Science.gov

Sample records for aav capsid proteins

  1. Mutants at the 2-Fold Interface of Adeno-associated Virus Type 2 (AAV2) Structural Proteins Suggest a Role in Viral Transcription for AAV Capsids

    PubMed Central

    Aydemir, Fikret; Salganik, Maxim; Resztak, Justyna; Singh, Jasbir; Bennett, Antonette; Agbandje-McKenna, Mavis

    2016-01-01

    ABSTRACT We previously reported that an amino acid substitution, Y704A, near the 2-fold interface of adeno-associated virus (AAV) was defective for transcription of the packaged genome (M. Salganik, F. Aydemir, H. J. Nam, R. McKenna, M. Agbandje-McKenna, and N. Muzyczka, J Virol 88:1071–1079, 2013, doi: http://dx.doi.org/10.1128/JVI.02093-13). In this report, we have characterized the defect in 6 additional capsid mutants located in a region ∼30 Å in diameter on the surface of the AAV type 2 (AAV2) capsid near the 2-fold interface. These mutants, which are highly conserved among primate serotypes, displayed a severe defect (3 to 6 logs) in infectivity. All of the mutants accumulated significant levels of uncoated DNA in the nucleus, but none of the mutants were able to accumulate significant amounts of genomic mRNA postinfection. In addition, wild-type (wt) capsids that were bound to the conformational antibody A20, which is known to bind the capsid surface in the region of the mutants, were also defective for transcription. In all cases, the mutant virus particles, as well as the antibody-bound wild-type capsids, were able to enter the cell, travel to the nucleus, uncoat, and synthesize a second strand but were unable to transcribe their genomes. Taken together, the phenotype of these mutants provides compelling evidence that the AAV capsid plays a role in the transcription of its genome, and the mutants map this functional region on the surface of the capsid near the 2-fold interface. This appears to be the first example of a viral structural protein that is also involved in the transcription of the viral genome that it delivers to the nucleus. IMPORTANCE Many viruses package enzymes within their capsids that assist in expressing their genomes postinfection, e.g., retroviruses. A number of nonenveloped viruses, including AAV, carry proteases that are needed for capsid maturation or for capsid modification during infection. We describe here what appears to

  2. A 110-kDa nuclear shuttle protein, nucleolin, specifically binds to adeno-associated virus type 2 (AAV-2) capsid.

    PubMed

    Qiu, J; Brown, K E

    1999-05-10

    A 110-kDa protein was copurified with adeno-associated virus type 2 (AAV-2) virions after CsCl density gradient isopycnic centrifugation. Amino acid sequence of peptides derived from this protein after tryptic digestion, monoclonal antibody production, and Western blot analysis showed that the copurified protein was the major nucleolar phosphoprotein, human nucleolin. Virus overlay assays demonstrated that AAV-2 capsid specifically bound to the human nucleolin, and immunoprecipitation studies confirmed the in vitro binding of nucleolin and intact AAV-2 capsids but not denatured viral proteins. Double-immunofluorescence staining of infected cells showed that AAV capsid and nucleolin were colocalized in both cytoplasm and nucleus. In addition, when cytoplasmic and nuclear fractions were extracted from AAV-infected KB cells at different time points postinfection, immunoprecipitation data and Western blotting showed that AAV capsid formation and nucleolin interact specifically and share their subcellular localization in infected cells. With the known functions of nucleolin in the synthesis of rRNA and ribosome assembly, binding to single-stranded DNA, and acting as a shuttle between cytoplasm and nucleolus, our data showing that AAV-2 capsid binds specifically to nucleolin both in vitro and in vivo suggest a key role of nucleolin in AAV-2 replication, particularly in capsid assembly. PMID:10329548

  3. Novel Caprine Adeno-Associated Virus (AAV) Capsid (AAV-Go.1) Is Closely Related to the Primate AAV-5 and Has Unique Tropism and Neutralization Properties

    PubMed Central

    Arbetman, Alejandra E.; Lochrie, Michael; Zhou, Shangzhen; Wellman, Jennifer; Scallan, Ciaran; Doroudchi, Mohammad M.; Randlev, Britta; Patarroyo-White, Susannah; Liu, Tongyao; Smith, Peter; Lehmkuhl, Howard; Hobbs, Lea Ann; Pierce, Glenn F.; Colosi, Peter

    2005-01-01

    Preexisting humoral immunity to adeno-associated virus (AAV) vectors may limit their clinical utility in gene delivery. We describe a novel caprine AAV (AAV-Go.1) capsid with unique biological properties. AAV-Go.1 capsid was cloned from goat-derived adenovirus preparations. Surprisingly, AAV-Go.1 capsid was 94% identical to the human AAV-5, with differences predicted to be largely on the surface and on or under the spike-like protrusions. In an in vitro neutralization assay using human immunoglobulin G (IgG) (intravenous immune globulin [IVIG]), AAV-Go.1 had higher resistance than AAV-5 (100-fold) and resistance similar to that of AAV-4 or AAV-8. In an in vivo model, SCID mice were pretreated with IVIG to generate normal human IgG plasma levels prior to the administration of AAV human factor IX vectors. Protein expression after intramuscular administration of AAV-Go.1 was unaffected in IVIG-pretreated mice, while it was reduced 5- and 10-fold after administration of AAV-1 and AAV-8, respectively. In contrast, protein expression after intravenous administration of AAV-Go.1 was reduced 7.1-fold, similar to the 3.8-fold reduction observed after AAV-8administration in IVIG-pretreated mice, and protein expression was essentially extinguished after AAV-2 administration in mice pretreated with much less IVIG (15-fold). AAV-Go.1 vectors also demonstrated a marked tropism for lung when administered intravenously in SCID mice. The pulmonary tropism and high neutralization resistance to human preexisting antibodies suggest novel therapeutic uses for AAV-Go.1 vectors, including targeting diseases such as cystic fibrosis. Nonprimate sources of AAVs may be useful to identify additional capsids with distinct tropisms and high resistance to neutralization by human preexisting antibodies. PMID:16306595

  4. Targeted Gene Delivery to the Enteric Nervous System Using AAV: A Comparison Across Serotypes and Capsid Mutants

    PubMed Central

    Benskey, Matthew J; Kuhn, Nathan C; Galligan, James J; Garcia, Joanna; Boye, Shannon E; Hauswirth, William W; Mueller, Christian; Boye, Sanford L; Manfredsson, Fredric P

    2015-01-01

    Recombinant adeno-associated virus (AAV) vectors are one of the most widely used gene transfer systems in research and clinical trials. AAV can transduce a wide range of biological tissues, however to date, there has been no investigation on targeted AAV transduction of the enteric nervous system (ENS). Here, we examined the efficiency, tropism, spread, and immunogenicity of AAV transduction in the ENS. Rats received direct injections of various AAV serotypes expressing green fluorescent protein (GFP) into the descending colon. AAV serotypes tested included; AAV 1, 2, 5, 6, 8, or 9 and the AAV2 and AAV8 capsid mutants, AAV2-Y444F, AAV2-tripleY-F, AAV2-tripleY-F+T-V, AAV8-Y733F, and AAV8-doubeY-F+T-V. Transduction, as determined by GFP-positive cells, occurred in neurons and enteric glia within the myenteric and submucosal plexuses of the ENS. AAV6 and AAV9 showed the highest levels of transduction within the ENS. Transduction efficiency scaled with titer and time, was translated to the murine ENS, and produced no vector-related immune response. A single injection of AAV into the colon covered an area of ~47 mm2. AAV9 primarily transduced neurons, while AAV6 transduced enteric glia and neurons. This is the first report on targeted AAV transduction of neurons and glia in the ENS. PMID:25592336

  5. Microglia-specific targeting by novel capsid-modified AAV6 vectors

    PubMed Central

    Rosario, Awilda M; Cruz, Pedro E; Ceballos-Diaz, Carolina; Strickland, Michael R; Siemienski, Zoe; Pardo, Meghan; Schob, Keri-Lyn; Li, Andrew; Aslanidi, George V; Srivastava, Arun; Golde, Todd E; Chakrabarty, Paramita

    2016-01-01

    Recombinant adeno-associated viruses (rAAV) have been widely used in gene therapy applications for central nervous system diseases. Though rAAV can efficiently target neurons and astrocytes in mouse brains, microglia, the immune cells of the brain, are refractile to rAAV. To identify AAV capsids with microglia-specific transduction properties, we initially screened the most commonly used serotypes, AAV1–9 and rh10, on primary mouse microglia cultures. While these capsids were not permissive, we then tested the microglial targeting properties of a newly characterized set of modified rAAV6 capsid variants with high tropism for monocytes. Indeed, these newly characterized rAAV6 capsid variants, specially a triply mutated Y731F/Y705F/T492V form, carrying a self-complementary genome and microglia-specific promoters (F4/80 or CD68) could efficiently and selectively transduce microglia in vitro. Delivery of these constructs in mice brains resulted in microglia-specific expression of green fluorescent protein, albeit at modest levels. We further show that CD68 promoter–driven expression of the inflammatory cytokine, interleukin-6, using this capsid variant leads to increased astrogliosis in the brains of wild-type mice. Our study describes the first instance of AAV-targeted microglial gene expression leading to functional modulation of the innate immune system in mice brains. This provides the rationale for utilizing these unique capsid/promoter combinations for microglia-specific gene targeting for modeling or functional studies. PMID:27308302

  6. Tyrosine capsid-mutant AAV vectors for gene delivery to the canine retina from a subretinal or intravitreal approach

    PubMed Central

    Mowat, FM; Gornik, KR; Dinculescu, A; Boye, SL; Hauswirth, WW; Petersen-Jones, SM; Bartoe, JT

    2013-01-01

    Recombinant adeno-associated viruses are important vectors for retinal gene delivery. Currently utilized vectors have relatively slow onset and for efficient transduction it is necessary to deliver treatment subretinally, with the potential for damage to the retina. Amino-acid substitutions in the viral capsid improve efficiency in rodent eyes by evading host responses. As dogs are important large animal models for human retinitis pigmentosa, we evaluated the speed and efficiency of retinal transduction using capsid-mutant vectors injected both subretinally and intravitreally. We evaluated AAV serotypes 2 and 8 with amino-acid substitutions of surface exposed capsid tyrosine residues. The chicken beta-actin promoter was used to drive green fluorescent protein expression. Twelve normal adult beagles were injected, 4 dogs received intravitreal injections, 8 dogs received subretinal injections. Capsid-mutant viruses tested included AAV2(quad Y-F) (intravitreal and subretinal), and self-complementary scAAV8(Y733F) (subretinal only). Contralateral control eyes received injections of scAAV5 (subretinal) or scAAV2 (intravitreal). Subretinally delivered vectors had a faster expression onset than intravitreally delivered vectors. Subretinally delivered scAAV8(Y733F) had a faster onset of expression than scAAV5. All subretinally injected vector types transduced the outer retina with high efficiency, and the inner retina with moderate efficiency. Intravitreally delivered AAV2(quad Y-F) had a marginally higher efficiency of transduction of both outer retinal and inner retinal cells than scAAV2. Because of their rapid expression onset and efficient transduction, subretinally delivered capsid-mutant AAV8 vectors may increase the efficacy of gene therapy treatment for rapid photoreceptor degenerative diseases. With further refinement, capsid-mutant AAV2 vectors show promise for retinal gene delivery from an intravitreal approach. PMID:24225638

  7. AAV8 capsid variable regions at the two-fold symmetry axis contribute to high liver transduction by mediating nuclear entry and capsid uncoating

    SciTech Connect

    Tenney, Rebeca M.; Bell, Christie L.; Wilson, James M.

    2014-04-15

    Adeno-associated virus serotype 8 (AAV8) is a promising vector for liver-directed gene therapy. Although efficient uncoating of viral capsids has been implicated in AAV8's robust liver transduction, much about the biology of AAV8 hepatotropism remains unclear. Our study investigated the structural basis of AAV8 liver transduction efficiency by constructing chimeric vector capsids containing sequences derived from AAV8 and AAV2 – a highly homologous yet poorly hepatotropic serotype. Engineered vectors containing capsid variable regions (VR) VII and IX from AAV8 in an AAV2 backbone mediated near AAV8-like transduction in mouse liver, with higher numbers of chimeric genomes detected in whole liver cells and isolated nuclei. Interestingly, chimeric capsids within liver nuclei also uncoated similarly to AAV8 by 6 weeks after administration, in contrast with AAV2, of which a significantly smaller proportion were uncoated. This study links specific AAV capsid regions to the transduction ability of a clinically relevant AAV serotype. - Highlights: • We construct chimeric vectors to identify determinants of AAV8 liver transduction. • An AAV2-based vector with 17 AAV8 residues exhibited high liver transduction in mice. • This vector also surpassed AAV2 in cell entry, nuclear entry and onset of expression. • Most chimeric vector particles were uncoated at 6 weeks, like AAV8 and unlike AAV2. • Chimera retained heparin binding and was antigenically distinct from AAV2 and AAV8.

  8. AAV capsid CD8+ T-cell epitopes are highly conserved across AAV serotypes

    PubMed Central

    Hui, Daniel J; Edmonson, Shyrie C; Podsakoff, Gregory M; Pien, Gary C; Ivanciu, Lacramioara; Camire, Rodney M; Ertl, Hildegund; Mingozzi, Federico; High, Katherine A; Basner-Tschakarjan, Etiena

    2015-01-01

    Adeno-associated virus (AAV) has become one of the most promising vectors in gene transfer in the last 10 years with successful translation to clinical trials in humans and even market approval for a first gene therapy product in Europe. Administration to humans, however, revealed that adaptive immune responses against the vector capsid can present an obstacle to sustained transgene expression due to the activation and expansion of capsid-specific T cells. The limited number of peripheral blood mononuclear cells (PBMCs) obtained from samples within clinical trials allows for little more than monitoring of T-cell responses. We were able to identify immunodominant major histocompatibility complex (MHC) class I epitopes for common human leukocyte antigen (HLA) types by using spleens isolated from subjects undergoing splenectomy for non-malignant indications as a source of large numbers of lymphocytes and restimulating them with single AAV capsid peptides in vitro. Further experiments confirmed that these epitopes are naturally processed and functionally relevant. The design of more effective and less immunogenic AAV vectors, and precise immune monitoring of vector-infused subjects, are facilitated by these findings. PMID:26445723

  9. Reprogramming Immune Response With Capsid-Optimized AAV6 Vectors for Immunotherapy of Cancer.

    PubMed

    Pandya, Munjal; Britt, Kellee; Hoffman, Brad; Ling, Chen; Aslanidi, George V

    2015-09-01

    In the current studies we generated novel capsid-optimized adeno-associated virus (AAV) serotype 6 (AAV6) vectors expressing a tumor-associated antigen, and assessed their ability to activate a protective T-cell response in an animal model. First, we showed that specific mutations in the AAV6 capsid increase the transduction efficiency of these vectors in mouse bone marrow-derived dendritic cells in vitro for approximately 5-fold compared with the wild-type (WT) AAV6 vectors. Next, we evaluated the ability of the mutant AAV6 vectors to initiate specific T-cell clone proliferation in vivo. Our data indicate that the intramuscular administration of AAV6-S663V+T492V vectors expressing ovalbumin (OVA) led to a strong activation (approximately 9%) of specific T cells in peripheral blood compared with AAV6-WT treated animals (<1%). These OVA-specific T cells have a superior killing ability against mouse prostate cancer cell line RM1 stably expressing the OVA antigen when propagated in vitro. Finally, we evaluated the ability of capsid-optimized AAV6-S663V+T492V vectors to initiate a protective anticancer immune response in vivo. Our results document the suppression of subcutaneous tumor growth in animals immunized with AAV6-S663V+T492V vectors expressing prostatic acid phosphatase (PAP) for approximately 4 weeks in comparison with 1 week and 2 weeks for the negative controls, AAV6-EGFP, and AAV6-WT-PAP treated mice, respectively. These studies suggest that successful inhibition of tumor growth in an animal model would set the stage for potential clinical application of the capsid-optimized AAV6-S663V+T492V vectors. PMID:26261893

  10. Controlling AAV Tropism in the Nervous System with Natural and Engineered Capsids

    PubMed Central

    Castle, Michael J.; Turunen, Heikki T.; Vandenberghe, Luk H.; Wolfe, John H.

    2016-01-01

    More than one hundred naturally occurring variants of adeno-associated virus (AAV) have been identified, and this library has been further expanded by an array of techniques for modification of the viral capsid. AAV capsid variants possess unique antigenic profiles and demonstrate distinct cellular tropisms driven by differences in receptor binding. AAV capsids can be chemically modified to alter tropism, can be produced as hybrid vectors that combine the properties of multiple serotypes, and can carry peptide insertions that introduce novel receptor-binding activity. Furthermore, directed evolution of shuffled genome libraries can identify engineered variants with unique properties, and rational modification of the viral capsid can alter tropism, reduce blockage by neutralizing antibodies, or enhance transduction efficiency. This large number of AAV variants and engineered capsids provides a varied toolkit for gene delivery to the CNS and retina, with specialized vectors available for many applications, but selecting a capsid variant from the array of available vectors can be difficult. This chapter describes the unique properties of a range of AAV variants and engineered capsids, and provides a guide for selecting the appropriate vector for specific applications in the CNS and retina. PMID:26611584

  11. Engineering the AAV capsid to optimize vector-host-interactions.

    PubMed

    Büning, Hildegard; Huber, Anke; Zhang, Liang; Meumann, Nadja; Hacker, Ulrich

    2015-10-01

    Adeno-associated viral (AAV) vectors are the most widely used delivery system for in vivo gene therapy. Vectors developed from natural AAV isolates achieved clinical benefit for a number of patients suffering from monogenetic disorders. However, high vector doses were required and the presence of pre-existing neutralizing antibodies precluded a number of patients from participation. Further challenges are related to AAV's tropism that lacks cell type selectivity resulting in off-target transduction. Conversely, specific cell types representing important targets for gene therapy like stem cells or endothelial cells show low permissiveness. To overcome these limitations, elegant rational design- as well as directed evolution-based strategies were developed to optimize various steps of AAV's host interaction. These efforts resulted in next generation vectors with enhanced capabilities, that is increased efficiency of cell transduction, targeted transduction of previously non-permissive cell types, escape from antibody neutralization and off-target free in vivo delivery of vector genomes. These important achievements are expected to improve current and pave the way towards novel AAV-based applications in gene therapy and regenerative medicine. PMID:26302254

  12. In Vivo Selection Yields AAV-B1 Capsid for Central Nervous System and Muscle Gene Therapy.

    PubMed

    Choudhury, Sourav R; Fitzpatrick, Zachary; Harris, Anne F; Maitland, Stacy A; Ferreira, Jennifer S; Zhang, Yuanfan; Ma, Shan; Sharma, Rohit B; Gray-Edwards, Heather L; Johnson, Jacob A; Johnson, Aime K; Alonso, Laura C; Punzo, Claudio; Wagner, Kathryn R; Maguire, Casey A; Kotin, Robert M; Martin, Douglas R; Sena-Esteves, Miguel

    2016-08-01

    Adeno-associated viral (AAV) vectors have shown promise as a platform for gene therapy of neurological disorders. Achieving global gene delivery to the central nervous system (CNS) is key for development of effective therapies for many of these diseases. Here we report the isolation of a novel CNS tropic AAV capsid, AAV-B1, after a single round of in vivo selection from an AAV capsid library. Systemic injection of AAV-B1 vector in adult mice and cat resulted in widespread gene transfer throughout the CNS with transduction of multiple neuronal subpopulations. In addition, AAV-B1 transduces muscle, β-cells, pulmonary alveoli, and retinal vasculature at high efficiency. This vector is more efficient than AAV9 for gene delivery to mouse brain, spinal cord, muscle, pancreas, and lung. Together with reduced sensitivity to neutralization by antibodies in pooled human sera, the broad transduction profile of AAV-B1 represents an important improvement over AAV9 for CNS gene therapy. PMID:27117222

  13. Single tyrosine mutation in AAV8 and AAV9 capsids is insufficient to enhance gene delivery to skeletal muscle and heart.

    PubMed

    Qiao, Chunping; Yuan, Zhenhua; Li, Jianbin; Tang, Ruhang; Li, Juan; Xiao, Xiao

    2012-02-01

    Site-directed mutations of tyrosine (Y) to phenylalanine (F) on the surface of adeno-associated viral (AAV) capsids have been reported as a simple method to greatly enhance gene transfer in vitro and in vivo. To determine whether the Y-to-F mutation could also enhance AAV8 and AAV9 gene transfer in skeletal muscle and heart to facilitate muscular dystrophy gene therapy, we investigated four capsid mutants of AAV8 (Y447F or Y733F) and AAV9 (Y446F or Y731F). The mutants and their wild-type control AAV8 and AAV9 capsids were used to package reporter genes (luciferase or β-galactosidase) resulting in similar vector yields. To evaluate gene delivery efficiencies, especially in muscle and heart, the vectors were compared side by side in a series of experiments in vivo in two different strains of mice, the outbred ICR and the inbred C57BL/6. Because AAV8 and AAV9 are among the most effective in systemic gene delivery, we first examined the mutant and wild-type vectors in neonatal mice by intraperitoneal injection, or in adult mice by intravenous injection. To our surprise, no statistically significant differences in transgene expression were observed between the mutant and wild-type vectors, regardless of the reporter genes, vector doses, and the ages and strains of mice used. In addition, quantitative analyses of vector DNA copy number in various tissues from mice treated with mutant and wild-type vectors also showed similar results. Finally, direct intramuscular injection of the above-described vectors with the luciferase gene into the hind limb muscles revealed the same levels of gene expression between mutant and wild-type vectors. Our results thus demonstrate that a single mutation of Y447F or Y733F on capsids of AAV8, and of Y446F or Y731F on AAV9, is insufficient to enhance gene delivery to the skeletal muscle and heart. PMID:22428978

  14. Slow AAV2 clearance from the brain of nonhuman primates and anti-capsid immune response.

    PubMed

    Samaranch, L; Hadaczek, P; Kells, A P; Bringas, J R; Stockinger, D; San Sebastian, W; Macayan, M; Samineni, S; Pivirotto, P; Forsayeth, J; Bankiewicz, K S

    2016-04-01

    Adeno-associated virus serotype 2 (AAV2) has previously been reported to be a slowly uncoating virus in peripheral tissues, but persistence of intact vector in primate brain has not been explored. Because some neurological gene therapies may require re-administration of the same vector to patients, it seems important to understand the optimal timeframe in which to consider such repeat intervention. Surprisingly, convection-enhanced delivery of AAV2 into the thalamus of nonhuman primates (NHPs) resulted in robust staining of neurons with A20 antibody that detected intact AAV2 particles at ∼1.5 months after infusion. However, by 2.5 months, no A20 staining was visible. These data confirmed earlier findings of persistence of intact AAV2 particles in ocular and hepatic tissues. In order to probe the potential consequences of this persistence, we infused AAV2-human aromatic L-amino acid decarboxylase into left and right thalamus of three NHPs, with a 3-month delay between infusions. During that interval, we immunized each animal subcutaneously with AAV2 virus-like particles (empty vector) in order to induce strong anti-capsid humoral immunity. Various high neutralizing antibody titers were achieved. The lowest titer animal showed infiltration of B lymphocytes and CD8(+) T cells into both the secondary and primary infusion sites. In the other two animals, extremely high titers resulted in no transduction of the second site and, therefore, no lymphocytic infiltration. However, such infiltration was prominent at the primary infusion site in each animal and was associated with overt neuronal loss and inflammation. PMID:26510688

  15. Tyrosine-phosphorylation of AAV2 vectors and its consequences on viral intracellular trafficking and transgene expression

    SciTech Connect

    Zhong Li; Li Baozheng; Jayandharan, Giridhararao; Mah, Cathryn S.; Govindasamy, Lakshmanan; Agbandje-McKenna, Mavis; Herzog, Roland W.

    2008-11-25

    We have documented that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling negatively affects intracellular trafficking and transduction efficiency of recombinant adeno-associated virus 2 (AAV2) vectors. Specifically, inhibition of EGFR-PTK signaling leads to decreased ubiquitination of AAV2 capsid proteins, which in turn, facilitates viral nuclear transport by limiting proteasome-mediated degradation of AAV2 vectors. In the present studies, we observed that AAV capsids can indeed be phosphorylated at tyrosine residues by EGFR-PTK in in vitro phosphorylation assays and that phosphorylated AAV capsids retain their structural integrity. However, although phosphorylated AAV vectors enter cells as efficiently as their unphosphorylated counterparts, their transduction efficiency is significantly reduced. This reduction is not due to impaired viral second-strand DNA synthesis since transduction efficiency of both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors is decreased by {approx} 68% and {approx} 74%, respectively. We also observed that intracellular trafficking of tyrosine-phosphorylated AAV vectors from cytoplasm to nucleus is significantly decreased, which results from ubiquitination of AAV capsids followed by proteasome-mediated degradation, although downstream consequences of capsid ubiquitination may also be affected by tyrosine-phosphorylation. These studies provide new insights into the role of tyrosine-phosphorylation of AAV capsids in various steps in the virus life cycle, which has implications in the optimal use of recombinant AAV vectors in human gene therapy.

  16. Theoretical studies of viral capsid proteins.

    PubMed

    Phelps, D K; Speelman, B; Post, C B

    2000-04-01

    Recent results in structural biology and increases in computer power have prompted initial theoretical studies on capsids of nonenveloped icosahedral viruses. The macromolecular assembly of 60 to 180 protein copies into a protein shell results in a structure of considerable size for molecular dynamics simulations. Nonetheless, progress has been made in examining these capsid assemblies from molecular dynamics calculations and kinetic models. The goals of these studies are to understand capsid function and structural properties, including quarternary structural stability, effects of antiviral compounds that bind the capsid and the self-assembly process. The insight that can be gained from the detailed information provided by simulations is demonstrated in studies of human rhinovirus; an entropic basis for the antiviral activity of hydrophobic compounds, predicted from calculated compressibility values, has been corroborated by experimental measurements on poliovirus. PMID:10753813

  17. Formation of empty B19 parvovirus capsids by the truncated minor capsid protein.

    PubMed Central

    Wong, S; Momoeda, M; Field, A; Kajigaya, S; Young, N S

    1994-01-01

    We previously reported that empty capsids of B19 parvovirus were formed by the major capsid protein (VP2) alone expressed in a baculovirus system, but the minor capsid protein (VP1), longer by 227 amino acids, alone did not form empty capsids. We report here further investigations of the constraints on capsid formation by truncated versions of VP1. Studies were performed with recombinant baculoviruses expressed in Sf9 cells. Severely shortened VP1, extended beyond the VP2 core sequence by about 70 amino acids of the unique region, formed capsids normal in appearance; longer versions of VP1 also formed capsids but did so progressively less efficiently and produced capsids of more markedly dysmorphic appearance as the VP1-unique region was lengthened. Images PMID:8207846

  18. Adeno-associated virus (AAV) Rep proteins mediate complex formation between AAV DNA and its integration site in human DNA.

    PubMed Central

    Weitzman, M D; Kyöstiö, S R; Kotin, R M; Owens, R A

    1994-01-01

    AAV is unique among eukaryotic viruses in the ability of its DNA to integrate preferentially into a specific region of the human genome. Understanding AAV integration may aid in developing gene therapy systems with predictable integration sites. Using a gel mobility-shift assay, we have identified a DNA sequence within the AAV integration locus on human chromosome 19 which is specifically bound by the AAV Rep78 and Rep68 proteins. This Rep recognition sequence is a GCTC repeating motif very similar to sequences within the inverted terminal repeats of the AAV genome which are also bound by Rep78 and Rep68. Cloned oligonucleotides containing the recognition sequence can direct specific binding by Rep proteins. Binding assays with mutant Rep proteins show that the amino-terminal portion of Rep78 and Rep68 can direct binding to either the AAV terminal repeat hairpin DNA or chromosome 19. This human genomic DNA can be complexed with AAV DNA by Rep proteins as demonstrated by a dual-label (32P/biotin) assay. These results suggest a role for Rep in targeting viral integration. Images PMID:8016070

  19. Mechanostability of Proteins and Virus Capsids

    NASA Astrophysics Data System (ADS)

    Cieplak, Marek

    2013-03-01

    Molecular dynamics of proteins within coarse grained models have become a useful tool in studies of large scale systems. The talk will discuss two applications of such modeling. The first is a theoretical survey of proteins' resistance to constant speed stretching as performed for a set of 17134 simple and 318 multidomain proteins. The survey has uncovered new potent force clamps. They involve formation of cysteine slipknots or dragging of a cystine plug through the cystine ring and lead to characteristic forces that are significantly larger than the common shear-based clamp such as observed in titin. The second application involves studies of nanoindentation processes in virus capsids and elucidates their molecular aspects by showing deviations in behavior compared to the continuum shell model. Across the 35 capsids studied, both the collapse force and the elastic stiffness are observed to vary by a factor of 20. The changes in mechanical properties do not correlate simply with virus size or symmetry. There is a strong connection to the mean coordination number < z > , defined as the mean number of interactions to neighboring amino acids. The Young's modulus for thin shell capsids rises roughly quadratically with < z > - 6, where 6 is the minimum coordination for elastic stability in three dimensions. Supported by European Regional Development Fund, through Innovative Economy grant Nanobiom (POIG.01.01.02-00-008/08)

  20. Strategies to generate high-titer, high-potency recombinant AAV3 serotype vectors

    PubMed Central

    Ling, Chen; Yin, Zifei; Li, Jun; Zhang, Daniel; Aslanidi, George; Srivastava, Arun

    2016-01-01

    Although recombinant adeno-associated virus serotype 3 (AAV3) vectors were largely ignored previously, owing to their poor transduction efficiency in most cells and tissues examined, our initial observation of the selective tropism of AAV3 serotype vectors for human liver cancer cell lines and primary human hepatocytes has led to renewed interest in this serotype. AAV3 vectors and their variants have recently proven to be extremely efficient in targeting human and nonhuman primate hepatocytes in vitro as well as in vivo. In the present studies, we wished to evaluate the relative contributions of the cis-acting inverted terminal repeats (ITRs) from AAV3 (ITR3), as well as the trans-acting Rep proteins from AAV3 (Rep3) in the AAV3 vector production and transduction. To this end, we utilized two helper plasmids: pAAVr2c3, which carries rep2 and cap3 genes, and pAAVr3c3, which carries rep3 and cap3 genes. The combined use of AAV3 ITRs, AAV3 Rep proteins, and AAV3 capsids led to the production of recombinant vectors, AAV3-Rep3/ITR3, with up to approximately two to fourfold higher titers than AAV3-Rep2/ITR2 vectors produced using AAV2 ITRs, AAV2 Rep proteins, and AAV3 capsids. We also observed that the transduction efficiency of Rep3/ITR3 AAV3 vectors was approximately fourfold higher than that of Rep2/ITR2 AAV3 vectors in human hepatocellular carcinoma cell lines in vitro. The transduction efficiency of Rep3/ITR3 vectors was increased by ~10-fold, when AAV3 capsids containing mutations in two surface-exposed residues (serine 663 and threonine 492) were used to generate a S663V+T492V double-mutant AAV3 vector. The Rep3/ITR3 AAV3 vectors also transduced human liver tumors in vivo approximately twofold more efficiently than those generated with Rep2/ITR2. Our data suggest that the transduction efficiency of AAV3 vectors can be significantly improved both using homologous Rep proteins and ITRs as well as by capsid optimization. Thus, the combined use of homologous Rep

  1. Modeling virus capsids and their protein binding -- the search for weak regions within the HIV capsid

    NASA Astrophysics Data System (ADS)

    Sankey, Otto; Benson, Daryn

    2010-10-01

    Viruses remain a threat to the health of humans worldwide with 33 million infected with AIDS. Viruses are ubiquitous infecting animals, plants, and bacteria. Each virus infects in its own unique manner making the problem seem intractable. However, some general physical steps apply to many viruses and the application of basic physical modeling can potentially have great impact. The aim of this theoretical study is to investigate the stability of the HIV viral capsid (protein shell). The structural shell can be compromised by physical probes such as pulsed laser light. But what are the weakest regions of the capsid so that we can begin to understand vulnerabilities of these deadly materials? The atomic structure of HIV capsids is not precisely known and we begin by describing our work to model the capsid structure. Next we describe a course grained model to investigate protein interactions within the capsid.

  2. Efficient lysis of epithelial ovarian cancer cells by MAGE-A3-induced cytotoxic T lymphocytes using rAAV-6 capsid mutant vector.

    PubMed

    Batchu, Ramesh B; Gruzdyn, Oksana V; Moreno-Bost, Amberly M; Szmania, Susann; Jayandharan, Giridhararao; Srivastava, Arun; Kolli, Bala K; Weaver, Donald W; van Rhee, Frits; Gruber, Scott A

    2014-02-12

    MAGE-A3 is highly expressed in epithelial ovarian cancer (EOC), making it a promising candidate for immunotherapy. We investigated whether dendritic cells (DCs) transduced with a rAAV-6 capsid mutant vector Y445F could elicit effective MAGE-A3-specific anti-tumor cytotoxic T lymphocyte (CTL) responses in vitro. MAGE-A3 was cloned and rAAV-6-MAGE-A3 purified, followed by proviral genome detection using real-time PCR. Immunofluorescence detection of rAAV-6-Y445F-MAGE-A3-transduced DCs demonstrated 60% transduction efficiency. Fluorescent in situ hybridization analysis confirmed chromosomal integration of rAAV vectors. Flow cytometric analysis of transduced DCs showed unaltered expression of critical monocyte-derived surface molecules with retention of allo-stimulatory activity. Co-culture of autologous T lymphocytes with MAGE-A3-expressing DCs produced CTLs that secreted IFN-γ, and efficiently killed MAGE-A3+ EOC cells. This form of rAAV-based DC immunotherapy, either alone or more likely in combination with other immune-enhancing protocols, may prove useful in the clinical setting for management of EOC. PMID:24406390

  3. High Capsid–Genome Correlation Facilitates Creation of AAV Libraries for Directed Evolution

    PubMed Central

    Nonnenmacher, Mathieu; van Bakel, Harm; Hajjar, Roger J; Weber, Thomas

    2015-01-01

    Directed evolution of adeno-associated virus (AAV) through successive rounds of phenotypic selection is a powerful method to isolate variants with improved properties from large libraries of capsid mutants. Importantly, AAV libraries used for directed evolution are based on the “natural” AAV genome organization where the capsid proteins are encoded in cis from replicating genomes. This is necessary to allow the recovery of the capsid DNA after each step of phenotypic selection. For directed evolution to be used successfully, it is essential to minimize the random mixing of capsomers and the encapsidation of nonmatching viral genomes during the production of the viral libraries. Here, we demonstrate that multiple AAV capsid variants expressed from Rep/Cap containing viral genomes result in near-homogeneous capsids that display an unexpectedly high capsid–DNA correlation. Next-generation sequencing of AAV progeny generated by bulk transfection of a semi-random peptide library showed a strong counter-selection of capsid variants encoding premature stop codons, which further supports a strong capsid–genome identity correlation. Overall, our observations demonstrate that production of “natural” AAVs results in low capsid mosaicism and high capsid–genome correlation. These unique properties allow the production of highly diverse AAV libraries in a one-step procedure with a minimal loss in phenotype–genotype correlation. PMID:25586687

  4. Modeling virus capsids and their protein binding -- the search for weak regions within the HIV capsid

    NASA Astrophysics Data System (ADS)

    Sankey, Otto F.; Benson, Daryn E.; Gilbert, C. Michael

    2011-03-01

    Viruses remain a threat to the health of humans worldwide with 33 million infected with HIV. Viruses are ubiquitous, infecting animals, plants, and bacteria. Each virus infects in its own unique manner making the problem seem intractable. However, some general physical steps apply to many viruses and the application of basic physical modeling can potentially have great impact. The aim of this theoretical study is to investigate the stability of the HIV viral capsid (protein shell). The structural shell can be compromised by physical probes such as pulsed laser light [1,2]. But, what are the weakest regions of the capsid so that we can begin to understand vulnerabilities of these deadly materials? The atomic structure of HIV capsids is not precisely known and we begin by describing our work to model the capsid structure. We have constructed three representative viral capsids of different CA protein number -- HIV-900, HIV-1260 and HIV-1740. The complexity of the assembly requires a course grained model to investigate protein interactions within the capsid which we will describe.

  5. Modest truncation of the major capsid protein abrogates B19 parvovirus capsid formation.

    PubMed Central

    Kawase, M; Momoeda, M; Young, N S; Kajigaya, S

    1995-01-01

    In vitro studies have suggested an important role for the minor capsid protein (VP1) unique region and the junction between VP1 and the major capsid protein (VP2) in the neutralizing immune response to B19 parvovirus. We investigated the role of the NH2-terminal region of the major structural protein in capsid structure by expressing progressively more truncated versions of the VP2 gene followed by analysis using immunoblotting and electron microscopy of density gradient-purified particles. Deletion of the first 25 amino acids (aa) of VP2 did not affect capsid assembly. Altered VP2 with truncations to aa 26 to 30, including a single amino acid deletion at position 25, failed to self-assemble but did participate with normal VP2 in the capsid structure. The altered region corresponds to the beginning of the beta A antiparallel strand. Truncations beyond aa 30 were incompatible with either self-assembly or coassembly, probably because of deletion of the beta B strand, which helps to form the core structure of the virus. PMID:7666560

  6. A protein with simultaneous capsid scaffolding and dsRNA-binding activities enhances the birnavirus capsid mechanical stability

    PubMed Central

    Mertens, Johann; Casado, Santiago; Mata, Carlos P.; Hernando-Pérez, Mercedes; de Pablo, Pedro J.; Carrascosa, José L.; Castón, José R.

    2015-01-01

    Viral capsids are metastable structures that perform many essential processes; they also act as robust cages during the extracellular phase. Viruses can use multifunctional proteins to optimize resources (e.g., VP3 in avian infectious bursal disease virus, IBDV). The IBDV genome is organized as ribonucleoproteins (RNP) of dsRNA with VP3, which also acts as a scaffold during capsid assembly. We characterized mechanical properties of IBDV populations with different RNP content (ranging from none to four RNP). The IBDV population with the greatest RNP number (and best fitness) showed greatest capsid rigidity. When bound to dsRNA, VP3 reinforces virus stiffness. These contacts involve interactions with capsid structural subunits that differ from the initial interactions during capsid assembly. Our results suggest that RNP dimers are the basic stabilization units of the virion, provide better understanding of multifunctional proteins, and highlight the duality of RNP as capsid-stabilizing and genetic information platforms. PMID:26336920

  7. Nanoindentation studies of full and empty viral capsids and the effects of capsid protein mutations on elasticity and strength

    NASA Astrophysics Data System (ADS)

    Michel, J. P.; Ivanovska, I. L.; Gibbons, M. M.; Klug, W. S.; Knobler, C. M.; Wuite, G. J. L.; Schmidt, C. F.

    2006-04-01

    The elastic properties of capsids of the cowpea chlorotic mottle virus have been examined at pH 4.8 by nanoindentation measurements with an atomic force microscope. Studies have been carried out on WT capsids, both empty and containing the RNA genome, and on full capsids of a salt-stable mutant and empty capsids of the subE mutant. Full capsids resisted indentation more than empty capsids, but all of the capsids were highly elastic. There was an initial reversible linear regime that persisted up to indentations varying between 20% and 30% of the diameter and applied forces of 0.6-1.0 nN; it was followed by a steep drop in force that is associated with irreversible deformation. A single point mutation in the capsid protein increased the capsid stiffness. The experiments are compared with calculations by finite element analysis of the deformation of a homogeneous elastic thick shell. These calculations capture the features of the reversible indentation region and allow Young's moduli and relative strengths to be estimated for the empty capsids. atomic force microscopy | cowpea chlorotic mottle virus | finite element analysis | biomechanics

  8. Calibrating elastic parameters from molecular dynamics simulations of capsid proteins

    NASA Astrophysics Data System (ADS)

    Hicks, Stephen; Henley, Christopher

    2008-03-01

    Virus capsids are modeled with elastic network models in which a handful of parameters determine transitions in assembly [1] and morphology [2]. We introduce an approach to compute these parameters from the microscopic structure of the proteins involved. We consider each protein as one or a few rigid bodies with very general interactions, which we parameterize by fitting the simulated equilibrium fluctuations (relative translations and rotations) of a pair of proteins (or fragments) to a 6-dimensional Gaussian. We can then compose these generalized springs into the global capsid structure to determine the continuum elastic parameters. We demonstrate our approach on HIV capsid protein and compare our results with the observed lattice structure (from cryo-EM [3] and AFM indentation studies). [1] R. Zandi et al, PNAS 101 (2004) 15556. [2] J. Lidmar, L. Mirny, and D. R. Nelson, PRE 68 (2003) 051910. [3] B. K. Ganser-Pornillos et al, Cell 131 (2007) 70.

  9. [Structure, Immunogenicity and Clinical Value of Chlamydiaphage Capsid Protein 3].

    PubMed

    Yao, Weifeng; Li, Yiju; Yuan, Jing; Wang, Lei; Li, Qunyan; Song, Mengmeng; Lu, Guiling; Zhang, Litao

    2015-07-01

    We wished to assess the role of chlamydia micro virus capsid protein Vp3 in recombinant molecules, chart its molecular evolution, screen the wild-type strain, and reveal its value in clinical research. Using a protein BLAST multiple-alignment program, we compared various strains of Chlamydia micro virus capsid protein Vp3 sequences. Using a "distance tree" of those results, we created a phylogenetic tree. We applied the Karplus-Schulz method of flexible-region analyses for highly conserved alignments of amino-acid sequences. Gamier-Robson and Chou-Fasman methods were employed to analyze two-level structures of sequences. The Emini method was used for analyses of the accessibility of surface epitopes. Studies of hydrophilic proteins were undertaken using Kyte-Doolittle and Hopp-Woods methods. Analyses of antigen epitopes helped to reveal the antigen index using the Jameson-Wolf method. All sequences in the six strains of chlamydia micro virus capsid protein Vp3 were highly conserved, with the main differences being between Vp3 protein in Chp1 and the other five strains of the micro virus. The viral strain of Vp3 protein was based mainly on micro-alpha helix structures, and multiple epitopes were noted in highly conserved regions. Vp3 protein was highly conserved structurally, and was an important protein of the chlamydiaphage capsid. Vp3 protein has a complicated molecular structure, highly conserved regions with strong immunogenicity, and has considerable research value. PMID:26524915

  10. Analyzing Cellular Immunity to AAV in a Canine Model Using ELISpot Assay

    PubMed Central

    Wang, Zejing; Storb, Rainer; Tapscott, Stephen J; Riddell, Stanley

    2015-01-01

    Adeno-associated viral vector (AAV) mediated gene transfer represents a promising gene replacement strategy for treating various genetic diseases. One obstacle in using viral-derived vectors for in vivo gene delivery is the development of host immune responses to the vector. Recent studies have demonstrated cellular immune responses specific to capsid proteins of various AAV serotypes in animal models and in human trials for different diseases. We developed a canine specific ELISpot assay to detect such immunity in dogs received AAV treatment. Here, we describe in detail the use of a constructed panel of overlapping peptides spanning the entire VP1 sequence of AAV capsid protein to detect specific T cell responses in peripheral blood in dogs following intra-muscular injection of AAV. This high-throughput method allows the identification of T cell epitopes without the need for large cell numbers and the need for MHC matched cell lines. PMID:21956501

  11. Structure of the Small Outer Capsid Protein, Soc: A Clamp for Stabilizing Capsids of T4-like Phages

    SciTech Connect

    Qin, Li; Fokine, Andrei; O'Donnell, Erin; Rao, Venigalla B.; Rossmann, Michael G.

    2010-07-22

    Many viruses need to stabilize their capsid structure against DNA pressure and for survival in hostile environments. The 9-kDa outer capsid protein (Soc) of bacteriophage T4, which stabilizes the virus, attaches to the capsid during the final stage of maturation. There are 870 Soc molecules that act as a 'glue' between neighboring hexameric capsomers, forming a 'cage' that stabilizes the T4 capsid against extremes of pH and temperature. Here we report a 1.9 {angstrom} resolution crystal structure of Soc from the bacteriophage RB69, a close relative of T4. The RB69 crystal structure and a homology model of T4 Soc were fitted into the cryoelectron microscopy reconstruction of the T4 capsid. This established the region of Soc that interacts with the major capsid protein and suggested a mechanism, verified by extensive mutational and biochemical studies, for stabilization of the capsid in which the Soc trimers act as clamps between neighboring capsomers. The results demonstrate the factors involved in stabilizing not only the capsids of T4-like bacteriophages but also many other virus capsids.

  12. AAV Vectors for FRET-Based Analysis of Protein-Protein Interactions in Photoreceptor Outer Segments

    PubMed Central

    Becirovic, Elvir; Böhm, Sybille; Nguyen, Ong N. P.; Riedmayr, Lisa M.; Hammelmann, Verena; Schön, Christian; Butz, Elisabeth S.; Wahl-Schott, Christian; Biel, Martin; Michalakis, Stylianos

    2016-01-01

    Fluorescence resonance energy transfer (FRET) is a powerful method for the detection and quantification of stationary and dynamic protein-protein interactions. Technical limitations have hampered systematic in vivo FRET experiments to study protein-protein interactions in their native environment. Here, we describe a rapid and robust protocol that combines adeno-associated virus (AAV) vector-mediated in vivo delivery of genetically encoded FRET partners with ex vivo FRET measurements. The method was established on acutely isolated outer segments of murine rod and cone photoreceptors and relies on the high co-transduction efficiency of retinal photoreceptors by co-delivered AAV vectors. The procedure can be used for the systematic analysis of protein-protein interactions of wild type or mutant outer segment proteins in their native environment. Conclusively, our protocol can help to characterize the physiological and pathophysiological relevance of photoreceptor specific proteins and, in principle, should also be transferable to other cell types. PMID:27516733

  13. AAV Vectors for FRET-Based Analysis of Protein-Protein Interactions in Photoreceptor Outer Segments.

    PubMed

    Becirovic, Elvir; Böhm, Sybille; Nguyen, Ong N P; Riedmayr, Lisa M; Hammelmann, Verena; Schön, Christian; Butz, Elisabeth S; Wahl-Schott, Christian; Biel, Martin; Michalakis, Stylianos

    2016-01-01

    Fluorescence resonance energy transfer (FRET) is a powerful method for the detection and quantification of stationary and dynamic protein-protein interactions. Technical limitations have hampered systematic in vivo FRET experiments to study protein-protein interactions in their native environment. Here, we describe a rapid and robust protocol that combines adeno-associated virus (AAV) vector-mediated in vivo delivery of genetically encoded FRET partners with ex vivo FRET measurements. The method was established on acutely isolated outer segments of murine rod and cone photoreceptors and relies on the high co-transduction efficiency of retinal photoreceptors by co-delivered AAV vectors. The procedure can be used for the systematic analysis of protein-protein interactions of wild type or mutant outer segment proteins in their native environment. Conclusively, our protocol can help to characterize the physiological and pathophysiological relevance of photoreceptor specific proteins and, in principle, should also be transferable to other cell types. PMID:27516733

  14. RNA-binding region of Macrobrachium rosenbergii nodavirus capsid protein.

    PubMed

    Goh, Zee Hong; Mohd, Nur Azmina Syakirin; Tan, Soon Guan; Bhassu, Subha; Tan, Wen Siang

    2014-09-01

    White tail disease (WTD) kills prawn larvae and causes drastic losses to the freshwater prawn (Macrobrachium rosenbergii) industry. The main causative agent of WTD is Macrobrachium rosenbergii nodavirus (MrNV). The N-terminal end of the MrNV capsid protein is very rich in positively charged amino acids and is postulated to interact with RNA molecules. N-terminal and internal deletion mutagenesis revealed that the RNA-binding region is located at positions 20-29, where 80 % of amino acids are positively charged. Substitution of all these positively charged residues with alanine abolished the RNA binding. Mutants without the RNA-binding region still assembled into virus-like particles, suggesting that this region is not a part of the capsid assembly domain. This paper is, to the best of our knowledge, the first to report the specific RNA-binding region of MrNV capsid protein. PMID:24878641

  15. Cloning of adeno-associated virus type 4 (AAV4) and generation of recombinant AAV4 particles.

    PubMed Central

    Chiorini, J A; Yang, L; Liu, Y; Safer, B; Kotin, R M

    1997-01-01

    We have cloned and characterized the full-length genome of adeno-associated virus type 4 (AAV4). The genome of AAV4 is 4,767 nucleotides in length and contains an expanded p5 promoter region compared to AAV2 and AAV3. Within the inverted terminal repeat (ITR), several base changes were identified with respect to AAV2. However, these changes did not affect the ability of this region to fold into a hairpin structure. Within the ITR, the terminal resolution site and Rep binding sites were conserved; however, the Rep binding site was expanded from three GAGC repeats to four. The Rep gene product of AAV4 shows greater than 90% homology to the Rep products of serotypes 2 and 3, with none of the changes occurring in regions which had previously been shown to affect the known functions of Rep68 or Rep78. Most of the differences in the capsid proteins lie in regions which are thought to be on the exterior surface of the viral capsid. It is these unique regions which are most likely to be responsible for the lack of cross-reacting antibodies and the altered tissue tropism compared to AAV2. The results of our studies, performed with a recombinant version of AAV4 carrying a lacZ reporter gene, suggest that AAV4 can transduce human, monkey, and rat cells. Furthermore, comparison of transduction efficiencies in a number of cell lines, competition cotransduction experiments, and the effect of trypsin on transduction efficiency all suggest that the cellular receptor for AAV4 is distinct from that of AAV2. PMID:9261407

  16. Polymorphism in the assembly of polyomavirus capsid protein VP1.

    PubMed Central

    Salunke, D M; Caspar, D L; Garcea, R L

    1989-01-01

    Polyomavirus major capsid protein VP1, purified after expression of the recombinant gene in Escherichia coli, forms stable pentamers in low-ionic strength, neutral, or alkaline solutions. Electron microscopy showed that the pentamers, which correspond to viral capsomeres, can be self-assembled into a variety of polymorphic aggregates by lowering the pH, adding calcium, or raising the ionic strength. Some of the aggregates resembled the 500-A-diameter virus capsid, whereas other considerably larger or smaller capsids were also produced. The particular structures formed on transition to an environment favoring assembly depended on the pathway of the solvent changes as well as on the final conditions. Mass measurements from cryoelectron micrographs and image analysis of negatively stained specimens established that a distinctive 320-A-diameter particle consists of 24 close-packed pentamers arranged with octahedral symmetry. Comparison of this unexpected octahedral assembly with a 12-capsomere icosahedral aggregate and the 72-capsomere icosahedral virus capsid by computer graphics methods indicates that similar connections are made among trimers of pentamers in these shells of different size. The polymorphism in the assembly of VP1 pentamers can be related to the switching in bonding specificity required to build the virus capsid. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 5 FIGURE 6 PMID:2557933

  17. Antigenic properties of avian hepatitis E virus capsid protein.

    PubMed

    Zhao, Qin; Syed, Shahid Faraz; Zhou, En-Min

    2015-10-22

    Avian hepatitis E virus (HEV) is the main causative agent of big liver and spleen disease and hepatitis-splenomegaly syndrome in chickens, and is genetically and antigenically related to mammalian HEVs. HEV capsid protein contains immunodominant epitopes and induces a protective humoral immune response. A better understanding of the antigenic composition of this protein is critically important for the development of effective vaccine and sensitive and specific serological assays. To date, six linear antigenic domains (I-VI) have been characterized in avian HEV capsid protein and analyzed for their applications in the serological diagnosis and vaccine design. Domains I and V induce strong immune response in chickens and are common to avian, human, and swine HEVs, indicating that the shared epitopes hampering differential diagnosis of avian HEV infection. Domains III and IV are not immunodominant and elicit a weak immune response. Domain VI, located in the N-terminal region of the capsid protein, can also trigger an intense immune response, but the anti-domain VI antibodies are transient. The protection analysis showed that the truncated capsid protein containing the C-terminal 268 amino acid residues expressed by the bacterial system can provide protective immunity against avian HEV infection in chickens. However, the synthetic peptides incorporating the different linear antigenic domains (I-VI) and epitopes are non-protective. The antigenic composition of avian HEV capsid protein is altogether complex. To develop an effective vaccine and accurate serological diagnostic methods, more conformational antigenic domains or epitopes are to be characterized in detail. PMID:26340899

  18. Widespread Central Nervous System Gene Transfer and Silencing After Systemic Delivery of Novel AAV-AS Vector.

    PubMed

    Choudhury, Sourav R; Harris, Anne F; Cabral, Damien J; Keeler, Allison M; Sapp, Ellen; Ferreira, Jennifer S; Gray-Edwards, Heather L; Johnson, Jacob A; Johnson, Aime K; Su, Qin; Stoica, Lorelei; DiFiglia, Marian; Aronin, Neil; Martin, Douglas R; Gao, Guangping; Sena-Esteves, Miguel

    2016-04-01

    Effective gene delivery to the central nervous system (CNS) is vital for development of novel gene therapies for neurological diseases. Adeno-associated virus (AAV) vectors have emerged as an effective platform for in vivo gene transfer, but overall neuronal transduction efficiency of vectors derived from naturally occurring AAV capsids after systemic administration is relatively low. Here, we investigated the possibility of improving CNS transduction of existing AAV capsids by genetically fusing peptides to the N-terminus of VP2 capsid protein. A novel vector AAV-AS, generated by the insertion of a poly-alanine peptide, is capable of extensive gene transfer throughout the CNS after systemic administration in adult mice. AAV-AS is 6- and 15-fold more efficient than AAV9 in spinal cord and cerebrum, respectively. The neuronal transduction profile varies across brain regions but is particularly high in the striatum where AAV-AS transduces 36% of striatal neurons. Widespread neuronal gene transfer was also documented in cat brain and spinal cord. A single intravenous injection of an AAV-AS vector encoding an artificial microRNA targeting huntingtin (Htt) resulted in 33-50% knockdown of Htt across multiple CNS structures in adult mice. This novel AAV-AS vector is a promising platform to develop new gene therapies for neurodegenerative disorders. PMID:26708003

  19. L2, the minor capsid protein of papillomavirus

    SciTech Connect

    Wang, Joshua W.; Roden, Richard B.S.

