Viral Capsid Research Articles

Mimicry games: NPC-like MX2 condensates trap viruses

Recent findings suggest that HIV-1 capsids mimic nuclear transport receptors to engage FG-nucleoporins forentry into host nuclei. In this issue of Cell Host & Microbe, Moschonas etal. report that MX2 forms cytoplasmic condensates comprising FG-nucleoporins resembling nuclear pore complexes to capture viral capsids and hinder their nuclear transport.

Epidemiological investigation of equine rotavirus B outbreaks in horses in central Kentucky

Using metagenomic sequencing we identified equine rotavirus group B (ERVB) of ruminant origin in foal diarrhea outbreaks in the 2021 foaling season. To further investigate ERVB occurrence and determine its environmental stability, we collected mare and foal fecal samples from different farms in central Kentucky during the 2022 foaling season. The RT-qPCR-based analyses showed that ERVB genome was detected in 16.67% (42/252) of surveyed mare samples and 26.56% (34/128) of foal samples. Furthermore, 94.12% (16/17) of collected soil samples and 100% (13/13) of water samples obtained from the ERVB-positive farm premises also tested weakly positive. In addition, ERVB genome fragments were detected in 58.33% (7/12) of indoor samples collected from the equipment/barn/hospital wards during the outbreak period. Finally, the seroprevalence study showed 87% (113/130) of surveyed horse serum samples were positive for ERVB antibodies. Despite unsuccessful attempts in ERVB cultivation, phylogenetic analyses showed that fecal ERVB strains representing 2022 and 2023 foal diarrhea outbreaks, like 2021 strains, were more closely related to ruminant rotavirus B than other viruses. Further sequence analyses revealed that none of the three viral capsid proteins, the primary targets of virus-neutralizing antibodies, exhibited notable mutations among ERVB strains circulated over the past three years. Our data demonstrated that ERVB was widespread in horses on affected farms with extreme stability in the farm environment. These findings continue to support the need for future surveillance of ERVB in horses and the surrounding environment, and the development of effective countermeasures to protect horses against this new viral disease.

Development of bovine IgG3-specific assays using a novel recombinant single-domain binding reagent

Cattle express three subclasses of IgG antibody - IgG1, IgG2 and IgG3. Unlike IgG1 and IgG2, IgG3 was described relatively recently and the role of this subclass in immunity is unknown. Using recombinant bovine IgG1, IgG2 and IgG3 monoclonal antibodies (mAbs), we demonstrated that only one of the commercially available anti-bovine IgG mAbs tested was able to recognize IgG3, and no mAb exclusively bound IgG3. Here, we evaluated a small ankyrin repeat protein, called Ankyron™ AZS40101, that was generated to bind to bovine IgG3. Ankyron™ AZS40101 bound specifically to IgG3 with minimal reactivity to IgG1 and IgG2. Ankyron™ AZS40101 was shown to be useful in ELISA assays as either a capture or detection reagent. Utilisation of Ankyron™ AZS40101 alongside IgG1 and IgG2 specific mAbs to detect antigen-specific IgG subclasses in the serum of cattle sequentially vaccinated with heterologous foot-and-mouth disease virus capsid antigens revealed a low-level antigen-specific IgG3 response, in addition to IgG1 and IgG2 responses. Assessment of the total IgG1, IgG2 and IgG3 levels in healthy cattle plasma samples showed that IgG3 was measurable at a mean concentration of 0.19mg/mL, although this was significantly lower than those of IgG1 (mean 3.28mg/mL) and IgG2 (mean 3.41mg/mL). Thus, Ankyron™ AZS40101 is a new reagent that provides utility for measurement of bovine IgG3 responses to infection and vaccination.

A Novel and Simplified Anion Exchange Flow-Through Polishing Approach for the Separation of Full From Empty Adeno-Associated Virus Capsids.

