Dynamic Assembly Research Articles

Constructing Predictive Implicit Solvent Models: Insights from Exploring Potential Parameter Space and Property Predictions.

Implicit solvent (IS) models enhance the efficiency of simulating liquid systems, but those based on the potential of mean force calculations using explicit solvent (ES) models often fail to capture the solute structure and assembly dynamics under various conditions. This study examines the relationships between the parameter space of IS models and their predictive capabilities regarding solute structure and assembly dynamics, focusing on NaCl solutions and protein-bound silica colloidal nanoparticles. For NaCl solutions, models developed using concentration-dependent dielectric constants generally lack efficacy, and their effectiveness in predicting high-concentration liquid structures varies based on the ES model used. Adjusting the dielectric constant value or the shape of the short-range potentials can improve the accuracy of these models in predicting liquid structures. For protein-bound silica colloidal nanoparticles, we find that interaction site size and model resolution significantly influence the prediction of nanoparticle assembly dynamics, with different resolutions producing aggregates with distinct structures and connection modes. This study elucidates the complex relationship between parameter space in IS models and liquid structure and assembly kinetics, providing crucial insights for developing more accurate and predictive models.

A controlled co-assembly approach to tune temperature responsiveness of biomimetic proteins.

The controlled co-assembly of biomacromolecules through tuneable interactions offers a simple and fascinating opportunity to assemble multiple molecules into a single entity with enhanced complexity and unique properties. Herein, our study presents a dynamic co-assembled system using the multistimuli responsive intrinsically disordered protein Rec1-resilin and the adhesive hydrophilic protein silk sericin (SS). We utilized advanced characterization techniques including circular dichroism (CD) spectroscopy, dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), and small/ultra-small angle neutron scattering (SANS/USANS) to elucidate the detailed co-assembly behavior of the system and its evolution over time and temperature. To achieve sufficient neutron contrast, we successfully biosynthesised deuterium-labeled Rec1-resilin (D-Rec1). Our research demonstrates that this co-assembly allows the formation of a robust entity with dynamic conformational assembly and disassembly, exhibiting both the upper critical solution temperature (UCST) and lower critical solution temperature (LCST) with reversibility. The assembly and disassembly dynamics of the co-assembled entity at UCST are very fast, while the process is kinetically controlled at LCST. This study provides significant new insights into the interplay of a hydrophilic, multi-responsive IDP and a highly hydrophilic protein, shaping the thermoresponsive and stable properties of the co-assembled entity.

Cooperation between Coagulase and von Willebrand factor binding protein in Staphylococcus aureus fibrin pseudocapsule formation

The major human pathogen Staphylococcus aureus forms biofilms comprising of a fibrin network that increases attachment to surfaces and shields bacteria from the immune system. It secretes two coagulases, Coagulase (Coa) and von Willebrand factor binding protein (vWbp), which hijack the host coagulation cascade and trigger the formation of this fibrin clot. However, it is unclear how Coa and vWbp contribute differently to the localisation and dynamics of clot assembly in growing biofilms.Here, we address this question using high-precision time-resolved confocal microscopy of fluorescent fibrin to establish the spatiotemporal dynamics of fibrin clot formation in functional biofilms. We also use fluorescent fusion proteins to visualise the locations of Coa and vWbp in biofilms using both confocal laser scanning and high resolution highly inclined and laminated optical sheet microscopy. We visualise and quantify the spatiotemporal dynamics of fibrin production during initiation of biofilms in plasma amended with fluorescently labelled fibrinogen.We find that human serum stimulates coagulase production, and that Coa and vWbp loosely associate to the bacterial cell surface. Coa localises to cell surfaces to produce a surface-attached fibrin pseudocapsule but can diffuse from cells to produce matrix-associated fibrin. vWbp produces matrix-associated fibrin in the absence of Coa, and furthermore accelerates pseudocapsule production when Coa is present. Finally, we observe that fibrin production varies across the biofilm. A sub-population of non-dividing cells does not produce any pseudocapsule but remains within the protective extended fibrin network, which could be important for the persistence of S. aureus biofilm infections as antibiotics are more effective against actively growing cells.Our findings indicate a more cooperative role between Coa and vWbp in building fibrin networks than previously thought, and a bet-hedging cell strategy where some cells produce biofilm matrix while others do not, but instead assume a dormant phenotype that could be associated with antibiotic tolerance.