    2013-10-15

    The capsid protein L2 plays major roles in both papillomavirus assembly and the infectious process. While L1 forms the majority of the capsid and can self-assemble into empty virus-like particles (VLPs), L2 is a minor capsid component and lacks the capacity to form VLPs. However, L2 co-assembles with L1 into VLPs, enhancing their assembly. L2 also facilitates encapsidation of the ∼8 kbp circular and nucleosome-bound viral genome during assembly of the non-enveloped T=7d virions in the nucleus of terminally differentiated epithelial cells, although, like L1, L2 is not detectably expressed in infected basal cells. With respect to infection, L2 is not required for particles to bind to and enter cells. However L2 must be cleaved by furin for endosome escape. L2 then travels with the viral genome to the nucleus, wherein it accumulates at ND-10 domains. Here, we provide an overview of the biology of L2. - Highlights: • L2 is the minor antigen of the non-enveloped T=7d icosahedral Papillomavirus capsid. • L2 is a nuclear protein that can traffic to ND-10 and facilitate genome encapsidation. • L2 is critical for infection and must be cleaved by furin. • L2 is a broadly protective vaccine antigen recognized by neutralizing antibodies.

  20. CapsidMaps: Protein-protein interaction pattern discovery platform for the structural analysis of virus capsids using Google Maps

    PubMed Central

    Carrillo-Tripp, Mauricio; Montiel-García, Daniel Jorge; Brooks, Charles L.; Reddy, Vijay

    2016-01-01

    Structural analysis and visualization of protein-protein interactions is a challenging task since it is difficult to appreciate easily the extent of all contacts made by the residues forming the interfaces. In the case of viruses, structural analysis becomes even more demanding because several interfaces coexist and, in most cases, these are formed by hundreds of contacting residues that belong to multiple interacting coat proteins. CapsidMaps is an interactive analysis and visualization tool that is designed to benefit the structural virology community. Developed as an improved extension of the φ-ψ Explorer, here we describe the details of its design and implementation. We present results of analysis of a spherical virus to showcase the features and utility of the new tool. CapsidMaps also facilitates the comparison of quaternary interactions between two spherical virus particles by computing a similarity (S)-score. The tool can also be used to identify residues that are solvent exposed and in the process of locating antigenic epitope regions as well as residues forming the inside surface of the capsid that interact with the nucleic acid genome. CapsidMaps is part of the VIPERdb Science Gateway, and is freely available as a web-based and cross-browser compliant application at http://viperdb.scripps.edu. PMID:25697908

  1. Analysis of the protein-protein interactions in the parvovirus H-1 capsid.

    PubMed Central

    Paradiso, P R

    1983-01-01

    The structure of the icosahedral capsid of the H-1 parvovirus was probed by chemical cross-linking methods. Treatment of empty capsids with high-molecular-weight polyethylene glycols resulted in irreversible aggregation of the minor capsid protein VP1. Multimers of VP1 containing at least five and perhaps six molecules were obtained, but only with empty capsids and not with the full, DNA-containing virus. Cross-linking of the empty capsids with dimethylsuberimidate confirmed the assignments of the products formed after treatment with polyethylene glycol. With dimethylsuberimidate the most abundant product was a heterologous dimer containing VP1 and the major capsid protein VP2'. A small amount of homologous VP2' dimer was also obtained, but the majority of VP2' remained unreacted even at high concentrations of dimethylsuberimidate. The capsid proteins of the full virus, on the other hand, were completely unreactive to dimethylsuberimidate. The data suggest that the minor protein VP1 may be clustered in the capsid and perhaps composes one or two of the morphological units of the icosahedral shell. Images PMID:6827655

  2. Chemical Reactivity of Brome Mosaic Virus Capsid Protein

    PubMed Central

    Running, W. E.; Ni, P.; Kao, C. C.; Reilly, J. P.

    2012-01-01

    Viral particles are biological machines that have evolved to package, protect, and deliver the viral genome into the host via regulated conformational changes of virions. We have developed a procedure to modify lysine resides with S-methylthioacetimidate (SMTA) across the pH range from 5.5 to 8.5. Lysine residues that are not completely modified are involved in tertiary or quaternary structural interactions, and their extent of modification can be quantified as a function of pH. This procedure was applied to the pH-dependent structural transitions of Brome Mosaic Virus (BMV). As the reaction pH increases from 5.5 to 8.5, the average number of modified lysine residues in the BMV capsid protein increases from six to twelve, correlating well with the known pH dependent swelling behavior of BMV virions. The extent of reaction of each of the capsid protein’s lysine residues has been quantified at eight pH values using coupled liquid chromatography-tandem mass spectrometry. Each lysine can be assigned to one of three structural classes identified by inspection of the BMV virion crystal structure. Several lysine residues display reactivity that indicates their involvement in dynamic interactions that are not obvious in the crystal structure. The influence of several capsid protein mutants on the pH-dependent structural transition of BMV has also been investigated. Mutant H75Q exhibits an altered swelling transition accompanying solution pH increases. The H75Q capsids show increased reactivity at lysine residues 64 and 130, residues distal from the dimer interface occupied by H75, across the entire pH range. PMID:22750573

  3. Oversized AAV Transductifon Is Mediated via a DNA-PKcs-independent, Rad51C-dependent Repair Pathway

    PubMed Central

    Hirsch, Matthew L; Li, Chengwen; Bellon, Isabella; Yin, Chaoying; Chavala, Sai; Pryadkina, Marina; Richard, Isabelle; Samulski, Richard Jude

    2013-01-01

    A drawback of gene therapy using adeno-associated virus (AAV) is the DNA packaging restriction of the viral capsid (<4.7 kb). Recent observations demonstrate oversized AAV genome transduction through an unknown mechanism. Herein, AAV production using an oversized reporter (6.2 kb) resulted in chloroform and DNase-resistant particles harboring distinct “fragment” AAV (fAAV) genomes (5.0, 2.4, and 1.6 kb). Fractionation experiments determined that only the larger “fragments” mediated transduction in vitro, and relatively efficient transduction was also demonstrated in the muscle, the eye, and the liver. In contrast with concatemerization-dependent large-gene delivery by split AAV, fAAV transduction is independent of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in vitro and in vivo while disproportionately reliant on the DNA strand–annealing protein Rad51C. Importantly, fAAV's unique dependence on DNA repair proteins, compared with intact AAV, strongly suggests that the majority of oversized AAV transduction is mediated by fragmented genomes. Although fAAV transduction is less efficient than intact AAV, it is enhanced fourfold in muscle and sevenfold in the retina compared with split AAV transduction. Furthermore, fAAV carrying codon-optimized therapeutic dysferlin cDNA in a 7.5 kb expression cassette restored dysferlin levels in a dystrophic model. Collectively, oversized AAV genome transduction requires unique DNA repair pathways and offers an alternative, more efficient strategy for large-gene therapy. PMID:23939025

  4. Engineering AAV receptor footprints for gene therapy.

    PubMed

    Madigan, Victoria J; Asokan, Aravind

    2016-06-01

    Adeno-associated viruses (AAV) are currently at the forefront of human gene therapy clinical trials as recombinant vectors. Significant progress has been made in elucidating the structure, biology and tropisms of different naturally occurring AAV isolates in the past decade. In particular, a spectrum of AAV capsid interactions with host receptors have been identified and characterized. These studies have enabled a better understanding of key determinants of AAV cell recognition and entry in different hosts. This knowledge is now being applied toward engineering new, lab-derived AAV capsids with favorable transduction profiles. The current review conveys a structural perspective of capsid-glycan interactions and provides a roadmap for generating synthetic strains by engineering AAV receptor footprints. PMID:27262111

  5. Herpes simplex virus type 1 tegument proteins VP1/2 and UL37 are associated with intranuclear capsids

    SciTech Connect

    Bucks, Michelle A.; O'Regan, Kevin J.; Murphy, Michael A.; Wills, John W.; Courtney, Richard J. . E-mail: rcourtney@psu.edu

    2007-05-10

    The assembly of the tegument of herpes simplex virus type 1 (HSV-1) is a complex process that involves a number of events at various sites within virus-infected cells. Our studies focused on determining whether tegument proteins, VP1/2 and UL37, are added to capsids located within the nucleus. Capsids were isolated from the nuclear fraction of HSV-1-infected cells and purified by rate-zonal centrifugation to separate B capsids (containing the scaffold proteins and no viral DNA) and C capsids (containing DNA and no scaffold proteins). Western blot analyses of these capsids indicated that VP1/2 associated primarily with C capsids and UL37 associated with B and C capsids. The results demonstrate that at least two of the tegument proteins of HSV-1 are associated with capsids isolated from the nuclear fraction, and these capsid-tegument protein interactions may represent initial events of the tegumentation process.

  6. Primate TRIM5 proteins form hexagonal nets on HIV-1 capsids.

    PubMed

    Li, Yen-Li; Chandrasekaran, Viswanathan; Carter, Stephen D; Woodward, Cora L; Christensen, Devin E; Dryden, Kelly A; Pornillos, Owen; Yeager, Mark; Ganser-Pornillos, Barbie K; Jensen, Grant J; Sundquist, Wesley I

    2016-01-01

    TRIM5 proteins are restriction factors that block retroviral infections by binding viral capsids and preventing reverse transcription. Capsid recognition is mediated by C-terminal domains on TRIM5α (SPRY) or TRIMCyp (cyclophilin A), which interact weakly with capsids. Efficient capsid recognition also requires the conserved N-terminal tripartite motifs (TRIM), which mediate oligomerization and create avidity effects. To characterize how TRIM5 proteins recognize viral capsids, we developed methods for isolating native recombinant TRIM5 proteins and purifying stable HIV-1 capsids. Biochemical and EM analyses revealed that TRIM5 proteins assembled into hexagonal nets, both alone and on capsid surfaces. These nets comprised open hexameric rings, with the SPRY domains centered on the edges and the B-box and RING domains at the vertices. Thus, the principles of hexagonal TRIM5 assembly and capsid pattern recognition are conserved across primates, allowing TRIM5 assemblies to maintain the conformational plasticity necessary to recognize divergent and pleomorphic retroviral capsids. PMID:27253068

  7. Primate TRIM5 proteins form hexagonal nets on HIV-1 capsids

    PubMed Central

    Li, Yen-Li; Chandrasekaran, Viswanathan; Carter, Stephen D; Woodward, Cora L; Christensen, Devin E; Dryden, Kelly A; Pornillos, Owen; Yeager, Mark; Ganser-Pornillos, Barbie K; Jensen, Grant J; Sundquist, Wesley I

    2016-01-01

    TRIM5 proteins are restriction factors that block retroviral infections by binding viral capsids and preventing reverse transcription. Capsid recognition is mediated by C-terminal domains on TRIM5α (SPRY) or TRIMCyp (cyclophilin A), which interact weakly with capsids. Efficient capsid recognition also requires the conserved N-terminal tripartite motifs (TRIM), which mediate oligomerization and create avidity effects. To characterize how TRIM5 proteins recognize viral capsids, we developed methods for isolating native recombinant TRIM5 proteins and purifying stable HIV-1 capsids. Biochemical and EM analyses revealed that TRIM5 proteins assembled into hexagonal nets, both alone and on capsid surfaces. These nets comprised open hexameric rings, with the SPRY domains centered on the edges and the B-box and RING domains at the vertices. Thus, the principles of hexagonal TRIM5 assembly and capsid pattern recognition are conserved across primates, allowing TRIM5 assemblies to maintain the conformational plasticity necessary to recognize divergent and pleomorphic retroviral capsids. DOI: http://dx.doi.org/10.7554/eLife.16269.001 PMID:27253068

  8. Mutational analysis of the capsid protein of Leishmania RNA virus LRV1-4.

    PubMed Central

    Cadd, T L; MacBeth, K; Furlong, D; Patterson, J L

    1994-01-01

    The virion of Leishmania RNA virus is predicted to be composed of a 742-amino-acid major capsid protein and a small percentage of capsid-polymerase fusion molecules. Recently, the capsid protein alone was expressed and shown to spontaneously assemble into viruslike particles. Since the major structural protein of the virion shell self-assembles into viruslike particles when expressed in the baculovirus expression system, assembly of the virion can be studied by mutational analysis and expression of a single open reading frame. In this study, several deletions and one addition of the capsid protein of Leishmania RNA virus LRV1-4 were generated. These mutants show different degrees of assembly. Assembly domains are being identified such that the capsid protein may be used as a macromolecular packaging and delivery system for Leishmania species. Images PMID:7966563

  9. Purification of recombinant budgerigar fledgling disease virus VP1 capsid protein and its ability for in vitro capsid assembly

    NASA Technical Reports Server (NTRS)

    Rodgers, R. E.; Chang, D.; Cai, X.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    A recombinant system for the major capsid VP1 protein of budgerigar fledgling disease virus has been established. The VP1 gene was inserted into a truncated form of the pFlag-1 vector and expressed in Escherichia coli. The budgerigar fledgling disease virus VP1 protein was purified to near homogeneity by immunoaffinity chromatography. Fractions containing highly purified VP1 were pooled and found to constitute 3.3% of the original E. coli-expressed VP1 protein. Electron microscopy revealed that the VP1 protein was isolated as pentameric capsomeres. Electron microscopy also revealed that capsid-like particles were formed in vitro from purified VP1 capsomeres with the addition of Ca2+ ions and the removal of chelating and reducing agents.

  10. Purification of recombinant budgerigar fledgling disease virus VP1 capsid protein and its ability for in vitro capsid assembly.

    PubMed Central

    Rodgers, R E; Chang, D; Cai, X; Consigli, R A

    1994-01-01

    A recombinant system for the major capsid VP1 protein of budgerigar fledgling disease virus has been established. The VP1 gene was inserted into a truncated form of the pFlag-1 vector and expressed in Escherichia coli. The budgerigar fledgling disease virus VP1 protein was purified to near homogeneity by immunoaffinity chromatography. Fractions containing highly purified VP1 were pooled and found to constitute 3.3% of the original E. coli-expressed VP1 protein. Electron microscopy revealed that the VP1 protein was isolated as pentameric capsomeres. Electron microscopy also revealed that capsid-like particles were formed in vitro from purified VP1 capsomeres with the addition of Ca2+ ions and the removal of chelating and reducing agents. Images PMID:8151798

  11. C Terminus of Infectious Bursal Disease Virus Major Capsid Protein VP2 Is Involved in Definition of the T Number for Capsid Assembly

    PubMed Central

    Castón, José R.; Martínez-Torrecuadrada, Jorge L.; Maraver, Antonio; Lombardo, Eleuterio; Rodríguez, José F.; Casal, J. Ignacio; Carrascosa, José L.

    2001-01-01

    Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is a double-stranded RNA virus. The IBDV capsid is formed by two major structural proteins, VP2 and VP3, which assemble to form a T=13 markedly nonspherical capsid. During viral infection, VP2 is initially synthesized as a precursor, called VPX, whose C end is proteolytically processed to the mature form during capsid assembly. We have computed three-dimensional maps of IBDV capsid and virus-like particles built up by VP2 alone by using electron cryomicroscopy and image-processing techniques. The IBDV single-shelled capsid is characterized by the presence of 260 protruding trimers on the outer surface. Five classes of trimers can be distinguished according to their different local environments. When VP2 is expressed alone in insect cells, dodecahedral particles form spontaneously; these may be assembled into larger, fragile icosahedral capsids built up by 12 dodecahedral capsids. Each dodecahedral capsid is an empty T=1 shell composed of 20 trimeric clusters of VP2. Structural comparison between IBDV capsids and capsids consisting of VP2 alone allowed the determination of the major capsid protein locations and the interactions between them. Whereas VP2 forms the outer protruding trimers, VP3 is found as trimers on the inner surface and may be responsible for stabilizing functions. Since elimination of the C-terminal region of VPX is correlated with the assembly of T=1 capsids, this domain might be involved (either alone or in cooperation with VP3) in the induction of different conformations of VP2 during capsid morphogenesis. PMID:11602723

  12. Bioinformatics analysis of the epitope regions for norovirus capsid protein

    PubMed Central

    2013-01-01

    Background Norovirus is the major cause of nonbacterial epidemic gastroenteritis, being highly prevalent in both developing and developed countries. Despite of the available monoclonal antibodies (MAbs) for different sub-genogroups, a comprehensive epitope analysis based on various bioinformatics technology is highly desired for future potential antibody development in clinical diagonosis and treatment. Methods A total of 18 full-length human norovirus capsid protein sequences were downloaded from GenBank. Protein modeling was performed with program Modeller 9.9. The modeled 3D structures of capsid protein of norovirus were submitted to the protein antigen spatial epitope prediction webserver (SEPPA) for predicting the possible spatial epitopes with the default threshold. The results were processed using the Biosoftware. Results Compared with GI, we found that the GII genogroup had four deletions and two special insertions in the VP1 region. The predicted conformational epitope regions mainly concentrated on N-terminal (1~96), Middle Part (298~305, 355~375) and C-terminal (560~570). We find two common epitope regions on sequences for GI and GII genogroup, and also found an exclusive epitope region for GII genogroup. Conclusions The predicted conformational epitope regions of norovirus VP1 mainly concentrated on N-terminal, Middle Part and C-terminal. We find two common epitope regions on sequences for GI and GII genogroup, and also found an exclusive epitope region for GII genogroup. The overlapping with experimental epitopes indicates the important role of latest computational technologies. With the fast development of computational immunology tools, the bioinformatics pipeline will be more and more critical to vaccine design. PMID:23514273

  13. Targeted Modifications in Adeno-Associated Virus Serotype 8 Capsid Improves Its Hepatic Gene Transfer Efficiency In Vivo

    PubMed Central

    Sen, Dwaipayan; Gadkari, Rupali A; Sudha, Govindarajan; Gabriel, Nishanth; Kumar, Yesupatham Sathish; Selot, Ruchita; Samuel, Rekha; Rajalingam, Sumathi; Ramya, V.; Nair, Sukesh C.; Srinivasan, Narayanaswamy; Srivastava, Alok

    2013-01-01

    Abstract Recombinant adeno-associated virus vectors based on serotype 8 (AAV8) have shown significant promise for liver-directed gene therapy. However, to overcome the vector dose dependent immunotoxicity seen with AAV8 vectors, it is important to develop better AAV8 vectors that provide enhanced gene expression at significantly low vector doses. Since it is known that AAV vectors during intracellular trafficking are targeted for destruction in the cytoplasm by the host–cellular kinase/ubiquitination/proteasomal machinery, we modified specific serine/threonine kinase or ubiquitination targets on the AAV8 capsid to augment its transduction efficiency. Point mutations at specific serine (S)/threonine (T)/lysine (K) residues were introduced in the AAV8 capsid at the positions equivalent to that of the effective AAV2 mutants, generated successfully earlier. Extensive structure analysis was carried out subsequently to evaluate the structural equivalence between the two serotypes. scAAV8 vectors with the wild-type (WT) and each one of the S/T→Alanine (A) or K-Arginine (R) mutant capsids were evaluated for their liver transduction efficiency in C57BL/6 mice in vivo. Two of the AAV8-S→A mutants (S279A and S671A), and a K137R mutant vector, demonstrated significantly higher enhanced green fluorescent protein (EGFP) transcript levels (∼9- to 46-fold) in the liver compared to animals that received WT-AAV8 vectors alone. The best performing AAV8 mutant (K137R) vector also had significantly reduced ubiquitination of the viral capsid, reduced activation of markers of innate immune response, and a concomitant two-fold reduction in the levels of neutralizing antibody formation in comparison to WT-AAV8 vectors. Vector biodistribution studies revealed that the K137R mutant had a significantly higher and preferential transduction of the liver (106 vs. 7.7 vector copies/mouse diploid genome) when compared to WT-AAV8 vectors. To further study the utility of the K137R-AAV8

  14. The structure of avian polyomavirus reveals variably sized capsids, non-conserved inter-capsomere interactions, and a possible location of the minor capsid protein VP4

    SciTech Connect

    Shen, Peter S.; Enderlein, Dirk; Nelson, Christian D.S.; Carter, Weston S.; Kawano, Masaaki; Xing Li; Swenson, Robert D.; Olson, Norman H.; Baker, Timothy S.; Cheng, R. Holland; Atwood, Walter J.; Johne, Reimar; Belnap, David M.

    2011-03-01

    Avian polyomavirus (APV) causes a fatal, multi-organ disease among several bird species. Using cryogenic electron microscopy and other biochemical techniques, we investigated the structure of APV and compared it to that of mammalian polyomaviruses, particularly JC polyomavirus and simian virus 40. The structure of the pentameric major capsid protein (VP1) is mostly conserved; however, APV VP1 has a unique, truncated C-terminus that eliminates an intercapsomere-connecting {beta}-hairpin observed in other polyomaviruses. We postulate that the terminal {beta}-hairpin locks other polyomavirus capsids in a stable conformation and that absence of the hairpin leads to the observed capsid size variation in APV. Plug-like density features were observed at the base of the VP1 pentamers, consistent with the known location of minor capsid proteins VP2 and VP3. However, the plug density is more prominent in APV and may include VP4, a minor capsid protein unique to bird polyomaviruses.

  15. Hydroxyproline in the major capsid protein VP1 of polyomavirus

    SciTech Connect

    Ludlow, J.W.; Consigli, R.A.

    1989-06-01

    Amino acid analysis of (/sup 3/H)proline-labeled polyomavirus major capsid protein VP1 by two-dimensional paper chromatography of the acid-hydrolyzed protein revealed the presence of /sup 3/H-labeled hydroxyproline. Addition of the proline analog L-azetidine-2-carboxylic acid to infected mouse kidney cell cultures prevented or greatly reduced hydroxylation of proline in VP1. Immunofluorescence analysis performed on infected cells over a time course of analog addition revealed that virus proteins were synthesized but that transport from the cytoplasm to the nucleus was impeded. A reduction in the assembly of progeny virions demonstrated by CsCl gradient purification of virus from (/sup 35/S)methionine-labeled infected cell cultures was found to correlate with the time of analog addition. These results suggest that incorporation of this proline analog into VP1, accompanied by reduction of the hydroxyproline content of the protein, influences the amount of virus progeny produced by affecting transport of VP1 to the cell nucleus for assembly into virus particles.

  16. Characterization of the DNA binding properties of polyomavirus capsid protein

    NASA Technical Reports Server (NTRS)

    Chang, D.; Cai, X.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    The DNA binding properties of the polyomavirus structural proteins VP1, VP2, and VP3 were studied by Southwestern analysis. The major viral structural protein VP1 and host-contributed histone proteins of polyomavirus virions were shown to exhibit DNA binding activity, but the minor capsid proteins VP2 and VP3 failed to bind DNA. The N-terminal first five amino acids (Ala-1 to Lys-5) were identified as the VP1 DNA binding domain by genetic and biochemical approaches. Wild-type VP1 expressed in Escherichia coli (RK1448) exhibited DNA binding activity, but the N-terminal truncated VP1 mutants (lacking Ala-1 to Lys-5 and Ala-1 to Cys-11) failed to bind DNA. The synthetic peptide (Ala-1 to Cys-11) was also shown to have an affinity for DNA binding. Site-directed mutagenesis of the VP1 gene showed that the point mutations at Pro-2, Lys-3, and Arg-4 on the VP1 molecule did not affect DNA binding properties but that the point mutation at Lys-5 drastically reduced DNA binding affinity. The N-terminal (Ala-1 to Lys-5) region of VP1 was found to be essential and specific for DNA binding, while the DNA appears to be non-sequence specific. The DNA binding domain and the nuclear localization signal are located in the same N-terminal region.

  17. AAV-mediated RLBP1 gene therapy improves the rate of dark adaptation in Rlbp1 knockout mice.

    PubMed

    Choi, Vivian W; Bigelow, Chad E; McGee, Terri L; Gujar, Akshata N; Li, Hui; Hanks, Shawn M; Vrouvlianis, Joanna; Maker, Michael; Leehy, Barrett; Zhang, Yiqin; Aranda, Jorge; Bounoutas, George; Demirs, John T; Yang, Junzheng; Ornberg, Richard; Wang, Yu; Martin, Wendy; Stout, Kelly R; Argentieri, Gregory; Grosenstein, Paul; Diaz, Danielle; Turner, Oliver; Jaffee, Bruce D; Police, Seshidhar R; Dryja, Thaddeus P

    2015-01-01

    Recessive mutations in RLBP1 cause a form of retinitis pigmentosa in which the retina, before its degeneration leads to blindness, abnormally slowly recovers sensitivity after exposure to light. To develop a potential gene therapy for this condition, we tested multiple recombinant adeno-associated vectors (rAAVs) composed of different promoters, capsid serotypes, and genome conformations. We generated rAAVs in which sequences from the promoters of the human RLBP1, RPE65, or BEST1 genes drove the expression of a reporter gene (green fluorescent protein). A promoter derived from the RLBP1 gene mediated expression in the retinal pigment epithelium and Müller cells (the intended target cell types) at qualitatively higher levels than in other retinal cell types in wild-type mice and monkeys. With this promoter upstream of the coding sequence of the human RLBP1 gene, we compared the potencies of vectors with an AAV2 versus an AAV8 capsid in transducing mouse retinas, and we compared vectors with a self-complementary versus a single-stranded genome. The optimal vector (scAAV8-pRLBP1-hRLBP1) had serotype 8 capsid and a self-complementary genome. Subretinal injection of scAAV8-pRLBP1-hRLBP1 in Rlbp1 nullizygous mice improved the rate of dark adaptation based on scotopic (rod-plus-cone) and photopic (cone) electroretinograms (ERGs). The effect was still present after 1 year. PMID:26199951

  18. AAV-mediated RLBP1 gene therapy improves the rate of dark adaptation in Rlbp1 knockout mice

    PubMed Central

    Choi, Vivian W; Bigelow, Chad E; McGee, Terri L; Gujar, Akshata N; Li, Hui; Hanks, Shawn M; Vrouvlianis, Joanna; Maker, Michael; Leehy, Barrett; Zhang, Yiqin; Aranda, Jorge; Bounoutas, George; Demirs, John T; Yang, Junzheng; Ornberg, Richard; Wang, Yu; Martin, Wendy; Stout, Kelly R; Argentieri, Gregory; Grosenstein, Paul; Diaz, Danielle; Turner, Oliver; Jaffee, Bruce D; Police, Seshidhar R; Dryja, Thaddeus P

    2015-01-01

    Recessive mutations in RLBP1 cause a form of retinitis pigmentosa in which the retina, before its degeneration leads to blindness, abnormally slowly recovers sensitivity after exposure to light. To develop a potential gene therapy for this condition, we tested multiple recombinant adeno-associated vectors (rAAVs) composed of different promoters, capsid serotypes, and genome conformations. We generated rAAVs in which sequences from the promoters of the human RLBP1, RPE65, or BEST1 genes drove the expression of a reporter gene (green fluorescent protein). A promoter derived from the RLBP1 gene mediated expression in the retinal pigment epithelium and Müller cells (the intended target cell types) at qualitatively higher levels than in other retinal cell types in wild-type mice and monkeys. With this promoter upstream of the coding sequence of the human RLBP1 gene, we compared the potencies of vectors with an AAV2 versus an AAV8 capsid in transducing mouse retinas, and we compared vectors with a self-complementary versus a single-stranded genome. The optimal vector (scAAV8-pRLBP1-hRLBP1) had serotype 8 capsid and a self-complementary genome. Subretinal injection of scAAV8-pRLBP1-hRLBP1 in Rlbp1 nullizygous mice improved the rate of dark adaptation based on scotopic (rod-plus-cone) and photopic (cone) electroretinograms (ERGs). The effect was still present after 1 year. PMID:26199951

  19. Epitopes expressed in different adenovirus capsid proteins induce different levels of epitope-specific immunity.

    PubMed

    Krause, Anja; Joh, Ju H; Hackett, Neil R; Roelvink, Peter W; Bruder, Joseph T; Wickham, Thomas J; Kovesdi, Imre; Crystal, Ronald G; Worgall, Stefan

    2006-06-01

    On the basis of the concept that the capsid proteins of adenovirus (Ad) gene transfer vectors can be genetically manipulated to enhance the immunogenicity of Ad-based vaccines, the present study compared the antiantigen immunogenicity of Ad vectors with a common epitope of the hemagglutinin (HA) protein of the influenza A virus incorporated into the outer Ad capsid protein hexon, penton base, fiber knob, or protein IX. Incorporation of the same epitope into the different capsid proteins provided insights into the correlation between epitope position and antiepitope immunity. Following immunization of three different strains of mice (C57BL/6, BALB/c, and CBA) with either an equal number of Ad particles (resulting in a different total HA copy number) or different Ad particle numbers (to achieve the same HA copy number), the highest primary (immunoglobulin M [IgM]) and secondary (IgG) anti-HA humoral and cellular CD4 gamma interferon and interleukin-4 responses against HA were always achieved with the Ad vector carrying the HA epitope in fiber knob. These observations suggest that the immune response against an epitope inserted into Ad capsid proteins is not necessarily dependent on the capsid protein number and imply that the choice of incorporation site in Ad capsid proteins in their use as vaccines needs to be compared in vivo. PMID:16699033

  20. Understanding Dengue Virus Capsid Protein Interaction with Key Biological Targets

    PubMed Central

    Faustino, André F.; Martins, Ivo C.; Carvalho, Filomena A.; Castanho, Miguel A. R. B.; Maurer-Stroh, Sebastian; Santos, Nuno C.

    2015-01-01

    Dengue virus (DENV) causes over 500,000 hospitalizations and 20,000 deaths worldwide every year. Dengue epidemics now reach temperate regions due to globalization of trade and travel and climate changes. Currently, there are no successful therapeutic or preventive approaches. We previously developed a peptide drug lead, pep14-23, that inhibits the biologically relevant interaction of DENV capsid (C) protein with lipid droplets (LDs). Surprisingly, pep14-23 also inhibits DENV C interaction with very low-density lipoproteins (VLDL). We thus investigated the similarity between the proposed DENV C molecular targets in LDs and VLDL, respectively, the proteins perilipin 3 (PLIN3) and apolipoprotein E (APOE). APOE N-terminal and PLIN3 C-terminal regions are remarkably similar, namely APOE α-helix 4 (APOEα4) and PLIN3 α-helix 5 (PLIN3α5) sequences, which are also highly superimposable structurally. Interestingly, APOE α-helical N-terminal sequence and structure superimposes with DENV C α-helices α1 and α2. Moreover, the DENV C hydrophobic cleft can accommodate the structurally analogous APOEα4 and PLIN3α5 helical regions. Mirroring DENV C-LDs interaction (previously shown experimentally to require PLIN3), we experimentally demonstrated that DENV C-VLDL interaction requires APOE. Thus, the results fit well with previous data and suggest future drug development strategies targeting the above mentioned α-helical structures. PMID:26161501

  1. Photoreceptor-targeted gene delivery using intravitreally administered AAV vectors in dogs

    PubMed Central

    Boyd, RF; Sledge, DG; Boye, SL; Boye, SE; Hauswirth, WW; Komáromy, AM; Petersen-Jones, SM; Bartoe, JT

    2016-01-01

    Delivery of therapeutic transgenes to retinal photoreceptors using adeno-associated virus (AAV) vectors has traditionally required subretinal injection. Recently, photoreceptor transduction efficiency following intravitreal injection (IVT) has improved in rodent models through use of capsid-mutant AAV vectors; but remains limited in large animal models. Thickness of the inner limiting membrane (ILM) in large animals is thought to impair retinal penetration by AAV. Our study compared two newly developed AAV vectors containing multiple capsid amino acid substitutions following IVT in dogs. The ability of two promoter constructs to restrict reporter transgene expression to photoreceptors was also evaluated. AAV vectors containing the interphotoreceptor-binding protein (IRBP) promoter drove expression exclusively in rod and cone photoreceptors, with transduction efficiencies of ~ 4% of cones and 2% of rods. Notably, in the central region containing the cone-rich visual streak, 15.6% of cones were transduced. Significant regional variation existed, with lower transduction efficiencies in the temporal regions of all eyes. This variation did not correlate with ILM thickness. Vectors carrying a cone-specific promoter failed to transduce a quantifiable percentage of cone photoreceptors. The newly developed AAV vectors containing the IRBP promoter were capable of producing photoreceptor-specific transgene expression following IVT in the dog. PMID:26467396

  2. Intracellular Nucleic Acid Delivery by the Supercharged Dengue Virus Capsid Protein

    PubMed Central

    Freire, João Miguel; Veiga, Ana Salomé; Conceição, Thaís M.; Kowalczyk, Wioleta; Mohana-Borges, Ronaldo; Andreu, David; Santos, Nuno C.; Da Poian, Andrea T.; Castanho, Miguel A. R. B.

    2013-01-01

    Supercharged proteins are a recently identified class of proteins that have the ability to efficiently deliver functional macromolecules into mammalian cells. They were first developed as bioengineering products, but were later found in the human proteome. In this work, we show that this class of proteins with unusually high net positive charge is frequently found among viral structural proteins, more specifically among capsid proteins. In particular, the capsid proteins of viruses from the Flaviviridae family have all a very high net charge to molecular weight ratio (> +1.07/kDa), thus qualifying as supercharged proteins. This ubiquity raises the hypothesis that supercharged viral capsid proteins may have biological roles that arise from an intrinsic ability to penetrate cells. Dengue virus capsid protein was selected for a detailed experimental analysis. We showed that this protein is able to deliver functional nucleic acids into mammalian cells. The same result was obtained with two isolated domains of this protein, one of them being able to translocate lipid bilayers independently of endocytic routes. Nucleic acids such as siRNA and plasmids were delivered fully functional into cells. The results raise the possibility that the ability to penetrate cells is part of the native biological functions of some viral capsid proteins. PMID:24339931

  3. Reactive oxygen species promote heat shock protein 90-mediated HBV capsid assembly

    SciTech Connect

    Kim, Yoon Sik Seo, Hyun Wook Jung, Guhung

    2015-02-13

    Hepatitis B virus (HBV) infection induces reactive oxygen species (ROS) production and has been associated with the development of hepatocellular carcinoma (HCC). ROS are also an important factor in HCC because the accumulated ROS leads to abnormal cell proliferation and chromosome mutation. In oxidative stress, heat shock protein 90 (Hsp90) and glutathione (GSH) function as part of the defense mechanism. Hsp90 prevents cellular component from oxidative stress, and GSH acts as antioxidants scavenging ROS in the cell. However, it is not known whether molecules regulated by oxidative stress are involved in HBV capsid assembly. Based on the previous study that Hsp90 facilitates HBV capsid assembly, which is an important step for the packing of viral particles, here, we show that ROS enrich Hsp90-driven HBV capsid formation. In cell-free system, HBV capsid assembly was facilitated by ROS with Hsp90, whereas it was decreased without Hsp90. In addition, GSH inhibited the function of Hsp90 to decrease HBV capsid assembly. Consistent with the result of cell-free system, ROS and buthionine sulfoximine (BS), an inhibitor of GSH synthesis, increased HBV capsid formation in HepG2.2.15 cells. Thus, our study uncovers the interplay between ROS and Hsp90 during HBV capsid assembly. - Highlights: • We examined H{sub 2}O{sub 2} and GSH modulate HBV capsid assembly. • H{sub 2}O{sub 2} facilitates HBV capsid assembly in the presence of Hsp90. • GSH inhibits function of Hsp90 in facilitating HBV capsid assembly. • H{sub 2}O{sub 2} and GSH induce conformation change of Hsp90.

  4. The Amphipathic Helix of Adenovirus Capsid Protein VI Contributes to Penton Release and Postentry Sorting

    PubMed Central

    Martinez, Ruben; Schellenberger, Pascale; Vasishtan, Daven; Aknin, Cindy; Austin, Sisley; Dacheux, Denis; Rayne, Fabienne; Siebert, Alistair; Ruzsics, Zsolt; Gruenewald, Kay

    2014-01-01

    ABSTRACT Nuclear delivery of the adenoviral genome requires that the capsid cross the limiting membrane of the endocytic compartment and traverse the cytosol to reach the nucleus. This endosomal escape is initiated upon internalization and involves a highly coordinated process of partial disassembly of the entering capsid to release the membrane lytic internal capsid protein VI. Using wild-type and protein VI-mutated human adenovirus serotype 5 (HAdV-C5), we show that capsid stability and membrane rupture are major determinants of entry-related sorting of incoming adenovirus virions. Furthermore, by using electron cryomicroscopy, as well as penton- and protein VI-specific antibodies, we show that the amphipathic helix of protein VI contributes to capsid stability by preventing premature disassembly and deployment of pentons and protein VI. Thus, the helix has a dual function in maintaining the metastable state of the capsid by preventing premature disassembly and mediating efficient membrane lysis to evade lysosomal targeting. Based on these findings and structural data from cryo-electron microscopy, we suggest a refined disassembly mechanism upon entry. IMPORTANCE In this study, we show the intricate connection of adenovirus particle stability and the entry-dependent release of the membrane-lytic capsid protein VI required for endosomal escape. We show that the amphipathic helix of the adenovirus internal protein VI is required to stabilize pentons in the particle while coinciding with penton release upon entry and that release of protein VI mediates membrane lysis, thereby preventing lysosomal sorting. We suggest that this dual functionality of protein VI ensures an optimal disassembly process by balancing the metastable state of the mature adenovirus particle. PMID:25473051

  5. Reactive oxygen species promote heat shock protein 90-mediated HBV capsid assembly.

    PubMed

    Kim, Yoon Sik; Seo, Hyun Wook; Jung, Guhung

    2015-02-13

    Hepatitis B virus (HBV) infection induces reactive oxygen species (ROS) production and has been associated with the development of hepatocellular carcinoma (HCC). ROS are also an important factor in HCC because the accumulated ROS leads to abnormal cell proliferation and chromosome mutation. In oxidative stress, heat shock protein 90 (Hsp90) and glutathione (GSH) function as part of the defense mechanism. Hsp90 prevents cellular component from oxidative stress, and GSH acts as antioxidants scavenging ROS in the cell. However, it is not known whether molecules regulated by oxidative stress are involved in HBV capsid assembly. Based on the previous study that Hsp90 facilitates HBV capsid assembly, which is an important step for the packing of viral particles, here, we show that ROS enrich Hsp90-driven HBV capsid formation. In cell-free system, HBV capsid assembly was facilitated by ROS with Hsp90, whereas it was decreased without Hsp90. In addition, GSH inhibited the function of Hsp90 to decrease HBV capsid assembly. Consistent with the result of cell-free system, ROS and buthionine sulfoximine (BS), an inhibitor of GSH synthesis, increased HBV capsid formation in HepG2.2.15 cells. Thus, our study uncovers the interplay between ROS and Hsp90 during HBV capsid assembly. PMID:25576869

  6. Coat as a Dagger: The Use of Capsid Proteins to Perforate Membranes during Non-Enveloped DNA Viruses Trafficking

    PubMed Central

    Bilkova, Eva; Forstova, Jitka; Abrahamyan, Levon

    2014-01-01

    To get access to the replication site, small non-enveloped DNA viruses have to cross the cell membrane using a limited number of capsid proteins, which also protect the viral genome in the extracellular environment. Most of DNA viruses have to reach the nucleus to replicate. The capsid proteins involved in transmembrane penetration are exposed or released during endosomal trafficking of the virus. Subsequently, the conserved domains of capsid proteins interact with cellular membranes and ensure their efficient permeabilization. This review summarizes our current knowledge concerning the role of capsid proteins of small non-enveloped DNA viruses in intracellular membrane perturbation in the early stages of infection. PMID:25055856

  7. Production and characterization of monoclonal antibodies to budgerigar fledgling disease virus major capsid protein VP

    NASA Technical Reports Server (NTRS)

    Fattaey, A.; Lenz, L.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    Eleven hybridoma cell lines producing monoclonal antibodies (MAbs) against intact budgerigar fledgling disease (BFD) virions were produced and characterized. These antibodies were selected for their ability to react with BFD virions in an enzyme-linked immunosorbent assay. Each of these antibodies was reactive in the immunofluorescent detection of BFD virus-infected cells. These antibodies immunoprecipitated intact virions and specifically recognized the major capsid protein, VP1, of the dissociated virion. The MAbs were found to preferentially recognize native BFD virus capsid protein when compared with denatured virus protein. These MAbs were capable of detecting BFD virus protein in chicken embryonated cell-culture lysates by dot-blot analysis.

  8. In vivo encapsulation of nucleic acids using an engineered nonviral protein capsid.

    PubMed

    Lilavivat, Seth; Sardar, Debosmita; Jana, Subrata; Thomas, Geoffrey C; Woycechowsky, Kenneth J

    2012-08-15

    In Nature, protein capsids function as molecular containers for a wide variety of molecular cargoes. Such containers have great potential for applications in nanotechnology, which often require encapsulation of non-native guest molecules. Charge complementarity represents a potentially powerful strategy for engineering novel encapsulation systems. In an effort to explore the generality of this approach, we engineered a nonviral, 60-subunit capsid, lumazine synthase from Aquifex aeolicus (AaLS), to act as a container for nucleic acid. Four mutations were introduced per subunit to increase the positive charge at the inner surface of the capsid. Characterization of the mutant (AaLS-pos) revealed that the positive charges lead to the uptake of cellular RNA during production and assembly of the capsid in vivo. Surprisingly, AaLS-pos capsids were found to be enriched with RNA molecules approximately 200-350 bases in length, suggesting that this simple charge complementarity approach to RNA encapsulation leads to both high affinity and a degree of selectivity. The ability to control loading of RNA by tuning the charge at the inner surface of a protein capsid could illuminate aspects of genome recognition by viruses and pave the way for the development of improved RNA delivery systems. PMID:22827162

  9. The Impact of Capsid Proteins on Virus Removal and Inactivation During Water Treatment Processes

    PubMed Central

    Mayer, Brooke K; Yang, Yu; Gerrity, Daniel W; Abbaszadegan, Morteza

    2015-01-01

    This study examined the effect of the amino acid composition of protein capsids on virus inactivation using ultraviolet (UV) irradiation and titanium dioxide photocatalysis, and physical removal via enhanced coagulation using ferric chloride. Although genomic damage is likely more extensive than protein damage for viruses treated using UV, proteins are still substantially degraded. All amino acids demonstrated significant correlations with UV susceptibility. The hydroxyl radicals produced during photocatalysis are considered nonspecific, but they likely cause greater overall damage to virus capsid proteins relative to the genome. Oxidizing chemicals, including hydroxyl radicals, preferentially degrade amino acids over nucleotides, and the amino acid tyrosine appears to strongly influence virus inactivation. Capsid composition did not correlate strongly to virus removal during physicochemical treatment, nor did virus size. Isoelectric point may play a role in virus removal, but additional factors are likely to contribute. PMID:26604779

  10. Selective Cleavage of AAVS1 Substrates by the Adeno-Associated Virus Type 2 Rep68 Protein Is Dependent on Topological and Sequence Constraints

    PubMed Central

    Lamartina, Stefania; Ciliberto, Gennaro; Toniatti, Carlo

    2000-01-01

    The adeno-associated virus type 2 (AAV-2) Rep78 and Rep68 proteins are required for replication of the virus as well as its site-specific integration into a unique site, called AAVS1, of human chromosome 19. Rep78 and Rep68 initiate replication by binding to a Rep binding site (RBS) contained in the AAV-2 inverted terminal repeats (ITRs) and then specifically nicking at a nearby site called the terminal resolution site (trs). Similarly, Rep78 and Rep68 are postulated to trigger the integration process by binding and nicking RBS and trs homologues present in AAVS1. However, Rep78 and Rep68 cleave in vitro AAVS1 duplex-linear substrates much less efficiently than hairpinned ITRs. In this study, we show that the AAV-2 Rep68 endonuclease activity is affected by the topology of the substrates in that it efficiently cleaves in vitro in a site- and strand-specific manner the AAVS1 trs only if this sequence is in a supercoiled (SC) conformation. DNA sequence mutagenesis in the context of SC templates allowed us to elucidate for the first time the AAVS1 trs sequence and position requirements for Rep68-mediated cleavage. Interestingly, Rep68 did not cleave SC templates containing RBS from other sites of the human genome. These findings have intriguing implications for AAV-2 site-specific integration in vivo. PMID:10982325

  11. Eclipse Phase of Herpes Simplex Virus Type 1 Infection: Efficient Dynein-Mediated Capsid Transport without the Small Capsid Protein VP26

    PubMed Central

    Döhner, Katinka; Radtke, Kerstin; Schmidt, Simone; Sodeik, Beate

    2006-01-01

    Cytoplasmic dynein,together with its cofactor dynactin, transports incoming herpes simplex virus type 1 (HSV-1) capsids along microtubules (MT) to the MT-organizing center (MTOC). From the MTOC, capsids move further to the nuclear pore, where the viral genome is released into the nucleoplasm. The small capsid protein VP26 can interact with the dynein light chains Tctex1 (DYNLT1) and rp3 (DYNLT3) and may recruit dynein to the capsid. Therefore, we analyzed nuclear targeting of incoming HSV1-ΔVP26 capsids devoid of VP26 and of HSV1-GFPVP26 capsids expressing a GFPVP26 fusion instead of VP26. To compare the cell entry of different strains, we characterized the inocula with respect to infectivity, viral genome content, protein composition, and particle composition. Preparations with a low particle-to-PFU ratio showed efficient nuclear targeting and were considered to be of higher quality than those containing many defective particles, which were unable to induce plaque formation. When cells were infected with HSV-1 wild type, HSV1-ΔVP26, or HSV1-GFPVP26, viral capsids were transported along MT to the nucleus. Moreover, when dynein function was inhibited by overexpression of the dynactin subunit dynamitin, fewer capsids of HSV-1 wild type, HSV1-ΔVP26, and HSV1-GFPVP26 arrived at the nucleus. Thus, even in the absence of the potential viral dynein receptor VP26, HSV-1 used MT and dynein for efficient nuclear targeting. These data suggest that besides VP26, HSV-1 encodes other receptors for dynein or dynactin. PMID:16873277

  12. The HSV-1 tegument protein pUL46 associates with cellular membranes and viral capsids

    SciTech Connect

    Murphy, Michael A.; Bucks, Michelle A.; O'Regan, Kevin J.; Courtney, Richard J.

    2008-07-05

    The molecular mechanisms responsible for the addition of tegument proteins into nascent herpesvirus particles are poorly understood. To better understand the tegumentation process of herpes simplex virus type 1 (HSV-1) virions, we initiated studies that showed the tegument protein pUL46 (VP11/12) has a similar cellular localization to the membrane-associated tegument protein VP22. Using membrane flotation analysis we found that pUL46 associates with membranes in both the presence and absence of other HSV-1 proteins. However, when purified virions were stripped of their envelope, the majority of pUL46 was found to associate with the capsid fraction. This strong affinity of pUL46 for capsids was confirmed by an in vitro capsid pull-down assay in which purified pUL46-GST was able to interact specifically with capsids purified from the nuclear fraction of HSV-1 infected cells. These results suggest that pUL46 displays a dynamic interaction between cellular membranes and capsids.

  13. In Silico Studies of Medicinal Compounds Against Hepatitis C Capsid Protein from North India

    PubMed Central

    Mathew, Shilu; Faheem, Muhammad; Archunan, Govindaraju; Ilyas, Muhammad; Begum, Nargis; Jahangir, Syed; Qadri, Ishtiaq; Qahtani, Mohammad Al; Mathew, Shiny

    2014-01-01

    Hepatitis viral infection is a leading cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Over one million people are estimated to be persistently infected with hepatitis C virus (HCV) worldwide. As capsid core protein is the key element in spreading HCV; hence, it is considered to be the superlative target of antiviral compounds. Novel drug inhibitors of HCV are in need to complement or replace the current treatments such as pegylated interferon’s and ribavirin as they are partially booming and beset with various side effects. Our study was conducted to predict 3D structure of capsid core protein of HCV from northern part of India. Core, the capsid protein of HCV, handles the assembly and packaging of HCV RNA genome and is the least variable of all the ten HCV proteins among the six HCV genotypes. Therefore, we screened four phytochemicals inhibitors that are known to disrupt the interactions of core and other HCV proteins such as (a) epigallocatechin gallate (EGCG), (b) ladanein, (c) naringenin, and (d) silybin extracted from medicinal plants; targeted against active site of residues of HCV-genotype 3 (G3) (Q68867) and its subtypes 3b (Q68861) and 3g (Q68865) from north India. To study the inhibitory activity of the recruited flavonoids, we conducted a quantitative structure–activity relationship (QSAR). Furthermore, docking interaction suggests that EGCG showed a maximum number of hydrogen bond (H-bond) interactions with all the three modeled capsid proteins with high interaction energy followed by naringenin and silybin. Thus, our results strongly correlate the inhibitory activity of the selected bioflavonoid. Finally, the dynamic predicted capsid protein molecule of HCV virion provides a general avenue to target structure-based antiviral compounds that support the hypothesis that the screened inhibitors for viral capsid might constitute new class of potent agents but further confirmation is necessary using in vitro and in vivo

  14. In vitro assembly of polymorphic virus-like particles from the capsid protein of a nodavirus.

    PubMed

    Bajaj, Saumya; Banerjee, Manidipa

    2016-09-01

    Viral capsid proteins are programmed to assemble into homogeneous structures in native environments; but the molecular details of these assembly pathways are seldom clearly understood. In order to define the chain of events in the construction of a minimal system, we attempted controlled assembly of the capsid protein of a small insect nodavirus, Flock House Virus (FHV). Bacterial expression of the FHV capsid protein, and subsequent in vitro assembly, generated a heterogeneous population of closed particles. We show that in spite of the altered structure, these particles are capable of membrane disruption, like native viruses, and of incorporating and delivering foreign cargo to specific locations. The unique structure and characteristics of these particles extends our understanding of nodavirus assembly. Additionally, the establishment of a bacterial production system, and methods for in vitro assembly and packaging are of considerable benefit for biotechnological applications of FHV. PMID:27289029

  15. Porcine circovirus-2 capsid protein induces cell death in PK15 cells

    SciTech Connect

    Walia, Rupali; Dardari, Rkia Chaiyakul, Mark; Czub, Markus

    2014-11-15

    Studies have shown that Porcine circovirus (PCV)-2 induces apoptosis in PK15 cells. Here we report that cell death is induced in PCV2b-infected PK15 cells that express Capsid (Cap) protein and this effect is enhanced in interferon gamma (IFN-γ)-treated cells. We further show that transient PCV2a and 2b-Cap protein expression induces cell death in PK15 cells at rate similar to PCV2 infection, regardless of Cap protein localization. These data suggest that Cap protein may have the capacity to trigger different signaling pathways involved in cell death. Although further investigation is needed to gain deeper insights into the nature of the pathways involved in Cap-induced cell death, this study provides evidence that PCV2-induced cell death in kidney epithelial PK15 cells can be mapped to the Cap protein and establishes the need for future research regarding the role of Cap-induced cell death in PCV2 pathogenesis. - Highlights: • IFN-γ enhances PCV2 replication that leads to cell death in PK15 cells. • IFN-γ enhances nuclear localization of the PCV2 Capsid protein. • Transient PCV2a and 2b-Capsid protein expression induces cell death. • Cell death is not dictated by specific Capsid protein sub-localization.

  16. Point mutations in the potato leafroll virus major capsid protein alter virion stability and aphid transmission

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The coat protein (CP) of potato leafroll virus (PLRV) is the primary component of the capsid and is a multifunctional protein known to be involved in vector transmission and virus movement within plant hosts, in addition to particle assembly. Thirteen mutations were generated in various regions of ...

  17. Mapping the Structural Determinants Responsible for Enhanced T Cell Activation to the Immunogenic Adeno-Associated Virus Capsid from Isolate Rhesus 32.33

    PubMed Central

    Mays, Lauren E.; Wang, Lili; Tenney, Rebeca; Bell, Peter; Nam, Hyun-Joo; Lin, Jianping; Gurda, Brittney; Van Vliet, Kim; Mikals, Kyle; Agbandje-McKenna, Mavis

    2013-01-01

    Avoiding activation of immunity to vector-encoded proteins is critical to the safe and effective use of adeno-associated viral (AAV) vectors for gene therapy. While commonly used serotypes, such as AAV serotypes 1, 2, 7, 8, and 9, are often associated with minimal and/or dysfunctional CD8+ T cell responses in mice, the threshold for immune activation appears to be lower in higher-order species. We have modeled this discrepancy within the mouse by identifying two capsid variants with differential immune activation profiles: AAV serotype 8 (AAV8) and a hybrid between natural rhesus isolates AAVrh32 and AAVrh33 (AAVrh32.33). Here, we aimed to characterize the structural determinants of the AAVrh32.33 capsid that augment cellular immunity to vector-encoded proteins or those of AAV8 that may induce tolerance. We hypothesized that the structural domain responsible for differential immune activation could be mapped to surface-exposed regions of the capsid, such as hypervariable regions (HVRs) I to IX of VP3. To test this, a series of hybrid AAV capsids was constructed by swapping domains between AAV8 and AAVrh32.33. By comparing their ability to generate transgene-specific T cells in vivo versus the stability of transgene expression in the muscle, we confirmed that the functional domain lies within the VP3 portion of the capsid. Our studies were able to exclude the regions of VP3 which are not sufficient for augmenting the cellular immune response, notably, HVRs I, II, and V. We have also identified HVR IV as a region of interest in conferring the efficiency and stability of muscle transduction to AAVrh32.33. PMID:23720715

  18. Virus-specific interaction between the human cytomegalovirus major capsid protein and the C terminus of the assembly protein precursor.