Adeno-associated viruses (AAV) are widely used viral vectors for in vivo gene therapy. The purification of AAV, particularly the separation of genome-containing from empty AAV capsids, is usually time-consuming and requires expensive equipment. In this study, we present a novel laboratory scale anion exchange flow-through polishing method designed to separate full and empty AAV. Once the appropriate conditions are defined, this method eliminates the need for a chromatography system. Determination of optimal polishing conditions using a chromatography system revealed that the divalent salt MgCl2 resulted in better separation of full and empty AAV than the monovalent salt NaCl. The efficacy of the method was demonstrated for three distinct AAV serotypes (AAV8, AAV5, and AAV2) on two different stationary phases: a membrane adsorber and a monolith, resulting in a 4- to 7.5-fold enrichment of full AAV particles. Moreover, the method was shown to preserve the AAV capsids' functional potency and structural integrity. Following the successful establishment of the flow-through polishing approach, it was adapted to a manual syringe-based system. Manual flow-through polishing using the monolith or membrane adsorber achieved 3.6- and 5.4-fold enrichment of full AAV, respectively. This study demonstrates the feasibility of separating full and empty AAV without complex linear or step gradient elution and the necessity of specialized equipment. Flow-through polishing provides a rapid and easy-to-perform platform for polishing multiple vector preparations, addressing a critical aspect in the research and development of novel gene therapies.

Structural Basis for Alternative Self-Assembly Pathways Leading to Different Human Immunodeficiency Virus Capsid-Like Nanoparticles.

The mechanisms that underlie the spontaneous and faithful assembly of virus particles are guiding the design of self-assembling protein-based nanostructures for biomedical or nanotechnological uses. In this study, the human immunodeficiency virus (HIV-1) capsid was used as a model to investigate what molecular feature(s) may determine whether a protein nanoparticle with the intended architecture, instead of an aberrant particle, will be self-assembled in vitro. Attempts of using the HIV-1 capsid protein CA for achieving in vitro the self-assembly of cone-shaped nanoparticles that contain CA hexamers and pentamers, similar to authentic viral capsids, had typically yielded hexamer-only tubular particles. We hypothesized that a reduction in the stability of a transient major assembly intermediate, a trimer of CA dimers (ToD), will increase the propensity of CA to assemble in vitro into cone-shaped particles instead of tubes. Certain amino acid substitutions at CA-CA interfaces strongly favored in vitro the assembly of cone-shaped nanoparticles that resembled authentic HIV-1 capsids. All-atom MD simulations indicated that ToDs formed by CA mutants with increased propensity for assembly into cone-shaped particles are destabilized relative to ToDs formed by wt CA or by another mutant that assembles into tubes. The results also indicated that ToD destabilization is mediated by conformational distortion of different CA-CA interfaces, which removes some interprotein interactions within the ToD. A model is proposed to rationalize the linkage between reduced ToD stability and increased propensity for the formation of CA pentamers during particle growth in vitro, favoring the assembly of cone-shaped HIV-1 capsid-like nanoparticles.

The Epstein–Barr virus small capsid protein BFRF3 disrupts the NF‐кB signaling pathway by inhibiting p65 activity

AbstractEpstein–Barr virus (EBV), a common gamma herpesvirus, establishes a life‐long latent infection in the host to defend against innate immune recognition, which is closely related to a variety of malignant tumors, but its specific mechanism is unclear. BFRF3, an EBV‐encoded small capsid protein, is mainly involved in the assembly of the viral capsid structure and the maintenance of its stability. Here, we showed that BFRF3 can inhibit TNF‐α‐mediated NF‐кB promoter activation. Moreover, BFRF3 downregulates NF‐кB‐mediated promoter activation and transcription of inflammatory cytokines, including IL‐6 and IL‐8. Dual‐luciferase reporter assay demonstrated that BFRF3 restrains NF‐кB promoter activity at or below the p65 level, and coimmunoprecipitation analysis revealed that BFRF3 not only interacts with p65 but also binds to its critical truncated Rel homology domain (RHD) and transcriptional activation domain (TAD). However, BFRF3 does not affect the dimerization of p65‐p50, but overexpression of BFRF3 reduces the nuclear accumulation of p65, and the phosphorylation of p65 (Ser536) is repressed during BFRF3 transfection and EBV lytic infection, which promotes the proliferation of EBV. Overall, our study suggested that BFRF3 may play a crucial role in antiviral immunity to defend against EBV infection by inhibiting NF‐κB activity.

