Minor Protein Research Articles

Engineered M13-Derived Bacteriophages Capable of Gold Nanoparticle Synthesis and Nanogold Manipulations

For years, gold nanoparticles (AuNPs) have been widely used in medicine and industry. Although various experimental procedures have been reported for their preparation and manipulation, none of them is optimal for all purposes. In this work, we engineered the N-terminus of the pIII minor coat protein of bacteriophage (phage) M13 to expose a novel HLYLNTASTHLG peptide that effectively and specifically binds gold. In addition to binding gold, this engineered phage could synthesize spherical AuNPs of 20 nm and other sizes depending on the reaction conditions, aggregate them, and precipitate gold from a colloid, as revealed by transmission electron microscopy (TEM), atomic force microscopy (AFM), and scanning electron microscopy (SEM), as well as ultraviolet–visible (UV–vis) and Fourier-transform infrared (FTIR) spectroscopic methods. We demonstrated that the engineered phage exposing a foreign peptide selected from a phage-displayed library may serve as a sustainable molecular factory for both the synthesis of the peptide and the subsequent overnight preparation of AuNPs from gold ions at room temperature and neutral pH in the absence of strong reducing agents, such as commonly used NaBH4. Taken together, the results suggest the potential applicability of the engineered phage and the new, in vitro-identified gold-binding peptide in diverse biomimetic manipulations.

Complete genomes of two cluster AK Arthrobacter phages isolated from soil samples in Newburgh, NY, United States.

Two phages belonging to Arthrobacter phage cluster AK were isolated from soil samples collected in Newburgh, NY in 2021. Both are lytic with a genome organization typical of siphoviruses except for two genes encoding minor tail proteins with pyocin-knob domains found early in the genome, before the terminase gene.

The Feline calicivirus capsid protein VP1 is a client of the molecular chaperone Hsp90.

Feline calicivirus (FCV) icosahedral viral capsids are composed of dozens of structural subunits that rely on cellular chaperones to self-assemble in an orderly fashion. Here, we report that the heat shock protein 90 (Hsp90) inhibition significantly reduced FCV particle production, suggesting a role in the replicative cycle. We found that Hsp90 inhibition was not related to the synthesis or stability of the early proteins that translate from the gRNA nor to the minor capsid protein VP2 but with a reduction in the major capsid protein VP1 levels, both translated late in infection from the subgenomic RNAs. Reduction in VP1 levels was observed despite an augment of the leader of the capsid (LC)-VP1 precursor levels, from which the LC and VP1 proteins are produced after proteolytic processing by NS6/7. The direct interaction of VP1 with Hsp90 was observed in infected cells. These results suggest that upon release from the polyprotein precursor, VP1 becomes a client of Hsp90 and that this interaction is required for an efficient FCV replicative cycle.

Establishment of the Meyer-Overton correlation in an artificial membrane without protein

BackgroundThe potency of anesthetics with various structures increases exponentially with lipophilicity, which is the Meyer-Overton (MO) correlation discovered over 120 years ago. The MO correlation was also observed with various biological effects and chemicals, including alcohols; thus, the correlation represents a fundamental relationship between chemicals and organisms. The MO correlation was explained by the lipid and protein theories, although the principle remains unknown because these are still debating. MethodsThe gentle hydration method was used to form giant unilamellar vesicles (GUVs) consisting of high- and low-melting phospholipids and cholesterol in the presence of n-alcohols (C2-C12). Confocal fluorescence microscopy was used to determine the percentage of GUVs with domains in relation to the n-alcohol concentrations. Resultsn-Alcohols inhibited the domain formation of GUVs, and the half inhibitory concentration (IC50) in the aqueous phase (Cw) decreased exponentially with increasing chain length (lipophilicity). In contrast, the membrane concentrations (Cm) of alcohols for the inhibition, which is a product of the membrane-water partition coefficient and the IC50 values, remained constant irrespective of the chain length. ConclusionsThe MO correlation is established in GUVs, which supports the lipid theory. When alcohols reach the same critical concentration in the membrane, similar biological effects appear irrespective of the chain length, which is the principle underlying the MO correlation. General significanceThe protein theory states that a highly lipophilic compound targets minor membrane proteins due to the low Cw. However, our lipid theory states that the compound targets various membrane proteins due to the high Cm.