    PubMed Central

    Beaudet-Miller, M; Zhang, R; Durkin, J; Gibson, W; Kwong, A D; Hong, Z

    1996-01-01

    We previously identified a minimal 12-amino-acid domain in the C terminus of the herpes simplex virus type 1 (HSV-1) scaffolding protein which is required for interaction with the HSV-1 major capsid protein. An alpha-helical structure which maximizes the hydropathicity of the minimal domain is required for the interaction. To address whether cytomegalovirus (CMV) utilizes the same strategy for capsid assembly, several glutathione S-transferase fusion proteins to the C terminus of the CMV assembly protein precursor were produced and purified from bacterial cells. The study showed that the glutathione S-transferase fusion containing 16 amino acids near the C-terminal end was sufficient to interact with the major capsid protein. Interestingly, no cross-interaction between HSV-1 and CMV could be detected. Mutation analysis revealed that a three-amino-acid region at the N-terminal side of the central Phe residue of the CMV interaction domain played a role in determining the viral specificity of the interaction. When this region was converted so as to correspond to that of HSV-1, the CMV assembly protein domain lost its ability to interact with the CMV major capsid protein but gained full interaction with the HSV-1 major capsid protein. To address whether the minimal interaction domain of the CMV assembly protein forms an alpha-helical structure similar to that in HSV-1, peptide competition experiments were carried out. The results showed that a cyclic peptide derived from the interaction domain with a constrained (alpha-helical structure competed for interaction with the major capsid protein much more efficiently than the unconstrained linear peptide. In contrast, a cyclic peptide containing an Ala substitution for the critical Phe residue did not compete for the interaction at all. The results of this study suggest that (i) CMV may have developed a strategy similar to that of HSV-1 for capsid assembly; (ii) the minimal interaction motif in the CMV assembly protein

  19. Osmolyte-Mediated Encapsulation of Proteins inside MS2 Viral Capsids

    PubMed Central

    Glasgow, Jeff E.; Capehart, Stacy L.; Francis, Matthew B.; Tullman-Ercek, Danielle

    2012-01-01

    The encapsulation of enzymes in nanometer-sized compartments has the potential to enhance and control enzymatic activity, both in vivo and in vitro. Despite this potential, there are little quantitative data on the effect of encapsulation in a well-defined compartment under varying conditions. To gain more insight into these effects, we have characterized two improved methods for the encapsulation of heterologous molecules inside bacteriophage MS2 viral capsids. First, attaching DNA oligomers to a molecule of interest and incubating it with MS2 coat protein dimers yielded reassembled capsids that packaged the tagged molecules. The addition of a protein stabilizing osmolyte, trimethylamine-N-oxide (TMAO), significantly increased the yields of reassembly. Second, we found that expressed proteins with genetically encoded negatively charged peptide tags could also induce capsid reassembly, resulting in high yields of reassembled capsids containing the protein. This second method was used to encapsulate alkaline phosphatase tagged with a 16 amino acid peptide. The purified encapsulated enzyme was found to have the same Km value and a slightly lower kcat value than the free enzyme, indicating that this method of encapsulation had a minimal effect on enzyme kinetics. This method provides a practical and potentially scalable way of studying the complex effects of encapsulating enzymes in protein-based compartments. PMID:22953696

  20. Altering Tropism of rAAV by Directed Evolution.

    PubMed

    Marsic, Damien; Zolotukhin, Sergei

    2016-01-01

    Directed evolution represents an attractive approach to derive AAV capsid variants capable of selectively infect specific tissue or cell targets. It involves the generation of an initial library of high complexity followed by cycles of selection during which the library is progressively enriched for target-specific variants. Each selection cycle consists of the following: reconstitution of complete AAV genomes within plasmid molecules; production of virions for which each particular capsid variant is matched with the particular capsid gene encoding it; recovery of capsid gene sequences from target tissue after systemic administration. Prevalent variants are then analyzed and evaluated. PMID:26611585

  1. Antibody recognition of porcine circovirus type 2 capsid protein epitopes after vaccination, infection, and disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Open reading frame 2 (ORF2) of porcine circovirus type 2 (PCV2) codes for the 233-amino-acid capsid protein (CP). Baculovirus-based vaccines that express only ORF2 are protective against clinical disease following experimental challenge or natural infection. The goal of this study was to identify re...

  2. Hepatitis B Virus Core Protein Phosphorylation Sites Affect Capsid Stability and Transient Exposure of the C-terminal Domain.

    PubMed

    Selzer, Lisa; Kant, Ravi; Wang, Joseph C-Y; Bothner, Brian; Zlotnick, Adam

    2015-11-20

    Hepatitis B virus core protein has 183 amino acids divided into an assembly domain and an arginine-rich C-terminal domain (CTD) that regulates essential functions including genome packaging, reverse transcription, and intracellular trafficking. Here, we investigated the CTD in empty hepatitis B virus (HBV) T=4 capsids. We examined wild-type core protein (Cp183-WT) and a mutant core protein (Cp183-EEE), in which three CTD serines are replaced with glutamate to mimic phosphorylated protein. We found that Cp183-WT capsids were less stable than Cp183-EEE capsids. When we tested CTD sensitivity to trypsin, we detected two different populations of CTDs differentiated by their rate of trypsin cleavage. Interestingly, CTDs from Cp183-EEE capsids exhibited a much slower rate of proteolytic cleavage when compared with CTDs of Cp183-WT capsids. Cryo-electron microscopy studies of trypsin-digested capsids show that CTDs at five-fold symmetry vertices are most protected. We hypothesize that electrostatic interactions between glutamates and arginines in Cp183-EEE, particularly at five-fold, increase capsid stability and reduce CTD exposure. Our studies show that quasi-equivalent CTDs exhibit different rates of exposure and thus might perform distinct functions during the hepatitis B virus lifecycle. Our results demonstrate a structural role for CTD phosphorylation and indicate crosstalk between CTDs within a capsid particle. PMID:26405031

  3. Strong cortical and spinal cord transduction after AAV7 and AAV9 delivery into the cerebrospinal fluid of nonhuman primates.

    PubMed

    Samaranch, Lluis; Salegio, Ernesto A; San Sebastian, Waldy; Kells, Adrian P; Bringas, John R; Forsayeth, John; Bankiewicz, Krystof S

    2013-05-01

    The present study builds on previous work showing that infusion of adeno-associated virus type 9 (AAV9) into the cisterna magna (CM) of nonhuman primates resulted in widespread transduction throughout cortex and spinal cord. Transduction efficiency was severely limited, however, by the presence of circulating anti-AAV antibodies. Accordingly, we compared AAV9 to a related serotype, AAV7, which has a high capsid homology. CM infusion of either AAV7 or AAV9 directed high level of cell transduction with similar patterns of distribution throughout brain cortex and along the spinal cord. Dorsal root ganglia and corticospinal tracts were also transduced. Both astrocytes and neurons were transduced. Interestingly, little transduction was observed in peripheral organs. Our results indicate that intrathecal delivery of either AAV7 or AAV9 directs a robust and widespread cellular transduction in the central nervous system and other peripheral neural structures. PMID:23517473

  4. Herpes Simplex Virus Capsid-Organelle Association in the Absence of the Large Tegument Protein UL36p

    PubMed Central

    Kharkwal, Himanshu; Furgiuele, Sara Shanda; Smith, Caitlin G.

    2015-01-01

    ABSTRACT UL36p (VP1/2) is the largest protein encoded by herpes simplex virus 1 (HSV-1) and resides in the innermost layer of the viral tegument, lying between the capsid and the envelope. UL36p performs multiple functions in the HSV life cycle, including an essential role in cytoplasmic envelopment. We earlier described the isolation of a virion-associated cytoplasmic membrane fraction from HSV-infected cells. Biochemical and ultrastructural analyses showed that the organelles in this buoyant fraction contain enveloped infectious HSV particles in their lumens and naked capsids docked to their cytoplasmic surfaces. These organelles can also recruit molecular motors and transport their cargo virions along microtubules in vitro. Here we examine the properties of these HSV-associated organelles in the absence of UL36p. We find that while capsid envelopment is clearly defective, a subpopulation of capsids nevertheless still associate with the cytoplasmic faces of these organelles. The existence of these capsid-membrane structures was confirmed by subcellular fractionation, immunocytochemistry, lipophilic dye fluorescence microscopy, thin-section electron microscopy, and correlative light and electron microscopy. We conclude that capsid-membrane binding can occur in the absence of UL36p and propose that this association may precede the events of UL36p-driven envelopment. IMPORTANCE Membrane association and envelopment of the HSV capsid are essential for the assembly of an infectious virion. Envelopment involves the complex interplay of a large number of viral and cellular proteins; however, the function of most of them is unknown. One example of this is the viral protein UL36p, which is clearly essential for envelopment but plays a poorly understood role. Here we demonstrate that organelles utilized for HSV capsid envelopment still accumulate surface-bound capsids in the absence of UL36p. We propose that UL36p-independent binding of capsids to organelles occurs prior to

  5. Proof of concept study with an HER-2 mimotope anticancer vaccine deduced from a novel AAV-mimotope library platform

    PubMed Central

    Singer, Josef; Manzano-Szalai, Krisztina; Fazekas, Judit; Thell, Kathrin; Bentley-Lukschal, Anna; Stremnitzer, Caroline; Roth-Walter, Franziska; Weghofer, Margit; Ritter, Mirko; Pino Tossi, Kerstin; Hörer, Markus; Michaelis, Uwe; Jensen-Jarolim, Erika

    2016-01-01

    ABSTRACT Background: Anticancer vaccines could represent a valuable complementary strategy to established therapies, especially in settings of early stage and minimal residual disease. HER-2 is an important target for immunotherapy and addressed by the monoclonal antibody trastuzumab. We have previously generated HER-2 mimotope peptides from phage display libraries. The synthesized peptides were coupled to carriers and applied for epitope-specific induction of trastuzumab-like IgG. For simplification and to avoid methodological limitations of synthesis and coupling chemistry, we herewith present a novel and optimized approach by using adeno-associated viruses (AAV) as effective and high-density mimotope-display system, which can be directly used for vaccination. Methods: An AAV capsid display library was constructed by genetically incorporating random peptides in a plasmid encoding the wild-type AAV2 capsid protein. AAV clones, expressing peptides specifically reactive to trastuzumab, were employed to immunize BALB/c mice. Antibody titers against human HER-2 were determined, and the isotype composition and functional properties of these were tested. Finally, prophylactically immunized mice were challenged with human HER-2 transfected mouse D2F2/E2 cells. Results: HER-2 mimotope AAV-vaccines induced antibodies specific to human HER-2. Two clones were selected for immunization of mice, which were subsequently grafted D2F2/E2 cells. Both mimotope AAV clones delayed the growth of tumors significantly, as compared to controls. Conclusion: In this study, a novel mimotope AAV-based platform was created allowing the isolation of mimotopes, which can be directly used as anticancer vaccines. The example of trastuzumab AAV-mimotopes demonstrates that this vaccine strategy could help to establish active immunotherapy for breast-cancer patients. PMID:27622022

  6. Comparison of Transduction Efficiency of Recombinant AAV Serotypes 1, 2, 5, and 8 in the Rat Nigrostriatal System

    PubMed Central

    McFarland, Nikolaus R.; Lee, Jeng-Shin; Hyman, Bradley T.; McLean, Pamela J.

    2009-01-01

    Enhanced delivery and expression of genes in specific neuronal systems is critical for the development of genetic models of neurodegenerative disease and potential gene therapy. Recent discovery of new recombinant adeno-associated viral (rAAV) capsid serotypes has resulted in improved transduction efficiency, but expression levels, spread of transgene, and potential toxicity can differ depending on brain region and among species. We compared the transduction efficiency of titer-matched rAAV 2/1, 2/5 and 2/8 to the commonly used rAAV2/2 in the rat nigrostriatal system via expression of the reporter transgene, enhanced green fluorescent protein (EGFP). Newer rAAV serotypes 2/1, 2/5 and 2/8 demonstrated marked increase in transduction and spread of EGFP expression in dopaminergic nigrostriatal neurons and projections to the striatum and globus pallidus compared to rAAV2/2 at 2 weeks post injection. The number of nigral cells transduced was greatest for rAAV2/1, but for serotypes 2/5 and 2/8 was still 2 to 3-fold higher than that for 2/2. Enhanced transduction did not cause an increase in glial cell response or toxicity. New rAAV serotypes thus promise improved gene delivery to nigrostriatal system with the potential for better models and therapeutics for Parkinson disease and other neurodegenerative disorders. PMID:19250335

  7. A hydrophobic domain within the small capsid protein of Kaposi’s sarcoma-associated herpesvirus is required for assembly

    PubMed Central

    Capuano, Christopher M.; Grzesik, Peter; Kreitler, Dale; Pryce, Erin N.; Desai, Keshal V.; Coombs, Gavin; McCaffery, J. Michael

    2014-01-01

    Kaposi’s sarcoma-associated herpesvirus (KSHV) capsids can be produced in insect cells using recombinant baculoviruses for protein expression. All six capsid proteins are required for this process to occur and, unlike for alphaherpesviruses, the small capsid protein (SCP) ORF65 is essential for this process. This protein decorates the capsid shell by virtue of its interaction with the capsomeres. In this study, we have explored the SCP interaction with the major capsid protein (MCP) using GFP fusions. The assembly site within the nucleus of infected cells was visualized by light microscopy using fluorescence produced by the SCP–GFP polypeptide, and the relocalization of the SCP to these sites was evident only when the MCP and the scaffold protein were also present – indicative of an interaction between these proteins that ensures delivery of the SCP to assembly sites. Biochemical assays demonstrated a physical interaction between the SCP and MCP, and also between this complex and the scaffold protein. Self-assembly of capsids with the SCP–GFP polypeptide was evident. Potentially, this result can be used to engineer fluorescent KSHV particles. A similar SCP–His6 polypeptide was used to purify capsids from infected cell lysates using immobilized affinity chromatography and to directly label this protein in capsids using chemically derivatized gold particles. Additional studies with SCP–GFP polypeptide truncation mutants identified a domain residing between aa 50 and 60 of ORF65 that was required for the relocalization of SCP–GFP to nuclear assembly sites. Substitution of residues in this region and specifically at residue 54 with a polar amino acid (lysine) disrupted or abolished this localization as well as capsid assembly, whereas substitution with non-polar residues did not affect the interaction. Thus, this study identified a small conserved hydrophobic domain that is important for the SCP–MCP interaction. PMID:24824860

  8. A hydrophobic domain within the small capsid protein of Kaposi's sarcoma-associated herpesvirus is required for assembly.

    PubMed

    Capuano, Christopher M; Grzesik, Peter; Kreitler, Dale; Pryce, Erin N; Desai, Keshal V; Coombs, Gavin; McCaffery, J Michael; Desai, Prashant J

    2014-08-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) capsids can be produced in insect cells using recombinant baculoviruses for protein expression. All six capsid proteins are required for this process to occur and, unlike for alphaherpesviruses, the small capsid protein (SCP) ORF65 is essential for this process. This protein decorates the capsid shell by virtue of its interaction with the capsomeres. In this study, we have explored the SCP interaction with the major capsid protein (MCP) using GFP fusions. The assembly site within the nucleus of infected cells was visualized by light microscopy using fluorescence produced by the SCP-GFP polypeptide, and the relocalization of the SCP to these sites was evident only when the MCP and the scaffold protein were also present - indicative of an interaction between these proteins that ensures delivery of the SCP to assembly sites. Biochemical assays demonstrated a physical interaction between the SCP and MCP, and also between this complex and the scaffold protein. Self-assembly of capsids with the SCP-GFP polypeptide was evident. Potentially, this result can be used to engineer fluorescent KSHV particles. A similar SCP-His6 polypeptide was used to purify capsids from infected cell lysates using immobilized affinity chromatography and to directly label this protein in capsids using chemically derivatized gold particles. Additional studies with SCP-GFP polypeptide truncation mutants identified a domain residing between aa 50 and 60 of ORF65 that was required for the relocalization of SCP-GFP to nuclear assembly sites. Substitution of residues in this region and specifically at residue 54 with a polar amino acid (lysine) disrupted or abolished this localization as well as capsid assembly, whereas substitution with non-polar residues did not affect the interaction. Thus, this study identified a small conserved hydrophobic domain that is important for the SCP-MCP interaction. PMID:24824860

  9. DNA condensates organized by the capsid protein VP15 in White Spot Syndrome Virus

    SciTech Connect

    Liu Yingjie; Wu Jinlu; Chen Hu; Hew, Choy Leong; Yan Jie

    2010-12-20

    The White Spot Syndrome Virus (WSSV) has a large circular double-stranded DNA genome of around 300 kb and it replicates in the nucleus of the host cells. The machinery of how the viral DNA is packaged has been remained unclear. VP15, a highly basic protein, is one of the major capsid proteins found in the virus. Previously, it was shown to be a DNA binding protein and was hypothesized to participate in the viral DNA packaging process. Using Atomic Force Microscopy imaging, we show that the viral DNA is associated with a (or more) capsid proteins. The organized viral DNA qualitatively resembles the conformations of VP15 induced DNA condensates in vitro. Furthermore, single-DNA manipulation experiments revealed that VP15 is able to condense single DNA against forces of a few pico Newtons. Our results suggest that VP15 may aid in the viral DNA packaging process by directly condensing DNA.

  10. The Collagen-like Protein gp12 Is a Temperature-dependent Reversible Binder of SPP1 Viral Capsids*

    PubMed Central

    Zairi, Mohamed; Stiege, Asita C.; Nhiri, Naima; Jacquet, Eric; Tavares, Paulo

    2014-01-01

    Icosahedral capsids of viruses are lattices of defined geometry and homogeneous size. The (quasi-)equivalent organization of their protein building blocks provides, in numerous systems, the binding sites to assemble arrays of viral polypeptides organized with nanometer precision that protrude from the capsid surface. The capsid of bacterial virus (bacteriophage) SPP1 exposes, at its surface, the 6.6-kDa viral polypeptide gp12 that binds to the center of hexamers of the major capsid protein. Gp12 forms an elongated trimer with collagen-like properties. This is consistent with the fold of eight internal GXY repeats of gp12 to build a stable intersubunit triple helix in a prokaryotic setting. The trimer dissociates and unfolds at near physiological temperatures, as reported for eukaryotic collagen. Its structural organization is reacquired within seconds upon cooling. Interaction with the SPP1 capsid hexamers strongly stabilizes gp12, increasing its Tm to 54 °C. Above this temperature, gp12 dissociates from its binding sites and unfolds reversibly. Multivalent binding of gp12 trimers to the capsid is highly cooperative. The capsid lattice also provides a platform to assist folding and association of unfolded gp12 polypeptides. The original physicochemical properties of gp12 offer a thermoswitchable system for multivalent binding of the polypeptide to the SPP1 capsid surface. PMID:25074929

  11. The Staphylococcus aureus pathogenicity island 1 protein gp6 functions as an internal scaffold during capsid size determination

    PubMed Central

    Dearborn, Altaira D.; Spilman, Michael S.; Damle, Priyadarshan K.; Chang, Jenny R.; Monroe, Eric B.; Saad, Jamil S.; Christie, Gail E.; Dokland, Terje

    2011-01-01

    Staphylococcus aureus pathogenicity island 1 (SaPI1) is a mobile genetic element that carries genes for several superantigen toxins. SaPI1 is normally stably integrated into the host genome, but can become mobilized by “helper” bacteriophage 80α, leading to the packaging of SaPI1 genomes into phage-like transducing particles that are composed of structural proteins supplied by the helper phage, but having smaller capsids. We show that the SaPI1-encoded protein gp6 is necessary for efficient formation of small capsids. The NMR structure of gp6 reveals a dimeric protein with a helix-loop-helix motif similar to that of bacteriophage scaffolding proteins. The gp6 dimer matches internal densities that bridge capsid subunits in cryo-EM reconstructions of SaPI1 procapsids, suggesting that gp6 acts as an internal scaffolding protein in capsid size determination. PMID:21821042

  12. Insight into the Assembly of Viruses with Vertical Single β-barrel Major Capsid Proteins.

    PubMed

    Gil-Carton, David; Jaakkola, Salla T; Charro, Diego; Peralta, Bibiana; Castaño-Díez, Daniel; Oksanen, Hanna M; Bamford, Dennis H; Abrescia, Nicola G A

    2015-10-01

    Archaeal viruses constitute the least explored niche within the virosphere. Structure-based approaches have revealed close relationships between viruses infecting organisms from different domains of life. Here, using biochemical and cryo-electron microscopy techniques, we solved the structure of euryarchaeal, halophilic, internal membrane-containing Haloarcula hispanica icosahedral virus 2 (HHIV-2). We show that the density of the two major capsid proteins (MCPs) recapitulates vertical single β-barrel proteins and that disulfide bridges stabilize the capsid. Below, ordered density is visible close to the membrane and at the five-fold vertices underneath the host-interacting vertex complex underpinning membrane-protein interactions. The HHIV-2 structure exemplifies the division of conserved architectural elements of a virion, such as the capsid, from those that evolve rapidly due to selective environmental pressure such as host-recognizing structures. We propose that in viruses with two vertical single β-barrel MCPs the vesicle is indispensable, and membrane-protein interactions serve as protein-railings for guiding the assembly. PMID:26320579

  13. Synergistic inhibition of PARP-1 and NF-κB signaling downregulates immune response against recombinant AAV2 vectors during hepatic gene therapy.

    PubMed

    Hareendran, Sangeetha; Ramakrishna, Banumathi; Jayandharan, Giridhara R

    2016-01-01

    Host immune response remains a key obstacle to widespread application of adeno-associated virus (AAV) based gene therapy. Thus, targeted inhibition of the signaling pathways that trigger such immune responses will be beneficial. Previous studies have reported that DNA damage response proteins such as poly(ADP-ribose) polymerase-1 (PARP-1) negatively affect the integration of AAV in the host genome. However, the role of PARP-1 in regulating AAV transduction and the immune response against these vectors has not been elucidated. In this study, we demonstrate that repression of PARP-1 improves the transduction of single-stranded AAV vectors both in vitro (∼174%) and in vivo (two- to 3.4-fold). Inhibition of PARP-1, also significantly downregulated the expression of several proinflammatory and cytokine markers such as TLRs, ILs, NF-κB subunit proteins associated with the host innate response against self-complementary AAV2 vectors. The suppression of the inflammatory response targeted against these vectors was more effective upon combined inhibition of PARP-1 and NF-κB signaling. This strategy also effectively attenuated the AAV capsid-specific cytotoxic T-cell response, with minimal effect on vector transduction, as demonstrated in normal C57BL/6 and hemophilia B mice. These data suggest that targeting specific host cellular proteins could be useful to attenuate the immune barriers to AAV-mediated gene therapy. PMID:26443873

  14. Adeno-Associated Virus Capsid Proteins May Play a Role in Transcription and Second-Strand Synthesis of Recombinant Genomes

    PubMed Central

    Salganik, Maxim; Aydemir, Fikret; Nam, Hyun-Joo; McKenna, Robert; Agbandje-McKenna, Mavis

    2014-01-01

    A group of four interacting amino acids in adeno-associated virus type 8 (AAV8) called the pH quartet has been shown to undergo a structural change when subjected to acidic pH comparable to that seen in endosomal compartments. We examined the phenotypes of mutants with mutations in these amino acids as well as several nearby residues in the background of AAV2. We found that three of the mutations in this region (Y704A, E562A, and E564A) produce normal titers of mature capsids but are extremely defective for transduction (>107-fold). The remaining mutants were also defective for transduction, but the defect in these mutants (E563A, E561A, H526A, and R389A) is not as severe (3- to 22-fold). Two other mutants (Y700A and Y730A) were found to be defective for virus assembly. One of the extremely defective mutants (Y704A) was found to enter the cell, traffic to the nucleus, and uncoat its DNA nearly as efficiently as the wild type. This suggested that some step after nuclear entry and uncoating was defective. To see if the extremely defective mutants were impaired in second-strand synthesis, the Y704A, E562A, and E564A mutants containing self-complementary DNA were compared with virus containing single-stranded genomes. Two of the mutants (Y704A and E564A) showed 1-log and 3-log improvements in infectivity, respectively, while the third mutant (E562A) showed no change. This suggested that inhibition of second-strand synthesis was responsible for some but not most of the defect in these mutants. Comparison of Y704A mRNA synthesis with that of the wild-type capsid showed that accumulation of steady-state mRNA in the Y704A mutant was reduced 450-fold, even though equal genome numbers were uncoated. Our experiments have identified a novel capsid function. They suggest that AAV capsids may play a role in the initiation of both second-strand synthesis and transcription of the input genome. PMID:24198419

  15. Conversion of a dodecahedral protein capsid into pentamers via minimal point mutations.

    PubMed

    Chen, Hsiao-Nung; Woycechowsky, Kenneth J

    2012-06-12

    Protein self-assembly relies upon the formation of stabilizing noncovalent interactions across subunit interfaces. Identifying the determinants of self-assembly is crucial for understanding structure-function relationships in symmetric protein complexes and for engineering responsive nanoscale architectures for applications in medicine and biotechnology. Lumazine synthases (LS's) comprise a protein family that forms diverse quaternary structures, including pentamers and 60-subunit dodecahedral capsids. To improve our understanding of the basis for this difference in assembly, we attempted to convert the capsid-forming LS from Aquifex aeolicus (AaLS) into pentamers through a small number of rationally designed amino acid substitutions. Our mutations targeted side chains at ionic (R40), hydrogen bonding (H41), and hydrophobic (L121 and I125) interaction sites along the interfaces between pentamers. We found that substitutions at two or three of these positions could reliably generate pentameric variants of AaLS. Biophysical characterization indicates that this quaternary structure change is not accompanied by substantial changes in secondary or tertiary structure. Interestingly, previous homology-based studies of the assembly determinants in LS's had identified only one of these four positions. The ability to control assembly state in protein capsids such as AaLS could aid efforts in the development of new systems for drug delivery, biocatalysis, or materials synthesis. PMID:22606973

  16. Nature's favorite building block: Deciphering folding and capsid assembly of proteins with the HK97-fold.

    PubMed

    Suhanovsky, Margaret M; Teschke, Carolyn M

    2015-05-01

    For many (if not all) bacterial and archaeal tailed viruses and eukaryotic Herpesvirdae the HK97-fold serves as the major architectural element in icosahedral capsid formation while still enabling the conformational flexibility required during assembly and maturation. Auxiliary proteins or Δ-domains strictly control assembly of multiple, identical, HK97-like subunits into procapsids with specific icosahedral symmetries, rather than aberrant non-icosahedral structures. Procapsids are precursor structures that mature into capsids in a process involving release of auxiliary proteins (or cleavage of Δ-domains), dsDNA packaging, and conformational rearrangement of the HK97-like subunits. Some coat proteins built on the ubiquitous HK97-fold also have accessory domains or loops that impart specific functions, such as increased monomer, procapsid, or capsid stability. In this review, we analyze the numerous HK97-like coat protein structures that are emerging in the literature (over 40 at time of writing) by comparing their topology, additional domains, and their assembly and misassembly reactions. PMID:25864106

  17. Structure and Dynamics of Full Length HIV-1 Capsid Protein in Solution

    PubMed Central

    Deshmukh, Lalit; Schwieters, Charles D.; Grishaev, Alexander; Ghirlando, Rodolfo; Baber, James L.; Clore, G. Marius

    2013-01-01

    The HIV-1 capsid protein plays a crucial role in viral infectivity, assembling into a cone that encloses the viral RNA. In the mature virion, the N-terminal domain of the capsid protein forms hexameric and pentameric rings, while C-terminal domain homodimers connect adjacent N-terminal domain rings to one another. Structures of disulfide-linked hexamer and pentamer assemblies, as well as structures of the isolated domains have been solved previously. The dimer configuration in C-terminal domain constructs differs in solution (residues 144–231) and crystal (residues 146–231) structures by ~30°, and it has been postulated that the former connects the hexamers while the latter links pentamers to hexamers. Here we study the structure and dynamics of full-length capsid protein in solution, comprising a mixture of monomeric and dimeric forms in dynamic equilibrium, using ensemble simulated annealing driven by experimental NMR residual dipolar couplings and X-ray scattering data. The complexity of the system necessitated the development of a novel computational framework that should be generally applicable to many other challenging systems that currently escape structural characterization by standard application of mainstream techniques of structural biology. We show that the orientation of the C-terminal domains in dimeric full-length capsid and isolated C-terminal domain constructs is the same in solution, and obtain a quantitative description of the conformational space sampled by the N-terminal domain relative to the C-terminal domain on the nano- to millisecond time-scale. The positional distribution of the N-terminal domain relative to the C-terminal domain is large and modulated by the oligomerization state of the C-terminal domain. We also show that a model of the hexamer/pentamer assembly can be readily generated with a single configuration of the C-terminal domain dimer, and that capsid assembly likely proceeds via conformational selection of sparsely

  18. Functional characterization of Kaposi's sarcoma-associated herpesvirus small capsid protein by bacterial artificial chromosome-based mutagenesis

    SciTech Connect

    Sathish, Narayanan; Yuan Yan

    2010-11-25

    A systematic investigation of interactions amongst KSHV capsid proteins was undertaken in this study to comprehend lesser known KSHV capsid assembly mechanisms. Interestingly the interaction patterns of the KSHV small capsid protein, ORF65 suggested its plausible role in viral capsid assembly pathways. Towards further understanding this, ORF65-null recombinant mutants (BAC-{Delta}65 and BAC-stop65) employing a bacterial artificial chromosome (BAC) system were generated. No significant difference was found in both overall viral gene expression and lytic DNA replication between stable monolayers of 293T-BAC36 (wild-type) and 293T-BAC-ORF65-null upon induction with 12-O-tetradecanoylphorbol-13-acetate, though the latter released 30-fold fewer virions to the medium than 293T-BAC36 cells. Sedimentation profiles of capsid proteins of ORF65-null recombinant mutants were non-reflective of their organization into the KSHV capsids and were also undetectable in cytoplasmic extracts compared to noticeable levels in nuclear extracts. These observations collectively suggested the pivotal role of ORF65 in the KSHV capsid assembly processes.

  19. Retrovirus Restriction by TRIM5 Proteins Requires Recognition of Only a Small Fraction of Viral Capsid Subunits

    PubMed Central

    Shi, Jiong; Friedman, David B.

    2013-01-01

    The host restriction factors TRIM5α and TRIMCyp potently inhibit retrovirus infection by binding to the incoming retrovirus capsid. TRIM5 proteins are dimeric, and their association with the viral capsid appears to be enhanced by avidity effects owing to formation of higher-order oligomeric complexes. We examined the stoichiometric requirement for TRIM5 functional recognition by quantifying the efficiencies of restriction of HIV-1 and murine leukemia virus (MLV) particles containing various proportions of restriction-sensitive and -insensitive CA subunits. Both TRIMCyp and TRIM5α inhibited infection of retrovirus particles containing as little as 25% of the restriction-sensitive CA protein. Accordingly, we also observed efficient binding of TRIMCyp in vitro to capsid assemblies containing as little as one-fourth wild-type CA protein. Paradoxically, the ability of HIV-1 particles to abrogate TRIMCyp restriction in trans was more strongly dependent on the fraction of wild-type CA than was restriction of infection. Collectively, our results indicate that TRIM5 restriction factors bind to retroviral capsids in a highly cooperative manner and suggest that TRIM5 can engage a capsid lattice containing a minimum of three or fewer recognizable subunits per hexamer. Our study supports a model in which localized binding of TRIM5 to the viral capsid nucleates rapid polymerization of a TRIM5 lattice on the capsid surface. PMID:23785198

  20. Derivation of a triple mosaic adenovirus based on modification of the minor capsid protein IX

    SciTech Connect

    Tang Yizhe; Le, Long P.; Matthews, Qiana L.; Han Tie; Wu Hongju; Curiel, David T.

    2008-08-01

    Adenoviral capsid protein IX (pIX) has been shown to be a potential locale to insert targeting, imaging-related and therapeutic modalities by genetic modification. Recent evidences suggested that capsid protein mosaicism could be a promising strategy for improving the utility of Ad vector. In this study, we explored a method to genetically generate triple pIX mosaic Ad serotype 5 (Ad5) displaying three types of pIX on a single virion. pIXs were modified at their carboxy termini with a Flag sequence, a hexahistidine sequence (His{sub 6}) or a monomeric red fluorescent protein (mRFP1), respectively. Western blotting analysis and fluorescence microscopy of the purified recombinant viruses indicated that all three modified pIXs were incorporated into the viral particles. Immuno-gold electron microscopy (EM) further confirmed that three types of pIX indeed co-existed on an individual virion. These results firstly validated a triple mosaic capsid configuration on pIX, and demonstrated the possibility of further radical design.

  1. Hepatitis virus protein X-Phenylalanine Hydroxylase fusion proteins identified in PKU mice treated with AAV-WPRE vectors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Utilizing the Pahenu2 mouse model for phenylketonuria (PKU), we developed an improved expression vector containing the Woodchuck Hepatitis Virus post-transcriptional regulatory element inserted into a rAAV-mPAH construct (rAAV-mPAH-WPRE) for treatment of PKU. Following portal vein delivery of these ...

  2. The Minor Capsid Protein VP11 of Thermophilic Bacteriophage P23-77 Facilitates Virus Assembly by Using Lipid-Protein Interactions

    PubMed Central

    Moilanen, Anni M.; Rissanen, Ilona A.; Määttä, Juha A. E.; Hytönen, Vesa P.; Ihalainen, Janne A.

    2015-01-01

    ABSTRACT Thermus thermophilus bacteriophage P23-77 is the type member of a new virus family of icosahedral, tailless, inner-membrane-containing double-stranded DNA (dsDNA) viruses infecting thermophilic bacteria and halophilic archaea. The viruses have a unique capsid architecture consisting of two major capsid proteins assembled in various building blocks. We analyzed the function of the minor capsid protein VP11, which is the third known capsid component in bacteriophage P23-77. Our findings show that VP11 is a dynamically elongated dimer with a predominantly α-helical secondary structure and high thermal stability. The high proportion of basic amino acids in the protein enables electrostatic interaction with negatively charged molecules, including nucleic acid and large unilamellar lipid vesicles (LUVs). The plausible biological function of VP11 is elucidated by demonstrating the interactions of VP11 with Thermus-derived LUVs and with the major capsid proteins by means of the dynamic-light-scattering technique. In particular, the major capsid protein VP17 was able to link VP11-complexed LUVs into larger particles, whereas the other P23-77 major capsid protein, VP16, was unable to link VP11-comlexed LUVs. Our results rule out a previously suggested penton function for VP11. Instead, the electrostatic membrane association of VP11 triggers the binding of the major capsid protein VP17, thus facilitating a controlled incorporation of the two different major protein species into the assembling capsid. IMPORTANCE The study of thermophilic viruses with inner membranes provides valuable insights into the mechanisms used for stabilization and assembly of protein-lipid systems at high temperatures. Our results reveal a novel way by which an internal membrane and outer capsid shell are linked in a virus that uses two different major protein species for capsid assembly. We show that a positive protein charge is important in order to form electrostatic interactions with the

  3. Bioinformatic analysis of non-VP1 capsid protein of coxsackievirus A6.

    PubMed

    Liu, Hong-Bo; Yang, Guang-Fei; Liang, Si-Jia; Lin, Jun

    2016-08-01

    This study bioinformatically analyzed the non-VP1 capsid proteins (VP2-VP4) of Coxasckievirus A6 (CVA6), with an attempt to predict their basic physicochemical properties, structural/functional features and linear B cell eiptopes. The online tools SubLoc, TargetP and the others from ExPASy Bioinformatics Resource Portal, and SWISS-MODEL (an online protein structure modeling server), were utilized to analyze the amino acid (AA) sequences of VP2-VP4 proteins of CVA6. Our results showed that the VP proteins of CVA6 were all of hydrophilic nature, contained phosphorylation and glycosylation sites and harbored no signal peptide sequences and acetylation sites. Except VP3, the other proteins did not have transmembrane helix structure and nuclear localization signal sequences. Random coils were the major conformation of the secondary structure of the capsid proteins. Analysis of the linear B cell epitopes by employing Bepipred showed that the average antigenic indices (AI) of individual VP proteins were all greater than 0 and the average AI of VP4 was substantially higher than that of VP2 and VP3. The VP proteins all contained a number of potential B cell epitopes and some eiptopes were located at the internal side of the viral capsid or were buried. We successfully predicted the fundamental physicochemical properties, structural/functional features and the linear B cell eiptopes and found that different VP proteins share some common features and each has its unique attributes. These findings will help us understand the pathogenicity of CVA6 and develop related vaccines and immunodiagnostic reagents. PMID:27465341

  4. Stability of Norwalk Virus Capsid Protein Interfaces Evaluated by in Silico Nanoindentation

    PubMed Central

    Boyd, Kevin J.; Bansal, Prakhar; Feng, Jun; May, Eric R.

    2015-01-01

    Norwalk virus causes severe gastroenteritis for which there is currently no specific anti-viral therapy. A stage of the infection process is uncoating of the protein capsid to expose the viral genome and allow for viral replication. A mechanical characterization of the Norwalk virus may provide important information relating to the mechanism of uncoating. The mechanical strength of the Norwalk virus has previously been investigated using atomic force microscopy (AFM) nanoindentation experiments. Those experiments cannot resolve specific molecular interactions, and therefore, we have employed a molecular modeling approach to gain insights into the potential uncoating mechanism of the Norwalk capsid. In this study, we perform simulated nanoindentation using a coarse-grained structure-based model, which provides an estimate of the spring constant in good agreement with the experimentally determined value. We further analyze the fracture mechanisms and determine weak interfaces in the capsid structure, which are potential sites to inhibit uncoating by stabilization of these weak interfaces. We conclude by identifying potential target sites at the junction of a weak protein–protein interface. PMID:26284238

  5. Specific interaction of capsid protein and importin-{alpha}/{beta} influences West Nile virus production

    SciTech Connect

    Bhuvanakantham, Raghavan; Chong, Mun-Keat; Ng, Mah-Lee

    2009-11-06

    West Nile virus (WNV) capsid (C) protein has been shown to enter the nucleus of infected cells. However, the mechanism by which C protein enters the nucleus is unknown. In this study, we have unveiled for the first time that nuclear transport of WNV and Dengue virus C protein is mediated by their direct association with importin-{alpha}. This interplay is mediated by the consensus sequences of bipartite nuclear localization signal located between amino acid residues 85-101 together with amino acid residues 42 and 43 of C protein. Elucidation of biological significance of importin-{alpha}/C protein interaction demonstrated that the binding efficiency of this association influenced the nuclear entry of C protein and virus production. Collectively, this study illustrated the molecular mechanism by which the C protein of arthropod-borne flavivirus enters the nucleus and showed the importance of importin-{alpha}/C protein interaction in the context of flavivirus life-cycle.

  6. AAV-8 Is More Efficient than AAV-9 in Transducing Neonatal Dog Heart

    PubMed Central

    Pan, Xiufang; Yue, Yongping; Zhang, Keqing; Hakim, Chady H.; Kodippili, Kasun; McDonald, Thomas

    2015-01-01

    Abstract Adeno-associated virus serotype-8 and 9 (AAV-8 and 9) are the leading candidate vectors to test bodywide neonatal muscle gene therapy in large mammals. We have previously shown that systemic injection of 2–2.5×1014 viral genome (vg) particles/kg of AAV-9 resulted in widespread skeletal muscle gene transfer in newborn dogs. However, nominal transduction was observed in the heart. In contrast, robust expression was achieved in both skeletal muscle and heart in neonatal dogs with 7.14–9.06×1014 vg particles/kg of AAV-8. To determine whether superior cardiac transduction of AAV-8 is because of the higher vector dose, we delivered 6.14×1014 and 9.65×1014 vg particles/kg of AAV-9 to newborn puppies via the jugular vein. Transduction was examined 2.5 months later. Consistent with our previous reports, we observed robust bodywide transduction in skeletal muscle. However, increased AAV dose only moderately improved heart transduction. It never reached the level achieved by AAV-8. Our results suggest that differential cardiac transduction by AAV-8 and AAV-9 is likely because of the intrinsic property of the viral capsid rather than the vector dose. PMID:25763686

  7. scAAV-Mediated Gene Transfer of Interleukin 1-Receptor Antagonist to Synovium and Articular Cartilage in Large Mammalian Joints

    PubMed Central

    Watson, Rachael S.; Broome, Ted A.; Levings, Padraic P.; Rice, Bret L.; Kay, Jesse D.; Smith, Andrew D.; Gouze, Elvire; Gouze, Jean-Noel; Dacanay, E. Anthony; Hauswirth, William W.; Nickerson, David M.; Dark, Michael J.; Colahan, Patrick T.; Ghivizzani, Steven C.

    2012-01-01

    With the long-term goal of developing a gene-based treatment for osteoarthritis (OA), we performed studies to evaluate the equine joint as a model for AAV-mediated gene transfer to large, weight-bearing human joints. A self-complementary AAV2 vector containing the coding regions for human interleukin-1 receptor antagonist (hIL-1Ra) or green fluorescent protein (GFP) was packaged in AAV capsid serotypes 1, 2, 5, 8 and 9. Following infection of human and equine synovial fibroblasts in culture, we found that both were only receptive to transduction with AAV1, 2 and 5. For these serotypes, however, transgene expression from the equine cells was consistently at least 10-fold higher. Analyses of AAV surface receptor molecules and intracellular trafficking of vector genomes implicate enhanced viral uptake by the equine cells. Following delivery of 1 × 1011 vector genomes of serotypes 2, 5 and 8 into the forelimb joints of the horse, all three enabled hIL-1Ra expression at biologically relevant levels and effectively transduced the same cell types, primarily synovial fibroblasts and, to a lesser degree, chondrocytes in articular cartilage. These results provide optimism that AAV vectors can be effectively adapted for gene delivery to large human joints affected by OA. PMID:23151520

  8. AAV ancestral reconstruction library enables selection of broadly infectious viral variants.

    PubMed

    Santiago-Ortiz, J; Ojala, D S; Westesson, O; Weinstein, J R; Wong, S Y; Steinsapir, A; Kumar, S; Holmes, I; Schaffer, D V

    2015-12-01

    Adeno-associated virus (AAV) vectors have achieved clinical efficacy in treating several diseases. However, enhanced vectors are required to extend these landmark successes to other indications and protein engineering approaches may provide the necessary vector improvements to address such unmet medical needs. To generate new capsid variants with potentially enhanced infectious properties and to gain insights into AAV's evolutionary history, we computationally designed and experimentally constructed a putative ancestral AAV library. Combinatorial variations at 32 amino acid sites were introduced to account for uncertainty in their identities. We then analyzed the evolutionary flexibility of these residues, the majority of which have not been previously studied, by subjecting the library to iterative selection on a representative cell line panel. The resulting variants exhibited transduction efficiencies comparable to the most efficient extant serotypes and, in general, ancestral libraries were broadly infectious across the cell line panel, indicating that they favored promiscuity over specificity. Interestingly, putative ancestral AAVs were more thermostable than modern serotypes and did not use sialic acids, galactose or heparan sulfate proteoglycans for cellular entry. Finally, variants mediated 19- to 31-fold higher gene expression in the muscle compared with AAV1, a clinically used serotype for muscle delivery, highlighting their promise for gene therapy. PMID:26186661

  9. Location of the Bacteriophage P22 Coat Protein C-terminus Provides Opportunities for the Design of Capsid Based Materials

    PubMed Central

    Servid, Amy; Jordan, Paul; O’Neil, Alison; Prevelige, Peter; Douglas, Trevor

    2013-01-01

    Rational design of modifications to the interior and exterior surfaces of virus-like particles (VLPs) for future therapeutic and materials applications is based on structural information about the capsid. Existing cryo-electron microscopy based models suggest that the C-terminus of the bacteriophage P22 coat protein (CP) extends towards the capsid exterior. Our biochemical analysis through genetic manipulations of the C-terminus supports the model where the CP C-terminus is exposed on the exterior of the P22 capsid. Capsids displaying a 6xHis tag appended to the CP C-terminus bind to a Ni affinity column, and the addition of positively or negatively charged coiled coil peptides to the capsid results in association of these capsids upon mixing. Additionally, a single cysteine appended to the CP C-terminus results in the formation of intercapsid disulfide bonds and can serve as a site for chemical modifications. Thus, the C-terminus is a powerful location for multivalent display of peptides that facilitate nanoscale assembly and capsid modification. PMID:23957641

  10. Phosphorylation of the budgerigar fledgling disease virus major capsid protein VP1

    NASA Technical Reports Server (NTRS)

    Haynes, J. I. 2nd; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    The structural proteins of the budgerigar fledgling disease virus, the first known nonmammalian polyomavirus, were analyzed by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The major capsid protein VP1 was found to be composed of at least five distinct species having isoelectric points ranging from pH 6.45 to 5.85. By analogy with the murine polyomavirus, these species apparently result from different modifications of an initial translation product. Primary chicken embryo cells were infected in the presence of 32Pi to determine whether the virus structural proteins were modified by phosphorylation. SDS-PAGE of the purified virus structural proteins demonstrated that VP1 (along with both minor capsid proteins) was phosphorylated. Two-dimensional analysis of the radiolabeled virus showed phosphorylation of only the two most acidic isoelectric species of VP1, indicating that this posttranslational modification contributes to VP1 species heterogeneity. Phosphoamino acid analysis of 32P-labeled VP1 revealed that phosphoserine is the only phosphoamino acid present in the VP1 protein.

  11. Phosphorylation of the budgerigar fledgling disease virus major capsid protein VP1.

    PubMed Central

    Haynes, J I; Consigli, R A

    1992-01-01

    The structural proteins of the budgerigar fledgling disease virus, the first known nonmammalian polyomavirus, were analyzed by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The major capsid protein VP1 was found to be composed of at least five distinct species having isoelectric points ranging from pH 6.45 to 5.85. By analogy with the murine polyomavirus, these species apparently result from different modifications of an initial translation product. Primary chicken embryo cells were infected in the presence of 32Pi to determine whether the virus structural proteins were modified by phosphorylation. SDS-PAGE of the purified virus structural proteins demonstrated that VP1 (along with both minor capsid proteins) was phosphorylated. Two-dimensional analysis of the radiolabeled virus showed phosphorylation of only the two most acidic isoelectric species of VP1, indicating that this posttranslational modification contributes to VP1 species heterogeneity. Phosphoamino acid analysis of 32P-labeled VP1 revealed that phosphoserine is the only phosphoamino acid present in the VP1 protein. Images PMID:1318417

  12. Heat-shock protein 90 promotes nuclear transport of herpes simplex virus 1 capsid protein by interacting with acetylated tubulin.

    PubMed

    Zhong, Meigong; Zheng, Kai; Chen, Maoyun; Xiang, Yangfei; Jin, Fujun; Ma, Kaiqi; Qiu, Xianxiu; Wang, Qiaoli; Peng, Tao; Kitazato, Kaio; Wang, Yifei

    2014-01-01

    Although it is known that inhibitors of heat shock protein 90 (Hsp90) can inhibit herpes simplex virus type 1 (HSV-1) infection, the role of Hsp90 in HSV-1 entry and the antiviral mechanisms of Hsp90 inhibitors remain unclear. In this study, we found that Hsp90 inhibitors have potent antiviral activity against standard or drug-resistant HSV-1 strains and viral gene and protein synthesis are inhibited in an early phase. More detailed studies demonstrated that Hsp90 is upregulated by virus entry and it interacts with virus. Hsp90 knockdown by siRNA or treatment with Hsp90 inhibitors significantly inhibited the nuclear transport of viral capsid protein (ICP5) at the early stage of HSV-1 infection. In contrast, overexpression of Hsp90 restored the nuclear transport that was prevented by the Hsp90 inhibitors, suggesting that Hsp90 is required for nuclear transport of viral capsid protein. Furthermore, HSV-1 infection enhanced acetylation of α-tubulin and Hsp90 interacted with the acetylated α-tubulin, which is suppressed by Hsp90 inhibition. These results demonstrate that Hsp90, by interacting with acetylated α-tubulin, plays a crucial role in viral capsid protein nuclear transport and may provide novel insight into the role of Hsp90 in HSV-1 infection and offer a promising strategy to overcome drug-resistance. PMID:24901434

  13. Improved dual AAV vectors with reduced expression of truncated proteins are safe and effective in the retina of a mouse model of Stargardt disease

    PubMed Central

    Trapani, Ivana; Toriello, Elisabetta; de Simone, Sonia; Colella, Pasqualina; Iodice, Carolina; Polishchuk, Elena V.; Sommella, Andrea; Colecchi, Linda; Rossi, Settimio; Simonelli, Francesca; Giunti, Massimo; Bacci, Maria L.; Polishchuk, Roman S.; Auricchio, Alberto

    2015-01-01

    Stargardt disease (STGD1) due to mutations in the large ABCA4 gene is the most common inherited macular degeneration in humans. We have shown that dual adeno-associated viral (AAV) vectors effectively transfer ABCA4 to the retina of Abca4−/− mice. However, they express both lower levels of transgene compared with a single AAV and truncated proteins. To increase productive dual AAV concatemerization, which would overcome these limitations, we have explored the use of either various regions of homology or heterologous inverted terminal repeats (ITR). In addition, we tested the ability of various degradation signals to decrease the expression of truncated proteins. We found the highest levels of transgene expression using regions of homology based on either alkaline phosphatase or the F1 phage (AK). The use of heterologous ITR does not decrease the levels of truncated proteins relative to full-length ABCA4 and impairs AAV vector production. Conversely, the inclusion of the CL1 degradation signal results in the selective degradation of truncated proteins from the 5′-half without affecting full-length protein production. Therefore, we developed dual AAV hybrid ABCA4 vectors including homologous ITR2, the photoreceptor-specific G protein-coupled receptor kinase 1 promoter, the AK region of homology and the CL1 degradation signal. We show that upon subretinal administration these vectors are both safe in pigs and effective in Abca4−/− mice. Our data support the use of improved dual AAV vectors for gene therapy of STGD1. PMID:26420842

  14. Solid-State NMR Studies of HIV-1 Capsid Protein Assemblies

    PubMed Central

    Han, Yun; Ahn, Jinwoo; Concel, Jason; Byeon, In-Ja L.; Gronenborn, Angela M.; Yang, Jun; Polenova, Tatyana

    2010-01-01

    In mature HIV-1 virions, a 26.6 kDa CA protein is assembled into a characteristic cone shaped core (capsid) that encloses the RNA viral genome. The assembled capsid structure is best described by a fullerene cone model that is made up from a hexameric lattice containing a variable number of CA pentamers, thus allowing for closure of tubular or conical structures. In this report, we present a solid-state NMR analysis of the wild type HIV-1 CA protein, prepared as conical and spherical assemblies that are stable and are not affected by magic angle spinning of the samples at frequencies between 10 and 25 kHz. Multidimensional homo- and heteronuclear correlation spectra of CA assemblies of uniformly 13C,15N-labelled CA exhibit narrow lines, indicative of conformational homogeneity of the protein in these assemblies. For the conical assemblies, partial residue-specific resonance assignments were obtained. Analysis of the NMR spectra recorded for the conical and spherical assemblies indicates that the CA protein structure is not significantly different in the different morphologies. The present results demonstrate that the assemblies of CA protein are amenable to detailed structural analysis by solid-state NMR spectroscopy. PMID:20092249

  15. Solid-State NMR Studies of HIV-1 Capsid Protein Assemblies

    SciTech Connect

    Han, Yun; Ahn, Jinwoo; Concel, Jason; Byeon, In-Ja L.; Gronenborn, Angela M.; Yang, Jun; Polenova, Tatyana E.

    2010-02-17

    In mature HIV-1 virions, the 26.6 kDa CA protein is assembled into a characteristic cone-shaped core (capsid) that encloses the RNA viral genome. The assembled capsid structure is best described by a fullerene cone model that is made up from a hexameric lattice containing a variable number of CA pentamers, thus allowing for closure of tubular or conical structures. In this paper, we present a solid-state NMR analysis of the wild-type HIV-1 CA protein, prepared as conical and spherical assemblies that are stable and are not affected by magic angle spinning of the samples at frequencies between 10 and 25 kHz. Multidimensional homo- and heteronuclear correlation spectra of CA assemblies of uniformly 13C,15Nlabeled CA exhibit narrow lines, indicative of the conformational homogeneity of the protein in these assemblies. For the conical assemblies, partial residue-specific resonance assignments were obtained. Analysis of the NMR spectra recorded for the conical and spherical assemblies indicates that the CA protein structure is not significantly different in the different morphologies. The present results demonstrate that the assemblies of CA protein are amenable to detailed structural analysis by solid-state NMR spectroscopy.

  16. A functional nuclear localization sequence in the VP1 capsid protein of coxsackievirus B3

    SciTech Connect

    Wang, Tianying; Yu, Bohai; Lin, Lexun; Zhai, Xia; Han, Yelu; Qin, Ying; Guo, Zhiwei; Wu, Shuo; Zhong, Xiaoyan; Wang, Yan; Tong, Lei; Zhang, Fengmin; Si, Xiaoning; Zhao, Wenran; Zhong, Zhaohua

    2012-11-25

    The capsid proteins of some RNA viruses can translocate to the nucleus and interfere with cellular phenotypes. In this study we found that the VP1 capsid protein of coxsackievirus B3 (CVB3) was dominantly localized in the nucleus of the cells transfected with VP1-expressing plasmid. The VP1 nuclear localization also occurred in the cells infected with CVB3. Truncation analysis indicated that the VP1 nuclear localization sequence located near the C-terminal. The substitution of His220 with threonine completely abolished its translocation. The VP1 proteins of other CVB types might have the nuclear localization potential because this region was highly conserved. Moreover, the VP1 nuclear localization induced cell cycle deregulation, including a prolonged S phase and shortened G2-M phase. Besides these findings, we also found a domain between Ala72 and Phe106 that caused the VP1 truncates dotted distributed in the cytoplasm. Our results suggest a new pathogenic mechanism of CVB. - Highlights: Black-Right-Pointing-Pointer The VP1 protein of coxsackievirus B3 can specifically localize in the nucleus. Black-Right-Pointing-Pointer The nuclear localization signal of coxsackievirus B3 VP1 protein locates near its C-terminal. Black-Right-Pointing-Pointer The VP1 nuclear localization of coxsackievirus B3 can deregulate cell cycle. Black-Right-Pointing-Pointer There is a domain in the VP1 that determines it dotted distributed in the cytoplasm.

