30K Protein Research Articles

Cassava Brown Streak Viruses express second 6-kilodalton (6K2) protein with varied polarity and three dimensional (3D) structures: Basis for trait discrepancy between the virus species.

Cassava Brown Streak Virus (CBSV) and Ugandan Cassava Brown Streak Virus (UCBSV) are the two among six virus species speculated to cause the most catastrophic Brown Streak Disease of Cassava (CBSD) in Africa and Asia. Cassava Brown Streak Virus (CBSV) is hard to breed resistance for compared to Ugandan Cassava Brown Streak Virus (UCBSV) species. This is exemplified by incidences of CBSV species rather than UCBSV species in elite breeding line, KBH 2006/0026 at Bagamoyo, Tanzania. It is not yet understood as to why CBSV species could breakdown CBSD-resistance in the KBH 2006/0026 unlike the UCBSV species. This marks the first in silico study conducted to understand molecular basis for the trait discrepancy between CBSV and UCBSV species from structural biology view point. Following ab initio modelling and analysis of physical-chemical properties of second 6-kilodalton (6K2) protein encoded by CBSV and UCBSV species, using ROBETTA server and Protein Parameters tool, respectively we report that; three dimensional (3D) structures and polarity of the protein differs significantly between the two virus species. (95% and 5%) and (85% and 15%) strains of 20 CBSV and 20 UCBSV species respectively, expressed the protein in homo-trimeric and homo-tetrameric forms, correspondingly. 95% and 85% of studied strain population of the two virus species expressed hydrophilic and hydrophobic 6K2, respectively. Based on findings of the curent study, we hypothesize that; (i) The hydrophilic 6K2 expressed by the CBSV species, favour its faster systemic movement via vascular tissues of cassava host and hence result into higher tissue titres than the UCBSV species encoding hydrophobic form of the protein. t and (ii) The hydrophilic 6K2 expressed byCBSV species have additional interaction advantage with Nuclear Inclusion b protease domain (NIb) and Viral genome-linked protein (VPg), components of Virus Replication Complex (VRC) and hence contributing to faster replication of viral genome than the hydrophobic 6K2 expressed by the UCBSV species. Experimental studies are needed to resolve the 3D structures of the 6K2, VPg and NIb and comprehend complex molecular interactions between them. We suggest that, the 6K2 gene should be targeted for improvement of RNA interference (RNAi)-directed transgenesis of virus-resistant cassava as a more effective way to control the CBSD besides breeding.

Functional Analysis of Autoantibody Signatures in Rheumatoid Arthritis.

For the identification of antigenic protein biomarkers for rheumatoid arthritis (RA), we conducted IgG profiling on high density protein microarrays. Plasma IgG of 96 human samples (healthy controls, osteoarthritis, seropositive and seronegative RA, n = 24 each) and time-series plasma of a pristane-induced arthritis (PIA) rat model (n = 24 total) were probed on AIT’s 16k protein microarray. To investigate the analogy of underlying disease pathways, differential reactivity analysis was conducted. A total of n = 602 differentially reactive antigens (DIRAGs) at a significance cutoff of p < 0.05 were identified between seropositive and seronegative RA for the human samples. Correlation with the clinical disease activity index revealed an inverse correlation of antibodies against self-proteins found in pathways relevant for antigen presentation and immune regulation. The PIA model showed n = 1291 significant DIRAGs within acute disease. Significant DIRAGs for (I) seropositive, (II) seronegative and (III) PIA were subjected to the Reactome pathway browser which also revealed pathways relevant for antigen presentation and immune regulation; of these, seven overlapping pathways had high significance. We therefore conclude that the PIA model reflects the biological similarities of the disease pathogenesis. Our data show that protein array analysis can elucidate biological differences and pathways relevant in disease as well be a useful additional layer of omics information.

Complete genome sequence of a novel varicosavirus infecting tall morning glory (Ipomoea purpurea).

