An Elastin-like Polypeptide-fusion peptide targeting capsid-tegument interface as an antiviral against cytomegalovirus infection.

The tegument protein pp150 of Human Cytomegalovirus (HCMV) is known to be essential for the final stages of virus maturation and mediates its functions by interacting with capsid proteins. Our laboratory has previously identified the critical regions in pp150 important for pp150-capsid interactions and designed peptides similar in sequence to these regions, with a goal to competitively inhibit capsid maturation. Treatment with a specific peptide (PepCR2 or P10) targeted to pp150 conserved region 2 led to a significant reduction in murine CMV (MCMV) growth in cell culture, paving the way for in vivo testing in a mouse model of CMV infection. However, the general pharmacokinetic parameters of peptides, including rapid degradation and limited tissue and cell membrane permeability, pose a challenge to their successful use in vivo. Therefore, we designed a biopolymer-stabilized elastin-like polypeptide (ELP) fusion construct (ELP-P10) to enhance the bioavailability of P10. Antiviral efficacy and cytotoxic effects of ELP-P10 were studied in cell culture, and pharmacokinetics, biodistribution, and antiviral efficacy were studied in a mouse model of CMV infection. ELP-P10 maintained significant antiviral activity in cell culture, and this conjugation significantly enhanced P10 bioavailability in mouse tissues. The fluorescently labeled ELP-P10 accumulated to higher levels in mouse liver and kidneys as compared to the unconjugated P10. Moreover, viral titers from vital organs of MCMV-infected mice indicated a significant reduction of virus load upon ELP-P10 treatment. Therefore, ELP-P10 has the potential to be developed into an effective antiviral against CMV infection.

Natural history of eukaryotic DNA viruses with double jelly-roll major capsid proteins.

The phylum Preplasmiviricota (kingdom Bamfordvirae, realm Varidnaviria) is a broad assemblage of diverse viruses with comparatively short double-stranded DNA genomes (<50 kbp) that produce icosahedral capsids built from double jelly-roll major capsid proteins. Preplasmiviricots infect hosts from all cellular domains, testifying to their ancient origin, and, in particular, are associated with six of the seven supergroups of eukaryotes. Preplasmiviricots comprise four major groups of viruses, namely, polintons, polinton-like viruses (PLVs), virophages, and adenovirids. We used protein structure modeling and analysis to show that protein-primed DNA polymerases (pPolBs) of polintons, virophages, and cytoplasmic linear plasmids encompass an N-terminal domain homologous to the terminal proteins (TPs) of prokaryotic PRD1-like tectivirids and eukaryotic adenovirids that are involved in protein-primed replication initiation, followed by a viral ovarian tumor-like cysteine deubiquitinylase (vOTU) domain. The vOTU domain is likely responsible for the cleavage of the TP from the large pPolB polypeptide and is inactivated in adenovirids, in which TP is a separate protein. Many PLVs and transpovirons encode a distinct derivative of polinton-like pPolB that retains the TP, vOTU, and pPolB polymerization palm domains but lacks the exonuclease domain and instead contains a superfamily 1 helicase domain. Analysis of the presence/absence and inactivation of the vOTU domains and replacement of pPolB with other DNA polymerases in eukaryotic preplasmiviricots enabled us to outline a complete scenario for their origin and evolution.

Intrinsic disorder in flaviviral capsid proteins and its role in pathogenesis.

A high level of disorder in many viral proteins is a direct consequence of their small genomes, which makes interaction with multiple binding partners a necessity for infection and pathogenicity. A segment of the flaviviral capsid protein (C), also known as the molecular recognition feature (MoRF), undergoes a disorder-toorder transition upon binding to several protein partners. To understand their role in pathogenesis, MoRFs were identified and their occurrence across different flaviviral capsids were studied. Despite lack of sequence similarities, docking studies of Cs with the host proteins indicate conserved interactions involving MoRFs across members of phylogenetic subclades. Additionally, it was observed from the protein-protein networks that some MoRFs preferentially bind proteins that are involved in specialized functions such as ribosome biogenesis. The findings point to the importance of MoRFs in the flaviviral life cycle, with important consequences for disease progression and suppression of the host immune system. Potentially, they might have impacted the way flaviviruses evolved to infect varied hosts using multiple vectors.

TMV-CP based rational design and discovery of α-Amide phosphate derivatives as anti plant viral agents.