  17. Analysis of adeno-associated virus (AAV) wild-type and mutant Rep proteins for their abilities to negatively regulate AAV p5 and p19 mRNA levels.

    PubMed Central

    Kyöstiö, S R; Owens, R A; Weitzman, M D; Antoni, B A; Chejanovsky, N; Carter, B J

    1994-01-01

    The rep gene of adeno-associated virus type 2 (AAV) encodes four overlapping Rep proteins that are involved in gene regulation and replication of the virus. We studied here the regulation of mRNA transcribed from the AAV p5 and p19 promoters, using transient expression in human 293 cells followed by Northern (RNA) blot analysis of the mRNA. The p5 transcript encodes the larger Rep proteins, Rep78 and Rep68, while the p19 transcript encodes the smaller proteins, Rep52 and Rep40. A plasmid (pNTC3) containing the entire AAV genome with an amber mutation in the rep gene accumulated higher levels of p5 and p19 mRNA than a plasmid containing the wild-type AAV genome. Addition of increasing amounts of the wild-type rep gene in trans from a heterologous promoter inhibited p5 and p19 mRNA accumulation from pNTC3, indicating that the levels of both transcripts were decreased by the Rep proteins. Cotransfections with plasmids producing individual wild-type Rep proteins in trans showed that p5 and p19 mRNA accumulation was inhibited 5- to 10-fold by Rep78 and Rep68 and 2- to 3-fold by Rep52 and Rep40. Analysis of carboxyl-terminal truncation mutants of Rep78 showed that the ability of Rep78 to decrease p5 and p19 mRNA levels was lost when 159 or more amino acids were deleted. Rep78 and Rep68 mutants deleted for the methionine at residue 225 showed decreased abilities to down-regulate both p5 and p19 transcript levels, while mutants containing a substitution of glycine for the methionine resembled the wild-type Rep78. A Rep78 protein with a mutation in the putative nucleoside triphosphate binding site inhibited expression from p5 but not from p19, suggesting that the regulation of p5 transcript levels by Rep78 and Rep68 differs from that of p19. A deletion analysis of AAV cis sequences revealed that an intact terminal repeat was not required for negative regulation of p5 and p19 transcript levels and that the regulation of p19 mRNA levels by Rep78 did not require the presence

  18. Recombinant AAV Vectors for Enhanced Expression of Authentic IgG

    PubMed Central

    Fuchs, Sebastian P.; Martinez-Navio, José M.; Gao, Guangping; Desrosiers, Ronald C.

    2016-01-01

    Adeno-associated virus (AAV) has become a vector of choice for the treatment of a variety of genetic diseases that require safe and long-term delivery of a missing protein. Muscle-directed gene transfer for delivery of protective antibodies against AIDS viruses and other pathogens has been used experimentally in mice and monkeys. Here we examined a number of variations to AAV vector design for the ability to produce authentic immunoglobulin G (IgG) molecules. Expression of rhesus IgG from a single single-stranded AAV (ssAAV) vector (one vector approach) was compared to expression from two self-complementary AAV (scAAV) vectors, one for heavy chain and one for light chain (two vector approach). Both the one vector and the two vector approaches yielded considerable levels of expressed full-length IgG. A number of modifications to the ssAAV expression system were then examined for their ability to increase the efficiency of IgG expression. Inclusion of a furin cleavage sequence with a linker peptide just upstream of the 2A self-cleaving sequence from foot-and-mouth disease virus (F2A) increased IgG expression approximately 2 fold. Inclusion of these sequences also helped to ensure a proper sequence at the C-terminal end of the heavy chain. Inclusion of the post-transcriptional regulatory element from woodchuck hepatitis virus (WPRE) further increased IgG expression 1.5–2.0 fold. IgG1 versions of the two rhesus IgGs that were examined consistently expressed better than the IgG2 forms. In contrast to what has been reported for AAV2-mediated expression of other proteins, introduction of capsid mutations Y445F and Y731F did not increase ssAAV1-mediated expression of IgG as determined by transduction experiments in cell culture. Our findings provide a rational basis for AAV vector design for expression of authentic IgG. PMID:27332822

  19. Detection of Cryptosporidium parvum oocysts by dot-blotting using monoclonal antibodies to CPV40 capsid protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Monoclonal antibodies (MAb) were prepared against the 40 kDa capsid protein of Cryptosporidium parvum virus (CPV) by immunizing mice with purified recombinant CPV40 protein. By immunoblotting analysis, one MAb, designated MAbCPV40-1, bound to a 40 kDa protein in extracts of C. parvum oocysts, which...

  20. Identification of two functional nuclear localization signals in the capsid protein of duck circovirus

    SciTech Connect

    Xiang, Qi-Wang; Zou, Jin-Feng; Wang, Xin; Sun, Ya-Ni; Gao, Ji-Ming; Xie, Zhi-Jing; Wang, Yu; Zhu, Yan-Li; Jiang, Shi-Jin

    2013-02-05

    The capsid protein (CP) of duck circovirus (DuCV) is the major immunogenic protein and has a high proportion of arginine residues concentrated at the N terminus of the protein, which inhibits efficient mRNA translation in prokaryotic expression systems. In this study, we investigated the subcellular distribution of DuCV CP expressed via recombinant baculoviruses in Sf9 cells and the DNA binding activities of the truncated recombinant DuCV CPs. The results showed that two independent bipartite nuclear localization signals (NLSs) situated at N-terminal 1-17 and 18-36 amino acid residue of the CP. Moreover, two expression level regulatory signals (ELRSs) and two DNA binding signals (DBSs) were also mapped to the N terminus of the protein and overlapped with the two NLSs. The ability of CP to bind DNA, coupled with the karyophilic nature of this protein, strongly suggests that it may be responsible for nuclear targeting of the viral genome.

  1. Effect of internal cleavage site mutations in human immunodeficiency virus type 1 capsid protein on its structure and function.

    PubMed

    Tóth, Ferenc; Kádas, János; Mótyán, János András; Tőzsér, József

    2016-08-01

    The capsid protein of the human immunodeficiency virus type 1 has been found to be a substrate of the retroviral protease in vitro, and its processing was predicted to be strongly dependent on a pH-induced conformational change. Several protease cleavage sites have been identified within the capsid protein, but the importance of its cleavage by the viral protease at the early phase of infection is controversial. To confirm the relevance of this process, we aimed to design, produce, and characterize mutant capsid proteins, in which the protein susceptibility toward HIV-1 protease is altered without affecting other steps of the viral life cycle. Our results indicate that while the introduced mutations changed the cleavage rate at the mutated sites of the capsid protein by HIV-1 protease, some of them caused only negligible or moderate structural changes (A78V, L189F, and L189I). However, the effects of other mutations (W23A, A77P, and L189P) were dramatic, as assessed by secondary structure determination or cyclophilin A-binding assay. Based on our observations, the L189F mutant capsid remains structurally and functionally unchanged and may therefore be the best candidate for use in studies aimed at better understanding the role of the protease in the early postentry events of viral infection or retrovirus-mediated gene transduction. PMID:27516963

  2. Signals for Bidirectional Nucleocytoplasmic Transport in the Duck Hepatitis B Virus Capsid Protein

    PubMed Central

    Mabit, Helene; Breiner, Klaus M.; Knaust, Andreas; Zachmann-Brand, Beate; Schaller, Heinz

    2001-01-01

    Hepadnavirus genome replication involves cytoplasmic and nuclear stages, requiring balanced targeting of cytoplasmic nucleocapsids to the nuclear compartment. In this study, we analyze the signals determining capsid compartmentalization in the duck hepatitis B virus (DHBV) animal model, as this system also allows us to study hepadnavirus infection of cultured primary hepatocytes. Using fusions to the green fluorescent protein as a functional assay, we have identified a nuclear localization signal (NLS) that mediates nuclear pore association of the DHBV nucleocapsid and nuclear import of DHBV core protein (DHBc)-derived polypeptides. The DHBc NLS mapped is unique. It bears homology to repetitive NLS elements previously identified near the carboxy terminus of the capsid protein of hepatitis B virus, the human prototype of the hepadnavirus family, but it maps to a more internal position. In further contrast to the hepatitis B virus core protein NLS, the DHBc NLS is not positioned near phosphorylation target sites that are generally assumed to modulate nucleocytoplasmic transport. In functional assays with a knockout mutant, the DHBc NLS was found to be essential for nuclear pore association of the nucleocapsid. The NLS was found to be also essential for virus production from the full-length DHBV genome in transfected cells and from hepatocytes infected with transcomplemented mutant virus. Finally, the DHBc additionally displayed activity indicative of a nuclear export signal, presumably counterbalancing NLS function in the productive state of the infected cell and thereby preventing nucleoplasmic accumulation of nucleocapsids. PMID:11160696

  3. Nucleotide sequence and expression of the capsid protein gene of feline calicivirus.

    PubMed Central

    Neill, J D; Reardon, I M; Heinrikson, R L

    1991-01-01

    The sequence of the 3'-terminal 2,486 bases of the feline calicivirus (FCV) genome was determined. This region of the FCV genome, from which the 2.4-kb subgenomic RNA is derived, contained two open reading frames. The larger open reading frame, found in the 5' end of the subgenomic mRNA, contained 2,004 bases encoding a polypeptide of 73,467 Da. The smaller open reading frame, encoded in the 3' end of the mRNA, was composed of 318 bases, encoding a polypeptide of 12,185 Da. The AUG initiation codon of the second open reading frame overlapped the UGA termination codon of the first, with the sequence AUGA. The nucleotide sequence of the region containing this overlap resembles the -1 frameshift sequences of the retroviruses. The 5' end of the 2.4-kb subgenomic RNA was mapped by primer extension analysis. There were two apparent transcription initiation points, both of which were 5' to the AUG initiation codon of the large open reading frame. Transcription from these sites yielded RNA transcripts with 5' nontranslated leader regions of 17 and 18 bases. The total length of the 2.4-kb subgenomic RNA was 2,375 bases (from the 5'-most start site) excluding the poly(A) tail. Edman degradation of the purified capsid protein of FCV showed that the capsid protein was encoded by the large open reading frame. Western immunoblot analysis of FCV-infected cells using a feline anti-FCV antiserum demonstrated that translation of the capsid protein was detectable at 3 h postinfection and continued to accumulate until 8 h postinfection, the last time examined. Images PMID:1716692

  4. Expression and self-assembly of recombinant capsid protein from the antigenically distinct Hawaii human calicivirus.

    PubMed Central

    Green, K Y; Kapikian, A Z; Valdesuso, J; Sosnovtsev, S; Treanor, J J; Lew, J F

    1997-01-01

    The Norwalk and Hawaii viruses are antigenically distinct members of the family Caliciviridae and are considered to be important etiologic agents of epidemic gastroenteritis, with most studies focusing on the role of Norwalk virus. To further investigate the importance of Hawaii virus, Hawaii virus-like particles (VLPs) were produced by expression of its capsid protein in the baculovirus system and these VLPs were used as the antigen in an enzyme-linked immunosorbent assay that was efficient in the detection of a serologic response to Hawaii virus. The ready availability of Hawaii VLPs should enable larger-scale epidemiological studies to further elucidate the importance of this agent. PMID:9196224

  5. Polyvalent Display of Heme on Hepatitis B Virus Capsid Protein through Coordination to Hexahistidine Tags

    PubMed Central

    Prasuhn, Duane E.; Kuzelka, Jane; Strable, Erica; Udit, Andrew K.; Cho, So-Hye; Lander, Gabriel C.; Quispe, Joel D.; Diers, James R.; Bocian, David F.; Potter, Clint; Carragher, Bridget; Finn, M.G.

    2009-01-01

    SUMMARY The addition of a hexahistidine tag to the N terminus of the hepatitis B capsid protein gives rise to a self-assembled particle with 80 sites of high local density of histidine side chains. Iron protoporphyrin IX has been found to bind tightly at each of these sites, making a polyvalent system of well-defined spacing between metalloporphyrin complexes. The spectroscopic and redox properties of the resulting particle are consistent with the presence of 80 site-isolated bis(histidine)-bound heme centers, comprising a polyvalent b-type cytochrome mimic. PMID:18482703

  6. Stochastic Kinetics of Viral Capsid Assembly Based on Detailed Protein Structures

    PubMed Central

    Hemberg, Martin; Yaliraki, Sophia N.; Barahona, Mauricio

    2006-01-01

    We present a generic computational framework for the simulation of viral capsid assembly which is quantitative and specific. Starting from PDB files containing atomic coordinates, the algorithm builds a coarse-grained description of protein oligomers based on graph rigidity. These reduced protein descriptions are used in an extended Gillespie algorithm to investigate the stochastic kinetics of the assembly process. The association rates are obtained from a diffusive Smoluchowski equation for rapid coagulation, modified to account for water shielding and protein structure. The dissociation rates are derived by interpreting the splitting of oligomers as a process of graph partitioning akin to the escape from a multidimensional well. This modular framework is quantitative yet computationally tractable, with a small number of physically motivated parameters. The methodology is illustrated using two different viruses which are shown to follow quantitatively different assembly pathways. We also show how in this model the quasi-stationary kinetics of assembly can be described as a Markovian cascading process, in which only a few intermediates and a small proportion of pathways are present. The observed pathways and intermediates can be related a posteriori to structural and energetic properties of the capsid oligomers. PMID:16473916

  7. The Capsid Protein of Turnip Crinkle Virus Overcomes two Separate Defense Barriers to Facilitate Viral Systemic Movement in Arabidopsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The capsid protein (CP) of Turnip crinkle virus (TCV) is a multi-functional protein needed for virus assembly, suppression of RNA silencing-based antiviral defense, and long distance movement in infected plants. In this report, we have examined genetic requirements for the different functions of TCV...

  8. Development of an ELISA based on the baculovirus-expressed capsid protein of porcine circovirus type 2 as antigen.

    PubMed

    Liu, Changming; Ihara, Takeshi; Nunoya, Tetsuo; Ueda, Susumu

    2004-03-01

    The genome of porcine circovirus type 2 (PCV2) contains two major open reading frames, which have been shown to encode the virus capsid and replication-associated proteins. The capsid protein is a major structural protein of the virus; it can be a suitable target antigen for detecting PCV2-specific antibodies to monitor PCV2 infection. To produce the antigen, the capsid protein coding sequence was cloned into a baculovirus transfer vector, and a recombinant capsid (rC) protein of PCV2 was expressed as a combined fusion protein in frame with a C-terminal peptide of six histidines. The affinity-purified rC protein was used as coating antigen to develop an ELISA for detecting the virus-specific antibodies in swine sera. The rC protein-based ELISA (rcELISA) was evaluated by examining a panel of 49 PCV2-positive and 49 PCV2-negative swine sera. In comparative experiments of immunoperoxidase monolayer assay (IPMA) using 102 field sera, there was 89.2% coincidence between data obtained by the rcELISA and IPMA. The rcELISA achieved 88.5% specificity and 89.4% sensitivity for detection of PCV2 antibody in the field sera. The assay showed no cross-reactivity with antibodies to PCV type 1, porcine reproductive and respiratory syndrome virus and porcine parvovirus. The results suggest that the rcELISA is suitable for routine serodiagnosis and epidemiological surveys of PCV2-associated diseases. PMID:15107550

  9. RNase P Ribozymes Inhibit the Replication of Human Cytomegalovirus by Targeting Essential Viral Capsid Proteins

    PubMed Central

    Yang, Zhu; Reeves, Michael; Ye, Jun; Trang, Phong; Zhu, Li; Sheng, Jingxue; Wang, Yu; Zen, Ke; Wu, Jianguo; Liu, Fenyong

    2015-01-01

    An engineered RNase P-based ribozyme variant, which was generated using the in vitro selection procedure, was used to target the overlapping mRNA region of two proteins essential for human cytomegalovirus (HCMV) replication: capsid assembly protein (AP) and protease (PR). In vitro studies showed that the generated variant, V718-A, cleaved the target AP mRNA sequence efficiently and its activity was about 60-fold higher than that of wild type ribozyme M1-A. Furthermore, we observed a reduction of 98%–99% in AP/PR expression and an inhibition of 50,000 fold in viral growth in cells with V718-A, while a 75% reduction in AP/PR expression and a 500-fold inhibition in viral growth was found in cells with M1-A. Examination of the antiviral effects of the generated ribozyme on the HCMV replication cycle suggested that viral DNA encapsidation was inhibited and as a consequence, viral capsid assembly was blocked when the expression of AP and PR was inhibited by the ribozyme. Thus, our study indicates that the generated ribozyme variant is highly effective in inhibiting HCMV gene expression and blocking viral replication, and suggests that engineered RNase P ribozyme can be potentially developed as a promising gene-targeting agent for anti-HCMV therapy. PMID:26114473

  10. Different architectures in the assembly of infectious bursal disease virus capsid proteins expressed in insect cells.

    PubMed

    Martinez-Torrecuadrada, J L; Castón, J R; Castro, M; Carrascosa, J L; Rodriguez, J F; Casal, J I

    2000-12-20

    Infectious bursal disease virus (IBDV) capsid is formed by the processing of a large polyprotein and subsequent assembly of VPX/VP2 and VP3. To learn more about the processing of the polyprotein and factors affecting the correct assembly of the viral capsid in vitro, different constructs were made using two baculovirus transfer vectors, pFastBac and pAcYM1. Surprisingly, the expression of the capsid proteins gave rise to different types of particles in each system, as observed by electron microscopy and immunofluorescence. FastBac expression led to the production of only rigid tubular structures, similar to those described as type I in viral infection. Western blot analysis revealed that these rigid tubules are formed exclusively by VPX. These tubules revealed a hexagonal arrangement of units that are trimer clustered, similar to those observed in IBDV virions. In contrast, pAcYM1 expression led to the assembly of virus-like particles (VLPs), flexible tubules, and intermediate assembly products formed by icosahedral caps elongated in tubes, suggesting an aberrant morphogenesis. Processing of VPX to VP2 seems to be a crucial requirement for the proper morphogenesis and assembly of IBDV particles. After immunoelectron microscopy, VPX/VP2 was detected on the surface of tubules and VLPs. We also demonstrated that VP3 is found only on the inner surfaces of VLPs and caps of the tubular structures. In summary, assembly of VLPs requires the internal scaffolding of VP3, which seems to induce the closing of the tubular architecture into VLPs and, thereafter, the subsequent processing of VPX to VP2. PMID:11118356

  11. Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain.

    PubMed

    Deverman, Benjamin E; Pravdo, Piers L; Simpson, Bryan P; Kumar, Sripriya Ravindra; Chan, Ken Y; Banerjee, Abhik; Wu, Wei-Li; Yang, Bin; Huber, Nina; Pasca, Sergiu P; Gradinaru, Viviana

    2016-02-01

    Recombinant adeno-associated viruses (rAAVs) are commonly used vehicles for in vivo gene transfer. However, the tropism repertoire of naturally occurring AAVs is limited, prompting a search for novel AAV capsids with desired characteristics. Here we describe a capsid selection method, called Cre recombination-based AAV targeted evolution (CREATE), that enables the development of AAV capsids that more efficiently transduce defined Cre-expressing cell populations in vivo. We use CREATE to generate AAV variants that efficiently and widely transduce the adult mouse central nervous system (CNS) after intravenous injection. One variant, AAV-PHP.B, transfers genes throughout the CNS with an efficiency that is at least 40-fold greater than that of the current standard, AAV9 (refs. 14,15,16,17), and transduces the majority of astrocytes and neurons across multiple CNS regions. In vitro, it transduces human neurons and astrocytes more efficiently than does AAV9, demonstrating the potential of CREATE to produce customized AAV vectors for biomedical applications. PMID:26829320

  12. Synthesis of Capsid and Noncapsid Viral Proteins in Response to Encephalomyocarditis Virus Ribonucleic Acid in Animal Cell-Free Systems

    PubMed Central

    Dobos, P.; Kerr, Ian M.; Martin, E. M.

    1971-01-01

    The polypeptide products formed in two cell-free protein-synthetic systems programmed with encephalomyocarditis (EMC) virus ribonucleic acid (RNA) have been compared with the virus-specific proteins found in EMC-infected cells and with the capsid proteins of the purified virion. Tryptic peptides of 35S-methioninelabeled proteins from these three sources were compared by co-chromatography and electrophoresis and by isoelectric focussing. Fifty-two methionine-containing peptides were resolved in digests of material from infected cells, of which about one-third were also clearly present in digests of the virion capsid proteins. The product formed in response to EMC RNA in cell-free systems from Krebs mouse ascites tumor cells yielded 26 to 29 such peptides. Most of these peptides were shown to behave identically with virus-specific peptides from infected cells, whereas just under half of them appeared to be identical with peptides from the virion capsid proteins. The product formed in response to EMC RNA in the L-cell cell-free system was similar, whereas six additional EMC-specific peptides were detected in mixed Krebs L-cell systems. The results indicate that the EMC RNA genome is partially translated in the mouse cell-free systems used to yield products containing both virion capsid and virus-specific noncapsid polypeptides. Images PMID:4331652

  13. A Preclinical Animal Model to Assess the Effect of Pre-existing Immunity on AAV-mediated Gene Transfer

    PubMed Central

    Li, Hua; Lin, Shih-Wen; Giles-Davis, Wynetta; Li, Yan; Zhou, Dongming; Xiang, Zhi Quan; High, Katherine A; Ertl, Hildegund CJ

    2009-01-01

    Hepatic adeno-associated virus (AAV)-serotype 2–mediated gene transfer results in sustained transgene expression in experimental animals but not in human subjects. We hypothesized that loss of transgene expression in humans might be caused by immune memory mechanisms that become reactivated upon AAV vector transfer. Here, we tested the effect of immunological memory to AAV capsid on AAV-mediated gene transfer in a mouse model. Upon hepatic transfer of an AAV2 vector expressing human factor IX (hF.IX), mice immunized with adenovirus (Ad) vectors expressing AAV8 capsid before AAV2 transfer developed less circulating hF.IX and showed a gradual loss of hF.IX gene copies in liver cells as compared to control animals. This was not observed in mice immunized with an Ad vectors expressing AAV2 capsid before transfer of rAAV8-hF.IX vectors. The lower hF.IX expression was primarily linked to AAV-binding antibodies that lacked AAV-neutralizing activity in vitro rather than to AAV capsid–specific CD8+ T cells. PMID:19367258

  14. Immunogenicity of bacteriophage lambda particles displaying porcine Circovirus 2 (PCV2) capsid protein epitopes.

    PubMed

    Gamage, Lakshman N A; Ellis, John; Hayes, Sidney

    2009-11-01

    Phage lambda particles displaying four immunodominant regions of porcine Circovirus 2 (PCV2) capsid protein (LDP-D-CAP) was shown to be immunogenic in pigs. The immunodominant regions were fused to the carboxyl-terminal of lambda head protein D. Expression of D-CAP on lambda display particles was demonstrated by ELISA and Western blots. Pigs receiving LDP-D-CAP, without incorporating adjuvant, showed significant anti-PCV2 immune response following the primary vaccination. The LDP-D-CAP preparation induced PCV2 neutralizing antibodies. Delayed type hypersensitivity (DTH) reaction scores revealed that the immunized pigs were hypersensitive to both lambda phage and PCV2 antigens. The LDP-D-CAP elicited both cellular and humoral immune responses. Neither LDP-D-CAP nor the lambda control elicited any untoward local or systemic reactions following immunization. These studies produced the first potential phage vaccine to porcine Circovirus 2. PMID:19712770

  15. Open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein.

    PubMed

    Nawagitgul, P; Morozov, I; Bolin, S R; Harms, P A; Sorden, S D; Paul, P S

    2000-09-01

    Porcine circovirus 2 (PCV2), a single-stranded DNA virus associated with post-weaning multisystemic wasting syndrome of swine, has two potential open reading frames, ORF1 and ORF2, greater than 600 nucleotides in length. ORF1 is predicted to encode a replication-associated protein (Rep) essential for replication of viral DNA, while ORF2 contains a conserved basic amino acid sequence at the N terminus resembling that of the major structural protein of chicken anaemia virus. Thus far, the structural protein(s) of PCV2 have not been identified. In this study, a viral structural protein of 30 kDa was identified in purified PCV2 particles. ORF2 of PCV2 was cloned into a baculovirus expression vector and the gene product was expressed in insect cells. The expressed ORF2 gene product had a molecular mass of 30 kDa, similar to that detected in purified virus particles. The recombinant ORF2 protein self-assembled to form capsid-like particles when viewed by electron microscopy. Antibodies against the ORF2 protein were detected in samples of sera obtained from pigs as early as 3 weeks after experimental infection with PCV2. These results show that the major structural protein of PCV2 is encoded by ORF2 and has a molecular mass of 30 kDa. PMID:10950986

  16. Antivirals interacting with hepatitis B virus core protein and core mutations may misdirect capsid assembly in a similar fashion.

    PubMed

    Hacker, Hans Jörg; Deres, Karl; Mildenberger, Maria; Schröder, Claus H

    2003-12-15

    Recently, heteroarylpyrimidines (HAP) have been identified as potent inhibitors of capsid maturation. Here we discuss the HAP mode of action comparing the aggregation phenotype of wild-type and mutant core proteins with the respective phenotype imposed by HAP or other agents interacting with core protein. Pertinent tests include core fusion protein-mediated transactivation in a two-hybrid system and capsid formation. The finding that transactivation appeared to be unaffected by HAP, or by mutations preventing assembly, is surprising and raises the question for the structure of the interacting hybrid core proteins: Are they monomers, dimers or even oligomers? A direct activity of core fusion monomers is not excluded but considered to be highly unlikely due to rapid homodimerisation. A role of core fusion dimers in transactivation would indicate distinct interactions with a differential sensitivity to HAP. Regarding significance of data gained in two-hybrid systems, caution is necessary, since the site of transactivation is the nucleus, whereas the real site of the core protein interactions during replication is the cytoplasm. Apparently, HAP leave the monomer-monomer interface of HBV core protein unaffected but prevent capsid maturation by interacting with a region known to be crucial for dimer multimerisation and formation of stable capsids. It is suggested to use antivirals as tools for the elucidation of early steps in genome replication and capsid assembly. A frame for this could be the hypothesis that the virus uses soluble core protein, namely intracellular maturation intermediates of HbeAg for a core targeted self-restriction of replication. PMID:14637185

  17. AAV ANCESTRAL RECONSTRUCTION LIBRARY ENABLES SELECTION OF BROADLY INFECTIOUS VIRAL VARIANTS

    PubMed Central

    Santiago-Ortiz, Jorge; Ojala, David S.; Westesson, Oscar; Weinstein, John R.; Wong, Sophie Y.; Steinsapir, Andrew; Kumar, Sanjay; Holmes, Ian; Schaffer, David V.

    2015-01-01

    Adeno-associated virus (AAV) vectors have achieved clinical efficacy in treating several diseases. Enhanced vectors are required to extend these landmark successes to other indications, however, and protein engineering approaches may provide the necessary vector improvements to address such unmet medical needs. To generate new capsid variants with potentially enhanced infectious properties, and to gain insights into AAV’s evolutionary history, we computationally designed and experimentally constructed a putative ancestral AAV library. Combinatorial variations at 32 amino acid sites were introduced to account for uncertainty in their identities. We then analyzed the evolutionary flexibility of these residues, the majority of which have not been previously studied, by subjecting the library to iterative selection on a representative cell line panel. The resulting variants exhibited transduction efficiencies comparable to the most efficient extant serotypes, and in general ancestral libraries were broadly infectious across the cell line panel, indicating that they favored promiscuity over specificity. Interestingly, putative ancestral AAVs were more thermostable than modern serotypes and did not utilize sialic acids, galactose, or heparan sulfate proteoglycans for cellular entry. Finally, variants mediated 19–31 fold higher gene expression in muscle compared to AAV1, a clinically utilized serotype for muscle delivery, highlighting their promise for gene therapy. PMID:26186661

  18. RNA packaging of MRFV virus-like particles: The interplay between RNA pools and capsid coat protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virus-like particles (VLPs) can be produced through self-assembly of capsid protein (CP) into particles with discrete shapes and sizes and containing different types of RNA molecules. The general principle that governs particle assembly and RNA packaging is determined by unique interactions between ...

  19. Single-site cleavage in the 5'-untranslated region of Leishmaniavirus RNA is mediated by the viral capsid protein.

    PubMed Central

    MacBeth, K J; Patterson, J L

    1995-01-01

    Leishmaniavirus (LRV) is a double-stranded RNA virus that persistently infects the protozoan parasite Leishmania. LRV produces a short RNA transcript, corresponding to the 5' end of positive-sense viral RNA, both in vivo and in in vitro polymerase assays. The short transcript is generated by a single site-specific cleavage event in the 5' untranslated region of the 5.3-kb genome. This cleavage event can be reproduced in vitro with purified viral particles and a substrate RNA transcript possessing the viral cleavage site. A region of nucleotides required for cleavage was identified by analyzing the cleavage sites yielding the short transcripts of various LRV isolates. A 6-nt deletion at this cleavage site completely abolished RNA processing. In an in vitro cleavage assay, baculovirus-expressed capsid protein possessed an endonuclease activity identical to that of native virions, showing that the viral capsid protein is the RNA endonuclease. Identification of the LRV capsid protein as an RNA endonuclease is unprecedented among known viral capsid proteins. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7568059

  20. Continuum Theory of Retroviral Capsids

    NASA Astrophysics Data System (ADS)

    Nguyen, T. T.; Bruinsma, R. F.; Gelbart, W. M.

    2006-02-01

    We present a self-assembly phase diagram for the shape of retroviral capsids, based on continuum elasticity theory. The spontaneous curvature of the capsid proteins drives a weakly first-order transition from spherical to spherocylindrical shapes. The conical capsid shape which characterizes the HIV-1 retrovirus is never stable under unconstrained energy minimization. Only under conditions of fixed volume and/or fixed spanning length can the conical shape be a minimum energy structure. Our results indicate that, unlike the capsids of small viruses, retrovirus capsids are not uniquely determined by the molecular structure of the constituent proteins but depend in an essential way on physical constraints present during assembly.

  1. Specific interaction between hnRNP H and HPV16 L1 proteins: Implications for late gene auto-regulation enabling rapid viral capsid protein production

    SciTech Connect

    Zheng, Zi-Zheng; Sun, Yuan-Yuan; Zhao, Min; Huang, Hui; Zhang, Jun; Xia, Ning-Shao; Miao, Ji; Zhao, Qinjian

    2013-01-18

    Highlights: ► The RNA-binding hnRNP H regulates late viral gene expression. ► hnRNP H activity was inhibited by a late viral protein. ► Specific interaction between HPV L1 and hnRNP H was demonstrated. ► Co-localization of HPV L1 and hnRNP H inside cells was observed. ► Viral capsid protein production, enabling rapid capsid assembly, was implicated. -- Abstract: Heterogeneous nuclear ribonucleoproteins (hnRNPs), including hnRNP H, are RNA-binding proteins that function as splicing factors and are involved in downstream gene regulation. hnRNP H, which binds to G triplet regions in RNA, has been shown to play an important role in regulating the staged expression of late proteins in viral systems. Here, we report that the specific association between hnRNP H and a late viral capsid protein, human papillomavirus (HPV) L1 protein, leads to the suppressed function of hnRNP H in the presence of the L1 protein. The direct interaction between the L1 protein and hnRNP H was demonstrated by complex formation in solution and intracellularly using a variety of biochemical and immunochemical methods, including peptide mapping, specific co-immunoprecipitation and confocal fluorescence microscopy. These results support a working hypothesis that a late viral protein HPV16 L1, which is down regulated by hnRNP H early in the viral life cycle may provide an auto-regulatory positive feedback loop that allows the rapid production of HPV capsid proteins through suppression of the function of hnRNP H at the late stage of the viral life cycle. In this positive feedback loop, the late viral gene products that were down regulated earlier themselves disable their suppressors, and this feedback mechanism could facilitate the rapid production of capsid proteins, allowing staged and efficient viral capsid assembly.

  2. Genetic linkage of capsid protein-encoding RNA segments in group A equine rotaviruses.

    PubMed

    Miño, Samuel; Barrandeguy, María; Parreño, Viviana; Parra, Gabriel I

    2016-04-01

    Rotavirus virions are formed by three concentric protein layers that enclose the 11 dsRNA genome segments and the viral proteins VP1 and VP3. Interactions amongst the capsid proteins (VP2, VP6, VP7 and VP4) have been described to play a major role in viral fitness, whilst restricting the reassortment of the genomic segments during co-infection with different rotavirus strains. In this work we describe and characterize the linkage between VP6 and VP7 proteins based on structural and genomic analyses of group A rotavirus strains circulating in Argentinean horses. Strains with the VP7 genotype G3 showed a strong association with the VP6 genotype I6, whilst strains with G14 were associated with the I2 genotype. Most of the differences on the VP6 and VP7 proteins were observed in interactive regions between the two proteins, suggesting that VP6 : VP7 interactions may drive the co-evolution and co-segregation of their respective gene segments. PMID:26758293

  3. Multiple roles of the capsid protein in the early steps of HIV-1 infection.

    PubMed

    Fassati, Ariberto

    2012-12-01

    The early steps of HIV-1 infection starting after virus entry into cells up to integration of its genome into host chromosomes are poorly understood. From seminal work showing that HIV-1 and oncoretroviruses follow different steps in the early stages post-entry, significant advances have been made in recent years and an important role for the HIV-1 capsid (CA) protein, the constituent of the viral core, has emerged. CA appears to orchestrate several events, such as virus uncoating, recognition by restriction factors and the innate immune system. It also plays a role in nuclear import and integration of HIV-1 and has become a novel target for antiretroviral drugs. Here we describe the different functions of CA and how they may be integrated into one or more coherent models that illuminate the early events in HIV-1 infection and their relations with the host cell. PMID:23041358

  4. In vitro binding of anthrax protective antigen on bacteriophage T4 capsid surface through Hoc-capsid interactions: A strategy for efficient display of large full-length proteins

    SciTech Connect

    Shivachandra, Sathish B.; Rao, Mangala; Janosi, Laszlo; Sathaliyawala, Taheri; Matyas, Gary R.; Alving, Carl R.; Leppla, Stephen H.; Rao, Venigalla B. . E-mail: rao@cua.edu

    2006-02-05

    An in vitro binding system is described to display large full-length proteins on bacteriophage T4 capsid surface at high density. The phage T4 icosahedral capsid features 155 copies of a nonessential highly antigenic outer capsid protein, Hoc, at the center of each major capsid protein hexon. Gene fusions were engineered to express the 83-kDa protective antigen (PA) from Bacillus anthracis fused to the N-terminus of Hoc and the 130-kDa PA-Hoc protein was expressed in Escherichia coli and purified. The purified PA-Hoc was assembled in vitro on hoc {sup -} phage particles. Binding was specific, stable, and of high affinity. This defined in vitro system allowed manipulation of the copy number of displayed PA and imposed no significant limitation on the size of the displayed antigen. In contrast to in vivo display systems, the in vitro approach allows all the capsid binding sites to be occupied by the 130-kDa PA-Hoc fusion protein. The PA-T4 particles were immunogenic in mice in the absence of an adjuvant, eliciting strong PA-specific antibodies and anthrax lethal toxin neutralizing antibodies. The in vitro display on phage T4 offers a novel platform for potential construction of customized vaccines against anthrax and other infectious diseases.

  5. Identification of two functional nuclear localization signals in the capsid protein of duck circovirus.

    PubMed

    Xiang, Qi-Wang; Zou, Jin-Feng; Wang, Xin; Sun, Ya-Ni; Gao, Ji-Ming; Xie, Zhi-Jing; Wang, Yu; Zhu, Yan-Li; Jiang, Shi-Jin

    2013-02-01

    The capsid protein (CP) of duck circovirus (DuCV) is the major immunogenic protein and has a high proportion of arginine residues concentrated at the N terminus of the protein, which inhibits efficient mRNA translation in prokaryotic expression systems. In this study, we investigated the subcellular distribution of DuCV CP expressed via recombinant baculoviruses in Sf9 cells and the DNA binding activities of the truncated recombinant DuCV CPs. The results showed that two independent bipartite nuclear localization signals (NLSs) situated at N-terminal 1-17 and 18-36 amino acid residue of the CP. Moreover, two expression level regulatory signals (ELRSs) and two DNA binding signals (DBSs) were also mapped to the N terminus of the protein and overlapped with the two NLSs. The ability of CP to bind DNA, coupled with the karyophilic nature of this protein, strongly suggests that it may be responsible for nuclear targeting of the viral genome. PMID:23174505

  6. AAV Hybrid Serotypes: Improved Vectors for Gene Delivery

    PubMed Central

    Choi, Vivian W.; McCarty, Douglas M.; Samulski, R. Jude

    2006-01-01

    In recent years, significant efforts have been made on studying and engineering adeno-associated virus (AAV) capsid, in order to increase efficiency in targeting specific cell types that are non-permissive to wild type (wt) viruses and to improve efficacy in infecting only the cell type of interest. With our previous knowledge of the viral properties of the naturally occurring serotypes and the elucidation of their capsid structures, we can now generate capsid mutants, or hybrid serotypes, by various methods and strategies. In this review, we summarize the studies performed on AAV retargeting, and categorize the available hybrid serotypes to date, based on the type of modification: 1) transcapsidation, 2) adsorption of bi-specific antibody to capsid surface, 3) mosaic capsid, and 4) chimeric capsid. Not only these hybrid serotypes could achieve high efficiency of gene delivery to a specific targeted cell type, which can be better-tailored for a particular clinical application, but also serve as a tool for studying AAV biology such as receptor binding, trafficking and genome delivery into the nucleus. PMID:15975007

  7. Site-Specific Structural Variations Accompanying Tubular Assembly of the HIV-1 Capsid Protein

    PubMed Central

    Bayro, Marvin J.; Chen, Bo; Yau, Wai-Ming; Tycko, Robert

    2014-01-01

    The 231-residue capsid (CA) protein of HIV-1 spontaneously self-assembles into tubes with a hexagonal lattice that is believed to mimic the surface lattice of conical capsid cores within intact virions. We report the results of solid state nuclear magnetic resonance (NMR) measurements on HIV-1 CA tubes that provide new information regarding changes in molecular structure that accompany CA self-assembly, local dynamics within CA tubes, and possible mechanisms for the generation of lattice curvature. This information is contained in site-specific assignments of signals in two- and three-dimensional solid state NMR spectra, conformation-dependent 15N and 13C NMR chemical shifts, detection of highly dynamic residues under solution NMR conditions, measurements of local variations in transverse spin relaxation rates of amide 1H nuclei, and quantitative measurements of site-specific 15N-15N dipole-dipole couplings. Our data show that most of the CA sequence is conformationally ordered and relatively rigid in tubular assemblies and that the structures of N-terminal and C-terminal domains (NTD and CTD) observed in solution are largely retained. However, specific segments, including the N-terminal β-hairpin, the cyclophilin A binding loop, the inter-domain linker, segments involved in intermolecular NTD-CTD interactions, and the C-terminal tail, have substantial static or dynamical disorder in tubular assemblies. Other segments, including the 310-helical segment in CTD, undergo clear conformational changes. Structural variations associated with curvature of the CA lattice appear to be localized in the inter-domain linker and intermolecular NTD-CTD interface, while structural variations within NTD hexamers, around local three-fold symmetry axes, and in CTD-CTD dimerization interfaces are less significant. PMID:24370930

  8. Electrostatic Interaction between RNA and Protein Capsid in CCMV Simulated by a Coarse-grain RNA model and a Monte Carlo Approach

    PubMed Central

    Zhang, Deqiang; Konecny, Robert; Baker, Nathan A.; McCammon, J. Andrew

    2008-01-01

    Although many viruses have been crystallized and the protein capsid structures have been determined by X-ray crystallography, the nucleic acids often can not be resolved. This is especially true for RNA viruses. The lack of information about the conformation of DNA/RNA greatly hinders our understanding of the assembly mechanism of various viruses. Here we combine a coarse-grain model and a Monte Carlo method to simulate the distribution of viral RNA inside the capsid of Cowpea Chlorotic Mottle Virus (CCMV). Our results show that there is very strong interaction between the N-terminal residues of the capsid proteins, which are highly positive-charged, and the viral RNA. Without these residues, the binding energy disfavors the binding of RNA by the capsid. The RNA forms a shell close to the capsid with the highest densities associated with the capsid dimers. These high-density regions are connected to each other in the shape of a continuous net of triangles. The overall icosahedral shape of the net overlaps with the capsid subunit icosahedral organization. Medium density of RNA is found under the pentamers of the capsid. These findings are consistent with experimental observations. PMID:15386271

  9. Classic nuclear localization signals and a novel nuclear localization motif are required for nuclear transport of porcine parvovirus capsid proteins.

    PubMed

    Boisvert, Maude; Bouchard-Lévesque, Véronique; Fernandes, Sandra; Tijssen, Peter

    2014-10-01

    Nuclear targeting of capsid proteins (VPs) is important for genome delivery and precedes assembly in the replication cycle of porcine parvovirus (PPV). Clusters of basic amino acids, corresponding to potential nuclear localization signals (NLS), were found only in the unique region of VP1 (VP1up, for VP1 unique part). Of the five identified basic regions (BR), three were important for nuclear localization of VP1up: BR1 was a classic Pat7 NLS, and the combination of BR4 and BR5 was a classic bipartite NLS. These NLS were essential for viral replication. VP2, the major capsid protein, lacked these NLS and contained no region with more than two basic amino acids in proximity. However, three regions of basic clusters were identified in the folded protein, assembled into a trimeric structure. Mutagenesis experiments showed that only one of these three regions was involved in VP2 transport to the nucleus. This structural NLS, termed the nuclear localization motif (NLM), is located inside the assembled capsid and thus can be used to transport trimers to the nucleus in late steps of infection but not for virions in initial infection steps. The two NLS of VP1up are located in the N-terminal part of the protein, externalized from the capsid during endosomal transit, exposing them for nuclear targeting during early steps of infection. Globally, the determinants of nuclear transport of structural proteins of PPV were different from those of closely related parvoviruses. Importance: Most DNA viruses use the nucleus for their replication cycle. Thus, structural proteins need to be targeted to this cellular compartment at two distinct steps of the infection: in early steps to deliver viral genomes to the nucleus and in late steps to assemble new viruses. Nuclear targeting of proteins depends on the recognition of a stretch of basic amino acids by cellular transport proteins. This study reports the identification of two classic nuclear localization signals in the minor capsid

  10. Rubella virus capsid protein modulation of viral genomic and subgenomic RNA synthesis

    SciTech Connect

    Tzeng, W.-P.; Frey, Teryl K. . E-mail: tfrey@gsu.edu

    2005-07-05

    The ratio of the subgenomic (SG) to genome RNA synthesized by rubella virus (RUB) replicons expressing the green fluorescent protein reporter gene (RUBrep/GFP) is substantially higher than the ratio of these species synthesized by RUB (4.3 for RUBrep/GFP vs. 1.3-1.4 for RUB). It was hypothesized that this modulation of the viral RNA synthesis was by one of the virus structural protein genes and it was found that introduction of the capsid (C) protein gene into the replicons as an in-frame fusion with GFP resulted in an increase of genomic RNA production (reducing the SG/genome RNA ratio), confirming the hypothesis and showing that the C gene was the moiety responsible for the modulation effect. The N-terminal one-third of the C gene was required for the effect of be exhibited. A similar phenomenon was not observed with the replicons of Sindbis virus, a related Alphavirus. Interestingly, modulation was not observed when RUBrep/GFP was co-transfected with either other RUBrep or plasmid constructs expressing the C gene, demonstrating that modulation could occur only when the C gene was provided in cis. Mutations that prevented translation of the C protein failed to modulate RNA synthesis, indicating that the C protein was the moiety responsible for modulation; consistent with this conclusion, modulation of RNA synthesis was maintained when synonymous codon mutations were introduced at the 5' end of the C gene that changed the C gene sequence without altering the amino acid sequence of the C protein. These results indicate that C protein translated in proximity of viral replication complexes, possibly from newly synthesized SG RNA, participate in regulating the replication of viral RNA.

  11. Native display of complete foreign protein domains on the surface of hepatitis B virus capsids

    PubMed Central

    Kratz, Peter A.; Böttcher, Bettina; Nassal, Michael

    1999-01-01

    The nucleocapsid of hepatitis B virus (HBV), or HBcAg, is a highly symmetric structure formed by multiple dimers of a single core protein that contains potent T helper epitopes in its 183-aa sequence. Both factors make HBcAg an unusually strong immunogen and an attractive candidate as a carrier for foreign epitopes. The immunodominant c/e1 epitope on the capsid has been suggested as a superior location to convey high immunogenicity to a heterologous sequence. Because of its central position, however, any c/e1 insert disrupts the core protein’s primary sequence; hence, only peptides, or rather small protein fragments seemed to be compatible with particle formation. According to recent structural data, the epitope is located at the tips of prominent surface spikes formed by the very stable dimer interfaces. We therefore reasoned that much larger inserts might be tolerated, provided the individual parts of a corresponding fusion protein could fold independently. Using the green fluorescent protein (GFP) as a model insert, we show that the chimeric protein efficiently forms fluorescent particles; hence, all of its structurally important parts must be properly folded. We also demonstrate that the GFP domains are surface-exposed and that the chimeric particles elicit a potent humoral response against native GFP. Hence, proteins of at least up to 238 aa can be natively displayed on the surface of HBV core particles. Such chimeras may not only be useful as vaccines but may also open the way for high resolution structural analyses of nonassembling proteins by electron microscopy. PMID:10051569

  12. Expression and characterization of HPV-16 L1 capsid protein in Pichia pastoris

    PubMed Central

    Bazan, Silvia Boschi; de Alencar Muniz Chaves, Agtha; Aires, Karina Araújo; Cianciarullo, Aurora Marques; Garcea, Robert L.; Ho, Paulo Lee

    2013-01-01

    Human papillomaviruses (HPVs) are responsible for the most common human sexually transmitted viral infections. Infection with high-risk HPVs, particularly HPV16, is associated with the development of cervical cancer. The papillomavirus L1 major capsid protein, the basis of the currently marketed vaccines, self-assembles into virus-like particles (VLPs). Here, we describe the expression, purification and characterization of recombinant HPV16 L1 produced by a methylotrophic yeast. A codon-optimized HPV16 L1 gene was cloned into a non-integrative expression vector under the regulation of a methanol-inducible promoter and used to transform competent Pichia pastoris cells. Purification of L1 protein from yeast extracts was performed using heparin–sepharose chromatography, followed by a disassembly/reassembly step. VLPs could be assembled from the purified L1 protein, as demonstrated by electron microscopy. The display of conformational epitopes on the VLPs surface was confirmed by hemagglutination and hemagglutination inhibition assays and by immuno-electron microscopy. This study has implications for the development of an alternative platform for the production of a papillomavirus vaccine that could be provided by public health programs, especially in resource-poor areas, where there is a great demand for low-cost vaccines. PMID:19756360

  13. A novel inhibitor of dengue virus replication that targets the capsid protein.

    PubMed

    Byrd, Chelsea M; Dai, Dongcheng; Grosenbach, Douglas W; Berhanu, Aklile; Jones, Kevin F; Cardwell, Kara B; Schneider, Christine; Wineinger, Kristin A; Page, Jessica M; Harver, Chris; Stavale, Eric; Tyavanagimatt, Shanthakumar; Stone, Melialani A; Bartenschlager, Ralf; Scaturro, Pietro; Hruby, Dennis E; Jordan, Robert

    2013-01-01

    Dengue viruses (DENV) infect 50 to 100 million people worldwide per year, of which 500,000 develop severe life-threatening disease. This mosquito-borne illness is endemic in most tropical and subtropical countries and has spread significantly over the last decade. While there are several promising vaccine candidates in clinical trials, there are currently no approved vaccines or therapeutics available for treatment of dengue infection. Here, we describe a novel small-molecule compound, ST-148, that is a potent inhibitor of all four serotypes of DENV in vitro. ST-148 significantly reduced viremia and viral load in vital organs and tended to lower cytokine levels in the plasma in a nonlethal model of DENV infection in AG129 mice. Compound resistance mapped to the DENV capsid (C) gene, and a direct interaction of ST-148 with C protein is suggested by alterations of the intrinsic fluorescence of the protein in the presence of compound. Thus, ST-148 appears to interact with the DENV C protein and inhibits a distinct step(s) of the viral replication cycle. PMID:23070172

  14. Differential expression of two isolates of beak and feather disease virus capsid protein in Escherichia coli.

    PubMed

    Patterson, Edward I; Swarbrick, Crystall M D; Roman, Noelia; Forwood, Jade K; Raidal, Shane R

    2013-04-01

    Expression of recombinant beak and feather disease virus (BFDV) capsid-associated protein (Cap) has relied on inefficient techniques that typically produce low yields or use specialized expression systems, which greatly increase the cost and expertise required for mass production. An Escherichia coli system was used to express recombinant BFDV Cap derived from two isolates of BFDV, from a Long-billed Corella (Cacatua tenuirostris) and an Orange-bellied parrot (OBP; Neophema chrysogaster). Purification by affinity and size exclusion chromatography was optimized through an iterative process involving screening and modification of buffer constituents and pH. A buffer containing glycerol, β-mercaptoethanol, Triton X-100, and a high concentration of NaCl at pH 8 was used to increase solubility of the protein. The final concentration of the corella-isolated BFDV protein was fifteen- to twenty-fold greater than that produced in previous publications using E. coli expression systems. Immunoassays were used to confirm the specific antigenicity of recombinant Cap, verifying its validity for use in continued experimentation as a potential vaccine, a reagent in diagnostic assays, and as a concentrated sample for biological discoveries. PMID:23403150

  15. Several recombinant capsid proteins of equine rhinitis a virus show potential as diagnostic antigens.

    PubMed

    Li, Fan; Stevenson, Rachel A; Crabb, Brendan S; Studdert, Michael J; Hartley, Carol A

    2005-06-01

    Equine rhinitis A virus (ERAV) is a significant pathogen of horses and is also closely related to Foot-and-mouth disease virus (FMDV). Despite these facts, knowledge of the prevalence and importance of ERAV infections remains limited, largely due to the absence of a simple, robust diagnostic assay. In this study, we compared the antigenicities of recombinant full-length and fragmented ERAV capsid proteins expressed in Escherichia coli by using sera from experimentally infected and naturally exposed horses. We found that, from the range of antigens tested, recombinant proteins encompassing the C-terminal region of VP1, full-length VP2, and the N-terminal region of VP2 reacted specifically with antibodies present in sera from each of the five experimentally infected horses examined. Antibodies to epitopes on VP2 (both native and recombinant forms) persisted longer postinfection (>105 days) than antibodies specific for epitopes on other fragments. Our data also suggest that B-cell epitopes within the C terminus of VP1 and N terminus of VP2 contribute to a large proportion of the total reactivity of recombinant VP1 and VP2, respectively. Importantly, the reactivity of these VP1 and VP2 recombinant proteins in enzyme-linked immunosorbent assays (ELISAs) correlated well with the results from a range of native antigen-based serological assays using sera from 12 field horses. This study provides promising candidates for development of a diagnostic ERAV ELISA. PMID:15939754

  16. Properties of African Cassava Mosaic Virus Capsid Protein Expressed in Fission Yeast.

    PubMed

    Hipp, Katharina; Schäfer, Benjamin; Kepp, Gabi; Jeske, Holger

    2016-01-01

    The capsid proteins (CPs) of geminiviruses combine multiple functions for packaging the single-stranded viral genome, insect transmission and shuttling between the nucleus and the cytoplasm. African cassava mosaic virus (ACMV) CP was expressed in fission yeast, and purified by SDS gel electrophoresis. After tryptic digestion of this protein, mass spectrometry covered 85% of the amino acid sequence and detected three N-terminal phosphorylation sites (threonine 12, serines 25 and 62). Differential centrifugation of cell extracts separated the CP into two fractions, the supernatant and pellet. Upon isopycnic centrifugation of the supernatant, most of the CP accumulated at densities typical for free proteins, whereas the CP in the pellet fraction showed a partial binding to nucleic acids. Size-exclusion chromatography of the supernatant CP indicated high order complexes. In DNA binding assays, supernatant CP accelerated the migration of ssDNA in agarose gels, which is a first hint for particle formation. Correspondingly, CP shifted ssDNA to the expected densities of virus particles upon isopycnic centrifugation. Nevertheless, electron microscopy did not reveal any twin particles, which are characteristic for geminiviruses. PMID:27399762

  17. Properties of African Cassava Mosaic Virus Capsid Protein Expressed in Fission Yeast

    PubMed Central

    Hipp, Katharina; Schäfer, Benjamin; Kepp, Gabi; Jeske, Holger

    2016-01-01

    The capsid proteins (CPs) of geminiviruses combine multiple functions for packaging the single-stranded viral genome, insect transmission and shuttling between the nucleus and the cytoplasm. African cassava mosaic virus (ACMV) CP was expressed in fission yeast, and purified by SDS gel electrophoresis. After tryptic digestion of this protein, mass spectrometry covered 85% of the amino acid sequence and detected three N-terminal phosphorylation sites (threonine 12, serines 25 and 62). Differential centrifugation of cell extracts separated the CP into two fractions, the supernatant and pellet. Upon isopycnic centrifugation of the supernatant, most of the CP accumulated at densities typical for free proteins, whereas the CP in the pellet fraction showed a partial binding to nucleic acids. Size-exclusion chromatography of the supernatant CP indicated high order complexes. In DNA binding assays, supernatant CP accelerated the migration of ssDNA in agarose gels, which is a first hint for particle formation. Correspondingly, CP shifted ssDNA to the expected densities of virus particles upon isopycnic centrifugation. Nevertheless, electron microscopy did not reveal any twin particles, which are characteristic for geminiviruses. PMID:27399762

  18. Detection of subtle differences in analogous viral capsid proteins by allowing unrestricted specific interaction in solution competition ELISA.