A novel varicosa-like virus was identified in a tall morning glory (Ipomoea purpurea) plant by high-throughput sequencing and tentatively named "morning glory varicosavirus" (MGVV). The complete genome of MGVV contains two segments of negative-sense single-stranded RNA of 6409 (RNA1) and 5288 (RNA2) nucleotides. RNA1 encodes a 224.3-kDa large protein (224K), and RNA2 encodes four putative proteins of 48.6 kDa (49K), 46.4 kDa (46K), 35.7 kDa (36K), and 36.8 kDa (37K), respectively. The 224K and 49K proteins show amino acid sequence similarity to the large protein (39.4%) and the 49K protein (22.6%), respectively, of red clover-associated varicosavirus, and the 36K protein shares 19.6% amino acid sequence similarity with protein 3 of lettuce big-vein associated virus. The 46K and 37K proteins share no significant sequence similarity to known functional viral sequences. Phylogenetic analysis based on the large protein of MGVV and other rhabdoviruses showed that MGVV clustered with the varicosaviruses. These analyses indicate that MGVV is a novel member of the genus Varicosavirus in the family Rhabdoviridae.

Effect of modulating glutamate signaling on myelinating oligodendrocytes and their development-A study in the zebrafish model.

Myelination is crucial for the development and maintenance of axonal integrity, especially fast axonal action potential conduction. There is increasing evidence that glutamate signaling and release through neuronal activity modulates the myelination process. In this study, we examine the effect of manipulating glutamate signaling on myelination of oligodendrocyte (OL) lineage cells and their development in zebrafish (zf). We use the "intensity-based glutamate-sensing fluorescent reporter" (iGluSnFR) in the zf model (both sexes) to address the hypothesis that glutamate is implicated in regulation of myelinating OLs. Our results show that glial iGluSnFR expression significantly reduces OL lineage cell number and the expression of myelin markers in larvae (zfl) and adult brains. The specific glutamate receptor agonist, L-AP4, rescues this iGluSnFR effect by significantly increasing the expression of the myelin-related genes, plp1b and mbpa, and enhances myelination in L-AP4-injected zfl compared to controls. Furthermore, we demonstrate that degrading glutamate using Glutamat-Pyruvate Transaminase (GPT) or the blockade of glutamate reuptake by L-trans-pyrrolidine-2,4-dicarboxylate (PDC) significantly decreases myelin-related genes and drastically declines myelination in brain ventricle-injected zfl. Moreover, we found that myelin-specific ClaudinK (CldnK) and 36K protein expression is significantly decreased in iGluSnFR-expressing zfl and adult brains compared to controls. Taken together, this study confirms that glutamate signaling is directly required for the preservation of myelinating OLs and for the myelination process itself. These findings further suggest that glutamate signaling may provide novel targets to therapeutically boost remyelination in several demyelinating diseases of the CNS.

Five Silkworm 30K Proteins Are Involved in the Cellular Immunity against Fungi.

Simple SummaryThe molecular mechanism of 30K proteins in anti-fungal immunity remains unclear. Here, we examined the mRNA levels of 30K proteins, including BmLP1, BmLP2, BmLP3, BmLP4, and BmLP7, and found that all of these proteins were significantly upregulated after injection of pathogen-associated molecular patterns to the fifth instar larvae, implying their involvement in immune response. The binding assay results showed that only BmLP1 and BmLP4 can bind to both fungal cells and silkworm hemocytes. In vitro, the encapsulation of hemocytes on day 5 of the fifth instar larval stage was promoted by the coating of agarose beads with recombinant BmLP1 and BmLP4. Therefore, these results demonstrate that 30K proteins are involved in the cellular immunity of silkworms by acting as pattern recognition molecules to directly recruit hemocytes to the fungal surface. We believe that our study makes a significant contribution to the literature because it provides insights into the 30K-mediated cellular immunity in silkworms.Background: 30K proteins are a major group of nutrient storage proteins in the silkworm hemolymph. Previous studies have shown that 30K proteins are involved in the anti-fungal immunity; however, the molecular mechanism involved in this immunity remains unclear. Methods: We investigated the transcriptional expression of five 30K proteins, including BmLP1, BmLP2, BmLP3, BmLP4, and BmLP7. The five recombinant 30K proteins were expressed in an Escherichia coli expression system, and used for binding assays with fungal cells and hemocytes. Results: The transcriptional expression showed that the five 30K proteins were significantly upregulated after injection of pathogen-associated molecular patterns to the fifth instar larvae, indicating the possibility of their involvement in immune response. The binding assay showed that only BmLP1 and BmLP4 can bind to both fungal cells and silkworm hemocytes. Furthermore, we found that BmLP1-coated and BmLP4-coated agarose beads promote encapsulation of hemocytes in vitro. The hemocyte encapsulation was blocked when the BmLP1-coated beads were preincubated with BmLP1 specific polyclonal antibodies. Conclusions: These results demonstrate that 30K proteins are involved in the cellular immunity of silkworms by acting as pattern recognition molecules to directly recruit hemocytes to the fungal surface.