Dynamic fibrillar assembly of αB-crystallin induced by perturbation of the conserved NT-IXI motif resolved by cryo-EM

αB-crystallin is an archetypical member of the small heat shock proteins (sHSPs) vital for cellular proteostasis and mitigating protein misfolding diseases. Gaining insights into the principles defining their molecular organization and chaperone function have been hindered by intrinsic dynamic properties and limited high-resolution structural analysis. To disentangle the mechanistic underpinnings of these dynamical properties, we ablate a conserved IXI-motif located within the N-terminal (NT) domain of human αB-crystallin implicated in subunit exchange dynamics and client sequestration. This results in a profound structural transformation, from highly polydispersed caged-like native assemblies into an elongated fibril state amenable to high-resolution cryo-EM analysis. The reversible nature of this variant facilitates interrogation of functional effects due to perturbation of the NT-IXI motif in both the native-like oligomer and fibril states. Together, our investigations unveil several features thought to be key mechanistic attributes to sHSPs and point to a critical significance of the NT-IXI motif in αB-crystallin assembly, polydispersity, and chaperone activity.

A stacked graph neural network with self-exciting process for robotic cognitive strategy reasoning in proactive human-robot collaborative assembly

Proactive human-robot collaborative assembly, a cognitively driven human-robot collaboration, requires research into robot cognitive strategy reasoning to ensure that the robot actively collaborates with the operator in task completion. However, current methods primarily focus on the pairwise relationships of assembly components in discrete snapshots. They could fail to represent the interconnected status of dynamic assembly, leading to inaccurate task allocation, thereby affecting robotic cognitive strategy. To address this problem, we propose a stacked graph neural network (GNN) with self-exciting process to capture the correlation and triggering mechanisms between time-varying tasks. Firstly, a temporal hypergraph with assembly knowledge is constructed to represent the non-pairwise relationships among assembly components in time-varying tasks, aiming to reduce the redundant information brought by pairwise relationships. Then, considering the characteristic of mutual influence between assembly events, a Hawkes process is introduced into the stacked GNN architecture to learn the event correlation representation in the temporal hypergraph. This point process models the self-exciting process of assembly events for simultaneously capturing the individual and collective features of events, thereby revealing the triggering mechanisms of the dynamic events. Finally, the effectiveness of proposed method is demonstrated by comparative experiments and the results of robotic cognitive strategy reasoning on dynamic assembly.

Evaluation of Augmented Reality instructions based on initial and dynamic assembly tolerance allocation schemes in precise manual assembly

Augmented Reality (AR) assembly instructions can be used to guide precise manual assembly, providing detailed real-time information to assist in executing operations. Most AR instructions for precise manual assembly are based on initial assembly tolerance allocation schemes (ATASs), which provide a precision constrained zone and require assembly according to the initial zone. Dynamic ATASs can adjust the subsequent tolerances according to the completed assembly errors, thereby reducing the assembly difficulty. However, ATASs do not affect the AR cues for assembly guidance, and their effects on the usability of AR instructions are subtle and unclear. This study focuses on evaluating the effects of initial and dynamic ATASs on the usability of AR instructions. An AR instruction based on an initial ATAS and an AR instruction based on a dynamic ATAS were developed for a precise manual assembly task. A user study was conducted with 16 participants to compare the two AR instructions. The results showed significant differences in assembly speed, assembly quality, perceived ease-of-use, and perceived workload. We found that the AR instruction based on the dynamic ATAS can expedite assembly with better perceived ease-of-use and lower perceived workload, but may reduce assembly quality. Based on the results, several recommendations were provided to help researchers and developers design better AR instructions for precise manual assembly.

Universal salt-assisted assembly of MXene from suspension on polymer substrates

Two-dimensional carbides and nitrides, known as MXenes, are promising for water-processable coatings due to their excellent electrical, thermal, and optical properties. However, depositing hydrophilic MXene nanosheets onto inert or hydrophobic polymer surfaces requires plasma treatment or chemical modification. This study demonstrates a universal salt-assisted assembly method that produces ultra-thin, uniform MXene coatings with exceptional mechanical stability and washability on various polymers, including high-performance polymers for extreme temperatures. The salt in the Ti3C2Tx colloidal suspension reduces surface charges, enabling electrostatically hydrophobized MXene deposition on polymers. A library of salts was used to optimize assembly kinetics and coating morphology. A 170 nm MXene coating can reduce radiation temperature by ~200 °C on a 300 °C PEEK substrate, while the coating on Kevlar fabric provides comfort in extreme conditions, including outer space and polar regions.