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.

Out-of-sync evolutionary patterns and mutual interplay of major and minor capsid proteins in norovirus GII.2.

Human noroviruses are the most common cause of viral gastroenteritis, resulting annually in 219 000 deaths and a societal cost of $60 billion, and no antivirals or vaccines are available. The minor capsid protein may play a significant role in the evolution of norovirus. GII.4 is the predominant genotype of norovirus, and its VP2 undergoes epochal co-evolution with the major capsid protein VP1. Since the sudden emergence of norovirus GII.2[P16] in 2016, it has consistently remained a significant epidemic strain in recent years. In the construction of phylogenetic trees, the phylogenetic trees of VP2 closely parallel those of VP1 due to the shared tree topology of both proteins. To investigate the interaction patterns between the major and minor capsid proteins of norovirus GII.2, we chose five representative strains of GII.2 norovirus and investigated their evolutionary patterns using a yeast two-hybrid experiment. Our study shows VP1-VP2 interaction in GII.2, with critical interaction sites at 167-178 and 184-186 in the highly variable region. In the intra-within GII.2, we observed no temporal co-evolution between VP1 and VP2 of GII.2. Notable distinctions were observed in the interaction intensity of VP2 among inter-genotype (P<0.05), highlighting the divergent evolutionary patterns of VP2 within different norovirus genotypes. In summary, the interactions between VP2 and VP1 of GII.2 norovirus exhibit out-of-sync evolutionary patterns. This study offered valuable insights for further understanding and completing the evolutionary mechanism of norovirus.

From sequence to function: Exploring biophysical properties of bacteriophage BFK20 lytic transglycosylase domain from the minor tail protein gp15

Bacteriophages have evolved different mechanisms of infection and penetration of bacterial cell walls. In Siphoviridae-like viruses, the inner tail proteins have a pivotal role in these processes and often encode lytic protein domains which increase infection efficiency. A soluble lytic transglycosylase (SLT) domain was identified in the minor tail protein gp15 from the BFK20 bacteriophage. Six fragments containing this SLT domain with adjacent regions of different lengths were cloned, expressed and purified. The biophysical properties of the two best expressing fragments were characterized by nanoDSF and CD spectroscopy, which showed that both fragments had a high refolding ability of 90 %. 3D modeling indicated that the bacteriophage BFK20 SLT domain is structurally similar to lysozyme. The degradation activity of these SLT proteins was evaluated using a lysozyme activity assay. BFK20 might use its transglycosylase activity to allow efficient phage DNA entry into the host cell by degrading bacterial peptidoglycan.

Comparison of Antigen Retrieval Methods for Immunohistochemical Analysis of Cartilage Matrix Glycoproteins Using Cartilage Intermediate Layer Protein 2 (CILP-2) as an Example.

The aim of this study was to compare different antigen retrieval methods to improve the outcome of immunohistochemistry (IHC) performed on osteoarthritic (OA) cartilage obtained from total knee replacement operation. A voluminous and dense extracellular matrix of articular cartilage inhibits antibody penetration, and therefore, proteins present at low concentrations and masked during fixation may need antigen retrieval to enhance an IHC outcome. We focused on the IHC detection of a minor but diagnostically promising cartilage glycoprotein, CILP-2 (cartilage intermediate layer protein 2), to demonstrate the effect of four different protocols: (1) heat-induced epitope retrieval (HIER), (2) proteolytic-induced epitope retrieval applying proteinase K and hyaluronidase (PIER), (3) HIER combined with PIER, and (4) no antigen retrieval (control). A semi-quantitative staining assessment based on the CILP-2 staining extent was applied. Out of the tested antigen retrieval protocols, the best CILP-2 IHC staining results were achieved by PIER. Combining PIER with HIER did not improve CILP-2 staining in the given experimental setting. Rather the opposite, the application of heat reduced the positive effect of PIER on CILP-2 staining and resulted in the frequent detachment of sections from the slides. Our findings emphasize the need for proper adaptation of antigen retrieval protocols for IHC to maximize the quantitative evaluation of minor matrix proteins in OA articular cartilage samples.