Site-directed mutagenesis by biolistic transformation efficiently generates inheritable mutations in a targeted locus in soybean somatic embryos and transgene-free descendants in the T1 generation.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is being rapidly developed for mutagenesis in higher plants. Ideally, foreign DNA introduced by this system is removed in the breeding of edible crops and vegetables. Here, we report an efficient generation of Cas9-free mutants lacking an allergenic gene, Gly m Bd 30K, using biolistic transformation and the CRISPR/Cas9 system. Five transgenic embryo lines were selected on the basis of hygromycin resistance. Cleaved amplified polymorphic sequence analysis detected only two different mutations in e all of the lines. These results indicate that mutations were induced in the target gene immediately after the delivery of the exogenous gene into the embryo cells. Soybean plantlets (T0 plants) were regenerated from two of the transgenic embryo lines. The segregation pattern of the Cas9 gene in the T1 generation, which included Cas9-free plants, revealed that a single copy number of transgene was integrated in both lines. Immunoblot analysis demonstrated that no Gly m Bd 30K protein accumulated in the Cas9-free plants. Gene expression analysis indicated that nonsense mRNA decay might have occurred in mature mutant seeds. Due to the efficient induction of inheritable mutations and the low integrated transgene copy number in the T0 plants, we could remove foreign DNA easily by genetic segregation in the T1 generation. Our results demonstrate that biolistic transformation of soybean embryos is useful for CRISPR/Cas9-mediated site-directed mutagenesis of soybean for human consumption.

Association between flower stalk elongation, an Arabidopsis developmental trait, and the subcellular location and movement dynamics of the nonstructural protein P3 of Turnip mosaic virus.

Virus infections affect plant developmental traits but this aspect of the interaction has not been extensively studied so far. Two strains of Turnip mosaic virus differentially affect Arabidopsis development, especially flower stalk elongation, which allowed phenotypical, cellular, and molecular characterization of the viral determinant, the P3 protein. Transiently expressed wild‐type green fluorescent protein‐tagged P3 proteins of both strains and selected mutants of them revealed important differences in their behaviour as endoplasmic reticulum (ER)‐associated peripheral proteins flowing along the reticulum, forming punctate accumulations. Three‐dimensional (3D) model structures of all expressed P3 proteins were computationally constructed through I‐TASSER protein structure predictions, which were used to compute protein surfaces and map electrostatic potentials to characterize the effect of amino acid changes on features related to protein interactions and to phenotypical and subcellular results. The amino acid at position 279 was the main determinant affecting stalk development. It also determined the speed of ER‐flow of the expressed proteins and their final location. A marked change in the protein surface electrostatic potential correlated with changes in subcellular location. One single amino acid in the P3 viral protein determines all the analysed differential characteristics between strains differentially affecting flower stalk development. A model proposing a role of the protein in the intracellular movement of the viral replication complex, in association with the viral 6K2 protein, is proposed. The type of association between both viral proteins could differ between the strains.

Multiple ER-to-nucleus stress signaling pathways are activated during Plantago asiatica mosaic virus and Turnip mosaic virus infection in Arabidopsis thaliana.