Root hair developmental regulators orchestrate drought triggered microbiome changes and the interaction with beneficial Rhizobiaceae

Drought is one of the most serious abiotic stresses, and emerging evidence suggest plant microbiome affects plant drought tolerance. However, there is a lack of genetic evidence regarding whether and how plants orchestrate the dynamic assembly of the microbiome upon drought. By utilizing mutants with enhanced or decreased root hair densities, we find that root hair regulators also affect drought induced root microbiome changes. Rhizobiaceae is a key biomarker taxa affected by root hair related mutants. We isolated and sequenced 1479 root associated microbes, and confirmed that several Rhizobium strains presented stress-alleviating activities. Metagenome, root transcriptome and root metabolome studies further reveal the multi-omic changes upon drought stress. We knocked out an ornithine cyclodeaminase (ocd) gene in Rhizobium sp. 4F10, which significantly dampens its stress alleviating ability. Our genetic and integrated multi-omics studies confirm the involvement of host genetic effects in reshaping a stress-alleviating root microbiome during drought, and provide mechanistic insights into Rhizobiaceae mediated abiotic stress protection.

Assembly Characteristics and Influencing Factors of the Soil Microbial Community in the Typical Forest of Funiu Mountain.

Assessing the relationship between litter characteristics and soil microbial community traits across different forest types can enhance our understanding of the synergistic interactions among litter, soil, and microorganisms. This study focused on three representative forest types in the Funiu Mountains-Larix gmelinii (LG), Quercus aliena var. acutiserrata (QA), and Quercus aliena var. acutiserrata + Pinus armandii (QAPA). The findings indicated no significant differences in Chao1 among the three forests; however, the Shannon index exhibited an initial increase followed by a decline. NMDS and ANOSIM analyses revealed significant structural differences across these forest types. Network topological metrics (nodes, edges, average degree, and average path distance) for bacterial taxa were higher in LG and QA compared with QAPA. Additionally, LG and QA demonstrated significantly greater average niche breadth than QAPA. The results from the null models (the proportion occupied by dispersal limitation is 62.2%, 82.2%, and 64.4% in LG, QA, and QAPA), modified stochasticity ratio (LG: 0.708, QA: 0.664, and QAPA: 0.801), and neutral community models (LG: R2 = 0.665, QA: R2 = 0.630, and QAPA: R2 = 0.665) suggested that stochastic processes predominantly govern the assembly of soil bacterial communities. Random forest analysis alongside Mantel tests highlighted LTP (litter total phosphorus), STN (soil total nitrogen), MCP (carbon-to-phosphorus ratio of microbial biomass), and SCN (soil carbon-to-nitrogen ratio) as critical factors affecting bacterial niche width; conversely LCN (litter carbon-to-nitrogen ratio), RCP (ratio of dissolved carbon to phosphorus), MCP, and SCN emerged as key determinants influencing community assembly processes. Furthermore, the PLS-SEM results underscored how both litter characteristics along with soil properties-and their associated alpha diversity-impact variations in niche breadth while also shaping community assembly dynamics overall. This research provides vital insights into understanding synergistic relationships between litter quality, soil characteristics, and microbial community across diverse forest ecosystems.

Channel width modulates the permeability of DNA origami-based nuclear pore mimics.

Nucleoporins (nups) in the nuclear pore complex (NPC) form a selective barrier that suppresses the diffusion of most macromolecules while enabling rapid transport of nuclear transport receptor (NTR)-bound cargos. Recent studies have shown that the NPC may dilate and constrict, but how altering the NPC diameter affects its selective barrier properties remains unclear. Here, we build DNA nanopores with programmable diameters and nup arrangements to model the constricted and dilated NPCs. We find that Nup62 proteins form a dynamic cross-channel barrier impermeable to hepatitis B virus (HBV) capsids when grafted inside 60-nm-wide nanopores but not in 79-nm pores, where Nup62 cluster locally. Furthermore, importin-β1 substantially changes the dynamics of Nup62 assemblies and facilitates the passage of HBV capsids through the 60-nm NPC mimics containing Nup62 and Nup153. Our study shows that transport channel width is critical to the permeability of nup barriers and underscores NTRs' role in dynamically remodeling nup assemblies and mediating the nuclear entry of viruses.

