Genome-wide characterization and expression analysis of the HD-Zip II gene family in response to drought and GA3 stresses in Nicotiana tabacum.

BACKGROUND: Homeodomain-leucine ZIPper (HD-ZIP) transcription factors play crucial roles in plant growth, development, and stress responses. The HD-ZIP family is categorised into four groups (HD-ZIP I-IV). While extensive genome-wide studies have been conducted on the HD-ZIP I, III, and IV subfamily in Nicotiana tabacum (tobacco), comprehensive reports on the HD-ZIP II subfamily genes are limited. METHODS: Bioinformatics resources and tools were utilised to analyse molecular characteristics, phylogenetic homology, and protein interactions. Expression pattern analyses in various tissues and the relative expression of NtHD-ZIP II genes under drought and GA3 treatment were assessed by qRT-PCR. RESULTS: In this study, 24 HD-ZIP II members were systematically identified and categorised into seven independent clades through phylogenetic analysis involving tobacco and other plant species. We found that 19 NtHD-ZIP II genes exhibited tissue-specific expression. The transcripts of NtHD-ZIPII3, 4, 14, 23, 24 were notably induced under the drought treatments, while those of NtHD-ZIPII7, 11, 12, 20 were suppressed. Furthermore, NtHD-ZIPII15 transcripts decreased following GA3 treatment, whereas the transcripts of NtHD-ZIPII7, 8, 11, 12 were induced after GA3 treatment. Notably, an increase in trichomes was observed in tobacco leaves treated with GA3 and subjected to drought. CONCLUSIONS: The expression levels of some HD-ZIP II genes were altered, and an increase in glandular trichomes was induced under GA3 and drought treatments in tobacco. Overall, our findings provide insights into the expression patterns of NtHD-ZIP II genes and will facilitate their functional characterisation in future studies.

Genome-Wide Identification of Homeodomain Leucine Zipper (HD-ZIP) Transcription Factor, Expression Analysis, and Protein Interaction of HD-ZIP IV in Oil Palm Somatic Embryogenesis.

Understanding the molecular mechanisms underlying somatic embryogenesis is essential for resolving the problems related to the long duration of the process and a low rate of somatic embryo induction in oil palm tissue culture. In this study, we conducted genome-wide identification of the oil palm homeodomain leucine zipper (EgHD-ZIP) family, which is one of the plant-specific transcription factors reported to be involved in embryogenesis. EgHD-ZIP proteins can be divided into four subfamilies, which have similarities in gene structure and protein-conserved motifs within a group. In silico expression analysis showed that the expression of EgHD-ZIP gene members in the EgHD-ZIP I and II families, as well as most members in the EgHD-ZIP IV family, were up-regulated during the zygotic and somatic embryo developmental stages. In contrast, the expression of EgHD-ZIP gene members in the EgHD-ZIP III family was down-regulated during zygotic embryo development. Moreover, the expression of EgHD-ZIP IV genes was validated in the oil palm callus and at the somatic embryo stages (globular, torpedo, and cotyledon). The results revealed that EgHD-ZIP IV genes were up-regulated at the late stages of somatic embryogenesis (torpedo and cotyledon). While BABY BOOM (BBM) gene was up-regulated at the early stage of somatic embryogenesis (globular). In addition, the Yeast-two hybrid assay revealed the direct binding between all members of the oil palm HD-ZIP IV subfamily (EgROC2, EgROC3, EgROC5, EgROC8, and EgBBM). Our findings suggested that the EgHD-ZIP IV subfamily and EgBBM work together to regulate somatic embryogenesis in oil palms. This process is important because it is widely used in plant biotechnology to produce large quantities of genetically identical plants, which can be used for oil palm tissue culture improvement.

Basic Leucine Zipper Transcription Factors as Important Regulators of Leydig Cells' Functions.

Transcription factors members of the basic leucine zipper (bZIP) class play important roles in the regulation of genes and functions in testicular Leydig cells. Many of these factors, such as cAMP responsive element binding protein 1 (CREB1) and CCAAT enhancer binding protein beta (CEBPB), are regulated by the cAMP/protein kinase A (PKA) pathway, the main signaling pathway activated following the activation of the luteinizing hormone/choriogonadotropin membrane receptor LHCGR by the - hormone LH. Others, such as X-box binding protein 1 (XBP1) and members of the cAMP responsive element binding protein 3 (CREB3)-like superfamily, are implicated in the endoplasmic reticulum stress by regulating the unfolded protein response. In this review, the influences of bZIP transcription factors, including CREB1, CEBPB and activator protein 1 (AP-1) family members, on the regulation of genes important for cell proliferation, steroidogenesis and Leydig cell communication will be covered. In addition, unresolved questions regarding the mechanisms of actions of bZIP members in gene regulation will be identified.