    PubMed

    Cao, Lu; Wang, Xin; Fang, Mujin; Xia, Ningshao; Zhao, Qinjian

    2016-10-01

    Assay artifacts were reported in plate-based immuoassays during the assessment of specific molecular interactions owing to the surface induced aggregation/conformational changes. To circumvent surface adsorption and associated artifacts, we used a solution competition ELISA by allowing unrestricted interaction between binding partners to occur in solution for better discrimination between epitopes with subtle differences. A difference of two orders of magnitude in binding to neutralizing antibodies for two truncated versions of the hepatitis E virus capsid protein was observed, while other assays showed comparable antigenicity with the same monoclonal antibodies. Discrimination of epitopes with high degree resemblance in analogous viral capsid proteins was demonstrated quantitatively based on their specific interactions. Therefore, the solution competition ELISA is a method of choice when the detection of subtle differences of two highly analogous proteins is desired. PMID:27321427

  19. Structural Basis for the Development of Avian Virus Capsids That Display Influenza Virus Proteins and Induce Protective Immunity

    PubMed Central

    Pascual, Elena; Mata, Carlos P.; Gómez-Blanco, Josué; Moreno, Noelia; Bárcena, Juan; Blanco, Esther; Rodríguez-Frandsen, Ariel; Nieto, Amelia

    2014-01-01

    ABSTRACT Bioengineering of viruses and virus-like particles (VLPs) is a well-established approach in the development of new and improved vaccines against viral and bacterial pathogens. We report here that the capsid of a major avian pathogen, infectious bursal disease virus (IBDV), can accommodate heterologous proteins to induce protective immunity. The structural units of the ∼70-nm-diameter T=13 IBDV capsid are trimers of VP2, which is made as a precursor (pVP2). The pVP2 C-terminal domain has an amphipathic α helix that controls VP2 polymorphism. In the absence of the VP3 scaffolding protein, 466-residue pVP2 intermediates bearing this α helix assemble into genuine VLPs only when expressed with an N-terminal His6 tag (the HT-VP2-466 protein). HT-VP2-466 capsids are optimal for protein insertion, as they are large enough (cargo space, ∼78,000 nm3) and are assembled from a single protein. We explored HT-VP2-466-based chimeric capsids initially using enhanced green fluorescent protein (EGFP). The VLP assembly yield was efficient when we coexpressed EGFP-HT-VP2-466 and HT-VP2-466 from two recombinant baculoviruses. The native EGFP structure (∼240 copies/virion) was successfully inserted in a functional form, as VLPs were fluorescent, and three-dimensional cryo-electron microscopy showed that the EGFP molecules incorporated at the inner capsid surface. Immunization of mice with purified EGFP-VLPs elicited anti-EGFP antibodies. We also inserted hemagglutinin (HA) and matrix (M2) protein epitopes derived from the mouse-adapted A/PR/8/34 influenza virus and engineered several HA- and M2-derived chimeric capsids. Mice immunized with VLPs containing the HA stalk, an M2 fragment, or both antigens developed full protection against viral challenge. IMPORTANCE Virus-like particles (VLPs) are multimeric protein cages that mimic the infectious virus capsid and are potential candidates as nonliving vaccines that induce long-lasting protection. Chimeric VLPs can display

  20. IL12-mediated liver inflammation reduces the formation of AAV transcriptionally active forms but has no effect over preexisting AAV transgene expression.

    PubMed

    Gil-Fariña, Irene; Di Scala, Marianna; Vanrell, Lucia; Olagüe, Cristina; Vales, Africa; High, Katherine A; Prieto, Jesus; Mingozzi, Federico; Gonzalez-Aseguinolaza, Gloria

    2013-01-01

    Recombinant adenoassociated viral vectors (rAAV) have proven to be excellent candidates for gene therapy clinical applications. Recent results showed that cellular immunity to AAV represents a major challenge facing the clinical use of systemic administration of these vectors. Interestingly, no preclinical animal model has previously fully reproduced the clinical findings. The aim of the present work was to enhance the T cell immune response against AAV capsid in mice by the administration of a rAAV expressing the immunostimulatory cytokine IL-12. Our results indicate that although IL-12 expression enhanced the AAV capsid-specific immune response it failed to eliminate transduced hepatocytes and long-term expression was achieved. We found that AAV-mediated transgene expression is altered by IL-12-induced liver inflammation. However, IL-12 expression has no effect over preexisting AAV-mediated transgene expression. IL-12 down-regulates AAV mediated transgene expression via induction of IFN-γ production by NK and T cells, but without altering the transduction efficiency measured by viral genomes. Our results indicate that liver inflammation affects the formation of transcriptionally active AAV vector genomes through an unknown mechanism that can be avoided by the use of DNA-demethylating or anti-inflammatory agents. PMID:23844082

  1. IL12-Mediated Liver Inflammation Reduces the Formation of AAV Transcriptionally Active Forms but Has No Effect over Preexisting AAV Transgene Expression

    PubMed Central

    Gil-Fariña, Irene; Di Scala, Marianna; Vanrell, Lucia; Olagüe, Cristina; Vales, Africa; High, Katherine A.; Prieto, Jesus; Mingozzi, Federico; Gonzalez-Aseguinolaza, Gloria

    2013-01-01

    Recombinant adenoassociated viral vectors (rAAV) have proven to be excellent candidates for gene therapy clinical applications. Recent results showed that cellular immunity to AAV represents a major challenge facing the clinical use of systemic administration of these vectors. Interestingly, no preclinical animal model has previously fully reproduced the clinical findings. The aim of the present work was to enhance the T cell immune response against AAV capsid in mice by the administration of a rAAV expressing the immunostimulatory cytokine IL-12. Our results indicate that although IL-12 expression enhanced the AAV capsid-specific immune response it failed to eliminate transduced hepatocytes and long-term expression was achieved. We found that AAV-mediated transgene expression is altered by IL-12-induced liver inflammation. However, IL-12 expression has no effect over preexisting AAV-mediated transgene expression. IL-12 down-regulates AAV mediated transgene expression via induction of IFN-γ production by NK and T cells, but without altering the transduction efficiency measured by viral genomes. Our results indicate that liver inflammation affects the formation of transcriptionally active AAV vector genomes through an unknown mechanism that can be avoided by the use of DNA-demethylating or anti-inflammatory agents. PMID:23844082

  2. Antigenic relationships among human rotaviruses as determined by outer capsid protein VP4.

    PubMed

    Gorziglia, M; Larralde, G; Kapikian, A Z; Chanock, R M

    1990-09-01

    cDNA clones representing the VP4 gene of symptomatic human rotavirus strain KU (VP7 serotype 1) or DS-1 (VP7 serotype 2) or asymptomatic human rotavirus strain 1076 (VP7 serotype 2) were constructed and inserted into a baculovirus expression vector under the control of the polyhedrin promoter. The resulting recombinants expressed the appropriate authentic VP4 rotavirus outer capsid protein. Guinea pigs immunized with these VP4 proteins developed antibodies that neutralized infectivity of the rotavirus from which the immunizing VP4 was derived. These antisera were then used in neutralization tests to define the extent and distribution of VP4 antigenic polymorphism among human rotaviruses. Three distinct serotypes and one subtype of the VP4 outer capsid protein were identified among 17 human rotavirus strains that had previously been assigned to five distinct VP7 serotypes. For the most part, VP4 serotype segregated independently of VP7 serotype. Ten strains of human rotavirus that were associated with symptomatic infection and that exhibited VP7 serotype 1, 3, 4, or 9 specificity, each possessed a VP4 of the same serotype and subtype, designated VP4 serotype 1A. Both symptomatic human rotavirus strains with VP7 serotype 2 specificity were related by neutralization to the VP4 serotype 1A strains and were classified as a subtype of VP4 serotype 1--i.e., serotype 1B--since viruses of serotype 1A appeared to be prime strains. Four human rotavirus strains that were recovered from healthy infants in newborn nurseries in which virus transmission persisted over a long interval, belonged to VP7 serotype 1, 2, 3, or 4, but each strain possessed the same VP4 antigenic specificity that was designated VP4 serotype 2. Finally, a single strain of symptomatic human rotavirus of VP7 serotype 1 specificity possessed a unique VP4 that was provisionally classified as VP4 serotype 3 but this remains to be confirmed because neutralization tests were performed in only one direction. Among

  3. Synthesis and characterization of different immunogenic viral nanoconstructs from rotavirus VP6 inner capsid protein

    PubMed Central

    Bugli, Francesca; Caprettini, Valeria; Cacaci, Margherita; Martini, Cecilia; Paroni Sterbini, Francesco; Torelli, Riccardo; Della Longa, Stefano; Papi, Massimiliano; Palmieri, Valentina; Giardina, Bruno; Posteraro, Brunella; Sanguinetti, Maurizio; Arcovito, Alessandro

    2014-01-01

    In order to deliver low-cost viral capsomeres from a large amount of soluble viral VP6 protein from human rotavirus, we developed and optimized a biotechnological platform in Escherichia coli. Specifically, three different expression protocols were compared, differing in their genetic constructs, ie, a simple native histidine-tagged VP6 sequence, VP6 fused to thioredoxin, and VP6 obtained with the newly described small ubiquitin-like modifier (SUMO) fusion system. Our results demonstrate that the histidine-tagged protein does not escape the accumulation in the inclusion bodies, and that SUMO is largely superior to the thioredoxin-fusion tag in enhancing the expression and solubility of VP6 protein. Moreover, the VP6 protein produced according to the SUMO fusion tag displays well-known assembly properties, as observed in both transmission electron microscopy and atomic force microscopy images, giving rise to either VP6 trimers, 60 nm spherical virus-like particles, or nanotubes a few microns long. This different quaternary organization of VP6 shows a higher level of immunogenicity for the elongated structures with respect to the spheres or the protein trimers. Therefore, the expression and purification strategy presented here – providing a large amount of the viral capsid protein in the native form with relatively simple, rapid, and economical procedures – opens a new route toward large-scale production of a more efficient antigenic compound to be used as a vaccination tool or as an adjuvant, and also represents a top-quality biomaterial to be further modified for biotechnological purposes. PMID:24936129

  4. Identification of a nuclear localization sequence in the polyomavirus capsid protein VP2

    NASA Technical Reports Server (NTRS)

    Chang, D.; Haynes, J. I. 2nd; Brady, J. N.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    A nuclear localization signal (NLS) has been identified in the C-terminal (Glu307-Glu-Asp-Gly-Pro-Gln-Lys-Lys-Lys-Arg-Arg-Leu318) amino acid sequence of the polyomavirus minor capsid protein VP2. The importance of this amino acid sequence for nuclear transport of newly synthesized VP2 was demonstrated by a genetic "subtractive" study using the constructs pSG5VP2 (expressing full-length VP2) and pSG5 delta 3VP2 (expressing truncated VP2, lacking amino acids Glu307-Leu318). These constructs were transfected into COS-7 cells, and the intracellular localization of the VP2 protein was determined by indirect immunofluorescence. These studies revealed that the full-length VP2 was localized in the nucleus, while the truncated VP2 protein was localized in the cytoplasm and not transported to the nucleus. A biochemical "additive" approach was also used to determine whether this sequence could target nonnuclear proteins to the nucleus. A synthetic peptide identical to VP2 amino acids Glu307-Leu318 was cross-linked to the nonnuclear proteins bovine serum albumin (BSA) or immunoglobulin G (IgG). The conjugates were then labeled with fluorescein isothiocyanate and microinjected into the cytoplasm of NIH 3T6 cells. Both conjugates localized in the nucleus of the microinjected cells, whereas unconjugated BSA and IgG remained in the cytoplasm. Taken together, these genetic subtractive and biochemical additive approaches have identified the C-terminal sequence of polyoma-virus VP2 (containing amino acids Glu307-Leu318) as the NLS of this protein.

  5. Synthesis and characterization of different immunogenic viral nanoconstructs from rotavirus VP6 inner capsid protein.

    PubMed

    Bugli, Francesca; Caprettini, Valeria; Cacaci, Margherita; Martini, Cecilia; Paroni Sterbini, Francesco; Torelli, Riccardo; Della Longa, Stefano; Papi, Massimiliano; Palmieri, Valentina; Giardina, Bruno; Posteraro, Brunella; Sanguinetti, Maurizio; Arcovito, Alessandro

    2014-01-01

    In order to deliver low-cost viral capsomeres from a large amount of soluble viral VP6 protein from human rotavirus, we developed and optimized a biotechnological platform in Escherichia coli. Specifically, three different expression protocols were compared, differing in their genetic constructs, ie, a simple native histidine-tagged VP6 sequence, VP6 fused to thioredoxin, and VP6 obtained with the newly described small ubiquitin-like modifier (SUMO) fusion system. Our results demonstrate that the histidine-tagged protein does not escape the accumulation in the inclusion bodies, and that SUMO is largely superior to the thioredoxin-fusion tag in enhancing the expression and solubility of VP6 protein. Moreover, the VP6 protein produced according to the SUMO fusion tag displays well-known assembly properties, as observed in both transmission electron microscopy and atomic force microscopy images, giving rise to either VP6 trimers, 60 nm spherical virus-like particles, or nanotubes a few microns long. This different quaternary organization of VP6 shows a higher level of immunogenicity for the elongated structures with respect to the spheres or the protein trimers. Therefore, the expression and purification strategy presented here - providing a large amount of the viral capsid protein in the native form with relatively simple, rapid, and economical procedures - opens a new route toward large-scale production of a more efficient antigenic compound to be used as a vaccination tool or as an adjuvant, and also represents a top-quality biomaterial to be further modified for biotechnological purposes. PMID:24936129

  6. Simulations of HIV Capsid Protein Dimerization Reveal the Effect of Chemistry and Topography on the Mechanism of Hydrophobic Protein Association

    NASA Astrophysics Data System (ADS)

    Yu, Naiyin; Hagan, Michael F.

    2012-09-01

    Recent work has shown that the hydrophobic protein surfaces in aqueous solution sit near a drying transition. The tendency for these surfaces to expel water from their vicinity leads to self assembly of macromolecular complexes. In this article we show with a realistic model for a biologically pertinent system how this phenomenon appears at the molecular level. We focus on the association of the C-terminal domain (CA-C) of the human immunodeficiency virus (HIV) capsid protein. By combining all-atom simulations with specialized sampling techniques we measure the water density distribution during the approach of two CA-C proteins as a function of separation and amino acid sequence in the interfacial region. The simulations demonstrate that CA-C protein-protein interactions sit at the edge of a dewetting transition and that this mesoscopic manifestation of the underlying liquid-vapor phase transition can be readily manipulated by biology or protein engineering to significantly affect association behavior. While the wild type protein remains wet until contact, we identify a set of in silico mutations, in which three hydrophilic amino acids are replaced with nonpolar residues, that leads to dewetting prior to association. The existence of dewetting depends on the size and relative locations of substituted residues separated by nm length scales, indicating long range cooperativity and a sensitivity to surface topography. These observations identify important details which are missing from descriptions of protein association based on buried hydrophobic surface area.

  7. Understanding dengue virus capsid protein disordered N-Terminus and pep14-23-based inhibition.

    PubMed

    Faustino, André F; Guerra, Gabriela M; Huber, Roland G; Hollmann, Axel; Domingues, Marco M; Barbosa, Glauce M; Enguita, Francisco J; Bond, Peter J; Castanho, Miguel A R B; Da Poian, Andrea T; Almeida, Fabio C L; Santos, Nuno C; Martins, Ivo C

    2015-02-20

    Dengue virus (DENV) infection affects millions of people and is becoming a major global disease for which there is no specific available treatment. pep14-23 is a recently designed peptide, based on a conserved segment of DENV capsid (C) protein. It inhibits the interaction of DENV C with host intracellular lipid droplets (LDs), which is crucial for viral replication. Combining bioinformatics and biophysics, here, we analyzed pep14-23 structure and ability to bind different phospholipids, relating that information with the full-length DENV C. We show that pep14-23 acquires α-helical conformation upon binding to negatively charged phospholipid membranes, displaying an asymmetric charge distribution structural arrangement. Structure prediction for the N-terminal segment reveals four viable homodimer orientations that alternatively shield or expose the DENV C hydrophobic pocket. Taken together, these findings suggest a new biological role for the disordered N-terminal region, which may function as an autoinhibitory domain mediating DENV C interaction with its biological targets. The results fit with our current understanding of DENV C and pep14-23 structure and function, paving the way for similar approaches to understanding disordered proteins and improved peptidomimetics drug development strategies against DENV and similar Flavivirus infections. PMID:25412346

  8. Use of Cre/loxP recombination to swap cell binding motifs on the adenoviral capsid protein IX

    SciTech Connect

    Poulin, Kathy L.; Tong, Grace; Vorobyova, Olga; Pool, Madeline; Kothary, Rashmi; Parks, Robin J.

    2011-11-25

    We used Cre/loxP recombination to swap targeting ligands present on the adenoviral capsid protein IX (pIX). A loxP-flanked sequence encoding poly-lysine (pK-binds heparan sulfate proteoglycans) was engineered onto the 3'-terminus of pIX, and the resulting fusion protein allowed for routine virus propagation. Growth of this virus on Cre-expressing cells removed the pK coding sequence, generating virus that could only infect through alternative ligands, such as a tyrosine kinase receptor A (TrkA)-binding motif engineered into the capsid fibre protein for enhanced infection of neuronal cells. We used a similar approach to swap the pK motif on pIX for a sequence encoding a single-domain antibody directed towards CD66c for targeted infection of cancer cells; Cre-mediated removal of the pK-coding sequence simultaneously placed the single-domain antibody coding sequence in frame with pIX. Thus, we have developed a simple method to propagate virus lacking native viral tropism but containing cell-specific binding ligands. - Highlights: > We describe a method to grow virus lacking native tropism but containing novel cell-binding ligands. > Cre/loxP recombination was used to modify the adenovirus genome. > A targeting ligand present on capsid protein IX was removed or replaced using recombination. > Cre-loxP was also used to 'swap' the identity of the targeting ligand present on pIX.

  9. Nature’s favorite building block: Deciphering folding and capsid assembly of proteins with the HK97-fold

    PubMed Central

    Suhanovsky, Margaret M.; Teschke, Carolyn M.

    2015-01-01

    Summary For many (if not all) bacterial and archaeal tailed viruses and eukaryotic Herpesvirdae the HK97-fold serves as the major architectural element in icosahedral capsid formation while still enabling the conformational flexibility required during assembly and maturation. Auxiliary proteins or Δ-domains strictly control assembly of multiple, identical, HK97-like subunits into procapsids with specific icosahedral symmetries, rather than aberrant non-icosahedral structures. Procapsids are precursor structures that mature into capsids in a process involving release of auxiliary proteins (or cleavage of Δ-domains), dsDNA packaging, and conformational rearrangement of the HK97-like subunits. Some coat proteins built on the ubiquitous HK97-fold also have accessory domains or loops that impart specific functions, such as increased monomer, procapsid, or capsid stability. In this review, we analyze the numerous HK97-like coat protein structures that are emerging in the literature (over 40 at time of writing) by comparing their topology, additional domains, and their assembly and misassembly reactions. PMID:25864106

  10. Quantitation, with a new assay, and Theiler's virus capsid protein in the central nervous system of mice

    SciTech Connect

    Cash, E.; Chamorro, M.; Brahic, M.

    1986-11-01

    The authors developed a quantitative assay for antigens at the single-cell level. Tissue sections were reacted with (i) a primarily antibody , (ii) a biotinylated secondary antibody, or (iii) /sup 35/S-streptavidin. Binding of streptavidin to cells was quantitated by microscopic autoradiography. They showed that the number of autoradiographic grains was proportional to the amount of antigen per cell. With this assay, they studied the synthesis of Theiler's virus capsid proteins VP1, VP2, and VP3 in permissive BHK cells grown in vitro and in mouse central nervous system (CNS) cells during a persistent infection. They found that synthesis of the three capsid proteins was restricted in mouse CNS cells. Restricted virus replication could play a major role in the persistence of Theiler's virus in mouse CNS cells.

  11. Use of the baculovirus system to assemble polyomavirus capsid-like particles with different polyomavirus structural proteins: analysis of the recombinant assembled capsid-like particles.

    PubMed

    An, K; Gillock, E T; Sweat, J A; Reeves, W M; Consigli, R A

    1999-04-01

    The genes encoding the structural proteins (VP1, VP2 and VP3) of murine polyomavirus were cloned into the p2Bac dual multiple cloning site vector, individually or jointly, and the corresponding proteins were expressed in Spodoptera frugiperda (Sf9) insect cells by cotransfecting Sf9 cells with the constructed vector and the linear DNA of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV). Recombinant capsid-like particles could be purified 5 days post-infection from Sf9 cells infected with AcMNPV-VP1, with or without the involvement of minor protein (VP2 or VP3). Although VP2 and VP3 alone could not generate recombinant particles, they became incorporated into these particles when expressed with VP1 in Sf9 cells. Recombinant particles with different polyomavirus structural protein(s) were obtained by using different combined expression of these proteins in Sf9 cells. Cellular DNA of 5 kbp in size was packaged in all of the recombinant particles, which showed the same diameter as that of native virions. Agarose gel electrophoresis indicated that DNA packaged in these recombinant particles had a different pattern than that of native virions. Two-dimensional gel electrophoresis of the VP1 species of recombinant particles showed more VP1 species than those of the native virions from mouse cells, and an additional species of VP1 when VP2 was co-expressed with VP1. The recombinant particles were also compared for their ability to compete for polyomavirus infection. The competition assay indicated that the recombinant particles containing VP2 were the most efficient in inhibiting the native polyomavirus infection of 3T6 cells. PMID:10211971

  12. Analogs of LDL Receptor Ligand Motifs in Dengue Envelope and Capsid Proteins as Potential Codes for Cell Entry

    PubMed Central

    Guevara, Juan; Romo, Jamie; McWhorter, Troy; Guevara, Natalia Valentinova

    2016-01-01

    It is established that cell entry of low density lipoprotein particles (LLPs) containing Apo B100 and Apo E is mediated by receptors and GAGs. Receptor ligand motifs, XBBBXXBX, XBBXBX, and ΨBΨXB, and mono- and bipartite NLS sequences are abundant in Apo E and Apo B100 as well as in envelope and capsid proteins of Dengue viruses 1–4 (DENV1–4). Synthetic, fluorescence-labeled peptides of sequences in DENV2 envelope protein, and DENV3 capsid that include these motifs were used to conduct a qualitative assessment of cell binding and entry capacity using HeLa cells. DENV2 envelope peptide, Dsp2EP, 0564Gly-Gly0595, was shown to bind and remain at the cell surface. In contrast, DENV3 capsid protein peptide, Dsp3CP, 0002Asn-Gln0028, readily enters HeLa cells and accumulates at discrete loci in the nucleus. FITC-labeled dengue synthetic peptides colocalize with Low Density Lipoprotein-CM-DiI and Apo E-CM-DiI to a degree that suggests that Dengue viruses may utilize cell entry pathways used by LLPs. PMID:27123468

  13. Baculovirus expression of beak and feather disease virus (BFDV) capsid protein capable of self-assembly and haemagglutination.

    PubMed

    Stewart, Meredith E; Bonne, Nicolai; Shearer, Patrick; Khalesi, Bahman; Sharp, Margaret; Raidal, Shane

    2007-05-01

    Beak and feather disease virus (BFDV) is a common avian circovirus infection of wild Psittaciformes and is a recognised threat to endangered psittacine species. Currently, there is a requirement to develop BFDV antigen for diagnostic purposes and since efforts to propagate BFDV in vitro have so far been unsuccessful the entire coding region of BFDV ORF C1 was expressed in Sf9 insect cells using a baculovirus expression system. The entire coding region of BFDV ORF C1, the presumptive capsid, was expressed in Sf9 insect cells using baculovirus expression system. Electron microscopic examination of negatively stained material demonstrated that the recombinant protein self-assembled to produce virus-like particles (VLPs) thus confirming that ORF C1 is likely to be the sole determinant for capsid construction in vivo. BFDV VLPs also possessed haemagglutinating activity which provides further evidence that self-assembled BFDV VLPs retain receptor mediated biological activity and that the determinants for BFDV haemagglutination activity rely solely on the capsid protein. The recombinant protein reacted with anti-BFDV sera from naturally immune parrots and cockatoo and from chickens experimentally inoculated with native BFDV in both Western blots and haemagglutination inhibition (HI) assay. BFDV VLPs were also a suitable replacement antigen for serological detection of BFDV antibody by HI. PMID:17218022

  14. Genes VI, VII, and IX of phage M13 code for minor capsid proteins of the virion.

    PubMed Central

    Simons, G F; Konings, R N; Schoenmakers, J G

    1981-01-01

    The minor capsid proteins C and D from phage M13 have been characterized by differential amino acid labeling and amino-terminal sequence analysis. We demonstrate that D protein (Mr 12,260) is the product of gene VI, whereas the C component is composed of the products of both gene VII (Mr 3580) and gene IX (Mr 3650). Our data further show that the proteins of genes VI, VII, and IX are not subject to proteolytic processing but are packaged into mature virions as their primary translational products. On the basis of incorporation of specific amino acids, the copy numbers of these proteins in M13 virions could be estimated relative to the number of A protein molecules. The M13 phage contains on the average 5 molecules of A protein, 5 molecules of VI protein and 3-4 molecules of both VII protein and IX protein. These copy numbers remained unchanged in M13 recombinant phages of up to two times the length of wild-type phages, a fact that indicates that these minor capsid proteins are located at either one or both ends of the phage filament. Images PMID:6945579

  15. Directed Evolution of a Novel Adeno-associated Virus (AAV) Vector That Crosses the Seizure-compromised Blood–Brain Barrier (BBB)

    PubMed Central

    Gray, Steven J; Blake, Bonita L; Criswell, Hugh E; Nicolson, Sarah C; Samulski, R Jude; McCown, Thomas J

    2009-01-01

    DNA shuffling and directed evolution were employed to develop a novel adeno-associated virus (AAV) vector capable of crossing the seizure-compromised blood–brain barrier (BBB) and transducing cells in the brain. Capsid DNA from AAV serotypes 1–6, 8, and 9 were shuffled and recombined to create a library of chimeric AAVs. One day after kainic acid–induced limbic seizure activity in rats, the virus library was infused intravenously (i.v.), and 3 days later, neuron-rich cells were mechanically dissociated from seizure-sensitive brain sites, collected and viral DNA extracted. After three cycles of selection, green fluorescent protein (GFP)–packaged clones were administered directly into brain or i.v. 1 day after kainic acid–induced seizures. Several clones that were effective after intracranial administration did not transduce brain cells after the i.v. administration. However, two clones (32 and 83) transduced the cells after direct brain infusion and after i.v. administration transduced the cells that were localized to the piriform cortex and ventral hippocampus, areas exhibiting a seizure-compromised BBB. No transduction occurred in areas devoid of BBB compromise. Only one parental serotype (AAV8) exhibited a similar expression profile, but the biodistribution of 32 and 83 diverged dramatically from this parental serotype. Thus, novel AAV vectors have been created that can selectively cross the seizure-compromised BBB and transduce cells. PMID:20040913

  16. Is the major capsid protein of iridoviruses a suitable target for the study of viral evolution?

    PubMed

    Tidona, C A; Schnitzler, P; Kehm, R; Darai, G

    1998-01-01

    Iridoviruses are large cytoplasmic DNA viruses that are specific for different insect or vertebrate hosts. The major structural component of the non-enveloped icosahedral virus particles is the major capsid protein (MCP) which appears to be highly conserved among members of the family Iridoviridae, Phycodnaviridae, and African swine fever virus. The amino acid sequences of the known MCPs were used in comparative analyses to elucidate the phylogenic relationships between different cytoplasmic DNA viruses including three insect iridoviruses (Tipula iridescent virus, Simulium iridescent virus, Chilo iridescent virus), seven vertebrate iridoviruses isolated either from fish (lymphocystis disease virus, rainbow trout virus, European catfish virus, doctor fish virus), amphibians (frog virus 3), or reptiles (turtle virus 3, turtle virus 5), one member of the family Phycodnaviridae (Paramecium bursaria Chlorella virus type 1), and African swine fever virus. These analyses revealed that the amino acid sequence of the MCP is a suitable target for the study of viral evolution since it contains highly conserved domains, but is sufficiently diverse to distinguish closely related iridovirus isolates. Furthermore the results suggest that a substantial revision of the taxonomy of iridoviruses based on molecular phylogeny is required. PMID:9562891

  17. Antibody recognition of porcine circovirus type 2 capsid protein epitopes after vaccination, infection, and disease.

    PubMed

    Trible, Benjamin R; Kerrigan, Maureen; Crossland, Nicholas; Potter, Megan; Faaberg, Kay; Hesse, Richard; Rowland, Raymond R R

    2011-05-01

    Open reading frame 2 (ORF2) of porcine circovirus type 2 (PCV2) codes for the 233-amino-acid capsid protein (CP). Baculovirus-based vaccines that express only ORF2 are protective against clinical disease following experimental challenge or natural infection. The goal of this study was to identify regions in CP preferentially recognized by sera from experimentally infected and vaccinated pigs and to compare these responses to those of pigs diagnosed with porcine circovirus-associated disease (PCVAD), including porcine multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS). The approach was to react porcine sera with CP polypeptide fragments followed by finer mapping studies using overlapping oligopeptides that covered amino acids 141 to 200. The results showed that vaccinated pigs preferentially recognized only the largest polypeptide fragment, CP(43-233). A subset of experimentally infected pigs and pigs with PDNS showed strong reactivity against a CP oligopeptide, 169-STIDYFQPNNKR-180. Alanine scanning identified Y-173, F-174, Q-175, and K-179 as important for antibody recognition. The results from this study support the notion of PCV2 modulation of immunity, including antibody responses that may represent a precursor for disease. The recognition of CP(169-180) and other polypeptides provides opportunities to devise diagnostic tests for monitoring the immunological effectiveness of vaccination. PMID:21430122

  18. Nuclear import strategies of high-risk HPV18 L2 minor capsid protein

    SciTech Connect

    Klucevsek, K.; Daley, J.; Darshan, M.S.; Bordeaux, J.; Moroianu, J. . E-mail: moroianu@bc.edu

    2006-08-15

    We have investigated the nuclear import strategies of high-risk HPV18 L2 minor capsid protein. HPV18 L2 interacts with Kap {alpha}{sub 2} adapter, and Kap {beta}{sub 2} and Kap {beta}{sub 3} nuclear import receptors. Moreover, binding of RanGTP to either Kap {beta}{sub 2} or Kap {beta}{sub 3} inhibits their interaction with L2, suggesting that these Kap {beta}/L2 complexes are import competent. Mapping studies show that HPV18 L2 contains two NLSs: in the N-terminus (nNLS) and in the C-terminus (cNLS), both of which can independently mediate nuclear import. Both nNLS and cNLS form a complex with Kap {alpha}{sub 2}{beta}{sub 1} heterodimer and mediate nuclear import via a classical pathway. The nNLS is also essential for the interaction of HPV18 L2 with Kap {beta}{sub 2} and Kap {beta}{sub 3}. Interestingly, both nNLS and cNLS interact with the viral DNA and this DNA binding occurs without nucleotide sequence specificity. Together, the data suggest that HPV18 L2 can interact via its NLSs with several Kaps and the viral DNA and may enter the nucleus via multiple import pathways mediated by Kap {alpha}{sub 2}{beta}{sub 1} heterodimers, Kap {beta}{sub 2} and Kap {beta}{sub 3}.

  19. Engineering Bacterial Surface Displayed Human Norovirus Capsid Proteins: A Novel System to Explore Interaction Between Norovirus and Ligands

    PubMed Central

    Niu, Mengya; Yu, Qianqian; Tian, Peng; Gao, Zhiyong; Wang, Dapeng; Shi, Xianming

    2015-01-01

    Human noroviruses (HuNoVs) are major contributors to acute nonbacterial gastroenteritis outbreaks. Many aspects of HuNoVs are poorly understood due to both the current inability to culture HuNoVs, and the lack of efficient small animal models. Surrogates for HuNoVs, such as recombinant viral like particles (VLPs) expressed in eukaryotic system or P particles expressed in prokaryotic system, have been used for studies in immunology and interaction between the virus and its receptors. However, it is difficult to use VLPs or P particles to collect or isolate potential ligands binding to these recombinant capsid proteins. In this study, a new strategy was used to collect HuNoVs binding ligands through the use of ice nucleation protein (INP) to display recombinant capsid proteins of HuNoVs on bacterial surfaces. The viral protein-ligand complex could be easily separated by a low speed centrifugation step. This system was also used to explore interaction between recombinant capsid proteins of HuNoVs and their receptors. In this system, the VP1 capsid encoding gene (ORF2) and the protruding domain (P domain) encoding gene (3′ terminal fragment of ORF2) of HuNoVs GI.1 and GII.4 were fused with 5′ terminal fragment of INP encoding gene (inaQn). The results demonstrated that the recombinant VP1 and P domains of HuNoVs were expressed and anchored on the surface of Escherichia coli BL21 cells after the bacteria were transformed with the corresponding plasmids. Both cell surface displayed VP1 and P domains could be recognized by HuNoVs specific antibodies and interact with the viral histo-blood group antigens receptors. In both cases, displayed P domains had better binding abilities than VP1. This new strategy of using displayed HuNoVs capsid proteins on the bacterial surface could be utilized to separate HuNoVs binding components from complex samples, to investigate interaction between the virus and its receptors, as well as to develop an oral vaccine for HuNoVs. PMID

  20. Annexin II Binds to Capsid Protein VP1 of Enterovirus 71 and Enhances Viral Infectivity ▿

    PubMed Central

    Yang, Su-Lin; Chou, Ying-Ting; Wu, Cheng-Nan; Ho, Mei-Shang

    2011-01-01

    Enterovirus type 71 (EV71) causes hand, foot, and mouth disease (HFMD), which is mostly self-limited but may be complicated with a severe to fatal neurological syndrome in some children. Understanding the molecular basis of virus-host interactions might help clarify the largely unknown neuropathogenic mechanisms of EV71. In this study, we showed that human annexin II (Anx2) protein could bind to the EV71 virion via the capsid protein VP1. Either pretreatment of EV71 with soluble recombinant Anx2 or pretreatment of host cells with an anti-Anx2 antibody could result in reduced viral attachment to the cell surface and a reduction of the subsequent virus yield in vitro. HepG2 cells, which do not express Anx2, remained permissive to EV71 infection, though the virus yield was lower than that for a cognate lineage expressing Anx2. Stable transfection of plasmids expressing Anx2 protein into HepG2 cells (HepG2-Anx2 cells) could enhance EV71 infectivity, with an increased virus yield, especially at a low infective dose, and the enhanced infectivity could be reversed by pretreating HepG2-Anx2 cells with an anti-Anx2 antibody. The Anx2-interacting domain was mapped by yeast two-hybrid analysis to VP1 amino acids 40 to 100, a region different from the known receptor binding domain on the surface of the picornavirus virion. Our data suggest that binding of EV71 to Anx2 on the cell surface can enhance viral entry and infectivity, especially at a low infective dose. PMID:21900167

  1. Structural Studies of Adeno-Associated Virus Serotype 8 Capsid Transitions Associated with Endosomal Trafficking

    SciTech Connect

    Nam, Hyun-Joo; Gurda, Brittney L.; McKenna, Robert; Potter, Mark; Byrne, Barry; Salganik, Maxim; Muzyczka, Nicholas; Agbandje-McKenna, Mavis

    2012-09-17

    The single-stranded DNA (ssDNA) parvoviruses enter host cells through receptor-mediated endocytosis, and infection depends on processing in the early to late endosome as well as in the lysosome prior to nuclear entry for replication. However, the mechanisms of capsid endosomal processing, including the effects of low pH, are poorly understood. To gain insight into the structural transitions required for this essential step in infection, the crystal structures of empty and green fluorescent protein (GFP) gene-packaged adeno-associated virus serotype 8 (AAV8) have been determined at pH values of 6.0, 5.5, and 4.0 and then at pH 7.5 after incubation at pH 4.0, mimicking the conditions encountered during endocytic trafficking. While the capsid viral protein (VP) topologies of all the structures were similar, significant amino acid side chain conformational rearrangements were observed on (i) the interior surface of the capsid under the icosahedral 3-fold axis near ordered nucleic acid density that was lost concomitant with the conformational change as pH was reduced and (ii) the exterior capsid surface close to the icosahedral 2-fold depression. The 3-fold change is consistent with DNA release from an ordering interaction on the inside surface of the capsid at low pH values and suggests transitions that likely trigger the capsid for genome uncoating. The surface change results in disruption of VP-VP interface interactions and a decrease in buried surface area between VP monomers. This disruption points to capsid destabilization which may (i) release VP1 amino acids for its phospholipase A2 function for endosomal escape and nuclear localization signals for nuclear targeting and (ii) trigger genome uncoating.

  2. Grass carp Ctenopharyngodon idella Fibulin-4 as a potential interacting partner for grass carp reovirus outer capsid proteins.

    PubMed

    Yu, Fei; Wang, Hao; Liu, Weisha; Lu, Liqun

    2016-01-01

    Mammalian EGF containing fibulin-like extracellular matrix protein 2 (Fibulin-4/EFEMP2), an extracellular matrix(ECM) protein and a member of the fibulin family, is involved in elastic fiber formation, connective tissue development and some human diseases. In a yeast-two hybrid screening of host proteins interacting with outer capsid protein of grass carp reovirus (GCRV), a grass carp homologue of Fibulin-4 (designated as GcFibulin-4) is suggested to hold the potential to bind VP7, VP56 and VP55, the outer capsid protein encoded by type I, II, III GCRV, respectively. GcFibulin-4 gene of grass carp was cloned and sequenced from the cDNA library constructed for the yeast two-hybrid screening. Full-length cDNA of GcFibulin-4 contains an open reading frame (ORF) of 1323 bp encoding a putative protein of 440 amino acids. Phylogenetic analysis of GcFibulin-4 indicated that it shared a high homology with zebra fish Fibulin-4 protein. Transcriptional distribution analysis of GcFibulin-4 in various tissues of healthy grass carp showed that GcFibulin-4 was highly expressed in muscle, moderately expressed in the intestine and brain, and slightly expressed in other examined tissues; the expression pattern is consistent with tissue tropism of GCRV resulting in hemorrhage symptom in the corresponding tissues. Our results suggested that Fibulin-4 might enable free GCRV particles, the pathogen for grass carp hemorrhagic disease, to target fish tissues more efficiently by interacting with viral outer capsid proteins. PMID:26626583

  3. Structural role of the matrix protein of type D retroviruses in gag polyprotein stability and capsid assembly.

    PubMed Central

    Rhee, S S; Hunter, E

    1990-01-01

    To obtain a better understanding of the role of the gag gene-encoded matrix (MA) protein in the assembly and maturation of type D retroviruses, we have made five mutants with specific in-frame deletions within the p10-coding region by the use of oligonucleotide-directed mutagenesis. The changes in the Gag polyprotein made by these mutations resulted in almost identical phenotypes. In cells expressing mutant genomes, the mutant Gag polyproteins were synthesized and modified with myristic acid in a normal manner. However, they were so unstable that the bulk of the newly synthesized polyproteins was degraded within 1 h without being processed into mature structural polypeptides. In contrast, wild-type polyproteins have a processing half-life of 3.0 to 3.5 h. The mutant Gag polyproteins were assembled with very low efficiency into capsids in the cytoplasm of the mutant-infected cells. Moreover, the few capsids that formed were neither released from nor accumulated in the cells. These results suggest that the matrix protein plays an important role in guiding the correct folding of the Gag polyprotein, which is presumably crucial for both stabilizing the molecule and facilitating the intermolecular interactions that occur during assembly of immature capsids. Images PMID:2200887

  4. Identification of the Neutralizing Epitopes of Merkel Cell Polyomavirus Major Capsid Protein within the BC and EF Surface Loops

    PubMed Central

    Fleury, Maxime J. J.; Nicol, Jérôme T. J.; Samimi, Mahtab; Arnold, Françoise; Cazal, Raphael; Ballaire, Raphaelle; Mercey, Olivier; Gonneville, Hélène; Combelas, Nicolas; Vautherot, Jean-Francois; Moreau, Thierry; Lorette, Gérard; Coursaget, Pierre; Touzé, Antoine

    2015-01-01

    Merkel cell polyomavirus (MCPyV) is the first polyomavirus clearly associated with a human cancer, i.e. the Merkel cell carcinoma (MCC). Polyomaviruses are small naked DNA viruses that induce a robust polyclonal antibody response against the major capsid protein (VP1). However, the polyomavirus VP1 capsid protein epitopes have not been identified to date. The aim of this study was to identify the neutralizing epitopes of the MCPyV capsid. For this goal, four VP1 mutants were generated by insertional mutagenesis in the BC, DE, EF and HI loops between amino acids 88-89, 150-151, 189-190, and 296-297, respectively. The reactivity of these mutants and wild-type VLPs was then investigated with anti-VP1 monoclonal antibodies and anti-MCPyV positive human sera. The findings together suggest that immunodominant conformational neutralizing epitopes are present at the surface of the MCPyV VLPs and are clustered within BC and EF loops. PMID:25812141

  5. Identification of the neutralizing epitopes of Merkel cell polyomavirus major capsid protein within the BC and EF surface loops.

    PubMed

    Fleury, Maxime J J; Nicol, Jérôme T J; Samimi, Mahtab; Arnold, Françoise; Cazal, Raphael; Ballaire, Raphaelle; Mercey, Olivier; Gonneville, Hélène; Combelas, Nicolas; Vautherot, Jean-Francois; Moreau, Thierry; Lorette, Gérard; Coursaget, Pierre; Touzé, Antoine

    2015-01-01

    Merkel cell polyomavirus (MCPyV) is the first polyomavirus clearly associated with a human cancer, i.e. the Merkel cell carcinoma (MCC). Polyomaviruses are small naked DNA viruses that induce a robust polyclonal antibody response against the major capsid protein (VP1). However, the polyomavirus VP1 capsid protein epitopes have not been identified to date. The aim of this study was to identify the neutralizing epitopes of the MCPyV capsid. For this goal, four VP1 mutants were generated by insertional mutagenesis in the BC, DE, EF and HI loops between amino acids 88-89, 150-151, 189-190, and 296-297, respectively. The reactivity of these mutants and wild-type VLPs was then investigated with anti-VP1 monoclonal antibodies and anti-MCPyV positive human sera. The findings together suggest that immunodominant conformational neutralizing epitopes are present at the surface of the MCPyV VLPs and are clustered within BC and EF loops. PMID:25812141

  6. Using AAV vectors expressing the β2-adrenoceptor or associated Gα proteins to modulate skeletal muscle mass and muscle fibre size

    PubMed Central

    Hagg, Adam; Colgan, Timothy D.; Thomson, Rachel E.; Qian, Hongwei; Lynch, Gordon S.; Gregorevic, Paul

    2016-01-01

    Anabolic β2-adrenoceptor (β2-AR) agonists have been proposed as therapeutics for treating muscle wasting but concerns regarding possible off-target effects have hampered their use. We investigated whether β2-AR-mediated signalling could be modulated in skeletal muscle via gene delivery to the target tissue, thereby avoiding the risks of β2-AR agonists. In mice, intramuscular administration of a recombinant adeno-associated virus-based vector (rAAV vector) expressing the β2-AR increased muscle mass by >20% within 4 weeks. This hypertrophic response was comparable to that of 4 weeks’ treatment with the β2-AR agonist formoterol, and was not ablated by mTOR inhibition. Increasing expression of inhibitory (Gαi2) and stimulatory (GαsL) G-protein subunits produced minor atrophic and hypertrophic changes in muscle mass, respectively. Furthermore, Gαi2 over-expression prevented AAV:β2-AR mediated hypertrophy. Introduction of the non-muscle Gαs isoform, GαsXL elicited hypertrophy comparable to that achieved by AAV:β2-AR. Moreover, GαsXL gene delivery was found to be capable of inducing hypertrophy in the muscles of mice lacking functional β1- and β2-ARs. These findings demonstrate that gene therapy-based interventions targeting the β2-AR pathway can promote skeletal muscle hypertrophy independent of ligand administration, and highlight novel methods for potentially modulating muscle mass in settings of disease. PMID:26972746

  7. Using AAV vectors expressing the β2-adrenoceptor or associated Gα proteins to modulate skeletal muscle mass and muscle fibre size.

    PubMed

    Hagg, Adam; Colgan, Timothy D; Thomson, Rachel E; Qian, Hongwei; Lynch, Gordon S; Gregorevic, Paul

    2016-01-01

    Anabolic β2-adrenoceptor (β2-AR) agonists have been proposed as therapeutics for treating muscle wasting but concerns regarding possible off-target effects have hampered their use. We investigated whether β2-AR-mediated signalling could be modulated in skeletal muscle via gene delivery to the target tissue, thereby avoiding the risks of β2-AR agonists. In mice, intramuscular administration of a recombinant adeno-associated virus-based vector (rAAV vector) expressing the β2-AR increased muscle mass by >20% within 4 weeks. This hypertrophic response was comparable to that of 4 weeks' treatment with the β2-AR agonist formoterol, and was not ablated by mTOR inhibition. Increasing expression of inhibitory (Gαi2) and stimulatory (GαsL) G-protein subunits produced minor atrophic and hypertrophic changes in muscle mass, respectively. Furthermore, Gαi2 over-expression prevented AAV:β2-AR mediated hypertrophy. Introduction of the non-muscle Gαs isoform, GαsXL elicited hypertrophy comparable to that achieved by AAV:β2-AR. Moreover, GαsXL gene delivery was found to be capable of inducing hypertrophy in the muscles of mice lacking functional β1- and β2-ARs. These findings demonstrate that gene therapy-based interventions targeting the β2-AR pathway can promote skeletal muscle hypertrophy independent of ligand administration, and highlight novel methods for potentially modulating muscle mass in settings of disease. PMID:26972746

  8. Serotype-specific Binding Properties and Nanoparticle Characteristics Contribute to the Immunogenicity of rAAV1 Vectors.

    PubMed

    Ferrand, Maxime; Da Rocha, Sylvie; Corre, Guillaume; Galy, Anne; Boisgerault, Florence

    2015-06-01

    The immunogenic properties of recombinant adeno-associated virus (rAAV) gene transfer vectors remain incompletely characterized in spite of their usage as gene therapy vectors or as vaccines. Molecular interactions between rAAV and various types of antigen-presenting cells (APCs), as well as the impact of these interactions on transgene or capsid-specific immunization remain unclear. We herein show that binding motifs recognized by the capsid and which determine the vector tissue tropism are also critical for key immune activation processes. Using rAAV capsid serotype 1 (rAAV1) vectors which primary receptors on target cells are α2,3 and α2,6 N-linked sialic acids, we show that sialic acid-dependent binding of rAAV1 on APCs is essential to trigger CD4(+) T-cell responses by increasing rAAV1 uptake and contributing to antigenic presentation of both the capsid and transgene product although this involves different APCs. In addition, the nanoparticulate structure of the vector in itself appears to be sufficient to trigger mobilization and activation of some APCs. Therefore, combinations of structural and of serotype-specific cell-targeting properties of rAAV1 determine its complex immunogenicity. These findings may be useful to guide a selection of rAAV variants depending on the intended level of immunogenicity for either gene therapy or vaccination applications. PMID:25881000

  9. Global CNS gene delivery and evasion of anti-AAV-neutralizing antibodies by intrathecal AAV administration in non-human primates.

    PubMed

    Gray, S J; Nagabhushan Kalburgi, S; McCown, T J; Jude Samulski, R

    2013-04-01

    Injection of adeno-associated virus (AAV) into the cerebrospinal fluid (CSF) offers a means to achieve widespread transgene delivery to the central nervous system, where the doses can be readily translated from small to large animals. In contrast to studies with other serotypes (AAV2, AAV4 and AAV5) in rodents, we report that a naturally occurring capsid (AAV9) and rationally engineered capsid (AAV2.5) are able to achieve broad transduction throughout the brain and spinal cord parenchyma following a single injection into the CSF (via cisterna magna or lumbar cistern) in non-human primates (NHP). Using either vector at a dose of ∼2 × 10(12) vector genome (vg) per 3-6 kg animal, approximately 2% of the entire brain and spinal cord was transduced, covering all regions of the central nervous system (CNS). AAV9 in particular displayed efficient transduction of spinal cord motor neurons. The peripheral organ biodistribution was highly reduced compared with intravascular delivery, and the presence of circulating anti-AAV-neutralizing antibodies up to a 1:128 titer had no inhibitory effect on CNS gene transfer. Intra-CSF delivery effectively translates from rodents to NHPs, which provides encouragement for the use of this approach in humans to treat motor neuron and lysosomal storage diseases. PMID:23303281

  10. HIV Capsid Assembly, Mechanism, and Structure.

    PubMed

    Chen, Bo

    2016-05-10

    The HIV genome materials are encaged by a proteinaceous shell called the capsid, constructed from ∼1000-1500 copies of the capsid proteins. Because its stability and integrity are critical to the normal life cycle and infectivity of the virus, the HIV capsid is a promising antiviral drug target. In this paper, we review the studies shaping our understanding of the structure and dynamics of the capsid proteins and various forms of their assemblies, as well as the assembly mechanism. PMID:27074418

  11. Capsid is an important determinant for functional complementation of murine leukemia virus and spleen necrosis virus Gag proteins.

    PubMed

    Lee, Sook-Kyung; Boyko, Vitaly; Hu, Wei-Shau

    2007-04-10

    In this report, we examined the abilities and requirements of heterologous Gag proteins to functionally complement each other to support viral replication. Two distantly related gammaretroviruses, murine leukemia virus (MLV) and spleen necrosis virus (SNV), were used as a model system because SNV proteins can support MLV vector replication. Using chimeric or mutant Gag proteins that could not efficiently support MLV vector replication, we determined that a homologous capsid (CA) domain was necessary for the functional complementation of MLV and SNV Gag proteins. Findings from the bimolecular fluorescence complementation assay revealed that MLV and SNV Gag proteins were capable of colocalizing and interacting in cells. Taken together, our results indicated that MLV and SNV Gag proteins can interact in cells; however, a homologous CA domain is needed for functional complementation of MLV and SNV Gag proteins to complete virus replication. This requirement of homologous Gag most likely occurs at a postassembly step(s) of the viral replication. PMID:17156810

  12. Adjuvant effect of B domain of staphyloccocal protein A displayed on the surface of hepatitis B virus capsid.

    PubMed

    Kim, Hyun Jin; Ahn, Keum-Young; Bae, Kyung Dong; Lee, Jiyun; Sim, Sang Jun; Lee, Jeewon

    2016-02-01

    The hepatitis B virus (HBV) capsid-based recombinant particles, which display both major hydrophilic region of HBV surface antigen (HBV-MHR) and B domain of Staphylococcal protein A (SPAB ), were produced using Escherichia coli as expression host. SPAB was used as an adjuvant to elicit the immune response to HBV-MHR, and its adjuvant effect in the immunized mice was estimated with varying the position and amount of SPAB on the HBV capsid particles. Compared to the emulsified aluminum gel (alum gel) that is a currently commercialized vaccine adjuvant, SPAB caused the significantly higher level of anti-HBV immunoglobulin G (IgG) titer and seroconversion rate, and notably SPAB at the most surface-exposed position on the recombinant particle led to the highest immune response. Moreover, SPAB caused much lower ratio of IgG1 to IgG2a compared to alum gel, indicating that helper T-cell 1-mediated immune response (responsible for cytotoxic T-cell stimulation) is relatively more stimulated by SPAB , unlike alum gel that mainly stimulates helper T-cell 2-mediated immune response (responsible for B-cell stimulation). Although HBV-MHR and HBV capsid particle were used as proof-of-concept in this study, SPAB can be used as a highly effective adjuvant with other disease-specific antigens on the surface of other virus-like particles to produce various recombinant vaccines with high potency. PMID:26222886

  13. A sequence of basic residues in the porcine circovirus type 2 capsid protein is crucial for its co-expression and co-localization with the replication protein.

    PubMed

    Huang, Liping; Renne, Nicolaas Van; Liu, Changming; Nauwynck, Hans J

    2015-12-01

    Porcine circovirus type 2 (PCV2) encodes two major proteins: the replication protein (Rep) and the capsid protein (Cap). Cap displays a conserved stretch of basic residues situated on the inside of the capsid, whose role is so far unknown. We used a reverse-genetics approach to investigate its function and found that mutations in these amino acids hindered Cap mRNA translation and hampered Cap/Rep co-localization, yielding unfit viruses. Intriguingly, co-transfection with a WT PCV2 of a different genotype partially rescued mutant Cap expression, showing the importance of this basic pattern for efficient translation of Cap mRNA into protein. Our results show that Cap and Rep are expressed independently of each other, and that this amino acid sequence of Cap is vital for virus propagation. This study provides a method for studying unfit PCV2 virions and offers new insights into the intracellular modus vivendi of PCV2. PMID:26415571

  14. Delivering Transgenic DNA Exceeding the Carrying Capacity of AAV Vectors.

    PubMed

    Hirsch, Matthew L; Wolf, Sonya J; Samulski, R J

    2016-01-01

    Gene delivery using recombinant adeno-associated virus (rAAV) has emerged to the forefront demonstrating safe and effective phenotypic correction of diverse diseases including hemophilia B and Leber's congenital amaurosis. In addition to rAAV's high efficiency of transduction and the capacity for long-term transgene expression, the safety profile of rAAV remains unsoiled in humans with no deleterious vector-related consequences observed thus far. Despite these favorable attributes, rAAV vectors have a major disadvantage preventing widespread therapeutic applications; as the AAV capsid is the smallest described to date, it cannot package "large" genomes. Currently, the packaging capacity of rAAV has yet to be definitively defined but is approximately 5 kb, which has served as a limitation for large gene transfer. There are two main approaches that have been developed to overcome this limitation, split AAV vectors, and fragment AAV (fAAV) genome reassembly (Hirsch et al., Mol Ther 18(1):6-8, 2010). Split rAAV vector applications were developed based upon the finding that rAAV genomes naturally concatemerize in the cell post-transduction and are substrates for enhanced homologous recombination (HR) (Hirsch et al., Mol Ther 18(1):6-8, 2010; Duan et al., J Virol 73(1):161-169, 1999; Duan et al., J Virol 72(11):8568-8577, 1998; Duan et al., Mol Ther 4(4):383-391, 2001; Halbert et al., Nat Biotechnol 20(7):697-701, 2002). This method involves "splitting" the large transgene into two separate vectors and upon co-transduction, intracellular large gene reconstruction via vector genome concatemerization occurs via HR or nonhomologous end joining (NHEJ). Within the split rAAV approaches there currently exist three strategies: overlapping, trans-splicing, and hybrid trans-splicing (Duan et al., Mol Ther 4(4):383-391, 2001; Halbert et al., Nat Biotechnol 20(7):697-701, 2002; Ghosh et al., Mol Ther 16(1):124-130, 2008; Ghosh et al., Mol Ther 15(4):750-755, 2007). The other major

  15. Nucleolin interacts with the dengue virus capsid protein and plays a role in formation of infectious virus particles.