Structural Changes Likely Cause Chemical Differences between Empty and Full AAV Capsids.

Due to the success of adeno associated viruses (AAVs) in treating single-gene diseases, improved manufacturing technology is now needed to meet their demand. The largest challenge is creating a process to separate empty and full capsids. Patients received larger capsid doses than necessary due to the presence of empty capsids. By enabling the better separation of empty and full capsids, patients would receive the greatest therapeutic benefit with the least amount of virus capsids, thus limiting potential side effects from empty capsids. The two most common empty/full separation methods used in downstream processing are ultracentrifugation and anion exchange chromatography. Both processes have limitations, leading to a need for the identification of other structural differences that can be exploited to separate empty and full capsids. Here, we describe four possible theories of the structural changes that occur when AAV capsids envelop a genome. These theories include conformational changes occurring due to either the expansion or contraction of the capsid in the presence of nucleic acids, the constraining of the N-terminus into the five-fold pore when the genome is present, and the increased number of VP3 proteins in full capsids. These theories may reveal structural differences that can be exploited to separate full and empty capsids during manufacturing.

Subnanometer structure of medusavirus capsid during maturation using cryo-electron microscopy.

Medusavirus is a giant virus classified into an independent family of Mamonoviridae. Amoebae infected with medusavirus release immature particles in addition to virions. These particles were suggested to exhibit the maturation process of this virus, but the structure of these capsids during maturation remains unknown. Here, we apply a block-based reconstruction method in cryo-electron microscopy (cryo-EM) single particle analysis to these viral capsids, extending the resolution to 7-10 Å. The maps reveal a novel network composed of minor capsid proteins (mCPs) supporting major capsid proteins (MCPs). A predicted molecular model of the MCP fitted into the cryo-EM maps clarified the boundaries between the MCP and the underlining mCPs, as well as between the MCP and the outer spikes, and identified molecular interactions between the MCP and these components. Several structural changes of the mCPs under the fivefold vertices of the immature particles were observed, depending on the presence or absence of the underlying internal membrane. In addition, the lower part of the penton proteins on the fivefold vertices was also missing in mature virions. These dynamic conformational changes of mCPs indicate an important function in the maturation process of medusavirus.IMPORTANCEThe structural changes of giant virus capsids during maturation have not thus far been well clarified. Medusavirus is a unique giant virus in which infected amoebae release immature particles in addition to mature virus particles. In this study, we used cryo-electron microscopy to investigate immature and mature medusavirus particles and elucidate the structural changes of the viral capsid during the maturation process. In DNA-empty particles, the conformation of the minor capsid proteins changed dynamically depending on the presence or absence of the underlying internal membranes. In DNA-full particles, the lower part of the penton proteins was lost. This is the first report of structural changes of the viral capsid during the maturation process of giant viruses.

Nectin1 is a pivotal host factor involved in attachment and entry of red-spotted grouper nervous necrosis virus in the early stages of the viral life cycle.

Nervous necrosis virus (NNV) is a highly neurotropic virus that poses a persistent threat to the survival of multiple fish species. However, its inimitable neuropathogenesis remains largely elusive. To rummage potential partners germane to the nervous system, we investigated the interaction between red-spotted grouper NNV (RGNNV) and grouper brain by immunoprecipitation coupled with mass spectrometry and discerned Nectin1 as a novel host factor subtly involved in viral early invasion events. Nectin1 was abundant in neural tissues and implicated in the inception of tunnel nanotubes triggered by RGNNV. Its overexpression not only dramatically potentiated the replication dynamics of RGNNV in susceptible cells, but also empowered non-sensitive cells to expeditiously capture free virions within 2 min. This potency was impervious to low temperatures but was dose-dependently suppressed by soluble protein or specific antibody of Nectin1 ectodomain, indicating Nectin1 as an attachment receptor for RGNNV. Mechanistically, efficient hijacking of virions by Nectin1 strictly depended on intricate linkages to different modules of viral capsid protein, especially the direct binding between the IgC1 loop and P-domain. More strikingly, despite abortive proliferation in Nectin1-reconstructed CHSE-214 cells, a non-sensitive cell, RGNNV could gain access to the intracellular compartment by capitalizing on Nectin1, thereby inducing canonical cytoplasmic vacuolation. Altogether, our findings delineate a candidate entrance for RGNNV infiltration into the nervous system, which may shed unprecedented insights into the exploration and elucidation of RGNNV pathogenesis.IMPORTANCENervous necrosis virus (NNV) is one of the most virulent pathogens in the aquaculture industry, which inflicts catastrophic damage to ecology, environment, and economy annually around the world. Nevertheless, its idiosyncratic invasion and latency mechanisms pose enormous hardships to epidemic prevention and control. In this study, deploying grouper brain as a natural screening library, a single-transmembrane glycoprotein, Nectin1, was first identified as an emergent functional receptor for red-spotted grouper NNV (RGNNV) that widely allocated in nervous tissues and directly interacted with viral capsid protein through distinct Ig-like loops to bridge virus-host crosstalk, apprehend free virions, and concomitantly propel viral entry. Our findings illuminate the critical role of Nectin1 in RGNNV attachment and entry and provide a potential target for future clinical intervention strategies in the therapeutic race against RGNNV.