Genetic transformation and growth index determination of the Larix olgensis LoHDZ2 transcription factor gene in tobacco.

Homeodomain-leucine zippers (HD-Zip) are plant-specific transcription factors that participate in different plant development processes and differentially regulate metabolic processes. LoHDZ2 is an HD-ZipII subfamily transcription factor gene that we identified from a transcriptomic analysis of Larix olgensis. To understand its function, we built a LoHDZ2 expression vector and then inserted it into tobacco by genetic transformation. Transgenic plants were identified at the DNA and RNA levels. Phenotypic index analysis of transgenic tobacco showed dwarfed growth with larger leaves and earlier flowering than the wild type. LoHDZ2 was expressed differently after hormone treatment with IAA, MeJA and 2,4-D. The results suggested that LoHDZ2 may respond to hormones and be involved in regulating growth and metabolism. These results helped us better understand the function of LoHDZ2 and provided a candidate gene for Larix olgensis molecular breeding.

Methylprednisolone stimulated gene expression (GILZ, MCL-1) and basal cortisol levels in multiple sclerosis patients in relapse are associated with clinical response.

Glucocorticoids (GCs) are the main treatment of relapse in multiple sclerosis (MS). Decreased sensitivity to GCs in MS patients has been associated with lack of the suppressive effect of GCs on inflammatory molecules as well as increased resistance to apoptosis. We investigated GC-sensitivity by measuring the effect of intravenous methylprednisolone (IVMP) treatment on transactivation of anti-inflammatory and apoptotic genes (GILZ, MCL-1 and NOXA respectively), in accordance to clinical outcome. Thirty nine MS patients were studied: 15 with clinically isolated syndrome (CIS), 12 with relapsing remitting (RRMS) and 12 with secondary progressive (SPMS) under relapse. Patients underwent treatment with IVMP for 5 days. Blood was drawn before IVMP treatment on day 1 and 1 h after IVMP treatment on days 1 and 5. GIlZ, MCL-1 and NOXA were determined by qPCR. The Expanded Disability Status was evaluated and patients were divided according to their clinical response to IVMP. GILZ and MCL-1 gene expression were significantly higher following first IVMP treatment in responders, compared to non-responders. Furthermore, serum basal cortisol and 1,25-OH Vitamin D levels were significantly higher in clinical-responders as compared to non-clinical responders. Our findings suggest that the differential GILZ and MCL-1 gene expression between clinical-responders and non-clinical responders may implicate the importance of GILZ and MCL-1 as possible markers for predicting glucocorticoid sensitivity and response to GC-therapy in MS patients following first IVMP injection.

Metformin-Inducible Small Heterodimer Partner Interacting Leucine Zipper Protein Ameliorates Intestinal Inflammation.

Small heterodimer partner interacting leucine zipper protein (SMILE) is an orphan nuclear receptor and a member of the bZIP family of proteins. We investigated the mechanism by which SMILE suppressed the development of inflammatory bowel disease (IBD) using a DSS-induced colitis mouse model and peripheral blood mononuclear cells (PBMCs) from patients with ulcerative colitis (UC). Metformin, an antidiabetic drug and an inducer of AMPK, upregulated the level of SMILE in human intestinal epithelial cells and the number of SMILE-expressing cells in colon tissues from DSS-induced colitis mice compared to control mice. Overexpression of SMILE using a DNA vector reduced the severity of DSS-induced colitis and colitis-associated intestinal fibrosis compared to mock vector. Furthermore, SMILE transgenic mice showed ameliorated DSS-induced colitis compared with wild-type mice. The mRNA levels of SMILE and Foxp3 were downregulated and SMILE expression was positively correlated with Foxp3 in PBMCs from patients with UC and an inflamed mucosa. Metformin increased the levels of SMILE, AMPK, and Foxp3 but decreased the number of interleukin (IL)-17-producing T cells among PBMCs from patients with UC. These data suggest that SMILE exerts a therapeutic effect on IBD by modulating IL-17 production.

Biophysical and Structural Methods to Study the bHLHZip Region of Human c-MYC.

