Deletion Of Residues Research Articles

TRPV4-mediated Mechanotransduction Regulates the Differentiation of Valvular Interstitial Cells to Myofibroblasts: Implications for Aortic Stenosis.

As aortic valve stenosis (AVS) progresses, the valve tissue also stiffens. This increase in tissue stiffness causes the valvular interstitial cells (VICs) to transform into myofibroblasts in response. VIC-to-myofibroblast differentiation is critically involved in the development of AVS. Herein, we investigated the role of mechanosensitive Ca 2+ -permeant transient receptor potential vanilloid 4 (Trpv4) channels in matrix stiffness- and transforming growth factor β1 (TGFβ1)-induced VIC-myofibroblast activation. We confirmed Trpv4 functionality in primary mouse wild-type VICs compared to Trpv4 null VICs using live Ca 2+ influx detection during application of its selective agonist and antagonist. Using physiologically relevant hydrogels of varying stiffness that respectively mimic healthy or diseased aortic valve tissue stiffness, we found that genetic ablation of Trpv4 blocked matrix stiffness- and TGFβ1-induced VIC-myofibroblast activation as determined by changes in morphology, alterations of expression of α-smooth muscle actin, and modulations of F-actin generation. Our results showed that N-terminal residues 30-130 in Trpv4 were crucial for cellular force generation and VIC-myofibroblast activation, while deletion of residues 1-30 had no noticeable negative effect on these processes. Collectively, these data suggest a differential regulatory role for Trpv4 in stiffness/TGFβ1-induced VIC-myofibroblast activation. Our data further showed that Trpv4 regulates stiffness/TGFβ1-induced PI3K-AKT activity that is required for VIC-myofibroblast differentiation and cellular force generation, suggesting a mechanism by which Trpv4 activity regulates VIC-myofibroblast activation. Altogether, these data identify a novel role for Trpv4 mechanotransduction in regulating VIC-myofibroblast activation, implicating Trpv4 as a potential therapeutic target to slow and/or reverse AVS development.

Deletion within ameloblastin multitargeting domain reduces its interaction with artificial cell membrane

In human, mutations in the gene encoding the enamel matrix protein ameloblastin (Ambn) have been identified in cases of amelogenesis imperfecta. In mouse models, perturbations in the Ambn gene have caused loss of enamel and dramatic disruptions in enamel-making ameloblast cell function. Critical roles for Ambn in ameloblast cell signaling and polarization as well as adhesion to the nascent enamel matrix have been supported. Recently, we have identified a multitargeting domain (MTD) in Ambn that interacts with cell membrane, with the majority enamel matrix protein amelogenin, and with itself. This domain includes an amphipathic helix (AH) motif that directly interacts with cell membrane. In this study, we analyzed the sequence of the MTD for evolutionary conservation and found high conservation among mammals within the MTD and particularly within the AH motif. We computationally predicted that the AH motif lost its hydrophobic moment upon deleting hydrophobic but not hydrophilic residues from the motif. Furthermore, we rationally designed peptides that encompassed the Ambn MTD and contained deletions of largely hydrophobic or hydrophilic stretches of residues. To assess their AH-forming and membrane-binding abilities, we combined those peptides with synthetic phospholipid membrane vesicles and performed circular dichroism, membrane leakage, and vesicle clearance measurements. Circular dichroism showed retention of α-helix formation in all peptides except the one with the largest deletion of eleven amino acids including seven that were hydrophobic. This same peptide variant failed to cause leakage or clearance of synthetic membranes, while smaller deletions yielded intermediate membrane interaction as measured by leakage and clearance assays. Our data revealed that deletion of key hydrophobic residues from the AH leads to the most dramatic loss of Ambn–membrane interaction. Pinpointing roles of residues within the MTD has important implications for the multifunctionality of Ambn.

Liver CYP4A autophagic-lysosomal degradation (ALD): A major role for the autophagic receptor SQSTM1/p62 through an uncommon target interaction site.