Pathogens and other adverse environmental conditions can trigger endoplasmic reticulum (ER) stress. ER stress signaling increases the expression of cytoprotective ER-chaperones. The inositol-requiring enzyme (IRE1) is one ER stress sensor that is activated to splice the bZIP60 mRNA that produces a truncated transcription factor that activates gene expression in the nucleus. The IRE1/bZIP60 pathway is associated with restricting potyvirus and potexvirus infection. This study shows that the Plantago asiatica mosaic virus (PlAMV) triple gene block 3 (TGB3) and the Turnip mosaic virus (TuMV) 6K2 proteins activate alternative transcription pathways involving the bZIP17, bZIP28, BAG7, NAC089 and NAC103 factors in Arabidopsis thaliana. Using the corresponding knockout mutant lines, we show that bZIP17, bZIP60, BAG7 and NAC089 are factors in reducing PlAMV infection, whereas bZIP28 and bZIP60 are factors in reducing TuMV infection. We propose a model in which bZIP60 and bZIP17 synergistically induce genes restricting PlAMV infection, while bZIP60 and bZIP28 independently induce genes supporting PlAMV infection. Regarding TuMV-green fluorescent protein (GFP) infection, bZIP60 and bZIP28 serve to repress local and systemic infection. Finally, tauroursodeoxycholic acid treatments were used to demonstrate that the protein folding capacity significantly influences PlAMV accumulation.

John Peter Carr.

John Peter Carr.

The stability and antiapoptotic activity of Bm30K-3 can be improved by lysine acetylation in the silkworm, Bombyx mori.

Acetylation is an important, highly conserved, and reversible post-translational modification of proteins. Previously, we showed by nano-HPLC/MS/MS that many nutrient storage proteins in the silkworm are acetylated. Among these proteins, most of the known 30K proteins were shown to be acetylated, including 23 acetylated 30K proteins containing 49 acetylated sites (Kac), indicating the importance of the acetylation of 30K proteins in silkworm. In this study, Bm30K-3, a 30K protein containing three Kac sites, was further assessed in functional studies of its acetylation. Increasing the level of Bm30K-3 acetylation by adding the deacetylase inhibitor trichostatin A (TSA) increased the levels of this protein and further inhibited cellular apoptosis induced by H2 O2 . In contrast, decreasing the level of acetylation by adding the acetylase inhibitor C646 could reduce the level of Bm30K-3 and increase H2 O2 -induced apoptosis. Subsequently, BmN cells were treated with CHX and MG132, and increasing the acetylation level using TSA was shown to inhibit protein degradation and improve the stability of Bm30K-3. Furthermore, the acetylation of Bm30K-3 could compete with its ability to be ubiquitinated, suggesting that acetylation could inhibit the ubiquitin-mediated proteasome degradation pathway, improving the stability and accumulation of proteins in cells. These results further indicate that acetylation might regulate nutrition storage and utilization in Bombyx mori, which requires further study.

CNS myelin protein 36K regulates oligodendrocyte differentiation through Notch.

In contrast to humans and other mammals, zebrafish can successfully regenerate and remyelinate central nervous system (CNS) axons following injury. In addition to common myelin proteins found in mammalian myelin, 36K protein is a major component of teleost fish CNS myelin. Although 36K is one of the most abundant proteins in zebrafish brain, its function remains unknown. Here we investigate the function of 36K using translation-blocking Morpholinos. Morphant larvae showed fewer dorsally migrated oligodendrocyte precursor cells as well as upregulation of Notch ligand. A gamma secretase inhibitor, which prevents activation of Notch, could rescue oligodendrocyte precursor cell numbers in 36K morphants, suggesting that 36K regulates initial myelination through inhibition of Notch signaling. Since 36K like other short chain dehydrogenases might act on lipids, we performed thin layer chromatography and mass spectrometry of lipids and found changes in lipid composition in 36K morphant larvae. Altogether, we suggest that during early development 36K regulates membrane lipid composition, thereby altering the amount of transmembrane Notch ligands and the efficiency of intramembrane gamma secretase processing of Notch and thereby influencing oligodendrocyte precursor cell differentiation and further myelination. Further studies on the role of 36K short chain dehydrogenase in oligodendrocyte precursor cell differentiation during remyelination might open up new strategies for remyelination therapies in human patients.