Capsid structure of bacteriophage ΦKZ provides insights into assembly and stabilization of jumbo phages

Jumbo phages are a group of tailed bacteriophages with large genomes and capsids. As a prototype of jumbo phage, ΦKZ infects Pseudomonas aeruginosa, a multi-drug-resistant (MDR) opportunistic pathogen leading to acute or chronic infection in immunocompromised individuals. It holds potential to be used as an antimicrobial agent and as a model for uncovering basic phage biology. Although previous low-resolution structural studies have indicated that jumbo phages may have more complicated capsid structures than smaller phages such as HK97, the detailed structures and the assembly mechanism of their capsids remain largely unknown. Here, we report a 3.5-Å-resolution cryo-EM structure of the ΦKZ capsid. The structure unveiled ten minor capsid proteins, with some decorating the outer surface of the capsid and the others forming a complex network attached to the capsid’s inner surface. This network seems to play roles in driving capsid assembly and capsid stabilization. Similar mechanisms of capsid assembly and stabilization are probably employed by many other jumbo viruses.

The polinton-like supergroup of viruses: evolution, molecular biology, and taxonomy.

SUMMARYPolintons are 15-20 kb-long self-synthesizing transposons that are widespread in eukaryotic, and in particular protist, genomes. Apart from a transposase and a protein-primed DNA polymerase, polintons encode homologs of major and minor jelly-roll capsid proteins, DNA-packaging ATPases, and proteases involved in capsid maturation of diverse eukaryotic viruses of kingdom Bamfordvirae. Given the conservation of these structural and morphogenetic proteins among polintons, these elements are predicted to alternate between transposon and viral lifestyles and, although virions have thus far not been detected, are classified as viruses (class Polintoviricetes) in the phylum Preplasmiviricota. Related to polintoviricetes are vertebrate adenovirids; unclassified polinton-like viruses (PLVs) identified in various environments or integrated into diverse protist genomes; virophages (Maveriviricetes), which are part of tripartite hyperparasitic systems including protist hosts and giant viruses; and capsid-less derivatives, such as cytoplasmic linear DNA plasmids of fungi and transpovirons. Phylogenomic analysis indicates that the polinton-like supergroup of viruses bridges bacterial tectivirids (preplasmiviricot class Tectiliviricetes) to the phylum Nucleocytoviricota that includes large and giant eukaryotic DNA viruses. Comparative structural analysis of proteins encoded by polinton-like viruses led to the discovery of previously undetected functional domains, such as terminal proteins and distinct proteases implicated in DNA polymerase processing, and clarified the evolutionary relationships within Polintoviricetes. Here, we leverage these insights into the evolution of the polinton-like supergroup to develop an amended megataxonomy that groups Polintoviricetes, PLVs (new class 'Aquintoviricetes'), and virophages (renamed class 'Virophaviricetes') together with Adenoviridae (new class 'Pharingeaviricetes') in a preplasmiviricot subphylum 'Polisuviricotina' sister to a subphylum including Tectiliviricetes ('Prepoliviricotina').