The hepatic P450 hemoproteins CYPs 4A are typical N-terminally anchored Type I endoplasmic reticulum (ER)-proteins, that are inducible by hypolipidemic drugs and other "peroxisome proliferators". They are engaged in the ω-/ω-1-oxidation of various fatty acids including arachidonic acid, prostaglandins and leukotrienes and in the biotransformation of some therapeutic drugs. Herein we report that of the mammalian liver CYPs 4A, human CYP4A11 and mouse Cyp4a12a are preferential targets of the ER-lysosome-associated degradation (ERLAD). Consequently, these proteins are stabilized both as 1%Triton X100-soluble and -insoluble species in mouse hepatocytes and HepG2-cells deficient in the autophagic initiation ATG5-gene. Although these proteins exhibit surface LC3-interacting regions (LIRs) that would target them directly to the autophagosome, they nevertheless interact intimately with the autophagic receptor SQSTM1/p62. Through structural deletion analyses and site-directed mutagenesis, we have identified the Cyp4A-interacting p62 subdomain to lie between residues 170 and 233, which include its Traf6-binding and LIM-binding subdomains. Mice carrying a liver-specific genetic deletion of p62 residues 69-251 (p62Mut) that includes the CYP4A-interacting subdomain also exhibit Cyp4a-protein stabilization both as Triton X100-soluble and -insoluble species. Consistently, p62Mut mouse liver microsomes exhibit enhanced ω- and ω-1-hydroxylation of arachidonic acid to its physiologically active metabolites 19- and 20-HETEs relative to the corresponding wild-type mouse liver microsomes. Collectively, our findings suggest that any disruption of CYP4A ERLAD results in functionally active P450 protein and consequent production of proinflammatory metabolites on one hand, and insoluble aggregates on the other, which may contribute to pathological aggregates i.e. Mallory-Denk bodies/inclusions, hallmarks of many liver diseases.

Preformed mincle dimers stabilized by an interchain disulfide bond in the neck region.

The sugar-binding receptor mincle stimulates macrophages when it encounters surface glycans on pathogens, such as trehalose dimycolate glycolipid in the outer membrane of mycobacteria. Binding of oligosaccharide ligands to the extracellular C-type carbohydrate-recognition domain (CRD) in mincle initiates intracellular signaling through the common Fc receptor γ (FcRγ) adapter molecule associated with mincle. One potential mechanism for initiation of signaling involves clustering of receptors, so it is important to understand the oligomeric state of mincle. Affinity purification of mincle from transfected mammalian cells has been used to show that mincle exists as a pre-formed, disulfide-linked dimer. Deletion of cysteine residues and chemical crosslinking further demonstrate that the dimers of mincle are stabilized by a disulfide bond between cysteine residues in the neck sequence that links the CRD to the membrane. In contrast, cysteine residues in the transmembrane region of mincle are not required for dimer formation or association with FcRγ. A protocol has been developed for efficient production of a disulfide-linked extracellular domain fragment of mincle in a bacterial expression system by appending synthetic dimerization domains to guide dimer formation in the absence of the membrane anchor.

In Vitro Profiling of the Antiviral Peptide TAT-I24

The synthetic peptide TAT-I24 (GRKKRRQRRRPPQCLAFYACFC) exerts antiviral activity against several double-stranded (ds) DNA viruses, including herpes simplex viruses, cytomegalovirus, some adenoviruses, vaccinia virus and SV40 polyomavirus. In the present study, in vitro profiling of this peptide was performed with the aim of characterizing and improving its properties for further development. As TAT-I24 contains three free cysteine residues, a potential disadvantageous feature, peptide variants with replacements or deletions of specific residues were generated and tested in various cell systems and by biochemical analyses. Some cysteine replacements had no impact on the antiviral activity, such as the deletion of cysteine 14, which also showed improved biochemical properties, while the cyclization of cysteines 14 and 20 had the most detrimental effect on antiviral activity. At concentrations below 20 µM, TAT-I24 and selected variants did not induce hemolysis in red blood cells (RBCs) nor modulated lipopolysaccharide (LPS)-induced release of cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), in human peripheral blood mononuclear cells (PBMCs). These data indicate that TAT-I24 or its peptide variants are not expected to cause unwanted effects on blood cells.