    PubMed

    Balinsky, Corey A; Schmeisser, Hana; Ganesan, Sundar; Singh, Kavita; Pierson, Theodore C; Zoon, Kathryn C

    2013-12-01

    Dengue virus (DENV) is a mosquito-transmitted flavivirus that can cause severe disease in humans and is considered a reemerging pathogen of significant importance to public health. The DENV capsid (C) protein functions as a structural component of the infectious virion; however, it may have additional functions in the virus replicative cycle. Here, we show that the DENV C protein interacts and colocalizes with the multifunctional host protein nucleolin (NCL). Furthermore, we demonstrate that this interaction can be disrupted by the addition of an NCL binding aptamer (AS1411). Knockdown of NCL with small interfering RNA (siRNA) or treatment of cells with AS1411 results in a significant reduction of viral titers after DENV infection. Western blotting and quantitative RT-PCR (qRT-PCR) analysis revealed no differences in viral RNA or protein levels at early time points postinfection, suggesting a role for NCL in viral morphogenesis. We support this hypothesis by showing that treatment with AS1411 alters the migration characteristics of the viral capsid, as visualized by native electrophoresis. Here, we identify a critical interaction between DENV C protein and NCL that represents a potential new target for the development of antiviral therapeutics. PMID:24027323

  16. Nucleolin Interacts with the Dengue Virus Capsid Protein and Plays a Role in Formation of Infectious Virus Particles

    PubMed Central

    Balinsky, Corey A.; Schmeisser, Hana; Ganesan, Sundar; Singh, Kavita; Pierson, Theodore C.

    2013-01-01

    Dengue virus (DENV) is a mosquito-transmitted flavivirus that can cause severe disease in humans and is considered a reemerging pathogen of significant importance to public health. The DENV capsid (C) protein functions as a structural component of the infectious virion; however, it may have additional functions in the virus replicative cycle. Here, we show that the DENV C protein interacts and colocalizes with the multifunctional host protein nucleolin (NCL). Furthermore, we demonstrate that this interaction can be disrupted by the addition of an NCL binding aptamer (AS1411). Knockdown of NCL with small interfering RNA (siRNA) or treatment of cells with AS1411 results in a significant reduction of viral titers after DENV infection. Western blotting and quantitative RT-PCR (qRT-PCR) analysis revealed no differences in viral RNA or protein levels at early time points postinfection, suggesting a role for NCL in viral morphogenesis. We support this hypothesis by showing that treatment with AS1411 alters the migration characteristics of the viral capsid, as visualized by native electrophoresis. Here, we identify a critical interaction between DENV C protein and NCL that represents a potential new target for the development of antiviral therapeutics. PMID:24027323

  17. Pre-clinical immunogenicity of human papillomavirus alpha-7 and alpha-9 major capsid proteins

    PubMed Central

    Bissett, Sara L.; Mattiuzzo, Giada; Draper, Eve; Godi, Anna; Wilkinson, Dianna E.; Minor, Philip; Page, Mark; Beddows, Simon

    2014-01-01

    Human papillomavirus (HPV) vaccines confer protection against the oncogenic genotypes HPV16 and HPV18 through the generation of type-specific neutralizing antibodies raised against the constituent virus-like particles (VLP) based upon the major capsid proteins (L1) of these genotypes. The vaccines also confer a degree of cross-protection against some genetically related types from the Alpha-9 (HPV16-like: HPV31, HPV33, HPV35, HPV52, HPV58) and Alpha-7 (HPV18-like: HPV39, HPV45, HPV59, HPV68) species groups. The mechanism of cross-protection is unclear but may involve antibodies capable of recognizing shared inter-genotype epitopes. The relationship(s) between the genetic and antigenic diversity of the L1 protein, particularly for non-vaccine genotypes, is poorly understood. We carried out a comprehensive evaluation of the immunogenicity of L1 VLP derived from genotypes within the Alpha-7 and Alpha-9 species groups in New Zealand White rabbits and used L1L2 pseudoviruses as the target antigens in neutralization assays. The majority antibody response against L1 VLP was type-specific, as expected, but several instances of robust cross-neutralization were nevertheless observed including between HPV33 and HPV58 within the Alpha-9 species and between HPV39, HPV59 and HPV68 in the Alpha-7 species. Immunization with an experimental tetravalent preparation comprising VLP based upon HPV16, HPV18, HPV39 and HPV58 was capable of generating neutralizing antibodies against all the Alpha-7 and Alpha-9 genotypes. Competition of HPV31 and HPV33 cross-neutralizing antibodies in the tetravalent sera confirmed that these antibodies originated from HPV16 and HPV58 VLP, respectively, and suggested that they represent minority specificities within the antibody repertoire generated by the immunizing antigen. These data improve our understanding of the antigenic diversity of the L1 protein per se and may inform the rational design of a next generation vaccine formulation based upon

  18. Expression of Norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice.

    PubMed

    Mason, H S; Ball, J M; Shi, J J; Jiang, X; Estes, M K; Arntzen, C J

    1996-05-28

    Alternatives to cell culture systems for production of recombinant proteins could make very safe vaccines at a lower cost. We have used genetically engineered plants for expression of candidate vaccine antigens with the goal of using the edible plant organs for economical delivery of oral vaccines. Transgenic tobacco and potato plants were created that express the capsid protein of Norwalk virus, a calicivirus that causes epidemic acute gastroenteritis in humans. The capsid protein could be extracted from tobacco leaves in the form of 38-nm Norwalk virus-like particles. Recombinant Norwalk virus-like particle (rNV) was previously recovered when the same gene was expressed in recombinant baculovirus-infected insect cells. The capsid protein expressed in tobacco leaves and potato tubers cosedimented in sucrose gradients with insect cell-derived rNV and appeared identical to insect cell-derived rNV on immunoblots of SDS/polyacrylamide gels. The plant-expressed rNV was orally immunogenic in mice. Extracts of tobacco leaf expressing rNV were given to CD1 mice by gavage, and the treated mice developed both serum IgG and secretory IgA specific for rNV. Furthermore, when potato tubers expressing rNV were fed directly to mice, they developed serum IgG specific for rNV. These results indicate the potential usefulness of plants for production and delivery of edible vaccines. This is an appropriate technology for developing countries where vaccines are urgently needed. PMID:8643575

  19. Two proline residues are essential in the calcium-binding activity of rotavirus VP7 outer capsid protein.

    PubMed Central

    Gajardo, R; Vende, P; Poncet, D; Cohen, J

    1997-01-01

    Rotavirus maturation and stability of the outer capsid are calcium-dependent processes. It has been shown previously that the concentration of Ca2+-solubilizing outer capsid proteins from rotavirus particles is dependent on the virus strain. This property of viral particles has been associated with the gene coding for VP7 (gene 9). In this study the correlation between VP7 and resistance to low [Ca2+] was confirmed by analyzing the origin of gene 9 from reassortant viruses prepared under the selective pressure of low [Ca2+]. After chemical mutagenesis, we selected mutant viruses of the bovine strain RF that are more resistant to low [Ca2+]. The genes coding for the VP7 proteins of these independent mutants have been sequenced. Sequence analysis confirmed that these mutants are independent and revealed that all mutant VP7 proteins have proline 75 changed to leucine and have an outer capsid that solubilized at low [Ca2+]. The mutation of proline 279 to serine is found in all but two mutants. The phenotype of mutants having a single proline change can be distinguished from the phenotype of mutants having two proline changes. Sequence analysis showed that position 75 is in a region (amino acids 65 to 78) of great variability and that proline 75 is present in most of the bovine strains. In contrast, proline 279 is in a conserved region and is conserved in all the VP7 sequences in data banks. This region is rich in oxygenated residues that are correctly allocated in the metal-coordinating positions of the Ca2+-binding EF-hand structure pattern, suggesting that this region is important in the Ca2+ binding of VP7. PMID:9032355

  20. Chirality of Viral Capsids

    NASA Astrophysics Data System (ADS)

    Dharmavaram, Sanjay; Xie, Fangming; Bruinsma, Robijn; Klug, William; Rudnick, Joseph

    Most icosahedral viruses are classified by their T-number which identifies their capsid in terms of the number of capsomers and their relative arrangement. Certain T-numbers (T = 7 for instance) are inherently chiral (with no reflection planes) while others (e.g. T = 1) are achiral. We present a Landau-Brazovskii (LB) theory for weak crystallization in which a scalar order parameter that measures density of capsid proteins successfully predicts the various observed T-numbers and their respective chiralities. We find that chiral capsids gain stability by spontaneously breaking symmetry from an unstable chiral state. The inherently achiral LB-free energy does not preferentially select a particular chiral state from its mirror reflection. Based on the physical observation that proteins are inherently chiral molecules with directional interactions, we propose a new chiral term to the LB energy as a possible selection mechanism for chirality.

  1. Identification of residues in the hepatitis C virus core protein that are critical for capsid assembly in a cell-free system.

    PubMed

    Klein, Kevin C; Dellos, Sheri R; Lingappa, Jaisri R

    2005-06-01

    Significant advances have been made in understanding hepatitis C virus (HCV) replication through development of replicon systems. However, neither replicon systems nor standard cell culture systems support significant assembly of HCV capsids, leaving a large gap in our knowledge of HCV virion formation. Recently, we established a cell-free system in which over 60% of full-length HCV core protein synthesized de novo in cell extracts assembles into HCV capsids by biochemical and morphological criteria. Here we used mutational analysis to identify residues in HCV core that are important for capsid assembly in this highly reproducible cell-free system. We found that basic residues present in two clusters within the N-terminal 68 amino acids of HCV core played a critical role, while the uncharged linker domain between them was not. Furthermore, the aspartate at position 111, the region spanning amino acids 82 to 102, and three serines that are thought to be sites of phosphorylation do not appear to be critical for HCV capsid formation in this system. Mutation of prolines important for targeting of core to lipid droplets also failed to alter HCV capsid assembly in the cell-free system. In addition, wild-type HCV core did not rescue assembly-defective mutants. These data constitute the first systematic and quantitative analysis of the roles of specific residues and domains of HCV core in capsid formation. PMID:15890921

  2. Development and evaluation of an immunochromatographic strip for rapid detection of capsid protein antigen p27 of avian leukosis virus.

    PubMed

    Qian, Kun; Liang, You-zhi; Yin, Li-ping; Shao, Hong-xia; Ye, Jian-qiang; Qin, Ai-jian

    2015-09-01

    A rapid immunochromatographic strip for detecting capsid protein antigen p27 of avian leukosis virus was successfully developed based on two high-affinity monoclonal antibodies. The test strip could detect not only 600pg purified recombinant p27 protein but also quantified avian leukosis virus as low as 70 TCID50, which has comparative sensitivity to the commercial enzyme-linked immunosorbent assay (ELISA) kit. For the evaluation of this test strip, 1100 samples consisting of cloacal swabs, meconium collected from the earliest stool of one day old chicken and virus isolates were assessed both by the strip and by the commercial ELISA kit. The agreement between these two tests was 93.91%, 93.42% and 100%, respectively. The sensitivity and specificity of the strip were also calculated by using the ELISA kit as the standard. This immunochromatographic strip provides advantages of rapid and simple detection of capsid protein antigen p27 of avian leukosis virus, which could be applied as an on-site testing assay and used for control and eradication programs of avian leukosis disease. PMID:25977186

  3. Chondroitin Sulfate is the Primary Receptor for a Peptide-Modified AAV That Targets Brain Vascular Endothelium In Vivo

    PubMed Central

    Geoghegan, James C; Keiser, Nicholas W; Okulist, Anna; Martins, Inês; Wilson, Matthew S; Davidson, Beverly L

    2014-01-01

    Recently, we described a peptide-modified AAV2 vector (AAV-GMN) containing a capsid-displayed peptide that directs in vivo brain vascular targeting and transduction when delivered intravenously. In this study, we sought to identify the receptor that mediates transduction by AAV-GMN. We found that AAV-GMN, but not AAV2, readily transduces the murine brain endothelial cell line bEnd.3, a result that mirrors previously observed in vivo transduction profiles of brain vasculature. Studies in vitro revealed that the glycosaminoglycan, chondroitin sulfate C, acts as the primary receptor for AAV-GMN. Unlike AAV2, chondroitin sulfate expression is required for cell transduction by AAV-GMN, and soluble chondroitin sulfate C can robustly inhibit AAV-GMN transduction of brain endothelial cells. Interestingly, AAV-GMN retains heparin-binding properties, though in contrast to AAV2, it poorly transduces cells that express heparan sulfate but not chondroitin sulfate, indicating that the peptide insertion negatively impacts heparan-mediated transduction. Lastly, when delivered directly, this modified virus can transduce multiple brain regions, indicating that the potential of AAV-GMN as a therapeutic gene delivery vector for central nervous system disorders is not restricted to brain vascular endothelium. PMID:25313621

  4. Structure of the Dimerization Interface in the Mature HIV-1 Capsid Protein Lattice from Solid State NMR of Tubular Assemblies.

    PubMed

    Bayro, Marvin J; Tycko, Robert

    2016-07-13

    The HIV-1 capsid protein (CA) forms the capsid shell that encloses RNA within a mature HIV-1 virion. Previous studies by electron microscopy have shown that the capsid shell is primarily a triangular lattice of CA hexamers, with variable curvature that destroys the ideal symmetry of a planar lattice. The mature CA lattice depends on CA dimerization, which occurs through interactions between helix 9 segments of the C-terminal domain (CTD) of CA. Several high-resolution structures of the CTD-CTD dimerization interface have been reported, based on X-ray crystallography and multidimensional solution nuclear magnetic resonance (NMR), with significant differences in amino acid side chain conformations and helix 9-helix 9 orientations. In a structural model for tubular CA assemblies based on cryogenic electron microscopy (cryoEM) [Zhao et al. Nature, 2013, 497, 643-646], the dimerization interface is substantially disordered. The dimerization interface structure in noncrystalline CA assemblies and the extent to which this interface is structurally ordered within a curved lattice have therefore been unclear. Here we describe solid state NMR measurements on the dimerization interface in tubular CA assemblies, which contain the curved triangular lattice of a mature virion, including quantitative measurements of intermolecular and intramolecular distances using dipolar recoupling techniques, solid state NMR chemical shifts, and long-range side chain-side chain contacts. When combined with restraints on the distance and orientation between helix 9 segments from the cryoEM study, the solid state NMR data lead to a unique high-resolution structure for the dimerization interface in the noncrystalline lattice of CA tubes. These results demonstrate that CA lattice curvature is not dependent on disorder or variability in the dimerization interface. This work also demonstrates the feasibility of local structure determination within large noncrystalline assemblies formed by high

  5. Yeast Ty retrotransposons assemble into virus-like particles whose T-numbers depend on the C-terminal length of the capsid protein.

    PubMed

    AL-Khayat, H A; Bhella, D; Kenney, J M; Roth, J F; Kingsman, A J; Martin-Rendon, E; Saibil, H R

    1999-09-10

    The virus-like particles (VLPs) produced by the yeast Ty retrotransposons are structurally and functionally related to retroviral cores. Using cryo-electron microscopy (cryo-EM) and three-dimensional (3D) reconstruction, we have examined the structures of VLPs assembled from full-length and truncated forms of the capsid structural protein. The VLPs are highly polydisperse in their radius distribution. We have found that the length of the C-terminal region of the capsid structural protein dictates the T -number, and thus the size, of the assembled particles. Each construct studied appears to assemble into at least two or three size classes, with shorter C termini giving rise to smaller particles. This assembly property provides a model for understanding the variable assembly of retroviral core proteins. The particles are assembled from trimer-clustered units and there are holes in the capsid shells. PMID:10493857

  6. Structure of the Three N-Terminal Immunoglobulin Domains of the Highly Immunogenic Outer Capsid Protein from a T4-Like Bacteriophage

    SciTech Connect

    Fokine, Andrei; Islam, Mohammad Z.; Zhang, Zhihong; Bowman, Valorie D.; Rao, Venigalla B.; Rossmann, Michael G.

    2011-09-16

    The head of bacteriophage T4 is decorated with 155 copies of the highly antigenic outer capsid protein (Hoc). One Hoc molecule binds near the center of each hexameric capsomer. Hoc is dispensable for capsid assembly and has been used to display pathogenic antigens on the surface of T4. Here we report the crystal structure of a protein containing the first three of four domains of Hoc from bacteriophage RB49, a close relative of T4. The structure shows an approximately linear arrangement of the protein domains. Each of these domains has an immunoglobulin-like fold, frequently found in cell attachment molecules. In addition, we report biochemical data suggesting that Hoc can bind to Escherichia coli, supporting the hypothesis that Hoc could attach the phage capsids to bacterial surfaces and perhaps also to other organisms. The capacity for such reversible adhesion probably provides survival advantages to the bacteriophage.

  7. Orthogonal labeling of M13 minor capsid proteins with DNA to self-assemble end-to-end multi-phage structures

    PubMed Central

    Hess, Gaelen T.; Guimaraes, Carla P.; Spooner, Eric; Ploegh, Hidde L.; Belcher, Angela M.

    2014-01-01

    M13 bacteriophage has been used as a scaffold to organize materials for various applications. Building more complex multi-phage devices requires precise control of interactions between the M13 capsid proteins. Towards this end, we engineered a loop structure onto the pIII capsid protein of M13 bacteriophage to enable sortase-mediated labeling reactions for C-terminal display. Combining this with N-terminal sortase-mediated labeling, we thus created a phage scaffold that can be labeled orthogonally on three capsid proteins: the body and both ends. We show that covalent attachment of different DNA oligonucleotides at the ends of the new phage structure enables formation of multi-phage particles oriented in a specific order. These have potential as nanoscale scaffolds for multi–material devices. PMID:23713956

  8. Finding a needle in a haystack: detection of a small protein (the 12-kDa VP26) in a large complex (the 200-MDa capsid of herpes simplex virus).

    PubMed Central

    Booy, F P; Trus, B L; Newcomb, W W; Brown, J C; Conway, J F; Steven, A C

    1994-01-01

    Macromolecular complexes that consist of homopolymeric protein frameworks with additional proteins attached at strategic sites for a variety of structural and functional purposes are widespread in subcellular biology. One such complex is the capsid of herpes simplex virus type 1 whose basic framework consists of 960 copies of the viral protein, VP5 (149 kDa), arranged in an icosahedrally symmetric shell. This shell also contains major amounts of three other proteins, including VP26 (12 kDa), a small protein that is approximately equimolar with VP5 and accounts for approximately 6% of the capsid mass. With a view to inferring the role of VP26 in capsid assembly, we have localized it by quantitative difference imaging based on three-dimensional reconstructions calculated from cryo-electron micrographs. Purified capsids from which VP26 had been removed in vitro by treatment with guanidine hydrochloride were compared with preparations of the same depleted capsids to which purified VP26 had been rebound and with native (undepleted) capsids. The resulting three-dimensional density maps indicate that six VP26 subunits are distributed symmetrically around the outer tip of each hexon protrusion on VP26-containing capsids. Because VP26 may be readily dissociated from and reattached to the capsid, it does not appear to contribute significantly to structural stabilization. Rather, its exposed location suggests that VP26 may be involved in linking the capsid to the surrounding tegument and envelope at a later stage of viral assembly. Images PMID:8202543

  9. Perspective on Adeno-Associated Virus Capsid Modification for Duchenne Muscular Dystrophy Gene Therapy.

    PubMed

    Nance, Michael E; Duan, Dongsheng

    2015-12-01

    Duchenne muscular dystrophy (DMD) is a X-linked, progressive childhood myopathy caused by mutations in the dystrophin gene, one of the largest genes in the genome. It is characterized by skeletal and cardiac muscle degeneration and dysfunction leading to cardiac and/or respiratory failure. Adeno-associated virus (AAV) is a highly promising gene therapy vector. AAV gene therapy has resulted in unprecedented clinical success for treating several inherited diseases. However, AAV gene therapy for DMD remains a significant challenge. Hurdles for AAV-mediated DMD gene therapy include the difficulty to package the full-length dystrophin coding sequence in an AAV vector, the necessity for whole-body gene delivery, the immune response to dystrophin and AAV capsid, and the species-specific barriers to translate from animal models to human patients. Capsid engineering aims at improving viral vector properties by rational design and/or forced evolution. In this review, we discuss how to use the state-of-the-art AAV capsid engineering technologies to overcome hurdles in AAV-based DMD gene therapy. PMID:26414293

  10. Identification of a cytoplasmic interaction partner of the large regulatory proteins Rep78/Rep68 of adeno-associated virus type 2 (AAV-2)

    SciTech Connect

    Weger, Stefan . E-mail: stefan.weger@charite.de; Hammer, Eva; Goetz, Anne; Heilbronn, Regine

    2007-05-25

    Through yeast two-hybrid analysis and coimmunoprecipitation studies, we have identified a novel cellular AAV-2 Rep78/Rep68 interaction partner located predominantly in the cytoplasm. In public databases, it has been assigned as KCTD5, because of a region of high similarity to the cytoplasmic tetramerization domain of voltage-gated potassium channels. Whereas Rep/KCTD5 interaction relied on the region surrounding the Rep nuclear localization signal, nuclear accumulation of Rep was not required. Wildtype Rep78/Rep68 proteins induced the translocation of large portions of KCTD5 into the nucleus pointing to functional interactions both in the cytoplasm and the nucleus. In line with an anticipated functional interference in the cytoplasm, KCTD5 overexpression completely abrogated Rep68-mediated posttranscriptional activation of a HIV-LTR driven luciferase reporter gene. Our study expands the panel of already identified nuclear Rep interaction partners to a cytoplasmic protein, which raises the awareness that important steps in the AAV life cycle may be regulated in this compartment.

  11. Laminin receptor is an interacting partner for viral outer capsid protein VP5 in grass carp reovirus infection.

    PubMed

    Wang, Hao; Yu, Fei; Li, Jiale; Lu, Liqun

    2016-03-01

    Grass carp reovirus (GCRV) is responsible for viral hemorrhagic disease in cultured grass carp Ctenopharyngon idellus. Through yeast two-hybrid screen, laminin receptor (LamR) was identified as a potential interacting partner for the outer capsid protein VP5 of GCRV. We cloned and sequenced the gene encoding grass carp LamR. Viral attachment assay demonstrated the involvement of membrane-associated LamR in GCRV infection. Solid-phase overlay assays demonstrated that GCRV interacted with GST-tagged LamR in vitro. In contrast to VP7, GST-tagged VP5 was shown to associate with LamR in both pull-down and solid-phase blot overlay assays. With the reduction of LamR expression in CIK cells achieved by RNAi, remarkably reduced infection efficiency of GCRV was observed. CIK cells pretreated with polyclonal antibody against LamR resulted in dose-dependent inhibition of GCRV infection. These results collectively indicated that grass carp LamR was involved in GCRV infection by interacting with viral outer capsid protein VP5. PMID:26848829

  12. Crystal structure of a human rhinovirus neutralizing antibody complexed with a peptide derived from viral capsid protein VP2.

    PubMed Central

    Tormo, J; Blaas, D; Parry, N R; Rowlands, D; Stuart, D; Fita, I

    1994-01-01

    The three-dimensional structure of the complex between the Fab fragment of an anti-human rhinovirus neutralizing antibody (8F5) and a cross-reactive synthetic peptide from the viral capsid protein VP2 has been determined at 2.5 A resolution by crystallographic methods. The refinement is presently at an R factor of 0.18 and the antigen-binding site and viral peptide are well defined. The peptide antigen adopts a compact fold by two tight turns and interacts through hydrogen bonds, some with ionic character, and van der Waals contacts with antibody residues from the six hypervariable loops as well as several framework amino acids. The conformation adopted by the peptide is closely related to the corresponding region of the viral protein VP2 on the surface of human rhinovirus 1A whose three-dimensional structure is known. Implications for the cross-reactivity between peptides and the viral capsid are discussed. The peptide-antibody interactions, together with the analysis of mutant viruses that escape neutralization by 8F5 suggest two different mechanisms for viral escape. The comparison between the complexed and uncomplexed antibody structures shows important conformational rearrangements, especially in the hypervariable loops of the heavy chain. Thus, it constitutes a clear example of the 'induced fit' molecular recognition mechanism. Images PMID:8194515

  13. Preliminary crystallographic analysis of the major capsid protein P2 of the lipid-containing bacteriophage PM2

    SciTech Connect

    Abrescia, Nicola G. A.; Kivelä, Hanna M.; Grimes, Jonathan M.; Bamford, Jaana K. H.; Bamford, Dennis H.; Stuart, David I.

    2005-08-01

    The viral capsid protein P2 of bacteriophage PM2 has been crystallized. Preliminary X-ray analysis demonstrates the position and orientation of the two trimers in the asymmetric unit. PM2 (Corticoviridae) is a dsDNA bacteriophage which contains a lipid membrane beneath its icosahedral capsid. In this respect it resembles bacteriophage PRD1 (Tectiviridae), although it is not known whether the similarity extends to the detailed molecular architecture of the virus, for instance the fold of the major coat protein P2. Structural analysis of PM2 has been initiated and virus-derived P2 has been crystallized by sitting-nanodrop vapour diffusion. Crystals of P2 have been obtained in space group P2{sub 1}2{sub 1}2, with two trimers in the asymmetric unit and unit-cell parameters a = 171.1, b = 78.7, c = 130.1 Å. The crystals diffract to 4 Å resolution at the ESRF BM14 beamline (Grenoble, France) and the orientation of the non-crystallographic threefold axes, the spatial relationship between the two trimers and the packing of the trimers within the unit cell have been determined. The trimers form tightly packed layers consistent with the crystal morphology, possibly recapitulating aspects of the arrangement of subunits in the virus.

  14. Ranavirus phylogeny and differentiation based on major capsid protein, DNA polymerase and neurofilament triplet H1-like protein genes.

    PubMed

    Holopainen, R; Ohlemeyer, S; Schütze, H; Bergmann, S M; Tapiovaara, H

    2009-06-10

    In this study, we developed new methods for differentiation of ranaviruses based on polymerase chain reaction and restriction enzyme analysis of DNA polymerase and neurofilament triplet H1-like (NF-H1) protein gene. Using these methods, we were able to differentiate the 6 known ranaviruses--Bohle iridovirus (BIV), European catfish virus (ECV), epizootic haematopoietic necrosis virus (EHNV), European sheatfish virus (ESV), frog virus 3 (FV3) and Singapore grouper iridovirus (SGIV)--with 3 less characterised virus isolates: short-finned eel ranavirus (SERV), Rana esculenta virus Italy 282/I02 (REV 282/I02) and pike-perch iridovirus (PPIV). Doctor fish virus (DFV) and guppy virus 6 (GV6) were distinguished as a group from the other viruses. In addition, all 11 isolates were analysed and compared based on nucleotide sequences from 3 different genomic regions: major capsid protein (MCP), DNA polymerase and NF-H1. The partial DNA polymerase gene was sequenced from all analysed viruses. The complete sequence of the MCP and a fragment of the NF-H1 gene were obtained from BIV, ECV, EHNV, ESV, FV3, PPIV, REV 282/I02 and SERV. With the exception of GV6, DFV and SGIV, the sequence analyses showed only a few variations within the analysed viruses. The sequence data suggest that PPIV, REV 282/I02 and SERV are new members of the genus Ranavirus. The methods developed in this study provide tools to differentiate between closely related ranaviruses of different host and geographical origin. PMID:19694168

  15. Cloning and nucleotide sequence of the simian rotavirus gene 6 that codes for the major inner capsid protein.

    PubMed Central

    Estes, M K; Mason, B B; Crawford, S; Cohen, J

    1984-01-01

    The nucleotide sequence of the gene that codes for the major inner capsid protein of the simian rotavirus SA11 has been determined. A DNA copy of mRNA from gene 6 was cloned in the E. coli plasmid pBR322. The full-length gene is 1357 nucleotides long with a 5'-noncoding region of 23 nucleotides and a 3'-noncoding region of 140 nucleotides. The gene contains a single, long, open reading-frame of 1194 nucleotides capable of coding for a protein of 397 amino acids with a molecular weight of 44,816. The predicted protein product is relatively proline-rich with a net charge at neutral pH of -3.5. One stretch of 53 amino acids (encoded by nucleotides 327-485) is basic. Images PMID:6322125

  16. Safety and liver transduction efficacy of rAAV5-cohPBGD in nonhuman primates: a potential therapy for acute intermittent porphyria.

    PubMed

    Pañeda, Astrid; Lopez-Franco, Esperanza; Kaeppel, Christine; Unzu, Carmen; Gil-Royo, Ana Gloria; D'Avola, Delia; Beattie, Stuart G; Olagüe, Cristina; Ferrero, Roberto; Sampedro, Ana; Mauleon, Itsaso; Hermening, Stephan; Salmon, Florence; Benito, Alberto; Gavira, Juan Jose; Cornet, María Eugenia; del Mar Municio, María; von Kalle, Christof; Petry, Harald; Prieto, Jesus; Schmidt, Manfred; Fontanellas, Antonio; González-Aseguinolaza, Gloria

    2013-12-01

    Acute intermittent porphyria (AIP) results from haplo-insufficient activity of porphobilinogen deaminase (PBGD) and is characterized clinically by life-threatening, acute neurovisceral attacks. To date, liver transplantation is the only curative option for AIP. The aim of the present preclinical nonhuman primate study was to determine the safety and transduction efficacy of an adeno-associated viral vector encoding PBGD (recombinant AAV serotype 5-codon-optimized human porphobilinogen deaminase, rAAV5-cohPBGD) administered intravenously as part of a safety program to start a clinical study in patients with AIP. Macaques injected with either 1 × 10(13) or 5 × 10(13) vector genomes/kg of clinical-grade rAAV5-cohPBGD were monitored by standardized clinical parameters, and vector shedding was analyzed. Liver transduction efficacy, biodistribution, vector integration, and histopathology at day 30 postvector administration were determined. There was no evidence of acute toxicity, and no adverse effects were observed. The vector achieved efficient and homogenous hepatocellular transduction, reaching transgenic PBGD expression levels equivalent to 50% of the naturally expressed PBGD mRNA. No cellular immune response was detected against the human PBGD or AAV capsid proteins. Integration site analysis in transduced liver cells revealed an almost random integration pattern supporting the good safety profile of rAAV5-cohPBGD. Together, data obtained in nonhuman primates indicate that rAAV5-cohPBGD represents a safe therapy to correct the metabolic defect present in AIP patients. PMID:24070415

  17. Immune Responses to Intramuscular Administration of Alipogene Tiparvovec (AAV1-LPLS447X) in a Phase II Clinical Trial of Lipoprotein Lipase Deficiency Gene Therapy

    PubMed Central

    Twisk, Jaap; Kwikkers, Karin; Aronica, Eleonora; Brisson, Diane; Methot, Julie; Petry, Harald; Gaudet, Daniel

    2014-01-01

    Abstract Cellular immune responses to adeno-associated viral (AAV) vectors used for gene therapy have been linked to attenuated transgene expression and loss of efficacy. The impact of such cellular immune responses on the clinical efficacy of alipogene tiparvovec (Glybera; AAV1-LPLS447X; uniQure), a gene therapy consisting of intramuscular administration of a recombinant AAV1 mediating muscle-directed expression of lipoprotein lipase (LPL), was investigated. Five subjects with LPL deficiency (LPLD) were administered intramuscularly with a dose of 1×1012 gc/kg alipogene tiparvovec. All subjects were treated with immune suppression starting shortly before administration of alipogene tiparvovec and maintained until 12 weeks after administration. Systemic antibody and T cell responses against AAV1 and LPLS447X, as well as local cellular immune responses in the injected muscle, were investigated in five LPLD subjects. Long-term transgene expression was demonstrated despite a transient systemic cellular response and a stable humoral immune response against the AAV1 capsid protein. Cellular infiltrates were found in four of the five subjects but were not associated with adverse clinical events or elevation of inflammation markers. Consistent herewith, CD8+ T cells in the infiltrates lacked cytotoxic potential. Furthermore, FoxP3+/CD4+ T cells were found in the infiltrates, suggesting that multiple mechanisms contribute to local tolerance. Systemic and local immune responses induced by intramuscular injection of alipogene tiparvovec did not appear to have an impact on safety and did not prevent LPL transgene expression. These findings support the use of alipogene tiparvovec in individuals with LPLD and indicate that muscle-directed AAV-based gene therapy remains a promising approach for the treatment of human diseases. PMID:24299335

  18. Specific Inhibitors of HIV Capsid Assembly Binding to the C-Terminal Domain of the Capsid Protein: Evaluation of 2-Arylquinazolines as Potential Antiviral Compounds.

    PubMed

    Machara, Aleš; Lux, Vanda; Kožíšek, Milan; Grantz Šašková, Klára; Štěpánek, Ondřej; Kotora, Martin; Parkan, Kamil; Pávová, Marcela; Glass, Bärbel; Sehr, Peter; Lewis, Joe; Müller, Barbara; Kräusslich, Hans-Georg; Konvalinka, Jan

    2016-01-28

    Assembly of human immunodeficiency virus (HIV-1) represents an attractive target for antiretroviral therapy which is not exploited by currently available drugs. We established high-throughput screening for assembly inhibitors based on competition of small molecules for the binding of a known dodecapeptide assembly inhibitor to the C-terminal domain of HIV-1 CA (capsid). Screening of >70000 compounds from different libraries identified 2-arylquinazolines as low micromolecular inhibitors of HIV-1 capsid assembly. We prepared focused libraries of modified 2-arylquinazolines and tested their capacity to bind HIV-1 CA to compete with the known peptide inhibitor and to prevent the replication of HIV-1 in tissue culture. Some of the compounds showed potent binding to the C-terminal domain of CA and were found to block viral replication at low micromolar concentrations. PMID:26685880

  19. Transgenic Expression of Viral Capsid Proteins Predisposes to Axonal Injury in a Murine Model of Multiple Sclerosis

    PubMed Central

    Denic, Aleksandar; Zoecklein, Laurie; Kerkvliet, Jason; Papke, Louisa; Edukulla, Ramakrishna; Warrington, Arthur; Bieber, Allan; Pease, Larry R.; David, Chella S.; Rodriguez, Moses

    2010-01-01

    We used transgenic expression of capsid antigens to Theiler's murine encephalomyelitis virus (TMEV) to study the influence of VP1, VP2, or VP2121-130 to either protection or pathogenesis to chronic spinal cord demyelination, axonal loss and functional deficits during the acute and chronic phases of infection. We used both mice that are normally susceptible (FVB) and mice normally resistant (FVB.Db) to demyelination. Transgenic expression of VP2121-130 epitope in resistant FVB.Db mice caused spinal cord pathology and virus persistence because the VP2121-130 epitope is the dominant peptide recognized by Db, which is critical for virus clearance. In contrast, all three FVB TMEV transgenic mice showed more demyelination, inflammation, and axonal loss as compared to wild type FVB mice even though virus load was not increased. Motor function measured by rotarod showed weak correlation with total number of midthoracic axons, but a strong correlation with large caliber axons (>10μm2). This study supports the hypothesis that expression of viral capsid proteins as self influences the extent of axonal pathology following Theiler's virus-induced demyelination. The findings provide insight into the role of axonal injury in the development of functional deficits that may have relevance to human demyelinating disease. PMID:21314744

  20. Tabulation as a high-resolution alternative to coarse-graining protein interactions: Initial application to virus capsid subunits

    NASA Astrophysics Data System (ADS)

    Spiriti, Justin; Zuckerman, Daniel M.

    2015-12-01

    Traditional coarse-graining based on a reduced number of interaction sites often entails a significant sacrifice of chemical accuracy. As an alternative, we present a method for simulating large systems composed of interacting macromolecules using an energy tabulation strategy previously devised for small rigid molecules or molecular fragments [S. Lettieri and D. M. Zuckerman, J. Comput. Chem. 33, 268-275 (2012); J. Spiriti and D. M. Zuckerman, J. Chem. Theory Comput. 10, 5161-5177 (2014)]. We treat proteins as rigid and construct distance and orientation-dependent tables of the interaction energy between them. Arbitrarily detailed interactions may be incorporated into the tables, but as a proof-of-principle, we tabulate a simple α-carbon Gō-like model for interactions between dimeric subunits of the hepatitis B viral capsid. This model is significantly more structurally realistic than previous models used in capsid assembly studies. We are able to increase the speed of Monte Carlo simulations by a factor of up to 6700 compared to simulations without tables, with only minimal further loss in accuracy. To obtain further enhancement of sampling, we combine tabulation with the weighted ensemble (WE) method, in which multiple parallel simulations are occasionally replicated or pruned in order to sample targeted regions of a reaction coordinate space. In the initial study reported here, WE is able to yield pathways of the final ˜25% of the assembly process.

  1. Viral and Cellular Components of AAV2 Replication Compartments.

    PubMed

    Vogel, Rebecca; Seyffert, Michael; Pereira, Bruna de Andrade; Fraefel, Cornel

    2013-01-01

    Adeno-associated virus 2 (AAV2) is a helpervirus-dependent parvovirus with a bi-phasic life cycle comprising latency in absence and lytic replication in presence of a helpervirus, such as adenovirus (Ad) or herpes simplex virus type 1 (HSV-1). Helpervirus-supported AAV2 replication takes place in replication compartments (RCs) in the cell nucleus where virus DNA replication and transcription occur. RCs consist of a defined set of helper virus-, AAV2-, and cellular proteins. Here we compare the profile of cellular proteins recruited into AAV2 RCs or identified in Rep78-associated complexes when either Ad or HSV-1 is the helpervirus, and we discuss the potential roles of some of these proteins in AAV2 and helpervirus infection. PMID:24222808

  2. Phage display of intact domains at high copy number: a system based on SOC, the small outer capsid protein of bacteriophage T4.

    PubMed Central

    Ren, Z. J.; Lewis, G. K.; Wingfield, P. T.; Locke, E. G.; Steven, A. C.; Black, L. W.

    1996-01-01

    Peptides fused to the coat proteins of filamentous phages have found widespread applications in antigen display, the construction of antibody libraries, and biopanning. However, such systems are limited in terms of the size and number of the peptides that may be incorporated without compromising the fusion proteins' capacity to self-assemble. We describe here a system in which the molecules to be displayed are bound to pre-assembled polymers. The polymers are T4 capsids and polyheads (tubular capsid variants) and the display molecules are derivatives of the dispensable capsid protein SOC. In one implementation, SOC and its fusion derivatives are expressed at high levels in Escherichia coli, purified in high yield, and then bound in vitro to separately isolated polyheads. In the other, a positive selection vector forces integration of the modified soc gene into a soc-deleted T4 genome, leading to in vivo binding of the display protein to progeny virions. The system is demonstrated as applied to C-terminal fusions to SOC of (1) a tetrapeptide; (2) the 43-residue V3 loop domain of gp120, the human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein; and (3) poliovirus VP1 capsid protein (312 residues). SOC-V3 displaying phage were highly antigenic in mice and produced antibodies reactive with native gp120. That the fusion protein binds correctly to the surface lattice was attested in averaged electron micrographs of polyheads. The SOC display system is capable of presenting up to approximately 10(3) copies per capsid and > 10(4) copies per polyhead of V3-sized domains. Phage displaying SOC-VP1 were isolated from a 1:10(6) mixture by two cycles of a simple biopanning procedure, indicating that proteins of at least 35 kDa may be accommodated. PMID:8880907

  3. Vaccination of cats with an attenuated recombinant myxoma virus expressing feline calicivirus capsid protein.

    PubMed

    McCabe, Victoria J; Tarpey, Ian; Spibey, Norman

    2002-06-01

    Myxoma virus, a member of the Poxviridae family (genus Leporipoxvirus) is the agent responsible for myxomatosis in the European rabbit. Recombinant myxoma viruses expressing the capsid gene of an F9 strain of feline calicivirus (FCV) were constructed from an apathogenic, laboratory attenuated, isolate of myxoma virus. The FCV capsid genes were recombined into the myxoma growth factor (MGF) locus of the myxoma genome and expressed from synthetic poxvirus promoters. Myxoma virus is unable to replicate productively in feline cells in vitro, however, cells infected with recombinant viruses do express the heterologous antigens from both late and early/late synthetic promoters. Cats immunised with myxoma-FCV recombinant virus generated high levels of serum neutralising antibody and were protected from disease on subsequent challenge with virulent FCV. Furthermore, there was no evidence of transmission of myxoma-FCV recombinant virus from vaccinated to non-vaccinated cats. These results demonstrate the potential of myxoma virus as a safe vaccine vector for use in non-lepori species and in particular the cat. PMID:12057600

  4. The Pattern of Tegument-Capsid Interaction in the Herpes Simplex Virus Type 1 Virion Is Not Influenced by the Small Hexon-Associated Protein VP26

    PubMed Central

    Chen, Dong-Hua; Jakana, Joanita; McNab, David; Mitchell, Joyce; Zhou, Z. Hong; Dougherty, Matthew; Chiu, Wah; Rixon, Frazer J.

    2001-01-01

    Examination of the three-dimensional structure of intact herpes simplex virus type 1 (HSV-1) virions had revealed that the icosahedrally symmetrical interaction between the tegument and capsid involves the pentons but not the hexons (Z. H. Zhou, D. H. Chen, J. Jakana, F. J. Rixon, and W. Chiu, J. Virol. 73:3210–3218, 1999). To account for this, we postulated that the presence of the small capsid protein, VP26, on top of the hexons was masking potential binding sites and preventing tegument attachment. We have now tested this hypothesis by determining the structure of virions lacking VP26. Apart from the obvious absence of VP26 from the capsids, the structures of the VP26 minus and wild-type virions were essentially identical. Notably, they showed the same tegument attachment patterns, thereby demonstrating that VP26 is not responsible for the divergent tegument binding properties of pentons and hexons. PMID:11689667

  5. Development of novel AAV serotype 6 based vectors with selective tropism for human cancer cells.

    PubMed

    Sayroo, R; Nolasco, D; Yin, Z; Colon-Cortes, Y; Pandya, M; Ling, C; Aslanidi, G

    2016-01-01

    Viral vectors-based gene therapy is an attractive alternative to common anti-cancer treatments. In the present studies, AAV serotype 6 vectors were identified to be particularly effective in the transduction of human prostate (PC3), breast (T47D) and liver (Huh7) cancer cells. Next, we developed chimeric AAV vectors with Arg-Gly-Asp (RGD) peptide incorporated into the viral capsid to enable specific targeting of integrin-overexpressing malignant cells. These AAV6-RGD vectors improved transduction efficiency approximately 3-fold compared with wild-type AAV6 vectors by enhancing the viral entry into the cells. We also observed that transduction efficiency significantly improved, up to approximately 5-fold, by the mutagenesis of surface-exposed tyrosine and threonine residues involved in the intracellular trafficking of AAV vectors. Therefore, in our study, the AAV6-Y705-731F+T492V vector was identified as the most efficient. The combination of RGD peptide, tyrosine and threonine mutations on the same AAV6 capsid further increased the transduction efficiency, approximately 8-fold in vitro. In addition, we mutated lysine (K531E) to impair the affinity of AAV6 vectors to heparan sulfate proteoglycan. Finally, we showed a significant increase in both specificity and efficiency of AAV6-RGD-Y705-731F+T492V+K531E vectors in a xenograft animal model in vivo. In summary, the approach described here can lead to the development of AAV vectors with selective tropism to human cancer cells. PMID:26270885

  6. Role of a nuclear localization signal on the minor capsid Proteins VP2 and VP3 in BKPyV nuclear entry

    SciTech Connect

    Bennett, Shauna M.; Zhao, Linbo; Bosard, Catherine; Imperiale, Michael J.

    2015-01-01

    BK Polyomavirus (BKPyV) is a ubiquitous nonenveloped human virus that can cause severe disease in immunocompromised populations. After internalization into renal proximal tubule epithelial cells, BKPyV traffics through the ER and enters the cytosol. However, it is unclear how the virus enters the nucleus. In this study, we elucidate a role for the nuclear localization signal located on the minor capsid proteins VP2 and VP3 during infection. Site-directed mutagenesis of a single lysine in the basic region of the C-terminus of the minor capsid proteins abrogated their nuclear localization, and the analogous genomic mutation reduced infectivity. Additionally, through use of the inhibitor ivermectin and knockdown of importin β1, we found that the importin α/β pathway is involved during infection. Overall these data are the first to show the significance of the NLS of the BKPyV minor capsid proteins during infection in a natural host cell. - Highlights: • Polyomaviruses must deliver their genome to the nucleus to replicate. • The minor capsid proteins have a well-conserved nuclear localization signal. • Mutation of this NLS diminishes, but does not completely inhibit, infection.

  7. Recognition of the different structural forms of the capsid protein determines the outcome following infection with porcine circovirus type 2.

    PubMed

    Trible, Benjamin R; Suddith, Andrew W; Kerrigan, Maureen A; Cino-Ozuna, Ada G; Hesse, Richard A; Rowland, Raymond R R

    2012-12-01

    Porcine circovirus type 2 (PCV2) capsid protein (CP) is the only protein necessary for the formation of the virion capsid, and recombinant CP spontaneously forms virus-like particles (VLPs). Located within a single CP subunit is an immunodominant epitope consisting of residues 169 to 180 [CP(169-180)], which is exposed on the surface of the subunit, but, in the structural context of the VLP, the epitope is buried and inaccessible to antibody. High levels of anti-CP(169-180) activity are associated with porcine circovirus-associated disease (PCVAD). The purpose of this study was to investigate the role of the immune response to monomer CP in the development of PCVAD. The approach was to immunize pigs with CP monomer, followed by challenge with PCV2 and porcine reproductive and respiratory syndrome virus (PRRSV). To maintain the CP immunogen as a stable monomer, CP(43-233) was fused to ubiquitin (Ub-CP). Size exclusion chromatography showed that Ub-CP was present as a single 33-kDa protein. Pigs immunized with Ub-CP developed a strong antibody response to PCV2, including antibodies against CP(169-180). However, only low levels of virus neutralizing activity were detected, and viremia levels were similar to those of nonimmunized pigs. As a positive control, immunization with baculovirus-expressed CP (Bac-CP) resulted in high levels of virus neutralizing activity, small amounts of anti-CP(169-180) activity, and the absence of viremia in pigs following virus challenge. The data support the role of CP(169-180) as an immunological decoy and illustrate the importance of the structural form of the CP immunogen in determining the outcome following infection. PMID:23035215

  8. Recognition of the Different Structural Forms of the Capsid Protein Determines the Outcome following Infection with Porcine Circovirus Type 2

    PubMed Central

    Trible, Benjamin R.; Suddith, Andrew W.; Kerrigan, Maureen A.; Cino-Ozuna, Ada G.; Hesse, Richard A.

    2012-01-01

    Porcine circovirus type 2 (PCV2) capsid protein (CP) is the only protein necessary for the formation of the virion capsid, and recombinant CP spontaneously forms virus-like particles (VLPs). Located within a single CP subunit is an immunodominant epitope consisting of residues 169 to 180 [CP(169–180)], which is exposed on the surface of the subunit, but, in the structural context of the VLP, the epitope is buried and inaccessible to antibody. High levels of anti-CP(169–180) activity are associated with porcine circovirus-associated disease (PCVAD). The purpose of this study was to investigate the role of the immune response to monomer CP in the development of PCVAD. The approach was to immunize pigs with CP monomer, followed by challenge with PCV2 and porcine reproductive and respiratory syndrome virus (PRRSV). To maintain the CP immunogen as a stable monomer, CP(43–233) was fused to ubiquitin (Ub-CP). Size exclusion chromatography showed that Ub-CP was present as a single 33-kDa protein. Pigs immunized with Ub-CP developed a strong antibody response to PCV2, including antibodies against CP(169–180). However, only low levels of virus neutralizing activity were detected, and viremia levels were similar to those of nonimmunized pigs. As a positive control, immunization with baculovirus-expressed CP (Bac-CP) resulted in high levels of virus neutralizing activity, small amounts of anti-CP(169–180) activity, and the absence of viremia in pigs following virus challenge. The data support the role of CP(169–180) as an immunological decoy and illustrate the importance of the structural form of the CP immunogen in determining the outcome following infection. PMID:23035215

  9. C-Terminal Amino Acids 471-507 of Avian Hepatitis E Virus Capsid Protein Are Crucial for Binding to Avian and Human Cells.

    PubMed

    Zhang, Xinquan; Bilic, Ivana; Marek, Ana; Glösmann, Martin; Hess, Michael

    2016-01-01

    The infection of chickens with avian Hepatitis E virus (avian HEV) can be asymptomatic or induces clinical signs characterized by increased mortality and decreased egg production in adult birds. Due to the lack of an efficient cell culture system for avian HEV, the interaction between virus and host cells is still barely understood. In this study, four truncated avian HEV capsid proteins (ORF2-1 - ORF2-4) with an identical 338aa deletion at the N-terminus and gradual deletions from 0, 42, 99 and 136aa at the C-terminus, respectively, were expressed and used to map the possible binding site within avian HEV capsid protein. Results from the binding assay showed that three truncated capsid proteins attached to avian LMH cells, but did not penetrate into cells. However, the shortest construct, ORF2-4, lost the capability of binding to cells suggesting that the presence of amino acids 471 to 507 of the capsid protein is crucial for the attachment. The construct ORF2-3 (aa339-507) was used to study the potential binding of avian HEV capsid protein to human and other avian species. It could be demonstrated that ORF2-3 was capable of binding to QT-35 cells from Japanese quail and human HepG2 cells but failed to bind to P815 cells. Additionally, chicken serum raised against ORF2-3 successfully blocked the binding to LMH cells. Treatment with heparin sodium salt or sodium chlorate significantly reduced binding of ORF2-3 to LMH cells. However, heparinase II treatment of LMH cells had no effect on binding of the ORF2-3 construct, suggesting a possible distinct attachment mechanism of avian as compared to human HEV. For the first time, interactions between avian HEV capsid protein and host cells were investigated demonstrating that aa471 to 507 of the capsid protein are needed to facilitate interaction with different kind of cells from different species. PMID:27073893

  10. C-Terminal Amino Acids 471-507 of Avian Hepatitis E Virus Capsid Protein Are Crucial for Binding to Avian and Human Cells

    PubMed Central

    Zhang, Xinquan; Bilic, Ivana; Marek, Ana; Glösmann, Martin; Hess, Michael

    2016-01-01

    The infection of chickens with avian Hepatitis E virus (avian HEV) can be asymptomatic or induces clinical signs characterized by increased mortality and decreased egg production in adult birds. Due to the lack of an efficient cell culture system for avian HEV, the interaction between virus and host cells is still barely understood. In this study, four truncated avian HEV capsid proteins (ORF2-1 – ORF2-4) with an identical 338aa deletion at the N-terminus and gradual deletions from 0, 42, 99 and 136aa at the C-terminus, respectively, were expressed and used to map the possible binding site within avian HEV capsid protein. Results from the binding assay showed that three truncated capsid proteins attached to avian LMH cells, but did not penetrate into cells. However, the shortest construct, ORF2-4, lost the capability of binding to cells suggesting that the presence of amino acids 471 to 507 of the capsid protein is crucial for the attachment. The construct ORF2-3 (aa339-507) was used to study the potential binding of avian HEV capsid protein to human and other avian species. It could be demonstrated that ORF2-3 was capable of binding to QT-35 cells from Japanese quail and human HepG2 cells but failed to bind to P815 cells. Additionally, chicken serum raised against ORF2-3 successfully blocked the binding to LMH cells. Treatment with heparin sodium salt or sodium chlorate significantly reduced binding of ORF2-3 to LMH cells. However, heparinase II treatment of LMH cells had no effect on binding of the ORF2-3 construct, suggesting a possible distinct attachment mechanism of avian as compared to human HEV. For the first time, interactions between avian HEV capsid protein and host cells were investigated demonstrating that aa471 to 507 of the capsid protein are needed to facilitate interaction with different kind of cells from different species. PMID:27073893

  11. Adeno-associated virus rep protein synthesis during productive infection

    SciTech Connect

    Redemann, B.E.; Mendelson, E.; Carter, B.J.

    1989-02-01

    Adeno-associated virus (AAV) Rep proteins mediate viral DNA replication and can regulate expression from AAV genes. The authors studied the kinetics of synthesis of the four Rep proteins, Rep78, Rep68, Rep52, and Rep40, during infection of human 293 or KB cells with AAV and helper adenovirus by in vivo labeling with (/sup 35/S)methionine, immunoprecipitation, and immunoblotting analyses. Rep78 and Rep52 were readily detected concomitantly with detection of viral monomer duplex DNA replicating about 10 to 12 h after infection, and Rep68 and Rep40 were detected 2 h later. Rep78 and Rep52 were more abundant than Rep68 and Rep40 owing to a higher synthesis rate throughout the infectious cycle. In some experiments, very low levels of Rep78 could be detected as early as 4 h after infection. The synthesis rates of Rep proteins were maximal between 14 and 24 h and then decreased later after infection. Isotopic pulse-chase experiments showed that each of the Rep proteins was synthesized independently and was stable for at least 15 h. A slower-migrating, modified form of Rep78 was identified late after infection. AAV capsid protein synthesis was detected at 10 to 12 h after infection and also exhibited synthesis kinetics similar to those of the Rep proteins. AAV DNA replication showed at least two clearly defined stages. Bulk duplex replicating DNA accumulation began around 10 to 12 h and reached a maximum level at about 20 h when Rep and capsid protein synthesis was maximal. Progeny single-stranded DNA accumulation began about 12 to 13 h, but most of this DNA accumulated after 24 h when Rep and capsid protein synthesis had decreased.