Coordinate-based simulation of pair distance distribution functions for small and large molecular assemblies: implementation and applications.

X-ray scattering has become a major tool in the structural characterization of nanoscale materials. Thanks to the widely available experimental and computational atomic models, coordinate-based X-ray scattering simulation has played a crucial role in data interpretation in the past two decades. However, simulation of real-space pair distance distribution functions (PDDFs) from small- and wide-angle X-ray scattering, SAXS/WAXS, has been relatively less exploited. This study presents a comparison of PDDF simulation methods, which are applied to molecular structures that range in size from β-cyclo-dextrin [1 kDa molecular weight (MW), 66 non-hydrogen atoms] to the satellite tobacco mosaic virus capsid (1.1 MDa MW, 81 960 non-hydrogen atoms). The results demonstrate the power of interpretation of experimental SAXS/WAXS from the real-space view, particularly by providing a more intuitive method for understanding of partial structure contributions. Furthermore, the computational efficiency of PDDF simulation algorithms makes them attractive as approaches for the analysis of large nanoscale materials and biological assemblies. The simulation methods demonstrated in this article have been implemented in stand-alone software, SolX 3.0, which is available to download from https://12idb.xray.aps.anl.gov/solx.html.

Binding of Inhibitors to Nuclear Localization Signal Peptide from Venezuelan Equine Encephalitis Virus Capsid Protein Explored with All-Atom Replica Exchange Molecular Dynamics.

Several small molecule inhibitors have been designed to block binding of the Venezuelan equine encephalitis virus (VEEV) nuclear localization signal (NLS) sequence to the importin-α nuclear transport protein. To probe the inhibition mechanism on a molecular level, we used all-atom explicit water replica exchange molecular dynamics to study the binding of two inhibitors, I1 and I2, to the coreNLS peptide, representing the core fragment of the VEEV NLS sequence. Our objective was to evaluate the possibility of masking wherein binding of these inhibitors to the coreNLS occurs prior to its binding to importin-α. We found that the free energy of I1 and I2 binding to the coreNLS is less favorable than that to importin-α. This outcome argues against preemptive inhibitor binding to the coreNLS prior to importin-α. Instead, both inhibitors are expected to compete with the coreNLS peptide for binding to importin-α. The two factors responsible for the low affinities of the inhibitors to the coreNLS peptide are (i) the low cooperativity of binding to the peptide and (ii) the strong hydrophobic effect associated with binding to importin-α. Our results further show that upon binding to the coreNLS peptide, the inhibitors form multiple diverse binding poses. The coreNLS peptide coincubated with I1 and I2 adopts several conformational states, including open and collapsed, which underscores the fluidity of the coreNLS conformational ensemble as a target for inhibitors. Taken together with our prior investigations, this study sheds light on the molecular mechanism by which I1 and I2 ligands inhibit binding of the VEEV capsid protein to importin-α.

Receptors and Host Factors for Enterovirus Infection: Implications for Cancer Therapy.