Structural insight into broadening SARS-CoV-2 neutralization by an antibody cocktail harbouring both NTD and RBD potent antibodies

ABSTRACT The continual emergence of highly pathogenic novel coronaviruses and their variants has underscored the importance of neutralizing monoclonal antibodies (mAbs) as a pivotal therapeutic approach. In the present study, we report the specific neutralizing antibodies 13H7 and 9G11, which target the N-terminal domain (NTD) and receptor-binding domain (RBD) of the SARS-CoV-2, respectively. The comparative analysis observed that 13H7 not only neutralizes early variants of concern (VOCs) but also exhibits neutralizing activity against the Omicron sublineage, including BA.4, BA.5, BQ.1, and BQ.1.1. 9G11, as an RBD antibody, also demonstrated remarkable neutralizing efficacy. A cocktail antibody combining 13H7 and 9G11 with the previously reported 3E2 broaden the neutralization spectrum against new variants of the SARS-CoV-2. We elucidated the cryo-EM structure of the complex, clarifying the mechanism of action of the cocktail antibody combination. Additionally, we also emphasized the molecular mechanism between 13H7 and SARS-CoV-2 NTD, revealing the impact of Y144 and H146 residue deletions and mutations on the neutralizing efficacy of 13H7. Taken together, our findings not only offer novel insights into the combination therapy of NTD and RBD neutralizing mAbs but also lay a theoretical foundation for the development of vaccines targeting NTD antibodies, thus providing valuable understanding of alleviating the emergence of SARS-CoV-2 variants.

Design of Hepatitis C Protease Inhibitors Based on Knottin Fold

AbstractKnottins are very stable peptides (also called miniproteins) containing approximately 30 amino acid residues. Their structures are stabilized by three disulfide bonds forming a characteristic ‘pseudo‐knotted’ structure. This research was devoted to the design of potential inhibitors of human hepatitis C (HCV) NS3 protease by means of knottin fold using loop grafting, amino acid residues replacements and L/D amino acid epimerization. The initial structure used for design was a trypsin inhibitor from Momordica cochinchinensis seeds, MCOTI‐II. Modifications of the knottin template for optimization of the NS3 protease‐peptide interaction included deletion of several residues from the N‐terminus, replacement of residues in‐ and outside the inhibitor loop and replacement of certain L‐amino acids by their D‐stereoisomers. Stability of modified knottins and their complexes with HCV protease were evaluated by molecular dynamics simulation. Binding energy values for protein‐knottin complexes were calculated by MM‐GBSA method. The designed modified knottin characterized by the highest stability of the complex with HCV NS3 protease, was proposed for further experimental testing.

Abstract B061: Discovering co-driver genes and pathways of mutant TP53 in breast cancer by CRISPR screening and multi-omics approaches