Assembly of Silicate-Phenolic Network Coatings with Tunable Properties for Controlled Release of Small Molecules.

Engineered coatings are pivotal for tailoring the surface properties and release profiles of materials for applications across diverse areas. However, developing robust coatings that can both encapsulate and controllably release cargo is challenging. Herein, a dynamic covalent coordination assembly strategy is used to engineer robust silicate-based coatings, termed silicate-phenolic networks (SPNs), using sodium metasilicate and phenolic ligands (tannic acid, gallic acid, pyrogallol). The coatings are pH-responsive (owing to the dynamic covalent bonding), and their hydrophobicity can be tuned upon their post-functionalization with hydrophobic gallates (propyl, octyl, lauryl gallates). The potential of the SPN coatings for the controlled release of small molecules, such as urea (a widely used fertilizer), is demonstrated-controlled release of urea in soil is achieved in response to different pHs (up to 7 days) and different hydrophobicity (up to 14 days). Furthermore, leveraging the presence of silicon (within the coating) and post-functionalization of the SPN coatings with metal ions (Fe3+, Cu2+, Zn2+) generates a multipurpose delivery system for the sustained release of micronutrient fertilizers, and silicon and metal ions, over 28 and 14 days, respectively. These SPN coatings have potential applications beyond agriculture, including nutrient delivery, separations, food packaging, and medical device fabrication.

Capturing Dynamic Assembly of Nanoscale Proteins During Network Formation.

The structural evolution of hierarchical structures of nanoscale biomolecules is crucial for the construction of functional networks in vivo and in vitro. Despite the ubiquity of these networks, the physical mechanisms behind their formation and self-assembly remains poorly understood. Here, this study uses photochemically cross-linked folded protein hydrogels as a model biopolymer network system, with a combined time-resolved rheology and small-angle x-ray scattering (SAXS) approach to probe both the load-bearing structures and network architectures respectively thereby providing a cross-length scale understanding of the network formation. Combining SAXS, rheology, and kinetic modeling, a dual formation mechanism consisting of a primary formation phase is proposed, where monomeric folded proteins create the preliminary protein network scaffold; and a subsequent secondary formation phase, where both additional intra-network cross-links form and larger oligomers diffuse to join the preliminary network, leading to a denser more mechanically robust structure. Identifying this as the origin of the structural and mechanical properties of protein networks creates future opportunities to understand hierarchical biomechanics in vivo and develop functional, designed-for-purpose, biomaterials.

Tandem-Controlled Dynamic DNA Assembly Enables Temporally-Selective Orthogonal Regulation of cGAS-STING Stimulation.

Despite advances in the controlled reconfiguration of DNA structures for biological applications, the dearth of strategies that allow for orthogonal regulation of immune pathways remains a challenge. Here, we report for the first time an endogenous and exogenous tandem-regulated DNA assembly strategy that enables orthogonally controlled stimulation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway. A DNA motif containing two palindromic sequences is engineered with an abasic site (AP)-connected blocking sequence to inhibit its self-assembly function, while apurinic/apyrimidinic endonuclease 1 (APE1)-triggered enzymatic cleavage of the AP site enables the reconfiguration and self-assembly of DNA motif into long double-stranded structures, thus realizing allosteric activation of the catalytic activity of cGAS to produce 2'3'-cyclic-GMP-AMP for STING stimulation. Importantly, we demonstrate that APE1-regulated DNA assembly allows for cell-selective activation of cGAS-STING signaling. Furthermore, by re-engineering the DNA motif with a photocleavable group, enzyme-triggered DNA assembly allows the cGAS-STING stimulation to operate (switched "ON''), whereas light-mediated fragmentation of the double-stranded DNA enables termination of such stimulation (switched "OFF"), thereby achieving orthogonal control over immune regulation. This work highlights an endogenous and exogenous tandem regulated strategy to modulate the cGAS-STING pathway in an orthogonally controlled manner.