Enteroviruses, with their diverse clinical manifestations ranging from mild or asymptomatic infections to severe diseases such as poliomyelitis and viral myocarditis, present a public health threat. However, they can also be used as oncolytic agents. This review shows the intricate relationship between enteroviruses and host cell factors. Enteroviruses utilize specific receptors and coreceptors for cell entry that are critical for infection and subsequent viral replication. These receptors, many of which are glycoproteins, facilitate virus binding, capsid destabilization, and internalization into cells, and their expression defines virus tropism towards various types of cells. Since enteroviruses can exploit different receptors, they have high oncolytic potential for personalized cancer therapy, as exemplified by the antitumor activity of certain enterovirus strains including the bioselected non-pathogenic Echovirus type 7/Rigvir, approved for melanoma treatment. Dissecting the roles of individual receptors in the entry of enteroviruses can provide valuable insights into their potential in cancer therapy. This review discusses the application of gene-targeting techniques such as CRISPR/Cas9 technology to investigate the impact of the loss of a particular receptor on the attachment of the virus and its subsequent internalization. It also summarizes the data on their expression in various types of cancer. By understanding how enteroviruses interact with specific cellular receptors, researchers can develop more effective regimens of treatment, offering hope for more targeted and efficient therapeutic strategies.

Engineered AAV capsid transport mutants overcome transduction deficiencies in the aged CNS

Adeno-associated virus-based gene therapy has enjoyed great successes over the last decade, with FDA approved therapeutics and a robust clinical pipeline. Nonetheless, barriers to successful translation remain. For example, advanced age is associated with impaired brain transduction, with the diminution of infectivity depending on anatomical region and capsid. Given that CNS gene transfer is often associated with neurodegenerative diseases where age is the chief risk-factor, we sought to better understand the causes of this impediment. We assessed two AAV variants hypothesized to overcome factors negatively impacting transduction in the aged brain; specifically, changes in extracellular and cell-surface glycans, and intracellular transport. We evaluated a heparin sulfate proteoglycan null variant with or without mutations enhancing intracellular transport. Vectors were injected into the striatum of young-adult or aged rats to address whether improving extracellular diffusion, removing glycan receptor dependence, or improving intracellular transport are important factors in transducing the aged brain. We found that, regardless of the viral capsid, there was a reduction in many of our metrics of transduction in the aged brain. However, the transport mutant was less sensitive to age, suggesting that changes in the cellular transport of AAV capsids is a key factor in age-related transduction deficiency.

A Novel Method for Separating Full and Empty Adeno-Associated Viral Capsids Using Ultrafiltration.

Adeno-associated viral vectors (AAVs) are the predominant viral vectors used for gene therapy applications. A significant challenge in obtaining effective doses is removing non-therapeutic empty viral capsids lacking DNA cargo. Current methods for separating full (gene-containing) and empty capsids are challenging to scale, produce low product yields, are slow, and are difficult to operationalize for continuous biomanufacturing. This communication demonstrates the feasibility of separating full and empty capsids by ultrafiltration. Separation performance was quantified by measuring the sieving coefficients for full and empty capsids using ELISA, qPCR, and an infectivity assay based on the live cell imaging of green fluorescent protein expression. We demonstrated that polycarbonate track-etched membranes with a pore size of 30 nm selectively permeated empty capsids to full capsids, with a high recovery yield (89%) for full capsids. The average sieving coefficients of full and empty capsids obtained through ELISA/qPCR were calculated as 0.25 and 0.49, indicating that empty capsids were about twice as permeable as full capsids. Establishing ultrafiltration as a viable unit operation for separating full and empty AAV capsids has implications for developing the scale-free continuous purification of AAVs.

Elasticity of the HIV-1 core facilitates nuclear entry and infection.