Abstract Triple-Negative Breast Cancer (TNBC) is the most aggressive and highly heterogeneous molecular subtype of breast cancer lacking the progesterone, HER2 and estrogen receptors. The heterogeneity of these receptors makes it difficult to target and treat. The TP53 gene which encodes the p53 protein is mutated in 80% of TNBC cases. We hypothesized that, in addition to the neo-morphic activities of different p53 mutants themselves, the heterogeneity in clinical phenotypes observed in TNBC is due to functional interactions of the p53 driver mutants with other functionally important co-existing mutations called ‘co-drivers’. To identify co-driver mutations, our lab performed a genome-wide screening by using lentiviral CRISPR libraries on a cell line over-expressing p53-R273C (a prevalent mutant form of p53), and MYC (an oncogene). Upon injection of the library-transduced cells into mice, large tumor was formed, which was extracted and subjected to targeted PCR and whole exome sequencing. This identified truncation mutations in the EVC2 (EvC Ciliary Complex Subunit 2) and the NR1D1 (Nuclear Receptor 1D1) genes. This suggested that they are tumor suppressors in their wild type functions, and loss of this native function would co-drive the formation of tumors implicated with mutant p53-R273C to promote tumor formation in vivo. EVC2 positively modulates the Hedgehog signaling pathway. Interestingly, a majority of mutations in NR1D1 were an in-frame deletion of a tyrosine residue, which may be a critical regulatory residue of NR1D1. It is known to modulate the JAK/STAT3 pathway and, just like p53, NR1D1 can inhibit the circadian clock genes. We are testing whether deleting EVC2 and NR1D1 genes by CRISPR or expressing the mutant forms in the presence of p53-R273C increase invasion and/or other cancer hallmark phenotypes and also whether the amino acid, tyrosine is critical to the regulation of NR1D1, with the goal of understanding their contributions to the development of various hallmarks of cancer and the underlying mechanisms which will identify how these mutations contribute to the progression of TNBC and could potentially provide molecularly targeted therapies for TNBC. In parallel, our lab previously carried out RNA-Seq on cell lines expressing 10 different mutant p53 proteins, where pathway analysis identified the Hippo pathway, a tumor suppressor pathway, highly associated with aggressive cellular phenotypes in the mutant p53 contexts. In cancer, it is known that, upon suppression of the upstream Hippo pathway, unphosphorylated TAZ (or WWTR1) localizes in the nucleus binds to TEAD, leading to the activation of a series of genes to promote cancer hallmark phenotypes. We are investigating the molecular mechanisms of actions through which Hippo pathway drives the progression of TNBC and assess the pathway as the target for potential therapy for TNBC with specific TP53 mutations. This study will reveal how different mutant p53 proteins regulate Hippo pathway, TAZ activation/localization, and TEAD-mediated transcription to promote invasiveness. Citation Format: Lilian Nwekwo, Anasuya Pal, Yining Zheng, Lydia Sakala, Laura Gonzalez-Malerva, Dustin Grief, Dustin Lord, Jin Park, Joshua LaBaer. Discovering co-driver genes and pathways of mutant TP53 in breast cancer by CRISPR screening and multi-omics approaches [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B061.

Modified Knottins as Potential Inhibitors of HCV NS3 Protease

Knottins form a group of peptides containing approximately 30 amino acid residues. Their structures are stabilized by three disulfide bonds forming a characteristic “pseudo-knotted” structure. Several modifications in knottin from Momordica cochinchinensis were made to convert it to inhibitor of human hepatitis C (HCV) NS3 protease. These modifications of the knottin template included deletion of several residues from the N-terminus, replacement of residues in- and outside the inhibitor loop and replacement of certain L-amino acids by their D-stereoisomers. Binding energy values for protein-knottin complexes were estimated by MM-GBSA methods. Two designed knottins showed high stability in knottin-protease HCV complexes and values of binding energy comparable with known peptide inhibitors from crystal structures.

A Modified Recombinant DNA-Based SARS-CoV-2 Vaccine Expressing Stabilized Uncleavable Spike Protein Elicits Humoral and Cellular Immunity against Various SARS-CoV-2 Variants of Concern

The appearance of several variants of concern (VOCs) of SARS-CoV-2 affects the efficacy of currently available vaccines and causes continuous spread and reinfection between humans. These variants possess different spike (S) protein mutations, which could affect viral pathogenicity, transmission, and immune escape. Herein, we develop a synthetic codon-optimized DNA vaccine (VIU-1007) expressing full-length S protein. The developed vaccine is stabilized by two K986P and V987P proline substitutions and resistant to cleavage by proteases such as furin by deletion of arginine residues (R682, R683, and R685) in multibasic furin cleavage site (RRAR). Additionally, it carries K417N, E484K, N501Y, and D614G substitutions in the receptor binding domain (RBD) derived from the beta VOC. Following the validation and characterization of the in vitro S protein expression, the humoral and cellular immunogenicity of VIU-1007 was assessed in immunized Balb/c mice. While both regimens elicited a Th-1-biased immune response based on S1-specific binding IgG isotypes, three vaccine doses significantly enhanced IgG levels. Furthermore, CD4+ and CD8+ memory T cell responses in spleens and draining inguinal lymph nodes were significantly higher in mice received three doses of VIU-1007 when compared to those received two doses only. Importantly, sera from mice immunized with three doses showed broad neutralization breadth against several SARS-CoV-2 variants, including alpha, beta, gamma, delta, and omicron VOCs. Moreover, the sera showed limited neutralization capacity against SARS-CoV-1, Bat SARS-like coronavirus WIV1, and MERS-CoV. Together, while these data suggest the presence of common neutralizing-rich epitopes between SARS-CoV-2 variants and some other betacoronaviruses, the ongoing evolution of SARS-CoV-2 could result in escape from vaccine-induced immunity, which requires a continuous update of vaccines.