Tandem‐Controlled Dynamic DNA Assembly Enables Temporally‐Selective Orthogonal Regulation of cGAS–STING Stimulation

Despite advances in the controlled reconfiguration of DNA structures for biological applications, the dearth of strategies that allow for orthogonal regulation of immune pathways remains a challenge. Here, we report for the first time an endogenous and exogenous tandem‐regulated DNA assembly strategy that enables orthogonally controlled stimulation of the cyclic GMP‐AMP synthase (cGAS)–stimulator of interferon genes (STING) pathway. A DNA motif containing two palindromic sequences is engineered with an abasic site (AP)‐connected blocking sequence to inhibit its self‐assembly function, while apurinic/apyrimidinic endonuclease 1 (APE1)‐triggered enzymatic cleavage of the AP site enables the reconfiguration and self‐assembly of DNA motif into long double‐stranded structures, thus realizing allosteric activation of the catalytic activity of cGAS to produce 2’3’‐cyclic‐GMP‐AMP for STING stimulation. Importantly, we demonstrate that APE1‐regulated DNA assembly allows for cell‐selective activation of cGAS–STING signaling. Furthermore, by re‐engineering the DNA motif with a photocleavable group, enzyme‐triggered DNA assembly allows the cGAS–STING stimulation to operate (switched "ON''), whereas light‐mediated fragmentation of the double‐stranded DNA enables termination of such stimulation (switched "OFF"), thereby achieving orthogonal control over immune regulation. This work highlights an endogenous and exogenous tandem regulated strategy to modulate the cGAS–STING pathway in an orthogonally controlled manner.

Hydrogen Sulfide‐Triggered Artificial DNAzyme Switches for Precise Manipulation of Cellular Functions

AbstractThe development of synthetic molecular tools responsive to biological cues is crucial for advancing targeted cellular regulation. A significant challenge is the regulation of cellular processes in response to gaseous signaling molecules such as hydrogen sulfide (H2S). To address this, we present the design of Gas signaling molecule‐Responsive Artificial DNAzyme‐based Switches (GRAS) to manipulate cellular functions via H2S‐sensitive synthetic DNAzymes. By incorporating stimuli‐responsive moieties to the phosphorothioate backbone, DNAzymes are strategically designed with H2S‐responsive azide groups at cofactor binding locations within the catalytic core region. These modifications enable their activation through H2S‐reducing decaging, thereby initiating substrate cleavage activity. Our approach allows for the flexible customization of various DNAzymes to regulate distinct cellular processes in diverse scenarios. Intracellularly, the enzymatic activity of GRAS promotes H2S‐induced cleavage of specific mRNA sequences, enabling targeted gene silencing and inducing apoptosis in cancer cells. Moreover, integrating GRAS with dynamic DNA assembly allows for grafting these functional switches onto cell surface receptors, facilitating H2S‐triggered receptor dimerization. This extracellular activation transmits signals intracellularly to regulate cellular behaviors such as migration and proliferation. Collectively, synthetic switches are capable of rewiring cellular functions in response to gaseous cues, offering a promising avenue for advanced targeted cellular engineering.

The transmembrane domain of the desmosomal cadherin desmoglein-1 governs lipid raft association to promote desmosome adhesive strength.

Cholesterol- and sphingolipid-enriched domains called lipid rafts are hypothesized to selectively coordinate protein complex assembly within the plasma membrane to regulate cellular functions. Desmosomes are mechanically resilient adhesive junctions that associate with lipid raft membrane domains, yet the mechanisms directing raft association of the desmosomal proteins, particularly the transmembrane desmosomal cadherins, are poorly understood. We identified the desmoglein-1 (DSG1) transmembrane domain (TMD) as a key determinant of desmoglein lipid raft association and designed a panel of DSG1TMD variants to assess the contribution of TMD physicochemical properties (length, bulkiness, and palmitoylation) to DSG1 lipid raft association. Sucrose gradient fractionations revealed that TMD length and bulkiness, but not palmitoylation, govern DSG1 lipid raft association. Further, DSG1 raft association determines plakoglobin recruitment to raft domains. Super-resolution imaging and functional assays uncovered a strong relationship between the efficiency of DSG1TMD lipid raft association and the formation of morphologically and functionally robust desmosomes. Lipid raft association regulated both desmosome assembly dynamics and DSG1 cell surface stability, indicating that DSG1 lipid raft association is required for both desmosome formation and maintenance. These studies identify the biophysical properties of desmoglein transmembrane domains as key determinants of lipid raft association and desmosome adhesive function.