  12. Effect of capsid proteins to ICG mass ratio on fluorescent quantum yield of virus-resembling optical nano-materials

    NASA Astrophysics Data System (ADS)

    Gupta, Sharad; Ico, Gerardo; Matsumura, Paul; Rao, A. L. N.; Vullev, Valentine; Anvari, Bahman

    2012-03-01

    We recently reported construction of a new type of optical nano-construct composed of genome-depleted plant infecting brome mosaic virus (BMV) doped with Indocyanine green (ICG), an FDA-approved chromophore. We refer to these constructs as optical viral ghosts (OVGs) since only the capsid protein (CP) subunits of BMV remain to encapsulate ICG. To utilize OVGs as effective nano-probes in fluorescence imaging applications, their fluorescence quantum yield needs to be maximized. In this study, we investigate the effect of altering the CP to ICG mass ratio on the fluorescent quantum yield of OVGs. Results of this study provide the basis for construction of OVGs with optimal amounts of CP and ICG to yield maximal fluorescence quantum yield.

  13. Adeno-Associated Virus Serotype 1 (AAV1)- and AAV5-Antibody Complex Structures Reveal Evolutionary Commonalities in Parvovirus Antigenic Reactivity

    PubMed Central

    Tseng, Yu-Shan; Gurda, Brittney L.; Chipman, Paul; McKenna, Robert; Afione, Sandra; Chiorini, John A.; Muzyczka, Nicholas; Olson, Norman H.; Baker, Timothy S.; Kleinschmidt, Jürgen

    2014-01-01

    ABSTRACT The clinical utility of the adeno-associated virus (AAV) gene delivery system has been validated by the regulatory approval of an AAV serotype 1 (AAV1) vector for the treatment of lipoprotein lipase deficiency. However, neutralization from preexisting antibodies is detrimental to AAV transduction efficiency. Hence, mapping of AAV antigenic sites and engineering of neutralization-escaping vectors are important for improving clinical efficacy. We report the structures of four AAV-monoclonal antibody fragment complexes, AAV1-ADK1a, AAV1-ADK1b, AAV5-ADK5a, and AAV5-ADK5b, determined by cryo-electron microscopy and image reconstruction to a resolution of ∼11 to 12 Å. Pseudoatomic modeling mapped the ADK1a epitope to the protrusions surrounding the icosahedral 3-fold axis and the ADK1b and ADK5a epitopes, which overlap, to the wall between depressions at the 2- and 5-fold axes (2/5-fold wall), and the ADK5b epitope spans both the 5-fold axis-facing wall of the 3-fold protrusion and portions of the 2/5-fold wall of the capsid. Combined with the six antigenic sites previously elucidated for different AAV serotypes through structural approaches, including AAV1 and AAV5, this study identified two common AAV epitopes: one on the 3-fold protrusions and one on the 2/5-fold wall. These epitopes coincide with regions with the highest sequence and structure diversity between AAV serotypes and correspond to regions determining receptor recognition and transduction phenotypes. Significantly, these locations overlap the two dominant epitopes reported for autonomous parvoviruses. Thus, rather than the amino acid sequence alone, the antigenic sites of parvoviruses appear to be dictated by structural features evolved to enable specific infectious functions. IMPORTANCE The adeno-associated viruses (AAVs) are promising vectors for in vivo therapeutic gene delivery, with more than 20 years of intense research now realized in a number of successful human clinical trials that

  14. Circovirus Transport Proceeds via Direct Interaction of the Cytoplasmic Dynein IC1 Subunit with the Viral Capsid Protein

    PubMed Central

    Cao, Jingjing; Lin, Cui; Wang, Huijuan; Wang, Lun; Zhou, Niu; Jin, Yulan; Liao, Min

    2014-01-01

    ABSTRACT Microtubule transport of circovirus from the periphery of the cell to the nucleus is essential for viral replication in early infection. How the microtubule is recruited to the viral cargo remains unclear. In this study, we observed that circovirus trafficking is dependent on microtubule polymerization and that incoming circovirus particles colocalize with cytoplasmic dynein and endosomes. However, circovirus binding to dynein was independent of the presence of microtubular α-tubulin and translocation of cytoplasmic dynein into the nucleus. The circovirus capsid (Cap) subunit enhanced microtubular acetylation and directly interacted with intermediate chain 1 (IC1) of dynein. N-terminal residues 42 to 100 of the Cap viral protein were required for efficient binding to the dynein IC1 subunit and for retrograde transport. Knockdown of IC1 decreased virus transport and replication. These results demonstrate that Cap is a direct ligand of the cytoplasmic dynein IC1 subunit and an inducer of microtubule α-tubulin acetylation. Furthermore, Cap recruits the host dynein/microtubule machinery to facilitate transport toward the nucleus by an endosomal mechanism distinct from that used for physiological dynein cargo. IMPORTANCE Incoming viral particles hijack the intracellular trafficking machinery of the host in order to migrate from the cell surface to the replication sites. Better knowledge of the interaction between viruses and virus proteins and the intracellular trafficking machinery may provide new targets for antiviral therapies. Currently, little is known about the molecular mechanisms of circovirus transport. Here, we report that circovirus particles enter early endosomes and utilize the microtubule-associated molecular motor dynein to travel along microtubules. The circovirus capsid subunit enhances microtubular acetylation, and N-terminal residues 42 to 100 directly interact with the dynein IC1 subunit during retrograde transport. These findings

  15. Cell-free translation of Drosophila C virus RNA: identification of a virus protease activity involved in capsid protein synthesis and further studies on in vitro processing of cricket paralysis virus specified proteins.

    PubMed

    Reavy, B; Moore, N F

    1983-01-01

    Drosophila C virus RNA acted as mRNA in rabbit reticulocyte lysates and directed the synthesis of at least one capsid protein and a number of higher molecular weight proteins. Kinetic analysis by pulse-chase experiments showed that a number of high molecular weight products acted as precursors to the capsid protein(s). Various dilution experiments were performed which showed that the virus specified a protease activity essential for the correct processing of precursors to give the capsid protein(s). A similar result was obtained with Cricket paralysis virus, and mixing experiments showed that the protease activity specified by one virus could perform some of the cleavages resulting in the production of the capsid proteins of the other virus. Some of the cleavages involving the highest molecular weight precursors could not be performed by the protease activity of the other virus. We could find no evidence for intramolecular cleavage of the capsid precursors of either of the viruses. PMID:6307220

  16. Direct Binding of Retromer to Human Papillomavirus Type 16 Minor Capsid Protein L2 Mediates Endosome Exit during Viral Infection

    PubMed Central

    Harrison, Megan S.; Goodner, Kylia; Kazakov, Teymur; Goodwin, Edward C.; Lipovsky, Alex; Burd, Christopher G.; DiMaio, Daniel

    2015-01-01

    Trafficking of human papillomaviruses to the Golgi apparatus during virus entry requires retromer, an endosomal coat protein complex that mediates the vesicular transport of cellular transmembrane proteins from the endosome to the Golgi apparatus or the plasma membrane. Here we show that the HPV16 L2 minor capsid protein is a retromer cargo, even though L2 is not a transmembrane protein. We show that direct binding of retromer to a conserved sequence in the carboxy-terminus of L2 is required for exit of L2 from the early endosome and delivery to the trans-Golgi network during virus entry. This binding site is different from known retromer binding motifs and can be replaced by a sorting signal from a cellular retromer cargo. Thus, HPV16 is an unconventional particulate retromer cargo, and retromer binding initiates retrograde transport of viral components from the endosome to the trans-Golgi network during virus entry. We propose that the carboxy-terminal segment of L2 protein protrudes through the endosomal membrane and is accessed by retromer in the cytoplasm. PMID:25693203

  17. The A, B, Cs of Herpesvirus Capsids

    PubMed Central

    Tandon, Ritesh; Mocarski, Edward S.; Conway, James F.

    2015-01-01

    Assembly of herpesvirus nucleocapsids shares significant similarities with the assembly of tailed dsDNA bacteriophages; however, important differences exist. A unique feature of herpesviruses is the presence of different mature capsid forms in the host cell nucleus during infection. These capsid forms, referred to as A-, B-, and C-capsids, represent empty capsids, scaffold containing capsids and viral DNA containing capsids, respectively. The C-capsids are the closest in form to those encapsidated into mature virions and are considered precursors to infectious virus. The evidence supporting A- and B-capsids as either abortive forms or assembly intermediates has been lacking. Interaction of specific capsid forms with viral tegument proteins has been proposed to be a mechanism for quality control at the point of nuclear egress of mature particles. Here, we will review the available literature on these capsid forms and present data to debate whether A- and B-capsids play an important or an extraneous role in the herpesvirus life cycle. PMID:25730559

  18. Widespread AAV1- and AAV2-mediated transgene expression in the nonhuman primate brain: implications for Huntington's disease.

    PubMed

    Hadaczek, Piotr; Stanek, Lisa; Ciesielska, Agnieszka; Sudhakar, Vivek; Samaranch, Lluis; Pivirotto, Philip; Bringas, John; O'Riordan, Catherine; Mastis, Bryan; San Sebastian, Waldy; Forsayeth, John; Cheng, Seng H; Bankiewicz, Krystof S; Shihabuddin, Lamya S

    2016-01-01

    Huntington's disease (HD) is caused by a toxic gain-of-function associated with the expression of the mutant huntingtin (htt) protein. Therefore, the use of RNA interference to inhibit Htt expression could represent a disease-modifying therapy. The potential of two recombinant adeno-associated viral vectors (AAV), AAV1 and AAV2, to transduce the cortico-striatal tissues that are predominantly affected in HD was explored. Green fluorescent protein was used as a reporter in each vector to show that both serotypes were broadly distributed in medium spiny neurons in the striatum and cortico-striatal neurons after infusion into the putamen and caudate nucleus of nonhuman primates (NHP), with AAV1-directed expression being slightly more robust than AAV2-driven expression. This study suggests that both serotypes are capable of targeting neurons that degenerate in HD, and it sets the stage for the advanced preclinical evaluation of an RNAi-based therapy for this disease. PMID:27408903

  19. Drawing a high-resolution functional map of adeno-associated virus capsid by massively parallel sequencing

    PubMed Central

    Adachi, Kei; Enoki, Tatsuji; Kawano, Yasuhiro; Veraz, Michael; Nakai, Hiroyuki

    2014-01-01

    Adeno-associated virus (AAV) capsid engineering is an emerging approach to advance gene therapy. However, a systematic analysis on how each capsid amino acid contributes to multiple functions remains challenging. Here we show proof-of-principle and successful application of a novel approach, termed AAV Barcode-Seq, that allows us to characterize phenotypes of hundreds of different AAV strains in a high-throughput manner and therefore overcomes technical difficulties in the systematic analysis. In this approach, we generate DNA barcode-tagged AAV libraries and determine a spectrum of phenotypes of each AAV strain by Illumina barcode sequencing. By applying this method to AAV capsid mutant libraries tagged with DNA barcodes, we can draw a high-resolution map of AAV capsid amino acids important for the structural integrity and functions including receptor binding, tropism, neutralization and blood clearance. Thus, Barcode-Seq provides a new tool to generate a valuable resource for virus and gene therapy research. PMID:24435020

  20. AAV Gene Therapy for MPS1-associated Corneal Blindness.

    PubMed

    Vance, Melisa; Llanga, Telmo; Bennett, Will; Woodard, Kenton; Murlidharan, Giridhar; Chungfat, Neil; Asokan, Aravind; Gilger, Brian; Kurtzberg, Joanne; Samulski, R Jude; Hirsch, Matthew L

    2016-01-01

    Although cord blood transplantation has significantly extended the lifespan of mucopolysaccharidosis type 1 (MPS1) patients, over 95% manifest cornea clouding with about 50% progressing to blindness. As corneal transplants are met with high rejection rates in MPS1 children, there remains no treatment to prevent blindness or restore vision in MPS1 children. Since MPS1 is caused by mutations in idua, which encodes alpha-L-iduronidase, a gene addition strategy to prevent, and potentially reverse, MPS1-associated corneal blindness was investigated. Initially, a codon optimized idua cDNA expression cassette (opt-IDUA) was validated for IDUA production and function following adeno-associated virus (AAV) vector transduction of MPS1 patient fibroblasts. Then, an AAV serotype evaluation in human cornea explants identified an AAV8 and 9 chimeric capsid (8G9) as most efficient for transduction. AAV8G9-opt-IDUA administered to human corneas via intrastromal injection demonstrated widespread transduction, which included cells that naturally produce IDUA, and resulted in a >10-fold supraphysiological increase in IDUA activity. No significant apoptosis related to AAV vectors or IDUA was observed under any conditions in both human corneas and MPS1 patient fibroblasts. The collective preclinical data demonstrate safe and efficient IDUA delivery to human corneas, which may prevent and potentially reverse MPS1-associated cornea blindness. PMID:26899286

  1. AAV Gene Therapy for MPS1-associated Corneal Blindness

    PubMed Central

    Vance, Melisa; Llanga, Telmo; Bennett, Will; Woodard, Kenton; Murlidharan, Giridhar; Chungfat, Neil; Asokan, Aravind; Gilger, Brian; Kurtzberg, Joanne; Samulski, R. Jude; Hirsch, Matthew L.

    2016-01-01

    Although cord blood transplantation has significantly extended the lifespan of mucopolysaccharidosis type 1 (MPS1) patients, over 95% manifest cornea clouding with about 50% progressing to blindness. As corneal transplants are met with high rejection rates in MPS1 children, there remains no treatment to prevent blindness or restore vision in MPS1 children. Since MPS1 is caused by mutations in idua, which encodes alpha-L-iduronidase, a gene addition strategy to prevent, and potentially reverse, MPS1-associated corneal blindness was investigated. Initially, a codon optimized idua cDNA expression cassette (opt-IDUA) was validated for IDUA production and function following adeno-associated virus (AAV) vector transduction of MPS1 patient fibroblasts. Then, an AAV serotype evaluation in human cornea explants identified an AAV8 and 9 chimeric capsid (8G9) as most efficient for transduction. AAV8G9-opt-IDUA administered to human corneas via intrastromal injection demonstrated widespread transduction, which included cells that naturally produce IDUA, and resulted in a >10-fold supraphysiological increase in IDUA activity. No significant apoptosis related to AAV vectors or IDUA was observed under any conditions in both human corneas and MPS1 patient fibroblasts. The collective preclinical data demonstrate safe and efficient IDUA delivery to human corneas, which may prevent and potentially reverse MPS1-associated cornea blindness. PMID:26899286

  2. Detection of HIV-1 Tat and JCV capsid protein, VP1, in AIDS brain with progressive multifocal leukoencephalopathy.

    PubMed

    Del Valle, L; Croul, S; Morgello, S; Amini, S; Rappaport, J; Khalili, K

    2000-06-01

    HIV-1 infection can lead to severe central nervous system (CNS) clinical syndromes in more than 50% of HIV-1 positive individuals. Progressive multifocal leukoencephalopathy (PML) is the frequent opportunistic infection of the CNS which is seen in as high as 5% of AIDS patients. Results from previous cell culture studies showed that the HIV-1 regulatory protein, Tat can potentiate transcription of the human neurotropic virus, JCV, the causative agent for PML in cells derived from the human CNS. In this communication we examine the presence of the HIV-1 regulatory protein, Tat, as well as the HIV-1 and JCV structural proteins, p24 and VP1, respectively in AIDS/PML clinical samples. We demonstrate high level expression of the JCV capsid protein, VP1, in oligodendrocytes and to some degree in astrocytes of AIDS with PML. In HIV-1+ samples expression of HIV-1 core protein, p24 was detected in perivascular monocytic cells and to a lesser extent in astrocytes and endothelial cells. A lack of p24 expression in oligodendrocytes suggested no infection of these cells with HIV-1. Interestingly, HIV-1 Tat was detected in various infected cells as well as in uninfected oligodendrocytes from HIV-1+ tissue, supporting the earlier in vitro findings that secreted Tat from the infected cells can be localized in the neighboring uninfected cells. The presence of Tat in oligodendrocytes was particularly interesting as this protein can up-modulate JCV gene transcription and several key cell cycle regulatory proteins including cyclin E, Cdk2, and pRb. The data presented here provide in vivo evidence for a role of HIV-1 Tat in the pathogenesis of AIDS/PML by acting as a positive regulatory protein that affects the expression of JCV and other cell regulatory proteins in the CNS. PMID:10878711

  3. Quantification and modification of the equilibrium dynamics and mechanics of a viral capsid lattice self-assembled as a protein nanocoating

    NASA Astrophysics Data System (ADS)

    Valbuena, Alejandro; Mateu, Mauricio G.

    2015-09-01

    Self-assembling, protein-based bidimensional lattices are being developed as functionalizable, highly ordered biocoatings for multiple applications in nanotechnology and nanomedicine. Unfortunately, protein assemblies are soft materials that may be too sensitive to mechanical disruption, and their intrinsic conformational dynamism may also influence their applicability. Thus, it may be critically important to characterize, understand and manipulate the mechanical features and dynamic behavior of protein assemblies in order to improve their suitability as nanomaterials. In this study, the capsid protein of the human immunodeficiency virus was induced to self-assemble as a continuous, single layered, ordered nanocoating onto an inorganic substrate. Atomic force microscopy (AFM) was used to quantify the mechanical behavior and the equilibrium dynamics (``breathing'') of this virus-based, self-assembled protein lattice in close to physiological conditions. The results uniquely provided: (i) evidence that AFM can be used to directly visualize in real time and quantify slow breathing motions leading to dynamic disorder in protein nanocoatings and viral capsid lattices; (ii) characterization of the dynamics and mechanics of a viral capsid lattice and protein-based nanocoating, including flexibility, mechanical strength and remarkable self-repair capacity after mechanical damage; (iii) proof of principle that chemical additives can modify the dynamics and mechanics of a viral capsid lattice or protein-based nanocoating, and improve their applied potential by increasing their mechanical strength and elasticity. We discuss the implications for the development of mechanically resistant and compliant biocoatings precisely organized at the nanoscale, and of novel antiviral agents acting on fundamental physical properties of viruses.Self-assembling, protein-based bidimensional lattices are being developed as functionalizable, highly ordered biocoatings for multiple applications

  4. The C-Terminal Arm of the Human Papillomavirus Major Capsid Protein Is Immunogenic and Involved in Virus-Host Interaction.

    PubMed

    Li, Zhihai; Yan, Xiaodong; Yu, Hai; Wang, Daning; Song, Shuo; Li, Yunbing; He, Maozhou; Hong, Qiyang; Zheng, Qingbing; Zhao, Qinjian; Gu, Ying; Zhang, Jun; Janssen, Mandy E W; Cardone, Giovanni; Olson, Norman H; Baker, Timothy S; Li, Shaowei; Xia, Ningshao

    2016-06-01

    Cervical cancer is the second most prevalent malignant tumor among women worldwide. High-risk human papillomaviruses (HPVs) are believed to be the major causative pathogens of mucosal epithelial cancers including cervical cancer. The HPV capsid is made up of 360 copies of major (L1) and 72 copies of minor (L2) capsid proteins. To date, limited high-resolution structural information about the HPV capsid has hindered attempts to understand details concerning the mechanisms by which HPV assembles and infects cells. In this study, we have constructed a pseudo-atomic model of the HPV59 L1-only capsid and demonstrate that the C-terminal arm of L1 participates in virus-host interactions. Moreover, when conjugated to a scaffold protein, keyhole limpet hemocyanin (KLH), this arm is immunogenic in vivo. These results provide new insights that will help elucidate HPV biology, and hence pave a way for the design of next-generation HPV vaccines. PMID:27276427

  5. Assessment of Tropism and Effectiveness of New Primate-Derived Hybrid Recombinant AAV Serotypes in the Mouse and Primate Retina

    PubMed Central

    Lipinski, Daniel M.; Singh, Mandeep S.; Mouravlev, Alexandre; You, Qisheng; Barnard, Alun R.; Hankins, Mark W.; During, Matthew J.; MacLaren, Robert E.

    2013-01-01

    Adeno-associated viral vectors (AAV) have been shown to be safe in the treatment of retinal degenerations in clinical trials. Thus, improving the efficiency of viral gene delivery has become increasingly important to increase the success of clinical trials. In this study, structural domains of different rAAV serotypes isolated from primate brain were combined to create novel hybrid recombinant AAV serotypes, rAAV2/rec2 and rAAV2/rec3. The efficacy of these novel serotypes were assessed in wild type mice and in two models of retinal degeneration (the Abca4−/− mouse which is a model for Stargardt disease and in the Pde6brd1/rd1 mouse) in vivo, in primate tissue ex-vivo, and in the human-derived SH-SY5Y cell line, using an identical AAV2 expression cassette. We show that these novel hybrid serotypes can transduce retinal tissue in mice and primates efficiently, although no more than AAV2/2 and rAAV2/5 serotypes. Transduction efficiency appeared lower in the Abca4−/− mouse compared to wild type with all vectors tested, suggesting an effect of specific retinal diseases on the efficiency of gene delivery. Shuffling of AAV capsid domains may have clinical applications for patients who develop T-cell immune responses following AAV gene therapy, as specific peptide antigen sequences could be substituted using this technique prior to vector re-treatments. PMID:23593201

  6. Quantification and modification of the equilibrium dynamics and mechanics of a viral capsid lattice self-assembled as a protein nanocoating.

    PubMed

    Valbuena, Alejandro; Mateu, Mauricio G

    2015-09-28

    Self-assembling, protein-based bidimensional lattices are being developed as functionalizable, highly ordered biocoatings for multiple applications in nanotechnology and nanomedicine. Unfortunately, protein assemblies are soft materials that may be too sensitive to mechanical disruption, and their intrinsic conformational dynamism may also influence their applicability. Thus, it may be critically important to characterize, understand and manipulate the mechanical features and dynamic behavior of protein assemblies in order to improve their suitability as nanomaterials. In this study, the capsid protein of the human immunodeficiency virus was induced to self-assemble as a continuous, single layered, ordered nanocoating onto an inorganic substrate. Atomic force microscopy (AFM) was used to quantify the mechanical behavior and the equilibrium dynamics ("breathing") of this virus-based, self-assembled protein lattice in close to physiological conditions. The results uniquely provided: (i) evidence that AFM can be used to directly visualize in real time and quantify slow breathing motions leading to dynamic disorder in protein nanocoatings and viral capsid lattices; (ii) characterization of the dynamics and mechanics of a viral capsid lattice and protein-based nanocoating, including flexibility, mechanical strength and remarkable self-repair capacity after mechanical damage; (iii) proof of principle that chemical additives can modify the dynamics and mechanics of a viral capsid lattice or protein-based nanocoating, and improve their applied potential by increasing their mechanical strength and elasticity. We discuss the implications for the development of mechanically resistant and compliant biocoatings precisely organized at the nanoscale, and of novel antiviral agents acting on fundamental physical properties of viruses. PMID:26302823

  7. P22 coat protein structures reveal a novel mechanism for capsid maturation: Stability without auxiliary proteins or chemical cross-links

    PubMed Central

    Parent, Kristin N.; Khayat, Reza; Tu, Long H.; Suhanovsky, Margaret M.; Cortines, Juliana R.; Teschke, Carolyn M.; Johnson, John E.; Baker, Timothy S.

    2010-01-01

    Viral capsid assembly and stability in tailed, dsDNA phage and Herpesviridae are achieved by various means including chemical cross-links (unique to HK97), or auxiliary proteins (lambda, T4, ϕ29, and herpesviruses). All these viruses have coat proteins (CP) with a conserved, HK97-like core structure. We used a combination of trypsin digestion, gold-labeling, cryo-electron microscopy, 3D image reconstruction, and comparative modeling to derive two independent, pseudo-atomic models of bacteriophage P22 CP: before and after maturation. P22 capsid stabilization results from inter-subunit interactions among N-terminal helices and an extensive “P-loop”, which obviate the need for cross-links or auxiliary proteins. P22 CP also has a telokin-like, Ig domain that stabilizes the monomer fold so that assembly may proceed via individual subunit addition rather than via preformed capsomers as occurs in HK97. Hence, the P22 CP structure may be a paradigm for understanding how monomers assemble in viruses like ϕ29 and HSV-1. PMID:20223221

  8. Neutralizing antibodies against AAV2, AAV5 and AAV8 in healthy and HIV-1-infected subjects in China: implications for gene therapy using AAV vectors.

    PubMed

    Liu, Q; Huang, W; Zhang, H; Wang, Y; Zhao, J; Song, A; Xie, H; Zhao, C; Gao, D; Wang, Y

    2014-08-01

    Adeno-associated viruses (AAV) have attracted attention as potential vectors for gene therapy and vaccines against several diseases, including HIV-1 infection. However, the presence of neutralizing antibodies (NAbs) after natural AAV infections inhibits their transfection in re-exposed subjects. To identify candidate AAV vectors for therapeutic or prophylactic HIV vaccines, NAbs against AAV2, AAV5 and AAV8 were screened in the sera of healthy individuals in China and 10 developed countries and an HIV-1-infected Chinese population. Seroprevalence was higher for AAV2 (96.6%) and AAV8 (82.0%) than for AAV5 (40.2%) in normal Chinese subjects. Among individuals seropositive for AAV5, >80% had low NAb titers (<1:90). The prevalence and titers of NAbs against the three AAVs were significantly higher in China than in developed countries (P<0.01). The prevalence of NAbs against AAV5 did not differ significantly between healthy and HIV-1-infected Chinese subjects (P=0.39). Co-occurrence of NAbs against AAV2, AAV5, and AAV8 was observed in the healthy population, and 15, 41, and 41% of individuals were AAV2(+), AAV2(+)/AAV8(+), and AAV2(+)/AAV5(+)/AAV8(+), respectively. Therefore, AAV5 exposure is low in healthy and HIV-1-infected populations Chinese individuals, and vectors based on AAV5 may be appropriate for human gene therapy or vaccines. PMID:24849042

  9. Expressing Transgenes That Exceed the Packaging Capacity of Adeno-Associated Virus Capsids.

    PubMed

    Chamberlain, Kyle; Riyad, Jalish Mahmud; Weber, Thomas

    2016-02-01

    Recombinant adeno-associated virus vectors (rAAV) are being explored as gene delivery vehicles for the treatment of various inherited and acquired disorders. rAAVs are attractive vectors for several reasons: wild-type AAVs are nonpathogenic, and rAAVs can trigger long-term transgene expression even in the absence of genome integration-at least in postmitotic tissues. Moreover, rAAVs have a low immunogenic profile, and the various AAV serotypes and variants display broad but distinct tropisms. One limitation of rAAVs is that their genome-packaging capacity is only ∼5 kb. For most applications this is not of major concern because the median human protein size is 375 amino acids. Excluding the ITRs, for a protein of typical length, this allows the incorporation of ∼3.5 kb of DNA for the promoter, polyadenylation sequence, and other regulatory elements into a single AAV vector. Nonetheless, for certain diseases the packaging limit of AAV does not allow the delivery of a full-length therapeutic protein by a single AAV vector. Hence, approaches to overcome this limitation have become an important area of research for AAV gene therapy. Among the most promising approaches to overcome the limitation imposed by the packaging capacity of AAV is the use of dual-vector approaches, whereby a transgene is split across two separate AAV vectors. Coinfection of a cell with these two rAAVs will then-through a variety of mechanisms-result in the transcription of an assembled mRNA that could not be encoded by a single AAV vector because of the DNA packaging limits of AAV. The main purpose of this review is to assess the current literature with respect to dual-AAV-vector design, to highlight the effectiveness of the different methodologies and to briefly discuss future areas of research to improve the efficiency of dual-AAV-vector transduction. PMID:26757051

  10. Formation of AAV Single Stranded DNA Genome from a Circular Plasmid in Saccharomyces cerevisiae

    PubMed Central

    Cervelli, Tiziana; Backovic, Ana; Galli, Alvaro

    2011-01-01

    Adeno-associated virus (AAV)-based vectors are promising tools for targeted transfer in gene therapy studies. Many efforts have been accomplished to improve production and purification methods. We thought to develop a simple eukaryotic system allowing AAV replication which could provide an excellent opportunity for studying AAV biology and, more importantly, for AAV vector production. It has been shown that yeast Saccharomyces cerevisiae is able to replicate and form the capsid of many viruses. We investigated the ability of the yeast Saccharomyces cerevisiae to carry out the replication of a recombinant AAV (rAAV). When a plasmid containing a rAAV genome in which the cap gene was replaced with the S. cerevisiae URA3 gene, was co-transformed in yeast with a plasmid expressing Rep68, a significant number of URA3+ clones were scored (more than 30-fold over controls). Molecular analysis of low molecular weight DNA by Southern blotting revealed that single stranded DNA is formed and that the plasmid is entirely replicated. The ssDNA contains the ITRs, URA3 gene and also vector sequences suggesting the presence of two distinct molecules. Its formation was dependent on Rep68 expression and ITR. These data indicate that DNA is not obtained by the canonical AAV replication pathway. PMID:21853137

  11. Formation of AAV single stranded DNA genome from a circular plasmid in Saccharomyces cerevisiae.

    PubMed

    Cervelli, Tiziana; Backovic, Ana; Galli, Alvaro

    2011-01-01

    Adeno-associated virus (AAV)-based vectors are promising tools for targeted transfer in gene therapy studies. Many efforts have been accomplished to improve production and purification methods. We thought to develop a simple eukaryotic system allowing AAV replication which could provide an excellent opportunity for studying AAV biology and, more importantly, for AAV vector production. It has been shown that yeast Saccharomyces cerevisiae is able to replicate and form the capsid of many viruses. We investigated the ability of the yeast Saccharomyces cerevisiae to carry out the replication of a recombinant AAV (rAAV). When a plasmid containing a rAAV genome in which the cap gene was replaced with the S. cerevisiae URA3 gene, was co-transformed in yeast with a plasmid expressing Rep68, a significant number of URA3(+) clones were scored (more than 30-fold over controls). Molecular analysis of low molecular weight DNA by Southern blotting revealed that single stranded DNA is formed and that the plasmid is entirely replicated. The ssDNA contains the ITRs, URA3 gene and also vector sequences suggesting the presence of two distinct molecules. Its formation was dependent on Rep68 expression and ITR. These data indicate that DNA is not obtained by the canonical AAV replication pathway. PMID:21853137

  12. Identification of one critical amino acid that determines a conformational neutralizing epitope in the capsid protein of porcine circovirus type 2

    PubMed Central

    2011-01-01

    Background Porcine circovirus type 2 (PCV2) is associated with post-weaning multisystemic wasting syndrome (PMWS) in pigs. Currently, there is considerable interest in the immunology of PCV2; in particular, the immunological properties of the capsid protein. This protein is involved in PCV2 immunogenicity and is a potential target for vaccine development. In this study, we identified one critical amino acid that determines a conformational neutralizing epitope in the capsid protein of PCV2. Results One monoclonal antibody (mAb; 8E4), against the capsid protein of PCV2, was generated and characterized in this study. 8E4 reacted with the genotype PCV2a (CL, LG and JF2) strains but not PCV2b (YJ, SH and JF) strains by an immunoperoxidase monolayer assay (IPMA) and a capture ELISA. Furthermore, the mAb had the capacity to neutralize PCV2a (CL, LG and JF2) strains but not PCV2b (YJ, SH and JF) strains. One critical amino acid that determined a conformational neutralizing epitope was identified using mAb 8E4 and PCV2 infectious clone technique. Amino acid residues 47-72 in the capsid protein of PCV2a/CL were replaced with the corresponding region of PCV2b/YJ, and the reactivity of mAb 8E4 was lost. Further experiments demonstrated that one amino acid substitution, the alanine for arginine at position 59 (A59R) in the capsid protein of PCV2a (CL, LG and JF2) strains, inhibited completely the immunoreactivity of three PCV2a strains with mAb 8E4. Conclusions It is concluded that the alanine at position 59 in the capsid protein of PCV2a (CL, LG and JF2) strains is a critical amino acid, which determines one neutralizing epitope of PCV2a (CL, LG and JF2) strains. This study provides valuable information for further in-depth mapping of the conformational neutralizing epitope, understanding antigenic difference among PCV2 strains, and development of a useful vaccine for control of PCV2-associated disease. PMID:21859462

  13. Humoral and cell-mediated immune response, and growth factor synthesis after direct intraarticular injection of rAAV2-IGF-I and rAAV5-IGF-I in the equine middle carpal joint.

    PubMed

    Ortved, Kyla; Wagner, Bettina; Calcedo, Roberto; Wilson, James; Schaefer, Deanna; Nixon, Alan

    2015-03-01

    Intraarticular (IA) administration of viral vectors expressing a therapeutic transgene is an attractive treatment modality for osteoarthritis (OA) as the joint can be treated as a contained unit. Humoral and cell-mediated immune responses in vivo can limit vector effectiveness. Transduction of articular tissues has been investigated; however, the immune response to IA vectors remains largely unknown. We hypothesized that IA rAAV2 and rAAV5 overexpressing insulin-like growth factor-I (IGF-I) would result in long-term IGF-I formation but would also induce neutralizing antibodies (NAb) and anti-capsid effector T cells. Twelve healthy horses were assigned to treatment (rAAV2 or rAAV5) or control (saline) groups. Middle carpal joints were injected with 5×10(11) vector genomes/joint. Synovial fluid was analyzed for changes in composition, NAb titers, immunoglobulin isotypes, proinflammatory cytokines, and IGF-I. Serum was analyzed for antibody titers and cytokines. A T cell restimulation assay was used to assess T cell responses. Injection of rAAV2- or rAAV5-IGF-I did not induce greater inflammation compared with saline. Synovial fluid IGF-I was significantly increased in both rAAV2- and rAAV5-IGF-I joints by day 14 and remained elevated until day 56; however, rAAV5 achieved the highest concentrations. A capsid-specific T cell response was not noted although all virus-treated horses had increased NAbs in serum and synovial fluid after treatment. Taken together, our data show that IA injection of rAAV2- or rAAV5-IGF-I does not incite a clinically detectable inflammatory or cell-mediated immune response and that IA gene therapy using minimally immunogenic vectors represents a clinically relevant tool for treating articular disorders including OA. PMID:25705927

  14. Humoral and Cell-Mediated Immune Response, and Growth Factor Synthesis After Direct Intraarticular Injection of rAAV2-IGF-I and rAAV5-IGF-I in the Equine Middle Carpal Joint

    PubMed Central

    Wagner, Bettina; Calcedo, Roberto; Wilson, James; Schaefer, Deanna; Nixon, Alan

    2015-01-01

    Abstract Intraarticular (IA) administration of viral vectors expressing a therapeutic transgene is an attractive treatment modality for osteoarthritis (OA) as the joint can be treated as a contained unit. Humoral and cell-mediated immune responses in vivo can limit vector effectiveness. Transduction of articular tissues has been investigated; however, the immune response to IA vectors remains largely unknown. We hypothesized that IA rAAV2 and rAAV5 overexpressing insulin-like growth factor-I (IGF-I) would result in long-term IGF-I formation but would also induce neutralizing antibodies (NAb) and anti-capsid effector T cells. Twelve healthy horses were assigned to treatment (rAAV2 or rAAV5) or control (saline) groups. Middle carpal joints were injected with 5×1011 vector genomes/joint. Synovial fluid was analyzed for changes in composition, NAb titers, immunoglobulin isotypes, proinflammatory cytokines, and IGF-I. Serum was analyzed for antibody titers and cytokines. A T cell restimulation assay was used to assess T cell responses. Injection of rAAV2- or rAAV5-IGF-I did not induce greater inflammation compared with saline. Synovial fluid IGF-I was significantly increased in both rAAV2- and rAAV5-IGF-I joints by day 14 and remained elevated until day 56; however, rAAV5 achieved the highest concentrations. A capsid-specific T cell response was not noted although all virus-treated horses had increased NAbs in serum and synovial fluid after treatment. Taken together, our data show that IA injection of rAAV2- or rAAV5-IGF-I does not incite a clinically detectable inflammatory or cell-mediated immune response and that IA gene therapy using minimally immunogenic vectors represents a clinically relevant tool for treating articular disorders including OA. PMID:25705927

  15. Purification of recombinant virus-like particles of porcine circovirus type 2 capsid protein using ion-exchange monolith chromatography.

    PubMed

    Zaveckas, Mindaugas; Snipaitis, Simas; Pesliakas, Henrikas; Nainys, Juozas; Gedvilaite, Alma

    2015-06-01

    Diseases associated with porcine circovirus type 2 (PCV2) infection are having a severe economic impact on swine-producing countries. The PCV2 capsid (Cap) protein expressed in eukaryotic systems self-assemble into virus-like particles (VLPs) which can serve as antigens for diagnostics or/and as vaccine candidates. In this work, conventional adsorbents as well as a monolithic support with large pore sizes were examined for the chromatographic purification of PCV2 Cap VLPs from clarified yeast lysate. Q Sepharose XL was used for the initial separation of VLPs from residual host nucleic acids and some host cell proteins. For the further purification of PCV2 Cap VLPs, SP Sepharose XL, Heparin Sepharose CL-6B and CIMmultus SO3 monolith were tested. VLPs were not retained on SP Sepharose XL. The purity of VLPs after chromatography on Heparin Sepharose CL-6B was only 4-7% and the recovery of VLPs was 5-7%. Using ion-exchange chromatography on the CIMmultus SO3 monolith, PCV2 Cap VLPs with the purity of about 40% were obtained. The recovery of VLPs after chromatography on the CIMmultus SO3 monolith was 15-18%. The self-assembly of purified PCV2 Cap protein into VLPs was confirmed by electron microscopy. Two-step chromatographic purification procedure of PCV2 Cap VLPs from yeast lysate was developed using Q Sepharose XL and cation-exchange CIMmultus SO3 monolith. PMID:25910233

  16. New Structural Insights into the Genome and Minor Capsid Proteins of BK Polyomavirus using Cryo-Electron Microscopy

    PubMed Central

    Hurdiss, Daniel L.; Morgan, Ethan L.; Thompson, Rebecca F.; Prescott, Emma L.; Panou, Margarita M.; Macdonald, Andrew; Ranson, Neil A.

    2016-01-01

    Summary BK polyomavirus is the causative agent of several diseases in transplant patients and the immunosuppressed. In order to better understand the structure and life cycle of BK, we produced infectious virions and VP1-only virus-like particles in cell culture, and determined their three-dimensional structures using cryo-electron microscopy (EM) and single-particle image processing. The resulting 7.6-Å resolution structure of BK and 9.1-Å resolution of the virus-like particles are the highest-resolution cryo-EM structures of any polyomavirus. These structures confirm that the architecture of the major structural protein components of these human polyomaviruses are similar to previous structures from other hosts, but give new insight into the location and role of the enigmatic minor structural proteins, VP2 and VP3. We also observe two shells of electron density, which we attribute to a structurally ordered part of the viral genome, and discrete contacts between this density and both VP1 and the minor capsid proteins. PMID:26996963

  17. Subcellular localisation of Theiler's murine encephalomyelitis virus (TMEV) capsid subunit VP1 vis-á-vis host protein Hsp90.

    PubMed

    Ross, Caroline; Upfold, Nicole; Luke, Garry A; Bishop, Özlem Tastan; Knox, Caroline

    2016-08-15

    The VP1 subunit of the picornavirus capsid is the major antigenic determinant and mediates host cell attachment and virus entry. To investigate the localisation of Theiler's murine encephalomyelitis virus (TMEV) VP1 during infection, a bioinformatics approach was used to predict a surface-exposed, linear epitope region of the protein for subsequent expression and purification. This region, comprising the N-terminal 112 amino acids of the protein, was then used for rabbit immunisation, and the resultant polyclonal antibodies were able to recognise full length VP1 in infected cell lysates by Western blot. Following optimisation, the antibodies were used to investigate the localisation of VP1 in relation to Hsp90 in infected cells by indirect immunofluorescence and confocal microscopy. At 5h post infection, VP1 was distributed diffusely in the cytoplasm with strong perinuclear staining but was absent from the nucleus of all cells analysed. Dual-label immunofluorescence using anti-TMEV VP1 and anti-Hsp90 antibodies indicated that the distribution of both proteins colocalised in the cytoplasm and perinuclear region of infected cells. This is the first report describing the localisation of TMEV VP1 in infected cells, and the antibodies produced provide a valuable tool for investigating the poorly understood mechanisms underlying the early steps of picornavirus assembly. PMID:27269472

  18. Conservation of major and minor jelly-roll capsid proteins in Polinton (Maverick) transposons suggests that they are bona fide viruses

    PubMed Central

    2014-01-01

    Reviewers This article was reviewed by Lakshminarayan M. Iyer and I. King Jordan. For complete reviews, see the Reviewers’ Reports section. Polintons (also known as Mavericks) and Tlr elements of Tetrahymena thermophila represent two families of large DNA transposons widespread in eukaryotes. Here, we show that both Polintons and Tlr elements encode two key virion proteins, the major capsid protein with the double jelly-roll fold and the minor capsid protein, known as the penton, with the single jelly-roll topology. This observation along with the previously noted conservation of the genes for viral genome packaging ATPase and adenovirus-like protease strongly suggests that Polintons and Tlr elements combine features of bona fide viruses and transposons. We propose the name ‘Polintoviruses’ to denote these putative viruses that could have played a central role in the evolution of several groups of DNA viruses of eukaryotes. PMID:24773695

  19. The Lymantria dispar nucleopolyhedrovirus contains the capsid-associated p24 protein gene.

    PubMed

    Slavicek, James M; Hayes-Plazolles, Nancy

    2003-01-01

    During the course of investigations on a wild-type strain of Lymantria dispar multinucleocapsid nucleopolyhedrovirus (LdMNPV), a region of the viral genome was analyzed and found to contain 697 bp that is lacking in the sequenced strain (5-6) of LdMNPV (Kuzio et al., Virology 253, 17-34, 1999). The sequenced strain of LdMNPV contains a mutation in the 25 K few polyhedra (FP) gene, and exhibits the phenotype of a FP mutant. The additional sequence was located at approximately 81.4 map units within the viral genome, and was found in 10 different wild-type LdMNPV genotypic variants analyzed. Since the additional sequence wasfound in all wild-type virus strains analyzed, this sequence should be included in the representative LdMNPV genome. Sequence analysis of the genomic region containing the additional sequences revealed the presence of a homologue of the Autographa californica MNPV capsid-associated p24 gene (ORF 129). This gene, absent in LdMNPV isolate 5-6, is also present in the Orgyia pseudotsugata MNPV, Bombyx mori NPV, Spodoptera exigua MNPV, S. litura MNPV, Mamestra configurata MNPV, Helicoverpa armigera SNPV, H. zea SNPV, Buzura suppressaria SNPV, Xestia c-nigrum granulovirus, Plutella xylostella GV, and Cydia pomonella GV. PMID:12680688

  20. Roles of three amino acids of capsid proteins in mink enteritis parvovirus replication.

    PubMed

    Mao, Yaping; Su, Jun; Wang, Jigui; Zhang, Xiaomei; Hou, Qiang; Bian, Dawei; Liu, Weiquan

    2016-08-15

    Virulent mink enteritis parvovirus (MEV) strain MEV-LHV replicated to higher titers in feline F81 cells than attenuated strain MEV-L. Phylogenetic and sequence analyses of the VP2 gene of MEV-LHV, MEV-L and other strains in GenBank revealed two evolutionary branches separating virulent and attenuated strains. Three residues, 101, 232 and 411, differed between virulent and attenuated strains but were conserved within the two branches. Site-directed mutagenesis of the VP2 gene of infectious plasmids of attenuated strain MEV-L respectively replacing residues 101 Ile and 411 Ala with Thr and Glu of virulent strains (MEV-L I101T and MEV-L A411E) increased replication efficiency but still to lower levels than MEV-LHV. However, viruses with mutation of residue 232 (MEV-L I232V and MEV-L I101T/I232V/A411E) decreased viral transcription and replication levels. The three VP2 residues 101, 232 and 411, located on or near the capsid surface, played different roles in the infection processes of MEV. PMID:27212684

  1. AAV's Anatomy: Roadmap for Optimizing Vectors for Translational Success

    PubMed Central

    Samulski, R. Jude

    2014-01-01

    Adeno-Associated Virus based vectors (rAAV) are advantageous for human gene therapy due to low inflammatory responses, lack of toxicity, natural persistence, and ability to transencapsidate the genome allowing large variations in vector biology and tropism. Over sixty clinical trials have been conducted using rAAV serotype 2 for gene delivery with a number demonstrating success in immunoprivileged sites, including the retina and the CNS. Furthermore, an increasing number of trials have been initiated utilizing other serotypes of AAV to exploit vector tropism, trafficking, and expression efficiency. While these trials have demonstrated success in safety with emerging success in clinical outcomes, one benefit has been identification of issues associated with vector administration in humans (e.g. the role of pre-existing antibody responses, loss of transgene expression in non-immunoprivileged sites, and low transgene expression levels). For these reasons, several strategies are being used to optimize rAAV vectors, ranging from addition of exogenous agents for immune evasion to optimization of the transgene cassette for enhanced therapeutic output. By far, the vast majority of approaches have focused on genetic manipulation of the viral capsid. These methods include rational mutagenesis, engineering of targeting peptides, generation of chimeric particles, library and directed evolution approaches, as well as immune evasion modifications. Overall, these modifications have created a new repertoire of AAV vectors with improved targeting, transgene expression, and immune evasion. Continued work in these areas should synergize strategies to improve capsids and transgene cassettes that will eventually lead to optimized vectors ideally suited for translational success. PMID:20712583

  2. Biophysical and Ultrastructural Characterization of Adeno-Associated Virus Capsid Uncoating and Genome Release

    PubMed Central

    Horowitz, Eric D.; Rahman, K. Shefaet; Bower, Brian D.; Dismuke, David J.; Falvo, Michael R.; Griffith, Jack D.

    2013-01-01

    We describe biophysical and ultrastructural differences in genome release from adeno-associated virus (AAV) capsids packaging wild-type DNA, recombinant single-stranded DNA (ssDNA), or dimeric, self-complementary DNA (scDNA) genomes. Atomic force microscopy and electron microscopy (EM) revealed that AAV particles release packaged genomes and undergo marked changes in capsid morphology upon heating in physiological buffer (pH 7.2). When different AAV capsids packaging ss/scDNA varying in length from 72 to 123% of wild-type DNA (3.4 to 5.8 kb) were incrementally heated, the proportion of uncoated AAV capsids decreased with genome length as observed by EM. Genome release was further characterized by a fluorimetric assay, which demonstrated that acidic pH and high osmotic pressure suppress genome release from AAV particles. In addition, fluorimetric analysis corroborated an inverse correlation between packaged genome length and the temperature needed to induce uncoating. Surprisingly, scAAV vectors required significantly higher temperatures to uncoat than their ssDNA-packaging counterparts. However, externalization of VP1 N termini appears to be unaffected by packaged genome length or self-complementarity. Further analysis by tungsten-shadowing EM revealed striking differences in the morphologies of ssDNA and scDNA genomes upon release from intact capsids. Computational modeling and molecular dynamics simulations suggest that the unusual thermal stability of scAAV vectors might arise from partial base pairing and optimal organization of packaged scDNA. Our work further defines the biophysical mechanisms underlying adeno-associated virus uncoating and genome release. PMID:23269804

  3. Self-assembly of virus-like particles of porcine circovirus type 2 capsid protein expressed from Escherichia coli

    PubMed Central

    2010-01-01

    Background Porcine circovirus 2 (PCV2) is a serious problem to the swine industry and can lead to significant negative impacts on profitability of pork production. Syndrome associated with PCV2 is known as porcine circovirus closely associated with post-weaning multisystemic wasting syndrome (PMWS). The capsid (Cap) protein of PCV2 is a major candidate antigen for development of recombinant vaccine and serological diagnostic method. The recombinant Cap protein has the ability to self-assemble into virus-like particles (VLPs) in vitro, it is particularly opportunity to develop the PV2 VLPs vaccine in Escherichia coli,(E.coli ), because where the cost of the vaccine must be weighed against the value of the vaccinated pig, when it was to extend use the VLPs vaccine of PCV2. Results In this report, a highly soluble Cap-tag protein expressed in E.coli was constructed with a p-SMK expression vector with a fusion tag of small ubiquitin-like modifiers (SUMO). The recombinant Cap was purified using Ni2+ affinity resins, whereas the tag was used to remove the SUMO protease. Simultaneously, the whole native Cap protein was able to self-assemble into VLPs in vitro when viewed under an electron microscope. The Cap-like particles had a size and shape that resembled the authentic Cap. The result could also be applied in the large-scale production of VLPs of PCV2 and could be used as a diagnostic antigen or a potential VLP vaccine against PCV2 infection in pigs. Conclusion we have, for the first time, utilized the SUMO fusion motif to successfully express the entire authentic Cap protein of PCV2 in E. coli. After the cleavage of the fusion motif, the nCap protein has the ability to self-assemble into VLPs, which can be used as as a potential vaccine to protect pigs from PCV2-infection. PMID:20646322

  4. Stabilising the Herpes Simplex Virus capsid by DNA packaging

    NASA Astrophysics Data System (ADS)

    Wuite, Gijs; Radtke, Kerstin; Sodeik, Beate; Roos, Wouter

    2009-03-01

    Three different types of Herpes Simplex Virus type 1 (HSV-1) nuclear capsids can be distinguished, A, B and C capsids. These capsids types are, respectively, empty, contain scaffold proteins, or hold DNA. We investigate the physical properties of these three capsids by combining biochemical and nanoindentation techniques. Atomic Force Microscopy (AFM) experiments show that A and C capsids are mechanically indistinguishable whereas B capsids already break at much lower forces. By extracting the pentamers with 2.0 M GuHCl or 6.0 M Urea we demonstrate an increased flexibility of all three capsid types. Remarkably, the breaking force of the B capsids without pentamers does not change, while the modified A and C capsids show a large drop in their breaking force to approximately the value of the B capsids. This result indicates that upon DNA packaging a structural change at or near the pentamers occurs which mechanically reinforces the capsids structure. The reported binding of proteins UL17/UL25 to the pentamers of the A and C capsids seems the most likely candidate for such capsids strengthening. Finally, the data supports the view that initiation of DNA packaging triggers the maturation of HSV-1 capsids.

  5. Simple immunoblot and immunohistochemical detection of Penaeus stylirostris densovirus using monoclonal antibodies to viral capsid protein expressed heterologously.

    PubMed

    Sithigorngul, Paisarn; Hajimasalaeh, Warunee; Longyant, Siwaporn; Sridulyakul, Pattarin; Rukpratanporn, Sombat; Chaivisuthangkura, Parin

    2009-12-01

    Penaeus stylirostris densovirus (PstDNV), called formerly infectious hypodermal and hematopoietic necrosis virus (IHHNV), is an important shrimp pathogen which can cause mortality in the blue shrimp Penaeus (Litopenaeus) stylirostris and stunting in the whiteleg shrimp Penaeus (Litopenaeus) vannamei. Five monoclonal antibodies (MAbs) were produced against the 37kDa capsid protein 3 (CP3) of PstDNV expressed heterologously in the form of a fusion protein with glutathione-S-transferase called GST-CP3. All MAbs belonged to the IgG2b subclass and could bind to GST-CP3 at 300 pg/spot in immunodot-blot tests. They could detect CP3 in naturally infected shrimp extracts by Western blotting and dot blotting and in shrimp tissues by immunohistochemistry without cross-reactivity to extracts from uninfected shrimps or shrimps infected with several other viruses. Although dot blot assay sensitivity was approximately 1000 times lower than that of one step PCR for PstDNV, it easily detected PstDNV infections in field samples of Penaeus monodon and Penaeus vannamei. PMID:19654023

  6. Codon-optimized expression of fish iridovirus capsid protein in yeast and its application as an oral vaccine candidate.

    PubMed

    Seo, J Y; Chung, H J; Kim, T J

    2013-09-01

    Fish iridovirus causes systemic disease with high morbidity and mortality in various species of wild and farm-raised fish, resulting in severe economic losses. Recently, frequent outbreaks of iridovirus infection have occurred among cultured fish in many Asian countries, emphasizing the need for a protective vaccine programme or the development of a suitable therapy. In this study, we expressed a recombinant major capsid protein (rMCP) of rock bream iridovirus (RBIV) from yeast using codon optimization. The rMCP in yeast was added to feed in an attempt to induce intestinal mucosal immunity for protection against and/or to reduce the severity of fish iridovirus infection. We found that fish immunized orally with rMCP underwent a successful induction of antibodies (P < 0.05) and were protected (P = 0.0001) against viral challenge. Based upon these results, oral administration of immunogenic protein as an antigen can be considered a useful method for implementation of vaccine programmes against iridovirus as well as other marine viral diseases. PMID:23488597

  7. Bioprocessing of plant-derived virus-like particles of Norwalk virus capsid protein under current Good Manufacture Practice regulations

    PubMed Central

    Lai, Huafang; Chen, Qiang

    2012-01-01

    Despite the success in expressing a variety of subunit vaccine proteins in plants and the recent stride in improving vaccine accumulation levels by transient expression systems, there is still no plant-derived vaccine that has been licensed for human use. The lack of commercial success of plant-made vaccines lies in several technical and regulatory barriers that remain to be overcome. These challenges include the lack of scalable downstream processing procedures, the uncertainty of regulatory compliance of production processes, and the lack of demonstration of plant-derived products that meet the required standards of regulatory agencies in identity, purity, potency and safety. In this study, we addressed these remaining challenges and successfully demonstrate the ability of using plants to produce a pharmaceutical grade Norwalk virus (NV) vaccine under current Good Manufacture Practice (cGMP) guidelines at multiple gram scales. Our results demonstrate that an efficient and scalable extraction and purification scheme can established for processing virus-like particles (VLP) of NV capsid protein (NVCP). We successfully operated the upstream and downstream NVCP production processes under cGMP regulations. Furthermore, plant-derived NVCP VLP demonstrates the identity, purity, potency and safety that meet the preset release specifications. This material is being tested in a Phase I human clinical trial. This research provides the first report of producing a plant-derived vaccine at scale under cGMP regulations in an academic setting and an important step for plant-produced vaccines to become a commercial reality. PMID:22134876

  8. The complex subcellular distribution of satellite panicum mosaic virus capsid protein reflects its multifunctional role during infection

    SciTech Connect

    Qi Dong; Omarov, Rustem T.; Scholthof, Karen-Beth G.