The tobacco mosaic virus coat protein (TMV-CP) is indispensable for the virus's replication, movement and transmission, as well as for the host plant's immune system to recognize it. It constitutes the outermost layer of the virus particle, and serves as an essential component of the virus structure. TMV-CP is essential for initiating and extending viral assembly, playing a crucial role in the self-assembly process of Tobacco Mosaic Virus (TMV). This research employed TMV-CP as a primary target for virtual screening, from which a library of 43,417 compounds was sourced and SH-05 was chosen as the lead compound. Consequently, a series of α-amide phosphate derivatives were designed and synthesized, exhibiting remarkable anti-TMV efficacy. The synthesized compounds were found to be beneficial in treating TMV, with compound 3g displaying a slightly better curative effect than Ningnanmycin (NNM) (EC50 = 304.54 µg/mL) at an EC50 of 291.9 µg/mL. Additionally, 3g exhibited comparable inactivation activity (EC50 = 63.2 µg/mL) to NNM (EC50 = 67.5 µg/mL) and similar protective activity (EC50 = 228.9 µg/mL) to NNM (EC50 = 219.7 µg/mL). Microscale thermal analysis revealed that the binding of 3g (Kd = 4.5 ± 1.9 µM) to TMV-CP showed the same level with NNM (Kd = 5.5 ± 2.6 µM). Results from transmission electron microscopy indicated that 3g could disrupt the structure of TMV virus particles. The toxicity prediction indicated that 3g was low toxicity. Molecular docking showed that 3g interacted with TMV-CP through hydrogen bond, attractive charge interaction and π-Cation interaction. This research provided a novel α-amide phosphate structure target TMV-CP, which may help the discovery of new anti-TMV agents in the future.

Point mutation in a virus-like capsid drives symmetry reduction to form tetrahedral cages.

Protein capsids are a widespread form of compartmentalization in nature. Icosahedral symmetry is ubiquitous in capsids derived from spherical viruses, as this geometry maximizes the internal volume that can be enclosed within. Despite the strong preference for icosahedral symmetry, we show that simple point mutations in a virus-like capsid can drive the assembly of unique symmetry-reduced structures. Starting with the encapsulin from Myxococcus xanthus, a 180-mer bacterial capsid that adopts the well-studied viral HK97 fold, we use mass photometry and native charge detection mass spectrometry to identify a triple histidine point mutant that forms smaller dimorphic assemblies. Using cryoelectron microscopy, we determine the structures of a precedented 60-mer icosahedral assembly and an unexpected 36-mer tetrahedron that features significant geometric rearrangements around a new interaction surface between capsid protomers. We subsequently find that the tetrahedral assembly can be generated by triple-point mutation to various amino acids and that even a single histidine point mutation is sufficient to form tetrahedra. These findings represent a unique example of tetrahedral geometry when surveying all characterized encapsulins, HK97-like capsids, or indeed any virus-derived capsids reported in the Protein Data Bank, revealing the surprising plasticity of capsid self-assembly that can be accessed through minimal changes in the protein sequence.

Transformation of a Viral Capsid from Nanocages to Nanotubes and Then to Hydrogels: Redirected Self-Assembly and Effects on Immunogenicity.

The ability to manipulate the self-assembly of proteins is essential to understanding the mechanisms of life and beneficial to fabricating advanced nanomaterials. Here, we report the transformation of the MS2 phage capsid from nanocages to nanotubes and then to nanotube hydrogels through simple point mutations guided by interfacial interaction redesign. We demonstrate that site 70, which lies in the flexible FG loop of the capsid protein (CP), is a "magic" site that can largely dictate the final morphology of assemblies. By varying the amino acid at site 70, with the aid of a cysteine-to-alanine mutation at site 46, we achieved the assembly of double-helical or single-helical nanotubes in addition to nanocages. Furthermore, an additional cysteine substitution on the surface of nanotubes mediated their cross-linking to form hydrogels with reducing agent responsiveness. The hierarchical self-assembly system allowed for the investigation of morphology-related immunogenicity of MS2 CPs, which revealed dramatic differences among nanocages, nanotubes, and nanotube hydrogels in terms of immune response types, antibody levels and T cell functions. This study provides insights into the assembly manipulation of protein nanomaterials and the customized design of nanovaccines and drug delivery systems.