Alternaria TeA toxin activates a chloroplast retrograde signaling pathway to facilitate JA-dependent pathogenicity

The chloroplast is a critical battleground in the arms race between plants and pathogens. Among microbe-secreted mycotoxins, tenuazonic acid (TeA), produced by the genus Alternaria and other phytopathogenic fungi, inhibits photosynthesis, leading to a burst of photosynthetic singlet oxygen (1O2) that is implicated in damage and chloroplast-to-nucleus retrograde signaling. Despite the significant crop damage caused by Alternaria pathogens, our understanding of the molecular mechanism by which TeA promotes pathogenicity and cognate plant defense responses remains fragmentary. We now reveal that A. alternata induces necrotrophic foliar lesions by harnessing EXECUTER1 (EX1)/EX2-mediated chloroplast-to-nucleus retrograde signaling activated by TeA toxin–derived photosynthetic 1O2 in Arabidopsis thaliana. Mutation of the 1O2-sensitive EX1-W643 residue or complete deletion of the EX1 singlet oxygen sensor domain compromises expression of 1O2-responsive nuclear genes and foliar lesions. We also found that TeA toxin rapidly induces nuclear genes implicated in jasmonic acid (JA) synthesis and signaling, and EX1-mediated retrograde signaling appears to be critical for establishing a signaling cascade from 1O2 to JA. The present study sheds new light on the foliar pathogenicity of A. alternata, during which EX1-dependent 1O2 signaling induces JA-dependent foliar cell death.

Antibodies elicited by Plasmodium falciparum circumsporozoite proteins lacking sequentially deleted C-terminal amino acids reveal mouse strain and epitopes specific differences

Malaria affects ∼ ¼ billion people globally and requires the development of additional tools to aid in elimination efforts. The recently approved RTS,S/AS01 vaccine represents a positive step, however, the moderate efficacy necessitates the development of more efficacious vaccines. PfCSP is a key target antigen for pre-erythrocytic vaccines aimed at preventing Plasmodium falciparum malaria infections. Epitopes within the central repeat region and at the junction of the repeat and N-terminal domain are well documented as major protective B cell epitopes. On the other hand, a majority of antibodies against the epitopes in the C-terminal domain, have been shown to be non-protective against sporozoite challenge. The C-terminal domain, however, contains CD4+ and CD8+ T cell epitopes previously shown to be important for regulating immune responses. The present study was designed to further explore the immunomodulatory potential of the C-terminal domain using DNA vaccines encoding PfCSP with sequential C-terminal truncations following known T cell epitopes. Five DNA vaccines encoding different truncations of PfCSP within the C-terminal domain were administered via intramuscular route and in vivo electroporation for effective immunogenicity. Protection in mice was evaluated by challenge with transgenic P. berghei expressing PfCSP. In Balb/c mice, antibody responses and protective efficacy were both affected progressively with sequential deletion of C-terminal amino acid residues. Similar studies in C57Bl/6 mice revealed that immunizations with plasmids encoding truncated PfCSP showed partial protection from sporozoite challenge with no significant differences in antibody titers observed compared to full-length PfCSP DNA immunized mice. Further analysis revealed murine strain-specific differences in the recognition of specific epitopes.