The C-terminal region of the c-MYC transcription factor consists of approximately 100 amino acids that in its native state does not adopt a stable structure. When this region binds to the obligatory partner MAX via a coupled folding-and-binding mechanism, it forms a basic-helix-loop-helix-leucine zipper (bHLHZip) heterodimeric complex. The C-terminal region of MYC is the target for numerous drug discovery programs for direct MYC inhibition via blocking the dimerization event and/or binding to DNA, and a proper understanding of the partially folded, dynamic nature of the heterodimeric complex is essential to these efforts. The bHLHZip motif also drives protein-protein interactions with cofactors that are crucial for both transcriptional repression and activation of MYC target genes. Targeting these interactions could potentially provide a means of developing alternative approaches to halt MYC functions; however, the molecular mechanism of these regulatory interactions is poorly understood. Herein we provide methods to produce high-quality human c-MYC C-terminal by itself and in complex MAX, and how to study them using Nuclear Magnetic Resonance spectroscopy and X-ray crystallography. Our protein expression and purification protocols have already been used to study interactions with cofactors.

Methods of Expression, Purification, and Preparation of the c-Myc b-HLH-LZ for Its Biophysical Characterization.

The b-HLH-LZ domain of c-Myc is a key target for the development of cancer therapies by blunting its binding to DNA with cell penetrant b-HLH-LZs and/or by stabilizing it into a state that cannot recognize Max to activate and amplify transcription of oncogenic genes. Although recent milestones have been reached with DNA binding blunting of c-Myc with the cell penetrant b-HLH-LZ Omomyc, the targeting of its b-HLH-LZ with small molecules, peptides, or proteins is lagging. As reviewed recently, the main problem relies in the intrinsically disordered nature of the b-HLH-LZ of c-Myc. This greatly complicates the classical approach of targeting a docking site with inhibitors. The solution state methods such as NMR are progressing towards the characterization of the ensembles of structures or states the b-HLH-LZ can adopt. However, the delicate balance that dictates the population of these dynamically interchanging states relies on its primary structure and the weak polar, electrostatic and hydrophobic interactions allowed. In this context, it is of the utmost importance to study the b-HLH-LZ of c-Myc in its WT background and avoid the use of tags such as His-tags. These tags could disrupt the balance of forces which could alter the conformational and physical transitions and states it can undergo and adopt. Here, we describe a robust protocol to express the WT b-HLH-LZ in E. coli and purify it, without the need of tags, to obtain the required quantities for solution state biophysical characterization such as NMR.

c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1) attenuates curcumin-induced cell death differently from its family member, JNK-associated leucine zipper protein (JLP).

Curcumin, a major component of turmeric, is known to exhibit multiple biological functions including antitumor activity. We previously reported that the mitogen-activated protein kinase (MAPK) scaffold protein c-Jun NH2-terminal kinase (JNK)-associated leucine zipper protein (JLP) reduces curcumin-induced cell death by modulating p38 MAPK and autophagy through the regulation of lysosome positioning. In this study, we investigated the role of JNK/stress-activated protein kinase-associated protein 1 (JSAP1), a JLP family member, in curcumin-induced stress, and found that JSAP1 also attenuates curcumin-induced cell death. However, JSAP1 knockout showed no or little effect on the activation of JNK and p38 MAPKs in response to curcumin. In addition, small molecule inhibitors of JNK and p38 MAPKs did not increase curcumin-induced cell death. Furthermore, JSAP1 depletion did not impair lysosome positioning and autophagosome-lysosome fusion. Instead, we noticed substantial autolysosome accumulation accompanied by an inefficient autophagic flux in JSAP1 knockout cells. Taken together, these results indicate that JSAP1 is involved in curcumin-induced cell death differently from JLP, and may suggest that JSAP1 plays a role in autophagosome degradation and its dysfunction results in enhanced cell death. The findings of this study may contribute to the development of novel therapeutic approaches using curcumin for cancer.

Amino Acid Composition in Various Types of Nucleic Acid-Binding Proteins.

Nucleic acid-binding proteins are traditionally divided into two categories: With the ability to bind DNA or RNA. In the light of new knowledge, such categorizing should be overcome because a large proportion of proteins can bind both DNA and RNA. Another even more important features of nucleic acid-binding proteins are so-called sequence or structure specificities. Proteins able to bind nucleic acids in a sequence-specific manner usually contain one or more of the well-defined structural motifs (zinc-fingers, leucine zipper, helix-turn-helix, or helix-loop-helix). In contrast, many proteins do not recognize nucleic acid sequence but rather local DNA or RNA structures (G-quadruplexes, i-motifs, triplexes, cruciforms, left-handed DNA/RNA form, and others). Finally, there are also proteins recognizing both sequence and local structural properties of nucleic acids (e.g., famous tumor suppressor p53). In this mini-review, we aim to summarize current knowledge about the amino acid composition of various types of nucleic acid-binding proteins with a special focus on significant enrichment and/or depletion in each category.