HIV-1 infection requires passage of the viral core through the nuclear pore of the cell, a process that depends on functions of the viral capsid. Recent studies have shown that HIV-1 cores enter the nucleus prior to capsid disassembly. Interactions of the viral capsid with the nuclear pore complex are necessary but not sufficient for nuclear entry, and the mechanism by which the viral core traverses the comparably sized nuclear pore is unknown. Here we show that the HIV-1 core is highly elastic and that this property is linked to nuclear entry and infectivity. Using atomic force microscopy-based approaches, we found that purified wild type cores rapidly returned to their normal conical morphology following a severe compression. Results from independently performed molecular dynamic simulations of the mature HIV-1 capsid also revealed its elastic property. Analysis of four HIV-1 capsid mutants that exhibit impaired nuclear entry revealed that the mutant viral cores are brittle. Adaptation of two of the mutant viruses in cell culture resulted in additional substitutions that restored elasticity and rescued infectivity and nuclear entry. We also show that capsid-targeting compound PF74 and the antiviral drug Lenacapavir reduce core elasticity and block HIV-1 nuclear entry at concentrations that preserve interactions between the viral core and the nuclear envelope. Our results indicate that elasticity is a fundamental property of the HIV-1 core that enables nuclear entry, thereby facilitating infection. These results provide new insights into the role of the capsid in HIV-1 nuclear entry and the antiviral mechanisms of HIV-1 capsid inhibitors.

BRAF Inhibition and UVB Light Synergistically Promote Mus musculus Papillomavirus 1-Induced Skin Tumorigenesis.

The development of keratinocytic skin tumors, presumably attributable to paradoxical activation of the MAPK pathway, represents a relevant side effect of targeted therapies with BRAF inhibitors (BRAFis). The role of cutaneous papillomavirus infection in BRAFi-associated skin carcinogenesis, however, is still inconclusive. Employing the Mus musculus papillomavirus 1 (MmuPV1) skin infection model, the impact of BRAFis and UVB exposure on papillomavirus induced skin tumorigenesis was investigated in immunocompetent FVB/NCrl mice. Systemic BRAF inhibition in combination with UVB light induced skin tumors in 62% of the MmuPV1-infected animals. In contrast, significantly fewer tumors were observed in the absence of either BRAF inhibition, UVB irradiation or virus infection, as demonstrated by lesional outgrowth in 20%, 5% and 0% of the mice, respectively. Combinatory exposure to BRAFis and UVB favored productive viral infection, which was shown by high numbers of MmuPV1 genome copies and E1^E4 spliced transcripts and an abundance of E6/E7 oncogene mRNA and viral capsid proteins. BRAF inhibition, but not viral infection or UVB light, activated ERK1/2, whereas γH2AX expression, inducible by UVB light, remained unaltered by BRAFis. These results provide experimental evidence that BRAF inhibition and UVB irradiation synergistically promote MmuPV1-induced skin tumor development in vivo. This indicates an alternative pathway by which papillomavirus skin infection may contribute to BRAFi-associated skin tumorigenesis.

Application of chlorine dioxide and its disinfection mechanism.

Chlorine dioxide (ClO2) is a strong oxidizing agent and an efficient disinfectant. Due to its broad-spectrum bactericidal properties, good inactivation effect on the vast majority of bacteria and pathogenic microorganisms, low resistance to drugs, and low generation of halogenated by-products, chlorine dioxide is widely used in fields such as water purification, food safety, medical and public health, and living environment. This review introduced the properties and application status of chlorine dioxide, compared the action mode, advantages and disadvantages of various disinfectants. The mechanism of chlorine dioxide inactivating bacteria, fungi and viruses were reviewed. The lethal target of chlorine dioxide to bacteria and fungi is to destroy the structure of cell membrane, change the permeability of cell membrane, and make intracellular substances flow out, leading to their death. The lethal targets for viruses are the destruction of viral protein capsids and the degradation of RNA fragments. The purpose of this review is to provide more scientific guidance for the application of chlorine dioxide disinfectants.

Group-theoretic analysis of symmetry-preserving deployable structures and metamaterials.