Novel heterozygous VPS13A pathogenic variants in chorea-neuroacanthocytosis: a case report

BackgroundChorea-acanthocytosis (ChAc) is a rare hereditary autosomal recessive neurodegenerative disorder caused by pathogenic variants of the Vacuolar Protein Sorting 13 homolog A (VPS13A) gene. The variant spectrum of VPS13A has not been completely elucidated. This study reports two novel heterozygous VPS13A pathogenic variants in ChAc that expand the variant spectrum of VPS13A.Case presentationWe described a case of a 29-year-old man with typical clinical manifestations of ChAc, including chorea, orofacial lingual dyskinesia, vocal tics, elevated serum biochemical indicators, increased acanthocytes in peripheral blood, and caudate nucleus atrophy. Next-generation sequencing revealed two heterozygous variants of VPS13A: a nonsense variant (NM_033305.2: c.8215G > T, p. Glu2739Ter) and a deletion variant in the exons 25–31.ConclusionThe identified nonsense variant gives rise to premature translation termination, while the deletion variant is expected to cause a significant in-frame deletion of amino acid residues in the encoded protein. Both variants are considered to be pathogenic and result in loss-of-function proteins. These findings have implications for the genetic counseling of patients with VPS13A variants.

Construction of IgG–Fab2 bispecific antibody via intein-mediated protein trans-splicing reaction

A bispecific antibody (bsAb) is a class of engineered antibody molecules that simultaneously binds to two different antigens by having two kinds of antigen-binding domains. One of the major obstacles for the bsAb production is the incorrect chain-pairing problem, wherein each heavy and light chain should form pairings with the correct counterpart’s chains, but the structural similarity of the incorrect partners also forms the incorrect pairings. This study aimed to demonstrate a bsAb construction method using intein-mediated protein trans-splicing to create IgG–Fab2–type bsAbs, which is a modified antibody with a structure in which two additional Fabs are linked to the N-terminus of the heavy chain of an IgG molecule. The chain-paring problem between a heavy chain and a light chain is circumvented by separate expression and purification of the IgG part and the Fab part. We found that the deletion of a possible glycosylation residue improved the reaction yield and side-reaction cleavage in the protein ligation step. The resulting bsAb, IgG–Fab2 (Her2/CD3), demonstrated target binding activity and cytotoxicity mediated by activated T cells. These results indicate that the use of the protein ligation to produce the IgG–Fab2 type bsAb will expand the bsAb production method.

The phylogenetic analysis of the new emerging ALV-K revealing the co-prevailing of multiple clades in chickens and a proposal for the classification of ALV-K.

Subgroup K avian leukosis virus (ALV-K) is a new subgroup of avian leukosis virus (ALV) that was first defined in 2012 and has been become prevalent in Chinese native chickens in recent years. An in-depth analysis of the genetic diversity of ALV-K was performed in the study. By Blast analysis, the env gene and the sequences of the 25 ALV-K isolates we isolated were found to be closely related to the isolates from Guangdong, Hebei, Jiangsu, and Hubei provinces, China. Further eighty-nine sequences of the gp85 gene of ALV-K strains available were used in the phylogenetic and genetic distance analyses for the classification. ALV-K was divided into two second-order clades (Clades 1.1 and 1.2) and three third-order clades (Clades 1.2.1, 1.2.2, and 1.2.3), indicating that not only 1.1 and 1.2.3, the two old clades which are prevalent in Japan, but also two new clades (1.2.1, 1.2.2), are co-prevalent in China. The representative strains of each clade were defined for the first time. Notably, Clade 1.2.2 was found to have a deletion of an amino acid residue in the gp85 gene, which was obviously different from Clades 1.1, 1.2.1, and 1.2.3. The proposed classification method will facilitate future studies of ALV-K epidemiology and the comparison of sequences obtained across the world. The first global comprehensive molecular epidemiological analysis was accomplished on the emerging ALV-K.