The recurrent temporal restricted Boltzmann machine captures neural assembly dynamics in whole-brain activity

Animal behaviour alternates between stochastic exploration and goal-directed actions, which are generated by the underlying neural dynamics. Previously, we demonstrated that the compositional Restricted Boltzmann Machine (cRBM) can decompose whole-brain activity of larval zebrafish data at the neural level into a small number (∼100-200) of assemblies that can account for the stochasticity of the neural activity (van der Plas et al., eLife, 2023). Here, we advance this representation by extending to a combined stochastic-dynamical representation to account for both aspects using the recurrent temporal RBM (RTRBM) and transfer-learning based on the cRBM estimate. We demonstrate that the functional advantage of the RTRBM is captured in the temporal weights on the hidden units, representing neural assemblies, for both simulated and experimental data. Our results show that the temporal expansion outperforms the stochastic-only cRBM in terms of generalization error and achieves a more accurate representation of the moments in time. Lastly, we demonstrate that we can identify the original time-scale of assembly dynamics by estimating multiple RTRBMs at different temporal resolutions. Together, we propose that RTRBMs are a valuable tool for capturing the combined stochastic and time-predictive dynamics of large-scale data sets.

The recurrent temporal restricted Boltzmann machine captures neural assembly dynamics in whole-brain activity.

Animal behaviour alternates between stochastic exploration and goal-directed actions, which are generated by the underlying neural dynamics. Previously, we demonstrated that the compositional Restricted Boltzmann Machine (cRBM) can decompose whole-brain activity of larval zebrafish data at the neural level into a small number (∼100-200) of assemblies that can account for the stochasticity of the neural activity (van der Plas et al., eLife, 2023). Here, we advance this representation by extending to a combined stochastic-dynamical representation to account for both aspects using the recurrent temporal RBM (RTRBM) and transfer-learning based on the cRBM estimate. We demonstrate that the functional advantage of the RTRBM is captured in the temporal weights on the hidden units, representing neural assemblies, for both simulated and experimental data. Our results show that the temporal expansion outperforms the stochastic-only cRBM in terms of generalization error and achieves a more accurate representation of the moments in time. Lastly, we demonstrate that we can identify the original time-scale of assembly dynamics by estimating multiple RTRBMs at different temporal resolutions. Together, we propose that RTRBMs are a valuable tool for capturing the combined stochastic and time-predictive dynamics of large-scale data sets.

Dynamics of fluid–structure interaction in paintbrush

Fluid–structure interactions are fundamental in both natural phenomena and industrial applications, particularly in dip-coating processes where withdrawal velocity and drainage dynamics are crucial. Understanding these interactions is essential for optimizing various processes, from enhancing the efficiency of industrial coatings to developing advanced materials with tailored properties. Here, we examine the capillary-induced dynamics of fiber bundles using paintbrush-like structures. We submerge fiber bundles in water and withdraw them at various velocities, observing that water trapped within the bundles leads to capillary-driven fiber assembly. Our experiments show that the bundle diameter after emergence increases with higher withdrawal speed due to viscous effects. We develop a theoretical model that accurately predicts the dynamics of fiber assembly driven by capillary and viscous flows. The mathematical model agrees well with our experimental results, demonstrating the complex interplay between capillary forces and fiber packing. We anticipate that our findings improve the understanding of fluid-structure interactions in fibrous media, providing physical insights that can be applied to more complex systems such as nanopattern collapse and nano/micro-manufacturing.

Determinants in the HTLV-1 Capsid Major Homology Region that are Critical for Virus Particle Assembly

The Gag protein of retroviruses is the primary driver of virus particle assembly. Particle morphologies among retroviral genera are distinct, with intriguing differences observed relative to HIV-1, particularly that of human T-cell leukemia virus type 1 (HTLV-1). In contrast to HIV-1 and other retroviruses where the capsid (CA) carboxy-terminal domain (CTD) possesses the key amino acid determinants involved in driving Gag-Gag interactions, we have previously demonstrated that the amino-terminal domain (NTD) encodes the key residues crucial for Gag multimerization and immature particle production. Here in this study, we sought to thoroughly interrogate the conserved HTLV-1 major homology region (MHR) of the CACTD to determine whether this region harbors residues important for particle assembly. In particular, site-directed mutagenesis of the HTLV-1 MHR was conducted, and mutants were analyzed for their ability to impact Gag subcellular distribution, particle production and morphology, as well as the CA-CA assembly kinetics. Several key residues (i.e., Q138, E142, Y144, F147 and R150), were found to significantly impact Gag multimerization and particle assembly. Taken together, these observations imply that while the HTLV-1 CANTD acts as the major region involved in CA-CA interactions, residues in the MHR can impact Gag multimerization, particle assembly and morphology, and likely play an important role in the conformation the CACTD that is required for CA-CA interactions.