Role of small leucine zipper protein in hepatic gluconeogenesis and metabolic disorder.

Hepatic gluconeogenesis is the central pathway for glucose generation in the body. The imbalance between glucose synthesis and uptake leads to metabolic diseases such as obesity, diabetes, and cardiovascular diseases. Small leucine zipper protein (sLZIP) is an isoform of LZIP and it mainly functions as a transcription factor. Although sLZIP is known to regulate the transcription of genes involved in various cellular processes, the role of sLZIP in hepatic glucose metabolism is not known. In this study, we investigated the regulatory role of sLZIP in hepatic gluconeogenesis and its involvement in metabolic disorder. We found that sLZIP expression was elevated during glucose starvation, leading to the promotion of phosphoenolpyruvate carboxylase and glucose-6-phosphatase expression in hepatocytes. However, sLZIP knockdown suppressed the expression of the gluconeogenic enzymes under low glucose conditions. sLZIP also enhanced glucose production in the human liver cells and mouse primary hepatic cells. Fasting-induced cyclic adenosine monophosphate impeded sLZIP degradation. Results of glucose and pyruvate tolerance tests showed that sLZIP transgenic mice exhibited abnormal blood glucose metabolism. These findings suggest that sLZIP is a novel regulator of gluconeogenic enzyme expression and plays a role in blood glucose homeostasis during starvation.

Long non-coding RNA (lncRNA) PGM5P4-AS1 inhibits lung cancer progression by up-regulating leucine zipper tumor suppressor (LZTS3) through sponging microRNA miR-1275.

It is necessary to explore new molecules for the improvement of precise diagnosis and antitumor therapies in lung cancer. LncRNAs (long non-coding RNAs) play an important role in the regulation of cancer cell malignant behavior and tumor development. In this work, we found that a newly discovered lncRNA, lncRNA PGM5P4-AS1, was lower expressed in lung cancer tissues than adjacent tissues. Then, the lncRNA PGM5P4-AS1 was overexpressed or knocked-down in different lung cancer cells, and its effects on the malignant phenotypes were measured by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, cell cycle assay, wound healing assay, and transwell assay. The results showed that the overexpression of PGM5P4-AS1 inhibited lung cancer cell proliferation, migration, and invasion activities, while these abilities were prominently promoted by the interference of PGM5P4-AS1. Further, the growth of lung cancer tumors in nude mice was also inhibited by PGM5P4-AS1 overexpression. In mechanism, PGM5P4-AS1 has the binding site of miR-1275 and could positively regulate the expression of LZTS3 via sponging miR-1275. In conclusion, PGM5P4-AS1 could be a potential precise diagnosis and therapeutic target biomarker of lung cancer.

Site-Specific Post-translational Surface Modification of Adeno-Associated Virus Vectors Using Leucine Zippers.

Adeno-associated virus (AAV) is widely favored as a gene therapy vector, tested in over 200 clinical trials internationally. To improve targeted delivery a variety of genetic capsid modifications, such as insertion of targeting proteins/peptides into the capsid shell, have been explored with some success but larger insertions often have unpredictable deleterious impacts on capsid formation and gene delivery. Here, we demonstrate a modular platform for the integration of exogenous peptides and proteins onto the AAV capsid post-translationally while preserving vector functionality. We decorated the AAV capsid with leucine-zipper coiled-coil binding motifs that exhibit specific noncovalent heterodimerization. AAV capsids successfully display hexahistidine tagged-peptides using this approach, as demonstrated through nickel column affinity. This protein display platform may facilitate the incorporation of biological moieties on the AAV surface, expanding possibilities for vector enhancement and engineering.

Multitasking: Dual Leucine Zipper-Bearing Kinases in Neuronal Development and Stress Management.

The dual leucine zipper-bearing kinase (DLK) and leucine zipper-bearing kinase (LZK) are evolutionarily conserved MAPKKKs of the mixed-lineage kinase family. Acting upstream of stress-responsive JNK and p38 MAP kinases, DLK and LZK have emerged as central players in neuronal responses to a variety of acute and traumatic injuries. Recent studies also implicate their function in astrocytes, microglia, and other nonneuronal cells, reflecting their expanding roles in the multicellular response to injury and in disease. Of particular note is the potential link of these kinases to neurodegenerative diseases and cancer. It is thus critical to understand the physiological contexts under which these kinases are activated, as well as the signal transduction mechanisms that mediate specific functional outcomes. In this review we first provide a historical overview of the biochemical and functional dissection of these kinases. We then discuss recent findings on regulating their activity to enhance cellular protection following injury and in disease, focusing on but not limited to the nervous system.