Genetic Characteristics of Three Single-Farm-Isolated Porcine Reproductive and Respiratory Syndrome Viruses with Novel Recombination among NADC30-Like, JXA1-Like, and QYYZ-Like Strains

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically devastating pathogenic microorganism that greatly affects the pork industry in the world. The genetic variation and frequent emergence of novel strains greatly hinder the control efforts of PRRSV. Therefore, monitoring the evolutionary dynamics is long and rewarding work for PRRSV researchers and practitioners to make the control strategy. Here, three novel PRRSV strains named CHbj2101, CHbj2102, and CHbj2103 were isolated from different nursery barns with various mortality rates from 6% to 17%, belonging to the same farm, but at different periods of the outbreak. The genomic sequencing and phylogenetic analyses indicated that these three isolates were all clustered with NADC30-like virus CHsx1401, sharing higher genomic similarity from 87.3% to 89.8%, and having the same molecular marker of 131 amino acid residues deletion at their nsp2 coding region, but varied mutagenesis among the antigenic sites in the region of GP2 to GP5. Among the available PRRSV sequences in the GenBank, the isolates CHbj2101 and CHbj2102 display the highest genomic identity (90.1% and 89.8%) with NADC30-like recombinant strain 15LN3, and the rest CHbj2103 shows the highest genomic identity (90.8%) with NADC30-like virus strain 15SC3. The recombination analysis indicated that all three isolates are generated by multiple recombination events among the NADC30-like virus (major parent, Lineage 1), HP-PRRSV vaccine-like virus (minor parent, Lineage 8), and QYYZ-like virus (minor parent, Lineage 3). The isolates CHbj2101 and CHbj2102 shared a similar recombination pattern, but CHbj2103 has a different pattern in nonstructural protein coding regions. To further investigate the recombination characteristics of QYYZ-like strains, we analyzed all available whole genomic sequences of QYYZ-like PRRSV, submitted during the year 1991 and 2021 (n = 83) in China. The result shows that almost all QYYZ-like strains were products of recombination and their immunogenicity or protective protein fragments (nsp2–nsp7 and GP2–GP4) were mainly from QYYZ. These results provide us with some better insight into the evolution process of PRRSV strains in the field and warn us to pay more attention to monitoring and reducing the PRRSV variant on farms to reduce the risk of novel emergence and outbreak.

NS1 Protein N-Linked Glycosylation Site Affects the Virulence and Pathogenesis of Dengue Virus.

Live attenuated vaccine is one of the most effective vaccines against flavivirus. Recently, site-directed mutation of the flavivirus genome using reverse genetics techniques has been used for the rapid development of attenuated vaccines. However, this technique relies on basic research of critical virulence loci of the virus. To screen the attenuated sites in dengue virus, a total of eleven dengue virus type four mutant strains with deletion of N-glycosylation sites in the NS1 protein were designed and constructed. Ten of them (except for the N207-del mutant strain) were successfully rescued. Out of the ten strains, one mutant strain (N130del+207-209QQA) was found to have significantly reduced virulence through neurovirulence assay in suckling mice, but was genetically unstable. Further purification using the plaque purification assay yielded a genetically stable attenuated strain #11-puri9 with mutations of K129T, N130K, N207Q, and T209A in the NS1 protein and E99D in the NS2A protein. Identifying the virulence loci by constructing revertant mutant and chimeric viruses revealed that five amino acid adaptive mutations in the dengue virus type four non-structural proteins NS1 and NS2A dramatically affected its neurovirulence and could be used in constructing attenuated dengue chimeric viruses. Our study is the first to obtain an attenuated dengue virus strain through the deletion of amino acid residues at the N-glycosylation site, providing a theoretical basis for understanding the pathogenesis of the dengue virus and developing its live attenuated vaccines.

Arabidopsis COPT1 copper transporter uses a single histidine to regulate transport activity and protein stability

Copper acquisition and subsequent delivery to target proteins are essential for many biological processes. However, the cellular levels of this trace element must be controlled because of its potential toxicity. The COPT1 protein rich in potential metal-binding amino acids functions in high affinity copper uptake at the plasma membrane of Arabidopsis cells. The functional role of these putative metal-binding residues is largely unknown. Through truncations and site-directed mutagenesis, we identified His43, a single residue within the extracellular N-terminal domain as absolutely critical for copper uptake of COPT1. Substitution of this residue with leucine, methionine or cysteine almost inactivated transport function of COPT1, implying that His43 fails to serves as a copper ligand in the regulation of COPT1 activity. Deletion of all extracellular N-terminal metal-binding residues completely blocked copper-stimulated degradation but did not alter the subcellular distribution and multimerization of COPT1. Although mutation of His43 to alanine and serine retained the transporter activity in yeast cells, the mutant protein was unstable and degraded in the proteasome in Arabidopsis cells. Our results demonstrate a pivotal role for the extracellular residue His43 in high affinity copper transport activity, and suggest common molecular mechanisms for regulating both metal transport and protein stability of COPT1.