Ligand-based drug design against Herpes Simplex Virus-1 capsid protein by modification of limonene through in silico approaches.

The pharmacological effects of limonene, especially their derivatives, are currently at the forefront of research for drug development and discovery as well and structure-based drug design using huge chemical libraries are already widespread in the early stages of therapeutic and drug development. Here, various limonene derivatives are studied computationally for their potential utilization against the capsid protein of Herpes Simplex Virus-1. Firstly, limonene derivatives were designed by structural modification followed by conducting a molecular docking experiment against the capsid protein of Herpes Simplex Virus-1. In this research, the obtained molecular docking score exhibited better efficiency against the capsid protein of Herpes Simplex Virus-1 and hence we conducted further in silico investigation including molecular dynamic simulation, quantum calculation, and ADMET analysis. Molecular docking experiment has documented that Ligands 02 and 03 had much better binding affinities (- 7.4 kcal/mol and - 7.1 kcal/mol) to capsid protein of Herpes Simplex Virus-1 than Standard Acyclovir (- 6.5 kcal/mol). Upon further investigation, the binding affinities of primary limonene were observed to be slightly poor. But including the various functional groups also increases the affinities and capacity to prevent viral infection of the capsid protein of Herpes Simplex Virus-1. Then, the molecular dynamic simulation confirmed that the mentioned ligands might be stable during the formation of drug-protein complexes. Finally, the analysis of ADMET was essential in establishing them as safe and human-useable prospective chemicals. According to the present findings, limonene derivatives might be a promising candidate against the capsid protein of Herpes Simplex Virus-1 which ultimately inhibits Herpes Simplex Virus-induced encephalitis that causes interventions in brain inflammation. Our findings suggested further experimental screening to determine their practical value and utility.

Spectroscopic analysis of the bacterially expressed head domain of rotavirus VP6.

The rotavirus capsid protein VP6 forms the middle of three protein layers and is responsible for many critical steps in the viral life cycle. VP6 as a structural protein can be used in various applications including as a subunit vaccine component. The head domain of VP6 (VP6H) contains key sequences that allow the protein to trimerize and that represent epitopes that are recognized by human antibodies in the viral particle. The domain is rich in ß-sheet secondary structures. Here, VP6H was solubilised from bacterial inclusion bodies and purified using a single affinity chromatography step. Spectral (far-UV circular dichroism and intrinsic tryptophan fluorescence) analysis revealed that the purified domain had native-like secondary and tertiary structures. The domain could maintain structure up to 44°C during thermal denaturation following which structural changes result in an intermediate forming and finally irreversible aggregation and denaturation. The chemical denaturation with urea and guanidinium hydrochloride produces intermediates that represent a loss in the cooperativity. The VP6H domain is stable and can fold to produce its native structure in the absence of the VP6 base domain but cannot be defined as an independent folding unit.

Self-Powered Immunoassay of Norovirus in Human Stools by π-Electron-Rich Homojunction for Enhanced Charge Transfer.

Norovirus (NoV) stands as a significant causative agent of nonbacterial acute gastroenteritis on a global scale, presenting a substantial threat to public health. Hence, the development of simple and rapid analytical techniques for NoV detection holds great importance in preventing and controlling the outbreak of the epidemic. In this work, a self-powered photoelectrochemical (PEC) immunosensor of NoV capsid protein (VP1) was proposed by the π-electron-rich carbon nitride homojunction (ER-CNH) as the photoanode. C4N2 ring derived from π-rich locust bean gum was introduced into the tri-s-triazine structure, creating a large π-delocalized conjugated carbon nitride homojunction. This strategy enhances the C/N atomic ratio, which widens light utilization, narrows the bandgap, and optimizes the electronic band structure of carbon nitride. By introduction of a π-rich conjugated structure, p-type domains were induced within n-type domains to build the internal electric field at the interface, thus forming a p-n homojunction to boost carrier separation and transfer. The ER-CNH photoanode exhibited excellent photoelectric performance and water oxidation capacity. Since VP1 inhibits the water oxidation of the ER-CNH photoanode, the open-circuit potential of the as-prepared PEC immunosensor system was reduced for detecting NoV VP1. The self-powered PEC immunosensor achieved a remarkably low detection limit (∼5 fg mL-1) and displayed high stability and applicability for actual stool samples. This research serves as a foundation concept for constructing immunosensors to detect other viruses and promotes the application of self-powered systems for life safety.