BZW2 gene knockdown induces cell growth inhibition, G1 arrest and apoptosis in muscle-invasive bladder cancers: A microarray pathway analysis.

Bladder cancer is among the most common cancers all over the world. The function of basic leucine zipper and W2 domains 2 (BZW2) in tumour progression has been reported. However, the biological function of BZW2 in muscle-invasive bladder cancers (MIBCs) remains to be determined. The aim of the present study was to reveal the expression and roles of BZW2 in human MIBCs and to explore the molecular mechanisms underlying these functions. Clinically, BZW2 expression was higher in MIBC tissues than the adjacent non-tumour tissues. Knocking down BZW2 using shRNA inhibited cell proliferation and G1/S cell cycle progression in vitro, and induced apoptosis in both 5637 and T24 cells. Moreover, in vivo studies with mice xenograft models confirmed the anti-proliferative effects of BZW2-knockdown, providing a future therapeutic target. We also performed biochemical microarray analysis to identify the potential signalling pathways, disease states and functions which could be affected by suppressing BZW2 in MIBC cells. Collectively, our findings suggest BZW2 has an oncogenic role in MIBCs and serves as a promising target for molecular diagnosis and gene therapy.

Tumor penetrating peptides inhibiting MYC as a potent targeted therapeutic strategy for triple-negative breast cancers.

Overexpression of MYC oncogene is highly prevalent in many malignancies such as aggressive triple-negative breast cancers (TNBCs) and it is associated with very poor outcome. Despite decades of research, attempts to effectively inhibit MYC, particularly with small molecules, still remain challenging due to the featureless nature of its protein structure. Herein, we describe the engineering of the dominant-negative MYC peptide (OmoMYC) linked to a functional penetrating 'Phylomer' peptide (FPPa) as a therapeutic strategy to inhibit MYC in TNBC. We found FPPa-OmoMYC to be a potent inducer of apoptosis (with IC50 from 1-2 µM) in TNBC cells with negligible effects in non-tumorigenic cells. Transcriptome analysis of FPPa-OmoMYC-treated cells indicated that the fusion protein inhibited MYC-dependent networks, inducing dynamic changes in transcriptional, metabolic, and apoptotic processes. We demonstrated the efficacy of FPPa-OmoMYC in inhibiting breast cancer growth when injected orthotopically in TNBC allografts. Lastly, we identified strong pharmacological synergisms between FPPa-OmoMYC and chemotherapeutic agents. This study highlights a novel therapeutic approach to target highly aggressive and chemoresistant MYC-activated cancers.

Functional interplay between c-Myc and Max in B lymphocyte differentiation.

The Myc family of oncogenic transcription factors regulates myriad cellular functions. Myc proteins contain a basic region/helix-loop-helix/leucine zipper domain that mediates DNA binding and heterodimerization with its partner Max. Among the Myc proteins, c-Myc is the most widely expressed and relevant in primary B lymphocytes. There is evidence suggesting that c-Myc can perform some of its functions in the absence of Max in different cellular contexts. However, the functional in vivo interplay between c-Myc and Max during B lymphocyte differentiation is not well understood. Using in vivo and ex vivo models, we show that while c-Myc requires Max in primary B lymphocytes, several key biological processes, such as cell differentiation and DNA replication, can initially progress without the formation of c-Myc/Max heterodimers. We also describe that B lymphocytes lacking Myc, Max, or both show upregulation of signaling pathways associated with the B-cell receptor. These data suggest that c-Myc/Max heterodimers are not essential for the initiation of a subset of important biological processes in B lymphocytes, but are required for fine-tuning the initial response after activation.

Physiological and transcriptome response to cadmium in cosmos (Cosmos bipinnatus Cav.) seedlings.