Robust Systemic and Mucosal Immune Responses to Coxsackievirus B3 Elicited by Spider Silk Protein Based Nanovaccines via Subcutaneous Immunization

AbstractCoxsackievirus B3 (CVB3) is a member of the enterovirus genus and linked to several diseases, including myocarditis, which can progress to dilated cardiomyopathy. Despite ongoing preclinical efforts, no clinically approved vaccines against CVB3 are currently available, highlighting the urgent need for effective prophylactic solutions. In this study, a nanovaccine platform based on spider minor ampullate silk protein (MiSp) is introduced. This platform utilizes protein nanoparticles engineered from chimeric proteins that incorporate CVB3 antigenic peptides into customized MiSp, subsequently loaded with all‐trans retinoic acid (RA). These functional nanovaccines are capable of eliciting both mucosal and systemic immune responses following subcutaneous administration and demonstrate significant protective effects against CVB3 infection in mice. This study signifies an approach in peptide‐based parenteral vaccine strategies, utilizing engineered MiSp nanoparticles combined with RA. This methodology represents a promising pathway for preventing enterovirus infections by leveraging the unique immunomodulatory properties of spidroins and RA to combat these pathogens effectively.

Molecular characterization and identification of Stellaria aquatica virus C, a novel putative member of the genus Closterovirus infecting Stellaria aquatica in South Korea.

A novel virus infecting Stellaria aquatica plants, tentatively named "Stellaria aquatica virus C" (StAVC), was identified in Gangwon-do Province, South Korea. Its monopartite genome consists of a single-stranded RNA of 15,024 nucleotides, and it shares 38.24 to 56.2% nucleotide sequence identity with known closterovirus genome sequences. Its genome contains nine hypothetical open reading frames. These encode the multifunctional protein RNA-dependent RNA polymerase (RdRp), hydrophobic protein (P7), heat shock protein 70 homolog (HSP70h), coat protein homolog (CPh), minor coat protein (CPm), and major coat protein (CP), along with proteins involved in suppressing RNA silencing. Phylogenetic analysis reveals that, based on its HSP70h amino acid sequence, StAVC is closely related to members of the genus Closterovirus within the family Closteroviridae. This is the first record of the full genome sequence of StAVC in South Korea.

A peptide derived from sorting nexin 1 inhibits HPV16 entry, retrograde trafficking, and L2 membrane spanning

High risk human papillomavirus (HPV) infection is responsible for 99 % of cervical cancers and 5 % of all human cancers worldwide. HPV infection requires the viral genome (vDNA) to gain access to nuclei of basal keratinocytes of epithelium. After virion endocytosis, the minor capsid protein L2 dictates the subcellular retrograde trafficking and nuclear localization of the vDNA during mitosis. Prior work identified a cell-permeable peptide termed SNX1.3, derived from the BAR domain of sorting nexin 1 (SNX1), that potently blocks the retrograde and nuclear trafficking of EGFR in triple negative breast cancer cells. Given the importance of EGFR and retrograde trafficking pathways in HPV16 infection, we set forth to study the effects of SNX1.3 within this context. SNX1.3 inhibited HPV16 infection by both delaying virion endocytosis, as well as potently blocking virion retrograde trafficking and Golgi localization. SNX1.3 had no effect on cell proliferation, nor did it affect post-Golgi trafficking of HPV16. Looking more directly at L2 function, SNX1.3 was found to impair membrane spanning of the minor capsid protein. Future work will focus on mechanistic studies of SNX1.3 inhibition, and the role of EGFR signaling and SNX1-mediated endosomal tubulation, cargo sorting, and retrograde trafficking in HPV infection.

Backbone NMR resonance assignments for the VP1u N-terminal receptor-binding domain of the human parvovirus pathogen B19.

Parvovirus B19 (B19V) is a human pathogen that is the causative agent of several diseases in infants and adults. Due to a lack of antivirals against this virus, treatment options are limited. The minor capsid protein of B19V has a unique N terminus, named VP1u, which is essential for infection. The VP1u encodes a receptor binding domain (RBD), necessary for host cell entry, and a phospholipase A2 (PLA2) domain, crucial for endosomal escape during cellular trafficking. Both domains are indispensable for infection, making the RBD a plausible drug target for inhibitors against B19V, as it is located on the exterior surface of the virus. To date, no experimental structural information has been available for the VP1u component for any Parvovirus. Here we report the backbone NMR resonance assignments for the RBD of B19V and demonstrate it forms a stable structure. The backbone chemical shifts are in good agreement with a structure predicted by AlphaFold, validating that the RBD contains three helices connected by tight turns. This RBD construct can now be used for further NMR studies, including assignment of full-length VP1u, determination of protein-protein interaction interfaces, and development of B19 antivirals specific to the RBD domain.