Stimulus-responsive assembly of nonviral nucleocapsids.

Controlled assembly of a protein shell around a viral genome is a key step in the life cycle of many viruses. Here we report a strategy for regulating the co-assembly of nonviral proteins and nucleic acids into highly ordered nucleocapsids in vitro. By fusing maltose binding protein to the subunits of NC-4, an engineered protein cage that encapsulates its own encoding mRNA, we successfully blocked spontaneous capsid assembly, allowing isolation of the individual monomers in soluble form. To initiate RNA-templated nucleocapsid formation, the steric block can be simply removed by selective proteolysis. Analyses by transmission and cryo-electron microscopy confirmed that the resulting assemblies are structurally identical to their RNA-containing counterparts produced in vivo. Enzymatically triggered cage formation broadens the range of RNA molecules that can be encapsulated by NC-4, provides unique opportunities to study the co-assembly of capsid and cargo, and could be useful for studying other nonviral and viral assemblies.

Collision induced unfolding and molecular dynamics simulations of norovirus capsid dimers reveal strain-specific stability profiles.

Collision induced unfolding (CIU) is a method used with ion mobility mass spectrometry to examine protein structures and their stability. Such experiments yield information about higher order protein structures, yet are unable to provide details about the underlying processes. That information can however be provided using molecular dynamics simulations. Here, we investigate the gas-phase unfolding of norovirus capsid dimers from the Norwalk and Kawasaki strains by employing molecular dynamics simulations over a range of temperatures, representing different levels of activation, together with CIU experiments. The dimers have highly similar structures, but their CIU reveals different stability that can be explained by the different dynamics that arises in response to the activation seen in the simulations, including a part of the sequence with previously observed strain-specific dynamics in solution. Our findings show how similar protein variants can be examined using mass spectrometric techniques in conjunction with atomistic molecular dynamics simulations to reveal differences in stability as well as differences in how and where unfolding takes place upon activation.

DNA Released by Adeno-Associated Virus Strongly Alters Capsid Aggregation Kinetics in a Physiological Solution.

While adeno-associated virus is a leading vector for gene therapy, significant gaps remain in understanding AAV degradation and stability. In this work, we study the degradation of an engineered AAV serotype at physiological pH and ionic strength. Viral particles of varying fractions of encapsulated DNA were incubated between 30 and 60 °C, with changes in molecular weight measured by changes in total light scattering intensity at 90° over time. Mostly full vectors demonstrated a rapid decrease in molecular weight corresponding to the release of capsid DNA, followed by slow aggregation. In contrast, empty vectors demonstrated immediate, rapid colloid-type aggregation. Mixtures of full and empty capsids showed a pronounced decrease in initial aggregation that cannot be explained by a linear superposition of empty and full degradation scattering signatures, indicating interactions between capsids and ejected DNA that influenced aggregation mechanisms. This demonstrates key interactions between AAV capsids and their cargo that influence capsid degradation, aggregation, and DNA release mechanisms in a physiological solution.

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 2.8 Å, 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.

Structural morphing in the viral portal vertex of bacteriophage lambda.

The portal protein of tailed bacteriophage plays essential roles in various aspects of capsid assembly, motor assembly, genome packaging, connector formation, and infection processes. After DNA packaging is complete, additional proteins are assembled onto the portal to form the connector complex, which is crucial as it bridges the mature head and tail. In this study, we report high-resolution cryo-electron microscopy (cryo-EM) structures of the portal vertex from bacteriophage lambda in both its prohead and mature virion states. Comparison of these structures shows that during head maturation, in addition to capsid expansion, the portal protein undergoes conformational changes to establish interactions with the connector proteins. Additionally, the independently assembled tail undergoes morphological alterations at its proximal end, facilitating its connection to the head-tail joining protein and resulting in the formation of a stable portal-connector-tail complex. The B-DNA molecule spirally glides through the tube, interacting with the nozzle blade region of the middle-ring connector protein. These insights elucidate a mechanism for portal maturation and DNA translocation within the phage lambda system. IMPORTANCE: The tailed bacteriophages possess a distinct portal vertex that consists of a ring of 12 portal proteins associated with a 5-fold capsid shell. This portal protein is crucial in multiple stages of virus assembly and infection. Our research focused on examining the structures of the portal vertex in both its preliminary prohead state and the fully mature virion state of bacteriophage lambda. By analyzing these structures, we were able to understand how the portal protein undergoes conformational changes during maturation, the mechanism by which it prevents DNA from escaping, and the process of DNA spirally gliding.