Deletion of pentad residues in the N-terminal domain of spike protein attenuates porcine epidemic diarrhea virus in piglets

Our previous study revealed that tissue culture-adapted porcine epidemic diarrhea virus (PEDV) strains, namely KNU-141112-S DEL2/ORF3 and -S DEL5/ORF3, were attenuated to different extents in vivo, suggesting that their independent deletion (DEL) signatures, including 2-amino acid (aa; residues 56–57) or 5-aa (residues 56–60) DEL in the N-terminal domain (NTD) of the spike (S) protein, may contribute to the reduced virulence of each strain. To investigate whether each DEL in the NTD of the S1 subunit is a determinant for the virulence of PEDV, we generated two mutant viruses, named icS DEL2 and icS DEL5, by introducing the identical double or quintuple aa DEL into S1 using reverse genetics with an infectious cDNA clone of KNU-141112 (icKNU-141112). We then orally inoculated conventional suckling piglets with icKNU-141112, icS DEL2, or icS DEL5 to compare their pathogenicities. The virulence of both DEL mutant viruses was significantly diminished compared to that of icKNU-141112, which causes severe clinical signs and 100 % mortality. Interestingly, the degree of attenuation differed between the two mutant viruses: icS DEL5 caused neither diarrhea nor mortality, whereas icS DEL2 caused mild to moderate diarrhea, higher viral titers in feces and intestinal tissues, and 25 % mortality. Furthermore, the icS DEL5-infected piglets displayed no remarkable macroscopic and microscopic intestinal lesions, while the icS DEL2-infected piglets showed histopathological changes in small intestine tissues, including moderate-to-severe villous atrophy. Our data indicate that the loss of the pentad (56GENQG60) residues in S alone can be sufficient to give rise to an attenuated phenotype of PEDV.

A Porcine Epidemic Diarrhea Virus Isolated from a Sow Farm Vaccinated with CV777 Strain in Yinchuan, China: Characterization, Antigenicity, and Pathogenicity

Porcine epidemic diarrhea virus (PEDV) is a porcine enteric coronavirus globally, causing serious economic losses to the global pig industry since 2010. Here, a PEDV CH/Yinchuan/2021 strain was isolated in a CV777-vaccinated sow farm which experienced a large-scale PEDV invasion in Yinchuan, China, in 2021. Our results demonstrated that the CH/Yinchuan/2021 isolate could efficiently propagate in Vero cells, and its proliferation ability was weaker than that of CV777 at 10 passages (P10). Phylogenetic analysis of the S gene revealed that CH/Yinchuan/2021 was clustered into subgroup GIIa, forming an independent branch with 2020-2021 isolates in China. Moreover, GII was obviously allocated into four clades, showing regional and temporal differences in PEDV global isolates. Notably, CH/Yinchuan/2021 was analyzed as a recombinant originated from an American isolate and a Chinese isolate, with a big recombinant region spanning ORF1a and S1. Importantly, we found that CH/Yinchuan/2021 harbored multiple mutations relative to CV777 in neutralizing epitopes (S10, S1A, COE, and SS6). Homology modelling showed that these amino acid differences in S protein occur on the surface of its structure, especially the insertion and deletion of multiple consecutive residues at the S10 epitope. In addition, cross-neutralization analysis confirmed that the differences in the S protein of CH/Yinchuan/2021 changed its antigenicity compared with the CV777 strain, resulting in a different neutralization profile. Animal pathogenicity test showed that CH/Yinchuan/2021 caused PEDV-typified symptoms and 100% mortality in 3-day-old piglets. These data will provide valuable information to understand the epidemiology, molecular characteristics, evolution, and antigenicity of PEDV circulating in China.