A Peptide Derived from Sorting Nexin 1 Inhibits HPV16 Entry, Retrograde Trafficking, and L2 Membrane Spanning.

High risk human papillomavirus (HPV) infection is responsible for 99% of cervical cancers and 5% of all human cancers worldwide. HPV infection requires the viral genome (vDNA) to gain access to nuclei of basal keratinocytes of epithelium. After virion endocytosis, the minor capsid protein L2 dictates the subcellular retrograde trafficking and nuclear localization of the vDNA during mitosis. Prior work identified a cell-permeable peptide termed SNX1.3, derived from the BAR domain of sorting nexin 1 (SNX1), that potently blocks the retrograde and nuclear trafficking of EGFR in triple negative breast cancer cells. Given the importance of EGFR and retrograde trafficking pathways in HPV16 infection, we set forth to study the effects of SNX1.3 within this context. SNX1.3 inhibited HPV16 infection by both delaying virion endocytosis, as well as potently blocking virion retrograde trafficking and Golgi localization. SNX1.3 had no effect on cell proliferation, nor did it affect post-Golgi trafficking of HPV16. Looking more directly at L2 function, SNX1.3 was found to impair membrane spanning of the minor capsid protein. Future work will focus on mechanistic studies of SNX1.3 inhibition, and the role of EGFR signaling and SNX1- mediated endosomal tubulation, cargo sorting, and retrograde trafficking in HPV infection.

Spider minor ampullate silk protein nanoparticles: an effective protein delivery system capable of enhancing systemic immune responses.

Spider silk proteins (spidroins) are particularly attractive due to their excellent biocompatibility. Spider can produce up to seven different types of spidroins, each with unique properties and functions. Spider minor ampullate silk protein (MiSp) might be particularly interesting for biomedical applications, as the constituent silk is mechanically strong and does not super-contract in water, attributed to its amino acid composition. In this study, we evaluate the potential of recombinant nanoparticles derived from Araneus ventricosus MiSp as a protein delivery carrier. The MiSp-based nanoparticles were able to serve as an effective delivery system, achieving nearly 100% efficiency in loading the model protein lysozyme, and displayed a sustained release profile at physiological pH. These nanoparticles could significantly improve the delivery efficacy of the model proteins through different administration routes. Furthermore, nanoparticles loaded with model protein antigen lysozyme after subcutaneous or intramuscular administration could enhance antigen-specific immune responses in mouse models, through a mechanism involving antigen-depot effects at the injection site, long-term antigen persistence, and efficient uptake by dendritic cells as well as internalization by lymph nodes. These findings highlight the transnational potential of MiSp-based nanoparticle system for protein drug and vaccine delivery.

Complex regulation of mitochondrial signaling by human adenovirus minor capsid protein VI.

Human adenoviruses (HAdVs) are common pathogens causing various self-limiting diseases, including conjunctivitis and the common cold. HAdVs need to interfere with multiple cellular signaling pathways during the infection to gain control over the host cell. In this study, we identified human adenovirus type 5 (HAdV-C5) minor capsid protein VI as a factor modulating mitochondrial membrane integrity and mitochondrial signaling. We show that pVI-altered mitochondrial signaling impedes the cell's innate immune response, which may benefit HAdV growth. Overall, our study provides new detailed insights into the HAdV-mitochondria interactions and signaling. This knowledge is helpful when developing new anti-viral treatments against pathogenic HAdV infections and improving HAdV-based therapeutics.

Backbone NMR resonance assignments for the VP1u N-terminal receptor-binding domain of the human parvovirus pathogen B19.