Identification of Sugarcane Host Factors Interacting with the 6K2 Protein of the Sugarcane Mosaic Virus

The 6K2 protein of potyviruses plays a key role in the viral infection in plants. In the present study, the coding sequence of 6K2 was cloned from Sugarcane mosaic virus (SCMV) strain FZ1 into pBT3-STE to generate the plasmid pBT3-STE-6K2, which was used as bait to screen a cDNA library prepared from sugarcane plants infected with SCMV based on the DUALmembrane system. One hundred and fifty-seven positive colonies were screened and sequenced, and the corresponding full-length genes were cloned from sugarcane cultivar ROC22. Then, 24 genes with annotations were obtained, and the deduced proteins were classified into three groups, in which eight proteins were involved in the stress response, 12 proteins were involved in transport, and four proteins were involved in photosynthesis based on their biological functions. Of the 24 proteins, 20 proteins were verified to interact with SCMV-6K2 by yeast two-hybrid assays. The possible roles of these proteins in SCMV infection on sugarcane are analyzed and discussed. This is the first report on the interaction of SCMV-6K2 with host factors from sugarcane, and will improve knowledge on the mechanism of SCMV infection in sugarcane.

The effect of amantadine on an ion channel protein from Chikungunya virus.

Viroporins like influenza A virus M2, hepatitis C virus p7, HIV-1 Vpu and picornavirus 2B associate with host membranes, and create hydrophilic corridors, which are critical for viral entry, replication and egress. The 6K proteins from alphaviruses are conjectured to be viroporins, essential during egress of progeny viruses from host membranes, although the analogue in Chikungunya Virus (CHIKV) remains relatively uncharacterized. Using a combination of electrophysiology, confocal and electron microscopy, and molecular dynamics simulations we show for the first time that CHIKV 6K is an ion channel forming protein that primarily associates with endoplasmic reticulum (ER) membranes. The ion channel activity of 6K can be inhibited by amantadine, an antiviral developed against the M2 protein of Influenza A virus; and CHIKV infection of cultured cells can be effectively inhibited in presence of this drug. Our study provides crucial mechanistic insights into the functionality of 6K during CHIKV-host interaction and suggests that 6K is a potential therapeutic drug target, with amantadine and its derivatives being strong candidates for further development.

Molecular characterization of a new vitivirus discovered in a blueberry plant with green mosaic symptoms.

A new virus belonging to the genus Vitivirus in the family Betaflexiviridae was identified by next-generation sequencing of a blueberry plant showing green mosaic symptoms. The genome organization of the virus, which is tentatively named "blueberry green mosaic-associated virus" (BGMaV), is typical of vitiviruses, with five open reading frames (ORFs) and a polyadenylated 3' terminus. The ORFs code for the viral replicase, a 16K protein of unknown function, a movement protein, a coat protein (CP), and a nucleic acid binding protein. Phylogenetic analyses based on the deduced amino acid sequence of the CP and conserved motifs of the RNA-dependent RNA polymerase confirmed the taxonomic placement of BGMaV in the genus Vitivirus.

Interaction of a plant virus protein with the signature Cajal body protein coilin facilitates salicylic acid‐mediated plant defence responses

In addition to well-known roles in RNA metabolism, the nucleolus and Cajal bodies (CBs), both located within the nucleus, are involved in plant responses to biotic and abiotic stress. Previously we showed that plants in which expression of the CB protein coilin is downregulated are more susceptible to certain viruses including tobacco rattle virus (TRV), suggesting a role of coilin in antiviral defence. Experiments with coilin-deficient plants and the deletion mutant of the TRV 16K protein showed that both 16K and coilin are required for restriction of systemic TRV infection. The potential mechanisms of coilin-mediated antiviral defence were elucidated via experiments involving co-immunoprecipitation, use of NahG transgenic plants deficient in salicylic acid (SA) accumulation, measurement of endogenous SA concentrations and assessment of SA-responsive gene expression. Here we show that TRV 16K interacts with and relocalizes coilin to the nucleolus. In wild-type plants these events are accompanied by activation of SA-responsive gene expression and restriction of TRV systemic infection. By contrast, viral systemic spread was enhanced in NahG plants, implicating SA in these processes. Our findings suggest that coilin is involved in plant defence, responding to TRV infection by recognition of the TRV-encoded 16K protein and activating SA-dependent defence pathways.