Many deployable structures in nature, as well as human-made mechanisms, preserve symmetry as their configurations evolve. Examples in nature include blooming flowers, dilation of the iris within the human eye, viral capsid maturation and molecular and bacterial motors. Engineered examples include opening umbrellas, elongating scissor jacks, variable apertures in cameras, expanding Hoberman spheres and some kinds of morphing origami structures. In these cases, the structures either preserve a discrete symmetry group or are described as an evolution from one discrete symmetry group to another of the same type as the structure deploys. Likewise, elastic metamaterials built from lattice structures can also preserve symmetry type while passively deforming and changing lattice parameters. A mathematical formulation of such transitions/deployments is articulated here. It is shown that if [Formula: see text] is Euclidean space, [Formula: see text] is a continuous group of motions of Euclidean space and [Formula: see text] is the type of the discrete subgroup of [Formula: see text] describing the symmetries of the deploying structure, then the symmetry of the evolving structure can be described by time-dependent subgroups of [Formula: see text] of the form [Formula: see text], where [Formula: see text] is a time-dependent affine transformation. Then, instead of considering the whole structure in [Formula: see text], a 'sector' of it that lives in the orbit space [Formula: see text] can be considered at each instant in time, and instead of considering all motions in [Formula: see text], only representatives from right cosets in the space [Formula: see text] need to be considered. This article is part of the theme issue 'Current developments in elastic and acoustic metamaterials science (Part 1)'.

Templated trimerization of the phage L decoration protein on capsids.

The 134-residue phage L decoration protein (Dec) forms a capsid-stabilizing homotrimer that has an asymmetric tripod-like structure when bound to phage L capsids. The N-termini of the trimer subunits consist of spatially separated globular OB-fold domains that interact with the virions of phage L or the related phage P22. The C-termini of the trimer form a three-stranded intertwined spike structure that accounts for nearly all the interactions that stabilize the trimer. A Dec mutant with the spike residues 99-134 deleted (Dec 1-98 ) was used to demonstrate that the stable globular OB-fold domain folds independently of the C-terminal residues. However, Dec 1-98 was unable to bind phage P22 virions, indicating the C-terminal spike is essential for stable capsid interaction. The full-length Dec trimer is disassembled into monomers by acidification to pH <2. These monomers retain the folded globular OB-fold domain structure, but the spike is unfolded. Increasing the pH of the Dec monomer solution to pH 6 allowed for slow trimer formation in vitro over the course of days. The infectious cycle of phage L is only around an hour, however, implying Dec trimer assembly in vivo is templated by the phage capsid. The Thermodynamic Hypothesis holds that protein folding is determined by the amino acid sequence. Dec serves as an unusual example of an oligomeric folding step that is kinetically accelerated by a viral capsid template. The capsid templating mechanism could satisfy the flexibility needed for Dec to adapt to the unusual quasi-symmetric binding site on the mature phage L capsid.

Enhanced ER protein processing gene expression increases rAAV yield and full capsid ratio in HEK293 cells

The recombinant adeno-associated virus (rAAV) vector is among the most promising viral vectors in gene therapy. However, the limited manufacturing capacity in human embryonic kidney (HEK) cells is a barrier to rAAV commercialization. We investigated the impact of endoplasmic reticulum (ER) protein processing and apoptotic genes on transient rAAV production in HEK293 cells. We selected four candidate genes based on prior transcriptomic studies: XBP1, GADD34 / PPP1R15A, HSPA6, and BCL2. These genes were stably integrated into HEK293 host cells. Traditional triple-plasmid transient transfection was used to assess the vector production capability and the quality of both the overexpressed stable pools and the parental cells. We show that the overexpression of XBP1, HSPA6, and GADD34 increases rAAV productivity by up to 100% and increases specific rAAV productivity by up to 78% in HEK293T cells. Additionally, more prominent improvement associated with ER protein processing gene overexpression was observed when parental cell productivity was high, but no substantial variation was detected under low-producing conditions. We also confirmed genome titer improvement across different serotypes (AAV2 and AAV8) and different cell lines (HEK293T and HEK293); however, the extent of improvement may vary. This study unveiled the importance of ER protein processing pathways in viral particle synthesis, capsid assembly, and vector production.Key points• Upregulation of endoplasmic reticulum (ER) protein processing (XBP1, HSPA6, and GADD34) leads to a maximum 100% increase in rAAV productivity and a maximum 78% boost in specific rAAV productivity in HEK293T cells• The enhancement in productivity can be validated across different HEK293 cell lines and can be used for the production of various AAV serotypes, although the extent of the enhancement might vary slightly• The more pronounced improvements linked to overexpressing ER protein processing genes were observed when parental cell productivity was high, with minimal variation noted under low-producing conditions