Parvovirus B19 (B19V) is a human pathogen that is the causative agent of several diseases in infants and adults. Due to a lack of antivirals against this virus, treatment options are limited. The minor capsid protein of B19V has a unique N terminus, named VP1u, which is essential for infection. The VP1u encodes a receptor binding domain (RBD), necessary for host cell entry, and a phospholipase A2 (PLA2) domain, crucial for endosomal escape during cellular trafficking. Both domains are indispensable for infection, making the RBD a plausible drug target for inhibitors against B19V, as it is located on the exterior surface of the virus. To date, no experimental structural information has been available for the VP1u component for any Parvovirus. Here we report the backbone NMR resonance assignments for the RBD of B19V and demonstrate it forms a stable structure. The backbone chemical shifts are in good agreement with a structure predicted by AlphaFold, validating that the RBD contains three helices connected by tight turns. This RBD construct can now be used for further NMR studies, including assignment of full-length VP1u, determination of protein-protein interaction interfaces, and development of B19 antivirals specific to the RBD domain. Database: BMRB submission code: 52440.

Cloning, Expression, and Bioinformatics Modeling of Human Papillomavirus Type 52 L1/L2 Chimeric Protein in Escherichia coli BL21 (DE3)

Human papillomavirus (HPV) L1 major capsid protein generates a highly immunogenic virus like particles (VLPs), which have been used as the main component of its prophylactic vaccine. However, the neutralizing antibodies against L1 VLPs are mostly type specific and may not be effective to prevent infection from different strains of HPV. On the other hand, HPV L2 minor capsid protein has low antigenic variation, thus can induce cross-neutralization. This study aims to obtain HPV 52 L1/L2 chimeric protein, which is designed based on HPV type 52 as one of the most circulated high-risk types in Indonesia, to develop a broad-spectrum HPV vaccine. Substitution of HPV 52 H4 helix L1 region with an HPV 52 L2 epitope was carried out using overlap extension PCR. HPV 52 L1/L2 chimeric gene was constructed into pET-SUMO expression vector and expressed in Escherichia coli BL21 (DE3). Bioinformatics modeling suggested that L2 epitope was located inside of the loop region in monomer form, and on the contrary, it was located outside of the pentamer surface. Furthermore, B cell and T cell epitopes predictions were conducted using Immune Epitope Database (IEDB) analysis. The B cell epitopes prediction revealed eleven potential epitopes, whereas the T cell epitopes prediction showed seven potential epitopes for each MHC class I and MHC class II. This study showed that HPV 52 L1/L2 chimeric protein has the potential to induce cross-neutralizing antibodies and can be developed as a promising candidate for a new HPV vaccine.

Analysis of SARS-CoV-2 omicron mutations that emerged during long-term replication in a lung cancer xenograft mouse model.

SARS-CoV-2 Omicron has the largest number of mutations among all the known SARS-CoV-2 variants. The presence of these mutations might explain why Omicron is more infectious and vaccines have lower efficacy to Omicron than other variants, despite lower virulence of Omicron. We recently established a long-term in vivo replication model by infecting Calu-3 xenograft tumors in immunodeficient mice with parental SARS-CoV-2 and found that various mutations occurred majorly in the spike protein during extended replication. To investigate whether there are differences in the spectrum and frequency of mutations between parental SARS-CoV-2 and Omicron, we here applied this model to Omicron. At 30 days after infection, we found that the virus was present at high titers in the tumor tissues and had developed several rare sporadic mutations, mainly in ORF1ab with additional minor spike protein mutations. Many of the mutant isolates had higher replicative activity in Calu-3 cells compared with the original SARS-CoV-2 Omicron virus, suggesting that the novel mutations contributed to increased viral replication. Serial propagation of SARS-CoV-2 Omicron in cultured Calu-3 cells resulted in several rare sporadic mutations in various viral proteins with no mutations in the spike protein. Therefore, the genome of SARS-CoV-2 Omicron seems largely stable compared with that of the parental SARS-CoV-2 during extended replication in Calu-3 cells and xenograft model. The sporadic mutations and modified growth properties observed in Omicron might explain the emergence of Omicron sublineages. However, we cannot exclude the possibility of some differences in natural infection.