Structural analyses of the GI.4 norovirus by cryo-electron microscopy and X-ray crystallography revealing binding sites for human monoclonal antibodies.

Noroviruses are major causative agents of acute nonbacterial gastroenteritis in humans. There are neither antiviral therapeutic agents nor vaccines for noroviruses at this time. To evaluate the potential usefulness of two previously isolated human monoclonal antibody fragments, CV-1A1 and CV-2F5, we first conducted a single-particle analysis to determine the cryo-electron microscopy structure of virus-like particles (VLPs) from the genogroup I genotype 4 (GI.4) Chiba strain uniformly coated with CV-1A1 fragments. The results revealed that the GI.4-specific CV-1A1 antibody bound to the P2 subdomain, in which amino acids are less conserved and variable. Interestingly, a part of the CV-1A1 intrudes into the histo-blood group antigen-binding site, suggesting that this antibody might exert neutralizing activity. Next, we determined the crystal structure of the protruding (P) domain of the capsid protein in the complex form with the CV-2F5 antibody fragment. Consistent with the cross-reactivity, the CV-2F5 bound to the P1 subdomain, which is rich in amino acids conserved among the GI strains, and moreover induced a disruption of Chiba VLPs. These results suggest that the broadly reactive CV-2F5 antibody can be used as both a universal detection reagent and an antiviral drug for GI noroviruses. IMPORTANCE: We conducted the structural analyses of the VP1 protein from the GI.4 Chiba norovirus to identify the binding sites of the previously isolated human monoclonal antibodies CV-1A1 and CV-2F5. The cryo-electron microscopy of the Chiba virus-like particles (VLPs) complexed with the Fv-clasp forms of GI.4-specific CV-1A1 revealed that this antibody binds to the highly variable P2 subdomain, suggesting that this antibody may have neutralizing ability against the GI.4 strains. X-ray crystallography revealed that the CV-2F5 antibody bound to the P1 subdomain, which is rich in conserved amino acids. This result is consistent with the ability of the CV-2F5 antibody to react with a wide variety of GI norovirus strains. It is also found that the CV-2F5 antibody caused a disruption of VLPs. Our findings, together with previous reports on the structures of VP1 proteins and VLPs, are expected to open a path for the structure-based development of antivirals and vaccines against norovirus disease.

Multi-Gene Recombinant Baculovirus Expression Systems: From Inception to Contemporary Applications.

Many protein expression systems are primarily utilised to produce a single, specific recombinant protein. In contrast, most biological processes such as virus assembly rely upon a complex of several interacting proteins rather than the activity of a sole protein. The high complexity of the baculovirus genome, coupled with a multiphase replication cycle incorporating distinct transcriptional steps, made it the ideal system to manipulate for high-level expression of a single, or co-expression of multiple, foreign proteins within a single cell. We have developed and utilised a series of recombinant baculovirus systems to unravel the sequential assembly process of a complex non-enveloped model virus, bluetongue virus (BTV). The high protein yields expressed by the baculovirus system not only facilitated structure-function analysis of each viral protein but were also advantageous to crystallography studies and supported the first atomic-level resolution of a recombinant viral protein, the major BTV capsid protein. Further, the formation of recombinant double-shelled virus-like particles (VLPs) provided insights into the structure-function relationships among the four major structural proteins of the BTV whilst also representing a potential candidate for a viral vaccine. The baculovirus multi-gene expression system facilitated the study of structurally complex viruses (both non-enveloped and enveloped viruses) and heralded a new generation of viral vaccines.

Cell Culture Adaptive Amino Acid Substitutions in FMDV Structural Proteins: A Key Mechanism for Altered Receptor Tropism.