Proteomic Identification of Immune-Related Silkworm Proteins Involved in the Response to Bacterial Infection

Bombyx mori (Lepidoptera: Bombycidae) is an important economic insect and a classic Lepidopteran model system. Although immune-related genes have been identified at a genome-wide scale in the silkworm, proteins involved in immune defense of the silkworm have not been comprehensively characterized. In this study, two types of bacteria were injected into the silkworm larvae, Gram-negative Escherichia coli (Enterobacteriales: Enterobacteriaceae), or Gram-positive Staphylococcus aureus (Bacillales: Staphylococcaceae). After injection, proteomic analyses of hemolymph were performed by liquid chromatography—tandem mass spectrometry. In total, 514 proteins were identified in the uninduced control group, 540 were identified in the E. coli-induced group, and 537 were identified in the S. aureus-induced group. Based on Uniprot annotations, 32 immunological recognition proteins, 28 immunological signaling proteins, and 21 immunological effector proteins were identified. We found that 127 proteins showed significant upregulation, including 10 immunological recognition proteins, 4 immunological signaling proteins, 11 immunological effector proteins, and 102 other proteins. Using real-time quantitative polymerase chain reaction in the fat body, we verified that immunological recognition proteins, signaling proteins, and effector proteins also showed significant increases at the transcriptional level after infection with E. coli and S. aureus. Five newly identified proteins showed upregulation at both protein and transcription levels after infection, including 30K protein, yellow-d protein, chemosensory protein, and two uncharacterized proteins. This study identified many new immune-related proteins, deepening our understanding of the immune defense system in B. mori. The data have been deposited to the iProX with identifier IPX0001337000.

Evaluation of life cycle defective adenovirus mutants for production of adeno-associated virus vectors.

Adeno-associated virus-based vectors are efficient and safe drug candidates for different in vivo gene therapy applications. With increasing numbers of clinical studies based on AAV2 vectors that include not only rare, but also common diseases as a therapeutic target, there is an increased demand for the development of improved production technologies. In the present study, we compared two life cycle defective adenovirus mutants as helper viruses for AAV2 vector production. They had deletions either in the gene coding for the preterminal protein (pTP) that is expressed early in the viral life cycle and is essential for genome replication or in the gene coding for the 100K protein, a protein with many functions, one of which is involved in virus assembly. AAV2 vector production efficiencies were evaluated by analyzing genome-containing particles using a real-time polymerase chain reaction and functional units were investigated by transduction assays. Somewhat contrary to our expectations, the ∆100K mutant virus showed only a moderate efficiency as a helper virus for AAV2 vector production, whereas the replication-deficient ∆pTP mutant supported AAV2 production almost as efficiently as adenovirus wild-type. We also showed that a temperature shift to 32°C together with extended incubation times improved AAV2 vector productivity. The present study indicates the advantages of using a ∆pTP mutant adenovirus rather than adenovirus wild-type as a helper virus for AAV2 production and also indicates that temperature shifts to lower temperatures may improve AAV2 vector production rates.

GLUT1 and GLUT8 support lactose synthesis in Golgi of murine mammary epithelial cells.