To date, several species of Asteraceae have been considered as Cd-accumulators. However, little information on the Cd tolerance and associated mechanisms of Asteraceae species Cosmos bipinnatus, is known. Presently, several physiological indexes and transcriptome profiling under Cd stress were investigated. C. bipinnatus exhibited strong Cd tolerance and recommended as a Cd-accumulator, although the biomasses were reduced by Cd. Meanwhile, Cd stresses reduced Zn and Ca uptake, but increased Fe uptake. Subcellular distribution indicated that the vacuole sequestration in root mainly detoxified Cd under lower Cd stress. Whilst, cell wall binding and vacuole sequestration in root co-detoxified Cd under high Cd exposure. Meanwhile, 66,407 unigenes were assembled and 41,674 (62.75%) unigenes were annotated in at least one database. 2,658 DEGs including 1,292 up-regulated unigenes and 1,366 down-regulated unigenes were identified under 40 µmol/L Cd stress. Among of these DEGs, ZIPs, HMAs, NRAMPs and ABC transporters might participate in Cd uptake, translocation and accumulation. Many DEGs participating in several processes such as cell wall biosynthesis, GSH metabolism, TCA cycle and antioxidant system probably play critical roles in cell wall binding, vacuole sequestration and detoxification. These results provided a novel insight into the physiological and transcriptome response to Cd in C. bipinnatus seedlings.

A Conserved Leucine Zipper Motif in Gammaherpesvirus ORF52 Is Critical for Distinct Microtubule Rearrangements.

Productive viral infection often depends on the manipulation of the cytoskeleton. Herpesviruses, including rhesus monkey rhadinovirus (RRV) and its close homolog, the oncogenic human gammaherpesvirus Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV/HHV8), exploit microtubule (MT)-based retrograde transport to deliver their genomes to the nucleus. Subsequently, during the lytic phase of the life cycle, the maturing viral particles undergo orchestrated translocation to specialized regions within the cytoplasm, leading to tegumentation, secondary envelopment, and then egress. As a result, we hypothesized that RRV might induce changes in the cytoskeleton at both early and late stages of infection. Using confocal imaging, we found that RRV infection led to the thickening and acetylation of MTs emanating from the MT-organizing center (MTOC) shortly after viral entry and more pronounced and diffuse MT reorganization during peak stages of lytic gene expression and virion production. We subsequently identified open reading frame 52 (ORF52), a multifunctional and abundant tegument protein, as being the only virally encoded component responsible for these cytoskeletal changes. Mutational and modeling analyses indicated that an evolutionarily conserved, truncated leucine zipper motif near the N terminus as well as a strictly conserved arginine residue toward the C terminus of ORF52 play critical roles in its ability to rearrange the architecture of the MT cytoskeleton. Taken together, our findings combined with data from previous studies describing diverse roles for ORF52 suggest that it likely binds to different cellular components, thereby allowing context-dependent modulation of function.IMPORTANCE A thorough understanding of the processes governing viral infection includes knowledge of how viruses manipulate their intracellular milieu, including the cytoskeleton. Altering the dynamics of actin or MT polymerization, for example, is a common strategy employed by viruses to ensure efficient entry, maturation, and egress as well as the avoidance of antiviral defenses through the sequestration of key cellular factors. We found that infection with RRV, a homolog of the human pathogen KSHV, led to perinuclear wrapping by acetylated MT bundles and identified ORF52 as the viral protein underlying these changes. Remarkably, incoming virions were able to supply sufficient ORF52 to induce MT thickening and acetylation near the MTOC, potentially aiding in the delivery viral genomes to the nucleus. Although the function of MT alterations during late stages of infection requires further study, ORF52 shares functional and structural similarities with alphaherpesvirus VP22, underscoring the evolutionary importance of MT cytoskeletal manipulations for this virus family.

Engineering of a novel zipFv using leucine zipper motif against rabies virus glycoprotein G with improved protection potency in vivo.

Rabies is an acute zoonotic infectious disease with a high fatality rate but is preventable with vaccination and rabies immunoglobulin (RIG). The single-chain Fv fragment (scFv), a small engineered antigen-binding protein derived from antibody variable heavy (VH) and light (VL) chains connected by a peptide linker, can potentially be used to replace RIG. Here, we produced two peptides VH-JUN-HIS and VL-FOS-HA separately in Escherichia coli and assembled them to form zipFv successfully in vitro. The new zipFv utilizes FOS and JUN leucine zippers to form an antibody structure similar to the IgG counterpart with two free N-terminal ends of VH and VL. The zipFv protein showed notable improvement in binding ability and affinity over its corresponding scFv. The zipFv also demonstrated greater stability in serum and the same protective rate as RIG against challenge with a standard rabies virus (CVS-24) in mice. Our results indicated zipFv as a novel and efficient antibody form with enhanced neutralizing potency.