The foot-and-mouth disease virus is a highly contagious and economically devastating virus of cloven-hooved animals, including cattle, buffalo, sheep, and goats, causing reduced animal productivity and posing international trade restrictions. For decades, chemically inactivated vaccines have been serving as the most effective strategy for the management of foot-and-mouth disease. Inactivated vaccines are commercially produced in cell culture systems, which require successful propagation and adaptation of field isolates, demanding a high cost and laborious time. Cell culture adaptation is chiefly indebted to amino acid substitutions in surface-exposed capsid proteins, altering the necessity of RGD-dependent receptors to heparan sulfate macromolecules for virus binding. Several amino acid substations in VP1, VP2, and VP3 capsid proteins of FMDV, both at structural and functional levels, have been characterized previously. This literature review combines frequently reported amino acid substitutions in virus capsid proteins, their critical roles in virus adaptation, and functional characterization of the substitutions. Furthermore, this data can facilitate molecular virologists to develop new vaccine strains against the foot-and-mouth disease virus, revolutionizing vaccinology via reverse genetic engineering and synthetic biology.

Higher-Order Structure of Adeno-Associated Virus Serotype 8 by Hydrogen/Deuterium Exchange Mass Spectrometry.

The higher-order structure (HOS) is a critical quality attribute of recombinant adeno-associated viruses (rAAVs). Evaluating the HOS of the entire rAAV capsid is challenging because of the flexibility and/or less folded nature of the VP1 unique (VP1u) and VP1/VP2 common regions, which are structural features essential for these regions to exert their functions following viral infection. In this study, hydrogen/deuterium exchange mass spectrometry (HDX-MS) was used for the structural analysis of full and empty rAAV8 capsids. We obtained 486 peptides representing 85% sequence coverage. Surprisingly, the VP1u region showed rapid deuterium uptake even though this region contains the phospholipase A2 domain composed primarily of α-helices. The comparison of deuterium uptake between full and empty capsids showed significant protection from hydrogen/deuterium exchange in the full capsid at the channel structure of the 5-fold symmetry axis. This corresponds to cryo-electron microscopy studies in which the extended densities were observed only in the full capsid. In addition, deuterium uptake was reduced in the VP1u region of the full capsid, suggesting the folding and/or interaction of this region with the encapsidated genome. This study demonstrated HDX-MS as a powerful method for probing the structure of the entire rAAV capsid.

Virtual indentation of the empty capsid of the minute virus of mice using a minimal coarse-grained model.

A minimal coarse-grained model for T=1 viral capsids assembled from 20 protein rigid trimers has been designed by extending a previously proposed form of the interaction energy written as a sum of anisotropic pairwise interactions between the trimeric capsomers. The extension of the model has been performed to properly account for the coupling between two internal coordinates: the one that measures the intercapsomer distance and the other that gives the intercapsomer dihedral angle. The model has been able to fit with less than a 10% error the atomic force microscopy (AFM) indentation experimental data for the empty capsid of the minute virus of mice (MVM), providing in this way an admissible picture of the main mechanisms behind the capsid deformations. In this scenario, the bending of the intercapsomer dihedral angle is the angular internal coordinate that can support larger deformations away from its equilibrium values, determining important features of the AFM indentation experiments as the elastic constants along the three symmetry axes of the capsid and the critical indentations. From the value of one of the parameters of our model, we conclude that trimers in the MVM must be quite oblate tops, in excellent agreement with their known structure. The transition from the linear to the nonlinear regimes sampled in the indentation process appears to be an interesting topic for future research in physical virology.

The Nuclear Localization Signal of Porcine Circovirus Type 4 Affects the Subcellular Localization of the Virus Capsid and the Production of Virus-like Particles.

Porcine circovirus 4 (PCV4) is a newly identified virus belonging to PCV of the Circoviridae family, the Circovirus genus. We previously found that PCV4 is pathogenic in vitro, while the virus's replication in cells is still unknown. In this study, we evaluated the N-terminal of the PCV4 capsid (Cap) and identified an NLS at amino acid residues 4-37 of the N-terminus of the PCV4 Cap, 4RSRYSRRRRNRRNQRRRGLWPRASRRRYRWRRKN37. The NLS was further divided into two fragments (NLS-A and NLS-B) based on the predicted structure, including two α-helixes, which were located at 4RSRYSRRRRNRRNQRR19 and 24PRASRRRYRWRRK36, respectively. Further studies showed that the NLS, especially the first α-helixes formed by the NLS-A fragment, determined the nuclear localization of the Cap protein, and the amino acid 4RSRY7 in the NLS of the PCV4 Cap was the critical motif affecting the VLP packaging. These results will provide a theoretical basis for elucidating the infection mechanism of PCV4 and developing subunit vaccines based on VLPs.