The mammary gland increases energy requirements during pregnancy and lactation to support epithelial proliferation and milk nutrients synthesis. Lactose, the principal carbohydrate of the milk, is synthetized in the Golgi of mammary epithelial cells by lactose synthase from glucose and UPD galactose. We studied the temporal changes in the expression of GLUT1 and GLUT8 in mammary gland and their association with lactose synthesis and proliferation in BALB/c mice. Six groups were used: virgin, pregnant at 2 and 17days, lactating at 2 and 10days, and weaning at 2days. Temporal expression of GLUT1 and GLUT8 transporters by qPCR, western blot and immunohistochemistry, and its association with lactalbumin, Ki67, and cytokeratin 18 within mammary tissue was studied, along with subcellular localization. GLUT1 and GLUT8 transporters increased their expression during mammary gland progression, reaching 20-fold increasing in GLUT1 mRNA at lactation (p < 0.05) and 2-fold at protein level for GLUT1 and GLUT8 (p < 0.05 and 0.01, respectively). The temporal expression pattern was shared with cytokeratin 18 and Ki67 (p < 0.01). Endogenous GLUT8 partially co-localized with 58K protein and α-lactalbumin in mammary tissue and with Golgi membrane-associated protein 130in isolated epithelial cells. The spatial-temporal synchrony between expression of GLUT8/GLUT1 and alveolar cell proliferation, and its localization in cis-Golgi associated to lactose synthase complex, suggest that both transporters are involved in glucose uptake into this organelle, supporting lactose synthesis.

Mutagenesis Scanning Uncovers Evolutionary Constraints on Tobacco Etch Potyvirus Membrane-Associated 6K2 Protein.

RNA virus high mutation rate is a double-edged sword. At the one side, most mutations jeopardize proteins functions; at the other side, mutations are needed to fuel adaptation. The relevant question then is the ratio between beneficial and deleterious mutations. To evaluate this ratio, we created a mutant library of the 6K2 gene of tobacco etch potyvirus that contains every possible single-nucleotide substitution. 6K2 protein anchors the virus replication complex to the network of endoplasmic reticulum membranes. The library was inoculated into the natural host Nicotiana tabacum, allowing competition among all these mutants and selection of those that are potentially viable. We identified 11 nonsynonymous mutations that remain in the viral population at measurable frequencies and evaluated their fitness. Some had fitness values higher than the wild-type and some were deleterious. The effect of these mutations in the structure, transmembrane properties, and function of 6K2 was evaluated in silico. In parallel, the effect of these mutations in infectivity, virus accumulation, symptoms development, and subcellular localization was evaluated in the natural host. The α-helix H1 in the N-terminal part of 6K2 turned out to be under purifying selection, while most observed mutations affect the link between transmembrane α-helices H2 and H3, fusing them into a longer helix and increasing its rigidity. In general, these changes are associated with higher within-host fitness and development of milder or no symptoms. This finding suggests that in nature selection upon 6K2 may result from a tradeoff between within-host accumulation and severity of symptoms.

A novel myelin protein zero transgenic zebrafish designed for rapid readout of in vivo myelination.

Demyelination occurs following many neurological insults, most notably in multiple sclerosis (MS). Therapeutics that promote remyelination could slow the neurological decline associated with chronic demyelination; however, in vivo testing of candidate small molecule drugs and signaling cascades known to impact myelination is expensive and labor intensive. Here, we describe the development of a novel zebrafish line which uses the putative promoter of Myelin Protein Zero (mpz), a major structural protein in myelin, to drive expression of Enhanced Green Fluorescent Protein (mEGFP) specifically in the processes and nascent internodes of myelinating glia. We observe that changes in fluorescence intensity in Tg(mpz:mEGFP) larvae are a reliable surrogate for changes in myelin membrane production per se in live larvae following bath application of drugs. These changes in fluorescence are strongly predictive of changes in myelin-specific mRNAs [mpz, 36K and myelin basic protein (mbp)] and protein production (Mbp). Finally, we observe that certain drugs alter nascent internode number and length, impacting the overall amount of myelin membrane synthesized and a number of axons myelinated without significantly changing the number of myelinating oligodendrocytes. These studies demonstrate that the Tg(mpz:mEGFP) reporter line responds effectively to positive and negative small molecule regulators of myelination, and could be useful for identifying candidate drugs that specifically target myelin membrane production in vivo. Combined with high throughput cell-based screening of large chemical libraries and automated imaging systems, this transgenic line is useful for rapid large scale whole animal screening to identify novel myelinating small molecule compounds in vivo.