon mutagenesis of the VZV portal gene, ORF54. Subsequently, 65 VZVLUC recombinant viruses (TNs) were generated via recombineering. Insertions were mapped to predicted portal domains (clip, wing, stem, wall, crown, and β-hairpin tunnel-loop) and recombinant viruses were characterized for plaque morphology, replication kinetics, pORF54 expression, and classified based on replication in non-complementing (ARPE19) or complementing (ARPE54C50) cell lines. The N- and C-termini were tolerant to insertion mutagenesis, as were certain clip sub-domains. The majority of mutants mapping to the wing, wall, β-hairpin tunnel loop, and stem domains were lethal. Elimination of the predicted ORF54 start codon revealed that the first 40 amino acids of the N-terminus were not required for viral replication. Stop codon insertions in the C-terminus showed that the last 100 amino acids were not required for viral replication. Lastly, a putative protease cleavage site was identified in the C-terminus of pORF54. Cleavage was likely orchestrated by a viral protease; however, processing was not required for DNA encapsidation and viral replication. The panel of recombinants should prove valuable in future studies to dissect mammalian portal structure and function.IMPORTANCEThough nucleoside analogs and a live-attenuated vaccine are currently available to treat some human herpesvirus family members, alternate methods of combating herpesvirus infection could include blocking viral replication at the DNA encapsidation stage. The approval of Letermovir provided proof of concept regarding the use of encapsidation inhibitors to treat herpesvirus infections in the clinic. We propose that small-molecule compounds could be employed to interrupt portal oligomerization, assembly into the capsid vertex, or affect portal function/dynamics. Targeting portal at any of these steps would result in disruption of viral DNA packaging and a decrease or absence of mature infectious herpesvirus particles. The oligomeric portals of herpesviruses are structurally conserved, and therefore, it may be possible to find a single compound capable of targeting portals from one or more of the herpesvirus subfamilies. Drug candidates from such a series would be effective against viruses resistant to the currently available antivirals.

Abstract 7257: Examining the promise of precision medicine in immuno-oncology: Vectors for targeted therapeutic delivery

Abstract At the forefront of therapeutic delivery is an array of vectors and targeting methods that hold immense promise for precision medicine. Vehicles such as viral vectors (AAV and lentivirus), lipid nanoparticles (LNPs) or liposomes, and peptide micelles each possess distinct and inherent properties, empowering researchers with tools to navigate the intricate challenges of specificity and efficiency in drug transport. Herein, we discuss and demonstrate design principles underpinning these vectors, elucidating their unique mechanisms for targeted delivery including:- A high-throughput viral packaging process to enable rapid downstream CRISPR or shRNA screening- Proprietary AAV plasmid synthesis and preparation techniques to maintain ITR integrity and improve gene delivery- Rapid synthesis, in vitro transcription, and novel sequencing of mRNA constructs for complete characterization of critical components such as the polyA tailBy synthesizing advancements in vector technologies, this poster serves as a roadmap for researchers and practitioners navigating the evolving landscape of targeted drug delivery in immuno-oncology. Citation Format: Crystal Richardson, Prassanna Rao, Cassandra Koole, Michael J. Karney, Sumit Kumar. Examining the promise of precision medicine in immuno-oncology: Vectors for targeted therapeutic delivery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7257.

An ATP-free packaging of T4 DNA

Packaging of viral DNA into a capsid with liquid crystalline density is a crucial step in viral reproduction. DNA packaging with an adenosine triphosphate (ATP)-fueled molecular motor is an established viral DNA packaging mechanism system. On the other hand, DNA is compacted by the conformational change attributable to multivalent cations exclusively at valences higher than three. The conformational change of DNA was not considered as a mechanism of DNA packaging of viruses. Here, T4 DNA, ejected from a capsid in the ambient concentration of phosphate corresponding to the intra-cell concentration, is packaged into the capsid when the phosphate concentration decreases to the extra-cell concentration in coexistence with divalent cations, that is, Ca2+ and Mg2+. The compaction and packaging processes coincide with the conformational change of DNA. Divalent cations can compact T4 DNA when the counter anion is phosphate. The DNA-packaged and re-generated virions showed equivalent infective ability with the original populations. Fluorescent microscopy distinguished the conformational changes of DNA between compact forms and coil forms. Packaged or unpackaged DNA was confirmed enzymatically. Plaque-forming unit (Pfu) was used as the measure of infectious ability of virions. The concentration of ATP was measured by the luminometric method. The packaging process was proceeded in picomolar or lower concentration of ATP. This series of procedures may constitute a new ATP-free or low-ATP approach to packaging viral DNA with DNA conformational change underpinning the process. The results from this study may disclose an undiscovered facet of T4 life cycle.