    2008-06-20

    Satellite panicum mosaic virus (SPMV) depends on its helper Panicum mosaic virus for replication and movement in host plants. The positive-sense single-stranded genomic RNA of SPMV encodes a 17-kDa capsid protein (CP) to form 16-nm virions. We determined that SPMV CP accumulates in both cytosolic and non-cytosolic fractions, but cytosolic accumulation of SPMV CP is exclusively associated with virions. An N-terminal arginine-rich motif (N-ARM) on SPMV CP is used to bind its cognate RNA and to form virus particles. Intriguingly, virion formation is dispensable for successful systemic SPMV RNA accumulation, yet this process still depends on an intact N-ARM. In addition, a C-terminal domain on the SPMV CP is necessary for self-interaction. Biochemical fractionation and fluorescent microscopy of green fluorescent protein-tagged SPMV CP demonstrated that the non-cytosolic SPMV CP is associated with the cell wall, the nucleus and other membranous organelles. To our knowledge, this is the first report that a satellite virus CP not only accumulates exclusively as virions in the cytosol but also is directed to the nucleolus and membranes. That SPMV CP is found both in the nucleus and the cell wall suggests its involvement in viral nuclear import and cell-to-cell transport.

  9. Distinct binding sites for zinc and double-stranded RNA in the reovirus outer capsid protein sigma3

    SciTech Connect

    Schiff, L.A.; Nibert, M.L.; Co, M.S.; Brown, E.G.; Fields, B.N.

    1988-01-01

    By atomic absorption analysis, the authors determined that the reovirus outer capsid protein sigma3, which binds double-stranded RNA (dsRNA), is a zinc metalloprotein. Using Northwestern blots and a novel zinc blotting technique, the authors localized the zinc- and dsRNA-binding activities of sigma3 to distinct V8 protease-generated fragments. Zinc-binding activity was contained within an amino-terminal fragment that contained a transcription factor IIIA-like zinc-binding sequence, and dsRNA-binding activity was associated with a carboxy-terminal fragment. By these techniques, new zinc- and dsRNA-binding activities were also detected in reovirus core proteins. A sequence similarity was observed between the catalytic site of the picornavirus proteases and the transcription factor IIIA-like zinc-binding site within sigma3. The authors suggest that the zinc- and dsRNA-binding activities of sigma3 may be important for its proposed regulatory effects on viral and host cell transcription and translation.

  10. Amino acid motifs in both the major and minor capsid proteins of HPV51 impact antigenicity and infectivity.

    PubMed

    Godi, Anna; Epifano, Ilaria; Bissett, Sara L; Dell'Anna, Tiziana; Piana, Andrea; Cocuzza, Clementina; Beddows, Simon

    2015-07-01

    Persistent infection with oncogenic human papillomavirus (HPV) is a prerequisite for cervical disease development, yet data regarding the host immune response to infection at the genotype level are quite limited. We created pseudoviruses bearing the major (L1) and minor (L2) capsid proteins and L1 virus-like particles representing the reference sequence and a consensus of 34 European sequences of HPV51. Despite the formation of similarly sized particles, motifs in the reference L1 and L2 genes had a profound impact on the immunogenicity, antigenicity and infectivity of these antigens. The antibody status of women exhibiting low-grade disease was similar between HPV16 and the consensus HPV51, but both demonstrated discrepancies between binding and neutralizing antibody responses. These data support the use of pseudoviruses as the preferred target antigen in studies of natural HPV infection and the need to consider variation in both the L1 and L2 proteins for the appropriate presentation of antibody epitopes. PMID:25770119

  11. Major Variations in HIV-1 Capsid Assembly Morphologies Involve Minor Variations in Molecular Structures of Structurally Ordered Protein Segments.

    PubMed

    Lu, Jun-Xia; Bayro, Marvin J; Tycko, Robert

    2016-06-17

    We present the results of solid state nuclear magnetic resonance (NMR) experiments on HIV-1 capsid protein (CA) assemblies with three different morphologies, namely wild-type CA (WT-CA) tubes with 35-60 nm diameters, planar sheets formed by the Arg(18)-Leu mutant (R18L-CA), and R18L-CA spheres with 20-100 nm diameters. The experiments are intended to elucidate molecular structural variations that underlie these variations in CA assembly morphology. We find that multidimensional solid state NMR spectra of (15)N,(13)C-labeled CA assemblies are remarkably similar for the three morphologies, with only small differences in (15)N and (13)C chemical shifts, no significant differences in NMR line widths, and few differences in the number of detectable NMR cross-peaks. Thus, the pronounced differences in morphology do not involve major differences in the conformations and identities of structurally ordered protein segments. Instead, morphological variations are attributable to variations in conformational distributions within disordered segments, which do not contribute to the solid state NMR spectra. Variations in solid state NMR signals from certain amino acid side chains are also observed, suggesting differences in the intermolecular dimerization interface between curved and planar CA lattices, as well as possible differences in intramolecular helix-helix packing. PMID:27129282

  12. Humoral Immune Response to AAV

    PubMed Central

    Calcedo, Roberto; Wilson, James M.

    2013-01-01

    Adeno-associated virus (AAV) is a member of the family Parvoviridae that has been widely used as a vector for gene therapy because of its safety profile, its ability to transduce both dividing and non-dividing cells, and its low immunogenicity. AAV has been detected in many different tissues of several animal species but has not been associated with any disease. As a result of natural infections, antibodies to AAV can be found in many animals including humans. It has been shown that pre-existing AAV antibodies can modulate the safety and efficacy of AAV vector-mediated gene therapy by blocking vector transduction or by redirecting distribution of AAV vectors to tissues other than the target organ. This review will summarize antibody responses against natural AAV infections, as well as AAV gene therapy vectors and their impact in the clinical development of AAV vectors for gene therapy. We will also review and discuss the various methods used for AAV antibody detection and strategies to overcome neutralizing antibodies in AAV-mediated gene therapy. PMID:24151496

  13. Molecular Architecture of the Retroviral Capsid.

    PubMed

    Perilla, Juan R; Gronenborn, Angela M

    2016-05-01

    Retroviral capsid cores are proteinaceous containers that self-assemble to encase the viral genome and a handful of proteins that promote infection. Their function is to protect and aid in the delivery of viral genes to the nucleus of the host, and, in many cases, infection pathways are influenced by capsid-cellular interactions. From a mathematical perspective, capsid cores are polyhedral cages and, as such, follow well-defined geometric rules. However, marked morphological differences in shapes exist, depending on virus type. Given the specific roles of capsid in the viral life cycle, the availability of detailed molecular structures, particularly at assembly interfaces, opens novel avenues for targeted drug development against these pathogens. Here, we summarize recent advances in the structure and understanding of retroviral capsid, with particular emphasis on assemblies and the capsid cores. PMID:27039020

  14. Chlamydiaphage φCPG1 Capsid Protein Vp1 Inhibits Chlamydia trachomatis Growth via the Mitogen-Activated Protein Kinase Pathway

    PubMed Central

    Guo, Yuanli; Guo, Rui; Zhou, Quan; Sun, Changgui; Zhang, Xinmei; Liu, Yuanjun; Liu, Quanzhong

    2016-01-01

    Chlamydia trachomatis is the most common cause of curable bacterial sexually transmitted infections worldwide. Although the pathogen is well established, the pathogenic mechanisms remain unclear. Given the current challenges of antibiotic resistance and blocked processes of vaccine development, the use of a specific chlamydiaphage may be a new treatment solution. φCPG1 is a lytic phage specific for Chlamydia caviae, and shows over 90% nucleotide sequence identity with other chlamydiaphages. Vp1 is the major capsid protein of φCPG1. Purified Vp1 was previously confirmed to inhibit Chlamydia trachomatis growth. We here report the first attempt at exploring the relationship between Vp1-treated C. trachomatis and the protein and gene levels of the mitogen-activated/extracellular regulated protein kinase (MAPK/ERK) pathway by Western blotting and real-time PCR, respectively. Moreover, we evaluated the levels of pro-inflammatory cytokines interleukin (IL)-8 and IL-1 by enzyme-linked immunosorbent assay after Vp1 treatment. After 48 h of incubation, the p-ERK level of the Vp1-treated group decreased compared with that of the Chlamydia infection group. Accordingly, ERK1 and ERK2 mRNA expression levels of the Vp1-treated group also decreased compared with the Chlamydia infection group. IL-8 and IL-1 levels were also decreased after Vp1 treatment compared with the untreated group. Our results demonstrate that the inhibition effect of the chlamydiaphage φCPG1 capsid protein Vp1 on C. trachomatis is associated with the MAPK pathway, and inhibits production of the pro-inflammatory cytokines IL-8 and IL-1. The bacteriophages may provide insight into a new signaling transduction mechanism to influence their hosts, in addition to bacteriolysis. PMID:27089359

  15. Chlamydiaphage φCPG1 Capsid Protein Vp1 Inhibits Chlamydia trachomatis Growth via the Mitogen-Activated Protein Kinase Pathway.

    PubMed

    Guo, Yuanli; Guo, Rui; Zhou, Quan; Sun, Changgui; Zhang, Xinmei; Liu, Yuanjun; Liu, Quanzhong

    2016-04-01

    Chlamydia trachomatis is the most common cause of curable bacterial sexually transmitted infections worldwide. Although the pathogen is well established, the pathogenic mechanisms remain unclear. Given the current challenges of antibiotic resistance and blocked processes of vaccine development, the use of a specific chlamydiaphage may be a new treatment solution. φCPG1 is a lytic phage specific for Chlamydia caviae, and shows over 90% nucleotide sequence identity with other chlamydiaphages. Vp1 is the major capsid protein of φCPG1. Purified Vp1 was previously confirmed to inhibit Chlamydia trachomatis growth. We here report the first attempt at exploring the relationship between Vp1-treated C. trachomatis and the protein and gene levels of the mitogen-activated/extracellular regulated protein kinase (MAPK/ERK) pathway by Western blotting and real-time PCR, respectively. Moreover, we evaluated the levels of pro-inflammatory cytokines interleukin (IL)-8 and IL-1 by enzyme-linked immunosorbent assay after Vp1 treatment. After 48 h of incubation, the p-ERK level of the Vp1-treated group decreased compared with that of the Chlamydia infection group. Accordingly, ERK1 and ERK2 mRNA expression levels of the Vp1-treated group also decreased compared with the Chlamydia infection group. IL-8 and IL-1 levels were also decreased after Vp1 treatment compared with the untreated group. Our results demonstrate that the inhibition effect of the chlamydiaphage φCPG1 capsid protein Vp1 on C. trachomatis is associated with the MAPK pathway, and inhibits production of the pro-inflammatory cytokines IL-8 and IL-1. The bacteriophages may provide insight into a new signaling transduction mechanism to influence their hosts, in addition to bacteriolysis. PMID:27089359

  16. Packaging and structural phenotype of brome mosaic virus capsid protein with altered N-terminal {beta}-hexamer structure

    SciTech Connect

    Wispelaere, Melissanne de; Chaturvedi, Sonali; Wilkens, Stephan; Rao, A.L.N.

    2011-10-10

    The first 45 amino acid region of brome mosaic virus (BMV) capsid protein (CP) contains RNA binding and structural domains that are implicated in the assembly of infectious virions. One such important structural domain encompassing amino acids {sup 28}QPVIV{sup 32}, highly conserved between BMV and cowpea chlorotic mottle virus (CCMV), exhibits a {beta}-hexamer structure. In this study we report that alteration of the {beta}-hexamer structure by mutating {sup 28}QPVIV{sup 32} to {sup 28}AAAAA{sup 32} had no effect either on symptom phenotype, local and systemic movement in Chenopodium quinoa and RNA profile of in vivo assembled virions. However, sensitivity to RNase and assembly phenotypes distinguished virions assembled with CP subunits having {beta}-hexamer from those of wild type. A comparison of 3-D models obtained by cryo electron microscopy revealed overall similar structural features for wild type and mutant virions, with small but significant differences near the 3-fold axes of symmetry.

  17. Antibody Recognition of Porcine Circovirus Type 2 Capsid Protein Epitopes after Vaccination, Infection, and Disease▿†

    PubMed Central

    Trible, Benjamin R.; Kerrigan, Maureen; Crossland, Nicholas; Potter, Megan; Faaberg, Kay; Hesse, Richard; Rowland, Raymond R. R.

    2011-01-01

    Open reading frame 2 (ORF2) of porcine circovirus type 2 (PCV2) codes for the 233-amino-acid capsid protein (CP). Baculovirus-based vaccines that express only ORF2 are protective against clinical disease following experimental challenge or natural infection. The goal of this study was to identify regions in CP preferentially recognized by sera from experimentally infected and vaccinated pigs and to compare these responses to those of pigs diagnosed with porcine circovirus-associated disease (PCVAD), including porcine multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS). The approach was to react porcine sera with CP polypeptide fragments followed by finer mapping studies using overlapping oligopeptides that covered amino acids 141 to 200. The results showed that vaccinated pigs preferentially recognized only the largest polypeptide fragment, CP(43-233). A subset of experimentally infected pigs and pigs with PDNS showed strong reactivity against a CP oligopeptide, 169-STIDYFQPNNKR-180. Alanine scanning identified Y-173, F-174, Q-175, and K-179 as important for antibody recognition. The results from this study support the notion of PCV2 modulation of immunity, including antibody responses that may represent a precursor for disease. The recognition of CP(169-180) and other polypeptides provides opportunities to devise diagnostic tests for monitoring the immunological effectiveness of vaccination. PMID:21430122

  18. Absolute Quantification of Norovirus Capsid Protein in Food, Water, and Soil Using Synthetic Peptides with Electrospray and MALDI Mass Spectrometry

    PubMed Central

    Hartmann, Erica M.; Colquhoun, David R.; Schwab, Kellogg J.; Halden, Rolf U.

    2015-01-01

    Norovirus infections are one of the most prominent public health problems of microbial origin in the U.S. and other industrialized countries. Surveillance is necessary to prevent secondary infection, confirm successful cleanup after outbreaks, and track the causative agent. Quantitative mass spectrometry, based on absolute quantitation with stable-isotope labeled peptides, is a promising tool for norovirus monitoring because of its speed, sensitivity, and robustness in the face of environmental inhibitors. In the current study, we present two new methods for the detection of the norovirus genogroup I capsid protein using electrospray and matrixassisted laser desorption/ionization (MALDI) mass spectrometry. The peptide TLDPIEVPLEDVR was used to quantify norovirus-like particles down to 500 attomoles with electrospray and 100 attomoles with MALDI. With MALDI, we also demonstrate a detection limit of 1 femtomole and a quantitative dynamic range of 5 orders of magnitude in the presence of an environmental matrix effect. Due to the rapid processing time and applicability to a wide range of environmental sample types (bacterial lysate, produce, milk, soil, and groundwater), mass spectrometry-based absolute quantitation has a strong potential for use in public health and environmental sciences. PMID:25603302

  19. The Interdomain Linker Region of HIV-1 Capsid Protein is a Critical Determinant of Proper Core Assembly and Stability

    PubMed Central

    Jiang, Jiyang; Ablan, Sherimay; Derebail, Suchitra; Hercík, Kamil; Soheilian, Ferri; Thomas, James A.; Tang, Shixing; Hewlett, Indira; Nagashima, Kunio; Gorelick, Robert J.; Freed, Eric O.; Levin, Judith G.

    2011-01-01

    The HIV-1 capsid protein consists of two independently folded domains connected by a flexible peptide linker (residues 146–150), the function of which remains to be defined. To investigate the role of this region in virus replication, we made alanine or leucine substitutions in each linker residue and two flanking residues. Three classes of mutants were identified: (i) S146A and T148A behave like wild type (WT); (ii) Y145A, I150A, and L151A are noninfectious, assemble unstable cores with aberrant morphology, and synthesize almost no viral DNA; and (iii) P147L and S149A display a poorly infectious, attenuated phenotype. Infectivity of P147L and S149A is rescued specifically by pseudotyping with vesicular stomatitis virus envelope glycoprotein. Moreover, despite having unstable cores, these mutants assemble WT-like structures and synthesize viral DNA, although less efficiently than WT. Collectively, these findings demonstrate that the linker region is essential for proper assembly and stability of cores and efficient replication. PMID:22036671

  20. Absolute quantification of norovirus capsid protein in food, water, and soil using synthetic peptides with electrospray and MALDI mass spectrometry.

    PubMed

    Hartmann, Erica M; Colquhoun, David R; Schwab, Kellogg J; Halden, Rolf U

    2015-04-01

    Norovirus infections are one of the most prominent public health problems of microbial origin in the U.S. and other industrialized countries. Surveillance is necessary to prevent secondary infection, confirm successful cleanup after outbreaks, and track the causative agent. Quantitative mass spectrometry, based on absolute quantitation with stable-isotope labeled peptides, is a promising tool for norovirus monitoring because of its speed, sensitivity, and robustness in the face of environmental inhibitors. In the current study, we present two new methods for the detection of the norovirus genogroup I capsid protein using electrospray and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The peptide TLDPIEVPLEDVR was used to quantify norovirus-like particles down to 500 attomoles with electrospray and 100 attomoles with MALDI. With MALDI, we also demonstrate a detection limit of 1 femtomole and a quantitative dynamic range of 5 orders of magnitude in the presence of an environmental matrix effect. Due to the rapid processing time and applicability to a wide range of environmental sample types (bacterial lysate, produce, milk, soil, and groundwater), mass spectrometry-based absolute quantitation has a strong potential for use in public health and environmental sciences. PMID:25603302

  1. Controlling Viral Capsid Assembly with Templating

    PubMed Central

    Hagan, Michael F.

    2009-01-01

    We develop coarse-grained models that describe the dynamic encapsidation of functionalized nanoparticles by viral capsid proteins. We find that some forms of cooperative interactions between protein subunits and nanoparticles can dramatically enhance rates and robustness of assembly, as compared to the spontaneous assembly of subunits into empty capsids. For large core-subunit interactions, subunits adsorb onto core surfaces en masse in a disordered manner, and then undergo a cooperative rearrangement into an ordered capsid structure. These assembly pathways are unlike any identified for empty capsid formation. Our models can be directly applied to recent experiments in which viral capsid proteins assemble around the functionalized inorganic nanoparticles [Sun et al., Proc. Natl. Acad. Sci (2007) 104, 1354]. In addition, we discuss broader implications for understanding the dynamic encapsidation of single-stranded genomic molecules during viral replication and for developing multicomponent nanostructured materials. PMID:18643099

  2. High-Resolution X-Ray Structure and Functional Analysis of the Murine Norovirus 1 Capsid Protein Protruding Domain▿

    PubMed Central

    Taube, Stefan; Rubin, John R.; Katpally, Umesh; Smith, Thomas J.; Kendall, Ann; Stuckey, Jeanne A.; Wobus, Christiane E.

    2010-01-01

    Murine noroviruses (MNV) are closely related to the human noroviruses (HuNoV), which cause the majority of nonbacterial gastroenteritis. Unlike HuNoV, MNV grow in culture and in a small-animal model that represents a tractable model to study norovirus biology. To begin a detailed investigation of molecular events that occur during norovirus binding to cells, the crystallographic structure of the murine norovirus 1 (MNV-1) capsid protein protruding (P) domain has been determined. Crystallization of the bacterially expressed protein yielded two different crystal forms (Protein Data Bank identifiers [PDB ID], 3LQ6 and 3LQE). Comparison of the structures indicated a large degree of structural mobility in loops on the surface of the P2 subdomain. Specifically, the A′-B′ and E′-F′ loops were found in open and closed conformations. These regions of high mobility include the known escape mutation site for the neutralizing antibody A6.2 and an attenuation mutation site, which arose after serial passaging in culture and led to a loss in lethality in STAT1−/− mice, respectively. Modeling of a Fab fragment and crystal structures of the P dimer into the cryoelectron microscopy three-dimensional (3D) image reconstruction of the A6.2/MNV-1 complex indicated that the closed conformation is most likely bound to the Fab fragment and that the antibody contact is localized to the A′-B′ and E′-F′ loops. Therefore, we hypothesize that these loop regions and the flexibility of the P domains play important roles during MNV-1 binding to the cell surface. PMID:20335262

  3. Construction and immunogenicity of recombinant pseudotype baculovirus expressing the capsid protein of porcine circovirus type 2 in mice.

    PubMed

    Fan, Huiying; Pan, Yongfei; Fang, Liurong; Wang, Dang; Wang, Shengping; Jiang, Yunbo; Chen, Huanchun; Xiao, Shaobo

    2008-06-01

    Baculovirus has emerged recently as a novel and attractive gene delivery vehicle for mammalian cells. Porcine circovirus type 2 (PCV2) is known to be associated with post-weaning multisystemic wasting syndrome (PMWS), an emerging swine disease which results in tremendous economic losses. In this study, baculovirus pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) was used as a vector to express capsid (Cap) protein, the most important immunogen of PCV2, under the transcriptional control of cytomegalovirus immediate early (CMV-IE) enhancer/promoter. The resultant recombinant baculovirus (BV-G-ORF2) efficiently transduced and expressed the Cap protein in mammalian cells, as demonstrated by Western blot and flow cytometric analyses. After direct vaccination with 1x10(8) or 1x10(9)plaque forming units (PFU)/mouse of BV-G-ORF2, significant PCV2-specific ELISA antibodies, neutralizing antibodies, as well as cellular immune responses could be induced in mice. BV-G-ORF2 exhibited better immunogenicity than a DNA vaccine encoding the Cap protein, even at a dose of 1x10(8)PFU/mouse. Taken together, the improved immunogenicity of BV-G-ORF2, together with the unique advantages of pseudotype baculovirus, including easy manipulation, simple scale-up, lack of toxicity, and no pre-existing antibody against baculovirus in the hosts, indicate that pseudotype baculovirus-mediated gene delivery can be utilized as an alternative strategy to develop a new generation of vaccines against PCV2 infection. PMID:18394722

  4. Reduced retinal transduction and enhanced transgene-directed immunogenicity with intravitreal delivery of rAAV following posterior vitrectomy in dogs.

    PubMed

    Boyd, R F; Boye, S L; Conlon, T J; Erger, K E; Sledge, D G; Langohr, I M; Hauswirth, W W; Komáromy, A M; Boye, S E; Petersen-Jones, S M; Bartoe, J T

    2016-06-01

    Adeno-associated virus (AAV) vector-based gene therapy is a promising treatment strategy for delivery of neurotrophic transgenes to retinal ganglion cells (RGCs) in glaucoma patients. Retinal distribution of transgene expression following intravitreal injection (IVT) of AAV is variable in animal models and the vitreous humor may represent a barrier to initial vector penetration. The primary goal of our study was to investigate the effect of prior core vitrectomy with posterior hyaloid membrane peeling on pattern and efficiency of transduction of a capsid amino acid substituted AAV2 vector, carrying the green fluorescent protein (GFP) reporter transgene following IVT in dogs. When progressive intraocular inflammation developed starting 4 weeks post IVT, the study plan was modified to allow detailed characterization of the etiology as a secondary goal. Unexpectedly, surgical vitrectomy was found to significantly limit transduction, whereas in non-vitrectomized eyes transduction efficiency reached upwards to 37.3% of RGC layer cells. The developing retinitis was characterized by mononuclear cell infiltrates resulting from a delayed-type hypersensitivity reaction, which we suspect was directed at the GFP transgene. Our results, in a canine large animal model, support caution when considering surgical vitrectomy before IVT for retinal gene therapy in patients, as prior vitrectomy appears to significantly reduce transduction efficiency and may predispose the patient to development of vector-induced immune reactions. PMID:27052802

  5. Local and systemic responses following intravitreous injection of AAV2-encoded modified Volvox channelrhodopsin-1 in a genetically blind rat model.

    PubMed

    Sugano, E; Tabata, K; Takahashi, M; Nishiyama, F; Shimizu, H; Sato, M; Tamai, M; Tomita, H

    2016-02-01

    We previously designed a modified channelrhodopsin-1 (mVChR1) protein chimera with a broader action than that of Chlamydomonas channelrhodopsin-2 and reported that its transduction into retinal ganglion cells can restore visual function in genetically blind, dystrophic Royal College of Surgeons (RCS) rats, with photostimuli ranging from 486 to 640 nm. In the current study, we sought to investigate the safety and influence of mVChR1 transgene expression. Adeno-associated virus type 2 encoding mVChR1 was administered by intravitreous injection into dystrophic RCS rats. Reverse-transcription PCR was used to monitor virus and transgene dissemination and the results demonstrated that their expression was restricted specifically within the eye tissues, and not in non-target organs. Moreover, examination of the blood, plasma and serum revealed that no excess immunoreactivity was present, as determined using standard clinical hematological parameters. Serum antibodies targeting the recombinant adeno-associated virus (rAAV) capsid increased after the injection; however, no increase in mVChR1 antibody was detected during the observation period. In addition, retinal histological examination showed no signs of inflammation in rAAV-injected rats. In conclusion, our results demonstrate that mVChR1 can be exogenously expressed without harmful immunological reactions in vivo. These findings will aid in studies of AAV gene transfer to restore vision in late-stage retinitis pigmentosa. PMID:26440056

  6. Reduced retinal transduction and enhanced transgene-directed immunogenicity with intravitreal delivery of rAAV following posterior vitrectomy in dogs

    PubMed Central

    Boyd, RF; Boye, SL; Conlon, TJ; Erger, KE; Sledge, DG; Langohr, IM; Hauswirth, WW; Komáromy, AM; Boye, SE; Petersen-Jones, SM; Bartoe, JT

    2016-01-01

    Adeno-associated virus (AAV) vector-based gene therapy is a promising treatment strategy for delivery of neurotrophic transgenes to retinal ganglion cells (RGCs) in glaucoma patients. Retinal distribution of transgene expression following intravitreal injection (IVT) of AAV is variable in animal models and the vitreous humor may represent a barrier to initial vector penetration. The primary goal of our study was to investigate the effect of prior core vitrectomy with posterior hyaloid membrane peeling on pattern and efficiency of transduction of a capsid amino acid substituted AAV2 vector, carrying the green fluorescent protein (GFP) reporter transgene following IVT in dogs. When progressive intraocular inflammation developed starting 4 weeks post IVT, the study plan was modified to allow detailed characterization of the etiology as a secondary goal. Unexpectedly, surgical vitrectomy was found to significantly limit transduction, whereas in non-vitrectomized eyes transduction efficiency reached upwards to 37.3% of RGC layer cells. The developing retinitis was characterized by mononuclear cell infiltrates resulting from a delayed-type hypersensitivity reaction, which we suspect was directed at the GFP transgene. Our results, in a canine large animal model, support caution when considering surgical vitrectomy before IVT for retinal gene therapy in patients, as prior vitrectomy appears to significantly reduce transduction efficiency and may predispose the patient to development of vector-induced immune reactions. PMID:27052802

  7. Effective delivery of large genes to the retina by dual AAV vectors

    PubMed Central

    Trapani, Ivana; Colella, Pasqualina; Sommella, Andrea; Iodice, Carolina; Cesi, Giulia; de Simone, Sonia; Marrocco, Elena; Rossi, Settimio; Giunti, Massimo; Palfi, Arpad; Farrar, Gwyneth J; Polishchuk, Roman; Auricchio, Alberto

    2014-01-01

    Retinal gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans. However, AAV's limited cargo capacity prevents its application to therapies of inherited retinal diseases due to mutations of genes over 5 kb, like Stargardt's disease (STGD) and Usher syndrome type IB (USH1B). Previous methods based on ‘forced’ packaging of large genes into AAV capsids may not be easily translated to the clinic due to the generation of genomes of heterogeneous size which raise safety concerns. Taking advantage of AAV's ability to concatemerize, we generated dual AAV vectors which reconstitute a large gene by either splicing (trans-splicing), homologous recombination (overlapping), or a combination of the two (hybrid). We found that dual trans-splicing and hybrid vectors transduce efficiently mouse and pig photoreceptors to levels that, albeit lower than those achieved with a single AAV, resulted in significant improvement of the retinal phenotype of mouse models of STGD and USH1B. Thus, dual AAV trans-splicing or hybrid vectors are an attractive strategy for gene therapy of retinal diseases that require delivery of large genes. PMID:24150896

  8. NP1 Protein of the Bocaparvovirus Minute Virus of Canines Controls Access to the Viral Capsid Genes via Its Role in RNA Processing

    PubMed Central

    Fasina, Olufemi O.; Dong, Yanming

    2015-01-01

    ABSTRACT Minute virus of canines (MVC) is an autonomous parvovirus in the genus Bocaparvovirus. It has a single promoter that generates a single pre-mRNA processed via alternative splicing and alternative polyadenylation to produce at least 8 mRNA transcripts. MVC contains two polyadenylation sites, one at the right-hand end of the genome, (pA)d, and another complex site, (pA)p, within the capsid-coding region. During viral infection, the mRNAs must extend through (pA)p and undergo additional splicing of the immediately upstream 3D∕3A intron to access the capsid gene. MVC NP1 is a 22-kDa nuclear phosphoprotein unique to the genus Bocaparvovirus of the Parvovirinae which we have shown governs suppression of (pA)p independently of viral genome replication. We show here that in addition to suppression of (pA)p, NP1 is also required for the excision of the MVC 3D∕3A intron, independently of its effect on alternative polyadenylation. Mutations of the arginine∕serine (SR) di-repeats within the intrinsically disordered amino terminus of NP1 are required for splicing of the capsid transcript but not suppression of polyadenylation at (pA)p. 3′-end processing of MVC mRNAs at (pA)p is critical for viral genome replication and the optimal expression of NP1 and NS1. Thus, a finely tuned balance between (pA)p suppression and usage is necessary for efficient virus replication. NP1 is the first parvovirus protein implicated in RNA processing. Its characterization reveals another way that parvoviruses govern access to their capsid protein genes, namely, at the RNA level, by regulating the essential splicing of an intron and the suppression of an internal polyadenylation site. IMPORTANCE The Parvovirinae are small nonenveloped icosahedral viruses that are important pathogens in many animal species, including humans. Although parvoviruses have only subtle early-to-late expression shifts, they all regulate access to their capsid genes. Minute virus of canines (MVC) is an

  9. rAAV2/7 vector-mediated overexpression of alpha-synuclein in mouse substantia nigra induces protein aggregation and progressive dose-dependent neurodegeneration

    PubMed Central

    2013-01-01

    Background Alpha-synuclein is a key protein implicated in the pathogenesis of Parkinson's disease (PD). It is the main component of the Lewy bodies, a cardinal neuropathological feature in the disease. In addition, whole locus multiplications and point mutations in the gene coding for alpha-synuclein lead to autosomal dominant monogenic PD. Over the past decade, research on PD has impelled the development of new animal models based on alpha-synuclein. In this context, transgenic mouse lines have failed to reproduce several hallmarks of PD, especially the strong and progressive dopaminergic neurodegeneration over time that occurs in the patients. In contrast, viral vector-based models in rats and non-human primates display prominent, although highly variable, nigral dopaminergic neuron loss. However, the few studies available on viral vector-mediated overexpression of alpha-synuclein in mice report a weak neurodegenerative process and no clear Lewy body-like pathology. To address this issue, we performed a comprehensive comparative study of alpha-synuclein overexpression by means of recombinant adeno-associated viral vectors serotype 2/7 (rAAV2/7) at different doses in adult mouse substantia nigra. Results We noted a significant and dose-dependent alpha-synucleinopathy over time upon nigral viral vector-mediated alpha-synuclein overexpression. We obtained a strong, progressive and dose-dependent loss of dopaminergic neurons in the substantia nigra, reaching a maximum of 82% after 8 weeks. This effect correlated with a reduction in tyrosine hydroxylase immunoreactivity in the striatum. Moreover, behavioural analysis revealed significant motor impairments from 12 weeks after injection on. In addition, we detected the presence of alpha-synuclein-positive aggregates in the remaining surviving neurons. When comparing wild-type to mutant A53T alpha-synuclein at the same vector dose, both induced a similar degree of cell death. These data were supported by a biochemical

  10. A nuclear fraction of turnip crinkle virus capsid protein is important for elicitation of the host resistance response.

    PubMed

    Kang, Sung-Hwan; Qu, Feng; Morris, T Jack

    2015-12-01

    The N-terminal 25 amino acids (AAs) of turnip crinkle virus (TCV) capsid protein (CP) are recognized by the resistance protein HRT to trigger a hypersensitive response (HR) and systemic resistance to TCV infection. This same region of TCV CP also contains a motif that interacts with the transcription factor TIP, as well as a nuclear localization signal (NLS). However, it is not yet known whether nuclear localization of TCV CP is needed for the induction of HRT-mediated HR and resistance. Here we present new evidence suggesting a tight correlation between nuclear inclusions formed by CP and the manifestation of HR. We show that a fraction of TCV CP localized to cell nuclei to form discrete inclusion-like structures, and a mutated CP (R6A) known to abolish HR failed to form nuclear inclusions. Notably, TIP-CP interaction augments the inclusion-forming activity of CP by tethering inclusions to the nuclear membrane. This TIP-mediated augmentation is also critical for HR resistance, as another CP mutant (R8A) known to elicit a less restrictive HR, though still self-associated into nuclear inclusions, failed to direct inclusions to the nuclear membrane due to its inability to interact with TIP. Finally, exclusion of CP from cell nuclei abolished induction of HR. Together, these results uncovered a strong correlation between nuclear localization and nuclear inclusion formation by TCV CP and induction of HR, and suggest that CP nuclear inclusions could be the key trigger of the HRT-dependent, yet TIP-reinforced, resistance to TCV. PMID:26299399

  11. Evaluation of the immune response elicited by vaccination with viral vectors encoding FMDV capsid proteins and boosted with inactivated virus.

    PubMed

    Romanutti, Carina; D'Antuono, Alejandra; Palacios, Carlos; Quattrocchi, Valeria; Zamorano, Patricia; La Torre, Jose; Mattion, Nora

    2013-08-30

    The aim of the present study was to assess the effect of introducing a priming step with replication-defective viral vectors encoding the capsid proteins of FMDV, followed by a boost with killed virus vaccines, using a suitable BALB/c mice model. Additionally, the immune response to other combined vector immunization regimens was studied. For this purpose, we analyzed different prime-boost immunizations with recombinant adenovirus (Ad), herpesvirus amplicons (Hs) and/or killed virus (KV) vaccines. The highest antibody titers were found in the group that received two doses of adjuvanted KV (P<0.002). Antibody titers were higher in those groups receiving a mixed regimen of vectors, compared to immunization with either vector alone (P<0.0001). Priming with any of the viral vectors induced a shift of the cytokine balance toward a Th1 type immune response regardless of the delivery system used for boosting. The highest IgG1 titer was induced by two doses of adjuvanted KV (P=0.0002) and the highest IgG2a titer corresponded to the group primed with Ad and boosted with KV (P=0.01). Re-stimulation of all groups of mice with 0.5 μg of inactivated virus five months later resulted in a fast increase of antibody titers in all the groups tested. After virus stimulation, antibody titers in the groups that received KV alone or Ad prime-KV boost, were indistinguishable (P=0.800). Protection from challenge was similar (75%) in the groups of animals that received Ad prime-Hs boost or Ad prime-KV boost, or two doses of oil-adjuvanted KV. The data presented in this study suggest that sequential immunization with viral vectors-based vaccines combined with protein-based vaccines have the potential to enhance the quality of the immune response against FMDV. PMID:23683999

  12. Preclinical Refinements of a Broadly Protective VLP-based HPV Vaccine Targeting the Minor Capsid Protein, L2

    PubMed Central

    Tumban, Ebenezer; Muttil, Pavan; Escobar, Carolina Andrea A.; Peabody, Julianne; Wafula, Denis; Peabody, David S.; Chackerian, Bryce

    2015-01-01

    An ideal prophylactic human papillomavirus (HPV) vaccine would provide broadly protective and long-lasting immune responses against all high-risk HPV types, would be effective after a single dose, and would be formulated in such a manner to allow for long-term storage without the necessity for refrigeration. We have developed candidate HPV vaccines consisting of bacteriophage virus-like particles (VLPs) that display a broadly neutralizing epitope derived from the HPV16 minor capsid protein, L2. Immunization with 16L2 VLPs elicited high titer and broadly cross-reactive and cross-neutralizing antibodies against diverse HPV types. In this study we introduce two refinements for our candidate vaccines, with an eye towards enhancing efficacy and clinical applicability in the developing world. First, we assessed the role of antigen dose and boosting on immunogenicity. Mice immunized with 16L2-MS2 VLPs at doses ranging from 2–25 μg with or without alum were highly immunogenic at all doses; alum appeared to have an adjuvant effect at the lowest dose. Although boosting enhanced antibody titers, even a single immunization could elicit strong and long-lasting antibody responses. We also developed a method to enhance vaccine stability. Using a spray dry apparatus and a combination of sugars & an amino acid as protein stabilizers, we generated dry powder vaccine formulations of our L2 VLPs. Spray drying of our L2 VLPs did not affect the integrity or immunogenicity of VLPs upon reconstitution. Spray dried VLPs were stable at room temperature and at 37°C for over one month and the VLPs were highly immunogenic. Taken together, these enhancements are designed to facilitate implementation of a next-generation VLP-based HPV vaccine which addresses U.S. and global disparities in vaccine affordability and access in rural/remote populations. PMID:26003490

  13. Inhibition of Enterovirus 71 (EV-71) Infections by a Novel Antiviral Peptide Derived from EV-71 Capsid Protein VP1

    PubMed Central

    Tan, Chee Wah; Chan, Yoke Fun; Sim, Kooi Mow; Tan, Eng Lee; Poh, Chit Laa

    2012-01-01

    Enterovirus 71 (EV-71) is the main causative agent of hand, foot and mouth disease (HFMD). In recent years, EV-71 infections were reported to cause high fatalities and severe neurological complications in Asia. Currently, no effective antiviral or vaccine is available to treat or prevent EV-71 infection. In this study, we have discovered a synthetic peptide which could be developed as a potential antiviral for inhibition of EV-71. Ninety five synthetic peptides (15-mers) overlapping the entire EV-71 capsid protein, VP1, were chemically synthesized and tested for antiviral properties against EV-71 in human Rhabdomyosarcoma (RD) cells. One peptide, SP40, was found to significantly reduce cytopathic effects of all representative EV-71 strains from genotypes A, B and C tested, with IC50 values ranging from 6–9.3 µM in RD cells. The in vitro inhibitory effect of SP40 exhibited a dose dependent concentration corresponding to a decrease in infectious viral particles, total viral RNA and the levels of VP1 protein. The antiviral activity of SP40 peptide was not restricted to a specific cell line as inhibition of EV-71 was observed in RD, HeLa, HT-29 and Vero cells. Besides inhibition of EV-71, it also had antiviral activities against CV-A16 and poliovirus type 1 in cell culture. Mechanism of action studies suggested that the SP40 peptide was not virucidal but was able to block viral attachment to the RD cells. Substitutions of arginine and lysine residues with alanine in the SP40 peptide at positions R3A, R4A, K5A and R13A were found to significantly decrease antiviral activities, implying the importance of positively charged amino acids for the antiviral activities. The data demonstrated the potential and feasibility of SP40 as a broad spectrum antiviral agent against EV-71. PMID:22563456

  14. Cytomegalovirus Assembly Protein Precursor and Proteinase Precursor Contain Two Nuclear Localization Signals That Mediate Their Own Nuclear Translocation and That of the Major Capsid Protein

    PubMed Central

    Plafker, Scott M.; Gibson, Wade

    1998-01-01

    The cytomegalovirus (CMV) assembly protein precursor (pAP) interacts with the major capsid protein (MCP), and this interaction is required for nuclear translocation of the MCP, which otherwise remains in the cytoplasm of transfected cells (L. J. Wood et al., J. Virol. 71:179–190, 1997). We have interpreted this finding to indicate that the CMV MCP lacks its own nuclear localization signal (NLS) and utilizes the pAP as an NLS-bearing escort into the nucleus. The CMV pAP amino acid sequence has two clusters of basic residues (e.g., KRRRER [NLS1] and KARKRLK [NLS2], for simian CMV) that resemble the simian virus 40 large-T-antigen NLS (D. Kalderon et al., Cell 39:499–509, 1984) and one of these (NLS1) has a counterpart in the pAP homologs of other herpesviruses. The work described here establishes that NLS1 and NLS2 are mutually independent NLS that can act (i) in cis to translocate pAP and the related proteinase precursor (pNP1) into the nucleus and (ii) in trans to transport MCP into the nucleus. By using combinations of NLS mutants and carboxy-terminal deletion constructs, we demonstrated a self-interaction of pAP and cytoplasmic interactions of pAP with pNP1 and of pNP1 with itself. The relevance of these findings to early steps in capsid assembly, the mechanism of MCP nuclear transport, and the possible cytoplasmic formation of protocapsomeric substructures is discussed. PMID:9733808

  15. The capsid protein of beak and feather disease virus binds to the viral DNA and is responsible for transporting the replication-associated protein into the nucleus.

    PubMed

    Heath, Livio; Williamson, Anna-Lise; Rybicki, Edward P

    2006-07-01

    Circoviruses lack an autonomous DNA polymerase and are dependent on the replication machinery of the host cell for de novo DNA synthesis. Accordingly, the viral DNA needs to cross both the plasma membrane and the nuclear envelope before replication can occur. Here we report on the subcellular distribution of the beak and feather disease virus (BFDV) capsid protein (CP) and replication-associated protein (Rep) expressed via recombinant baculoviruses in an insect cell system and test the hypothesis that the CP is responsible for transporting the viral genome, as well as Rep, across the nuclear envelope. The intracellular localization of the BFDV CP was found to be directed by three partially overlapping bipartite nuclear localization signals (NLSs) situated between residues 16 and 56 at the N terminus of the protein. Moreover, a DNA binding region was also mapped to the N terminus of the protein and falls within the region containing the three putative NLSs. The ability of CP to bind DNA, coupled with the karyophilic nature of this protein, strongly suggests that it may be responsible for nuclear targeting of the viral genome. Interestingly, whereas Rep expressed on its own in insect cells is restricted to the cytoplasm, coexpression with CP alters the subcellular localization of Rep to the nucleus, strongly suggesting that an interaction with CP facilitates movement of Rep into the nucleus. PMID:16809327

  16. The Capsid Protein of Beak and Feather Disease Virus Binds to the Viral DNA and Is Responsible for Transporting the Replication-Associated Protein into the Nucleus

    PubMed Central

    Heath, Livio; Williamson, Anna-Lise; Rybicki, Edward P.

    2006-01-01

    Circoviruses lack an autonomous DNA polymerase and are dependent on the replication machinery of the host cell for de novo DNA synthesis. Accordingly, the viral DNA needs to cross both the plasma membrane and the nuclear envelope before replication can occur. Here we report on the subcellular distribution of the beak and feather disease virus (BFDV) capsid protein (CP) and replication-associated protein (Rep) expressed via recombinant baculoviruses in an insect cell system and test the hypothesis that the CP is responsible for transporting the viral genome, as well as Rep, across the nuclear envelope. The intracellular localization of the BFDV CP was found to be directed by three partially overlapping bipartite nuclear localization signals (NLSs) situated between residues 16 and 56 at the N terminus of the protein. Moreover, a DNA binding region was also mapped to the N terminus of the protein and falls within the region containing the three putative NLSs. The ability of CP to bind DNA, coupled with the karyophilic nature of this protein, strongly suggests that it may be responsible for nuclear targeting of the viral genome. Interestingly, whereas Rep expressed on its own in insect cells is restricted to the cytoplasm, coexpression with CP alters the subcellular localization of Rep to the nucleus, strongly suggesting that an interaction with CP facilitates movement of Rep into the nucleus. PMID:16809327

  17. Generation and characterization of potential dengue vaccine candidates based on domain III of the envelope protein and the capsid protein of the four serotypes of dengue virus.

    PubMed

    Suzarte, Edith; Marcos, Ernesto; Gil, Lázaro; Valdés, Iris; Lazo, Laura; Ramos, Yassel; Pérez, Yusleidi; Falcón, Viviana; Romero, Yaremis; Guzmán, María G; González, Sirenia; Kourí, Juan; Guillén, Gerardo; Hermida, Lisset

    2014-07-01

    Dengue is currently one of the most important arthropod-borne diseases, causing up to 25,000 deaths annually. There is currently no vaccine to prevent dengue virus infection, which needs a tetravalent vaccine approach. In this work, we describe the cloning and expression in Escherichia coli of envelope domain III-capsid chimeric proteins (DIIIC) of the four dengue serotypes as a tetravalent dengue vaccine candidate that is potentially able to generate humoral and cellular immunity. The recombinant proteins were purified to more than 85 % purity and were recognized by anti-dengue mouse and human sera. Mass spectrometry analysis verified the identity of the proteins and the correct formation of the intracatenary disulfide bond in the domain III region. The chimeric DIIIC proteins were also serotype-specific, and in the presence of oligonucleotides, they formed aggregates that were visible by electron microscopy. These results support the future use of DIIIC recombinant chimeric proteins in preclinical studies in mice for assessing their immunogenicity and efficacy. PMID:24420159

  18. Time-resolved spectroscopy of self-assembly of CCMV protein capsids

    NASA Astrophysics Data System (ADS)

    Moore, Jelyn; Aronzon, Dina; Manoharan, V. N.

    2008-10-01

    In order to gain a deeper understanding of the process a virus undergoes to assemble; the purpose of this study to time resolve the self-assembly of a virus. Cowpea Chlorotic Mottle virus (CCMV), an icosahedral type virus, can assemble without its genetic code (RNA) depending on its chemical and physical surroundings. The surface plasmon resonance (SPR) of colloidal gold particles is known to display a shift when the gold interacts with the proteins of a virus. Surface plasmon resonance is the free electron oscillation occurring at the surface of the gold particle resulting in a characteristic peak location at maximal absorbance and peak width. The shift results from the change in the refractive index of the particles as induced by the presence of the proteins. We hope to detect this shift through total internal reflection microscopy (TIRM). The accomplishments of this research are the completion of the TIR setup and the purification of the virus and its proteins.

  19. Identification of Positively Charged Residues in Enterovirus 71 Capsid Protein VP1 Essential for Production of Infectious Particles

    PubMed Central

    Yuan, Shilin; Li, Guiming; Wang, Ying; Gao, Qianqian; Wang, Yizhuo; Cui, Rui

    2015-01-01

    ABSTRACT Enterovirus 71 (EV71), a positive-stranded RNA virus, is the major cause of hand, foot, and mouth disease (HFMD) in children, which can cause severe central nervous system disease and death. The capsids of EV71 consist of 60 copies of each of four viral structural proteins (VP1 to VP4), with VP1, VP2, and VP3 exposed on the surface and VP4 arranged internally. VP1 plays a central role in particle assembly and cell entry. To gain insight into the role of positively charged residues in VP1 function in these processes, a charged-to-alanine scanning analysis was performed using an infectious cDNA clone of EV71. Twenty-seven mutants containing single charged-to-alanine changes were tested. Sixteen of them were not viable, seven mutants were replication defective, and the remaining four mutants were replication competent. By selecting revertants, second-site mutations which could at least partially restore viral infectivity were identified within VP1 for four defective mutations and two lethal mutations. The resulting residue pairs represent a network of intra- and intermolecular interactions of the VP1 protein which could serve as a potential novel drug target. Interestingly, mutation K215A in the VP1 GH loop led to a significant increase in thermal stability, demonstrating that conditional thermostable mutants can be generated by altering the charge characteristics of VP1. Moreover, all mutants were sensitive to the EV71 entry inhibitor suramin, which binds to the virus particle via the negatively charged naphthalenetrisulfonic acid group, suggesting that single charged-to-alanine mutation is not sufficient for suramin resistance. Taken together, these data highlight the importance of positively charged residues in VP1 for production of infectious particles. IMPORTANCE Infection with EV71 is more often associated with neurological complications in children and is responsible for the majority of fatalities. No licensed vaccines or antiviral therapies are

  20. Characterization of Mus musculus Papillomavirus 1 Infection In Situ Reveals an Unusual Pattern of Late Gene Expression and Capsid Protein Localization

    PubMed Central

    Handisurya, Alessandra; Day, Patricia M.; Thompson, Cynthia D.; Buck, Christopher B.; Pang, Yuk-Ying S.; Lowy, Douglas R.

    2013-01-01

    Full-length genomic DNA of the recently identified laboratory mouse papillomavirus 1 (MusPV1) was synthesized in vitro and was used to establish and characterize a mouse model of papillomavirus pathobiology. MusPV1 DNA, whether naked or encapsidated by MusPV1 or human papillomavirus 16 (HPV 16) capsids, efficiently induced the outgrowth of papillomas as early as 3 weeks after application to abraded skin on the muzzles and tails of athymic NCr nude mice. High concentrations of virions were extracted from homogenized papillomatous tissues and were serially passaged for >10 generations. Neutralization by L1 antisera confirmed that infectious transmission was capsid mediated. Unexpectedly, the skin of the murine back was much less susceptible to virion-induced papillomas than the muzzle or tail. Although reporter pseudovirions readily transduced the skin of the back, infection with native MusPV1 resulted in less viral genome amplification and gene expression on the back, including reduced expression of the L1 protein and very low expression of the L2 protein, results that imply skin region-specific control of postentry aspects of the viral life cycle. Unexpectedly, L1 protein on the back was predominantly cytoplasmic, while on the tail the abundant L1 was cytoplasmic in the lower epithelial layers and nuclear in the upper layers. Nuclear localization of L1 occurred only in cells that coexpressed the minor capsid protein, L2. The pattern of L1 protein staining in the infected epithelium suggests that L1 expression occurs earlier in the MusPV1 life cycle than in the life cycle of high-risk HPV and that virion assembly is regulated by a previously undescribed mechanism. PMID:24067981

  1. Dengue Virus Capsid Protein Binding to Hepatic Lipid Droplets (LD) Is Potassium Ion Dependent and Is Mediated by LD Surface Proteins

    PubMed Central

    Carvalho, Filomena A.; Carneiro, Fabiana A.; Martins, Ivo C.; Assunção-Miranda, Iranaia; Faustino, André F.; Pereira, Renata M.; Bozza, Patricia T.; Castanho, Miguel A. R. B.; Mohana-Borges, Ronaldo; Da Poian, Andrea T.

    2012-01-01

    Dengue virus (DENV) affects millions of people, causing more than 20,000 deaths annually. No effective treatment for the disease caused by DENV infection is currently available, partially due to the lack of knowledge on the basic aspects of the viral life cycle, including the molecular basis of the interaction between viral components and cellular compartments. Here, we characterized the properties of the interaction between the DENV capsid (C) protein and hepatic lipid droplets (LDs), which was recently shown to be essential for the virus replication cycle. Zeta potential analysis revealed a negative surface charge of LDs, with an average surface charge of −19 mV. The titration of LDs with C protein led to an increase of the surface charge, which reached a plateau at +13.7 mV, suggesting that the viral protein-LD interaction exposes the protein cationic surface to the aqueous environment. Atomic force microscopy (AFM)-based force spectroscopy measurements were performed by using C protein-functionalized AFM tips. The C protein-LD interaction was found to be strong, with a single (un)binding force of 33.6 pN. This binding was dependent on high intracellular concentrations of potassium ions but not sodium. The inhibition of Na+/K+-ATPase in DENV-infected cells resulted in the dissociation of C protein from LDs and a 50-fold inhibition of infectious virus production but not of RNA replication, indicating a biological relevance for the potassium-dependent interaction. Limited proteolysis of the LD surface impaired the C protein-LD interaction, and force measurements in the presence of specific antibodies indicated that perilipin 3 (TIP47) is the major DENV C protein ligand on the surface of LDs. PMID:22130547

  2. Rapid differentiation of Australian, European and American ranaviruses based on variation in major capsid protein gene sequence.

    PubMed

    Marsh, I B; Whittington, R J; O'Rourke, B; Hyatt, A D; Chisholm, O

    2002-04-01

    Epizootic haematopoietic necrosis virus (EHNV), Bohle iridovirus (BIV) and Wamena virus (WV) cause serious diseases in fish, amphibians and snakes, respectively but are restricted to Australasia. European catfish virus (ECV) and sheatfish virus (ESV) have caused epizootics in fish on farms in continental Europe. Currently there are no simple or readily available methods to distinguish these viruses, which are in the Iridoviridae. They are culturally, morphologically and antigenically very similar to Frog Virus 3 (FV3), the type species in Ranavirus in this family and Gutapo virus (GV), another amphibian ranavirus from America. The diseases caused by EHNV, ESV and ECV are so serious that they are internationally notifiable. Tests to distinguish these viruses are desirable to ensure that disease occurrences do not unnecessarily restrict trade in aquaculture products. The gene encoding the major capsid protein from two EHNV isolates from different fish species (Perca fluviatilis and Oncorhynchus mykiss) and one BIV isolate were sequenced and the data and deduced amino acid sequences were compared with those from FV3 and other iridoviruses. The sequences for the two EHNV isolates were identical, confirming suggestions from existing partial MCP sequence that the same type of EHNV infects wild redfin perch and farmed rainbow trout. Differences in restriction endonuclease patterns of specific PCR products were predicted and confirmed between EHNV, BIV, and WV and provided a basis for rapid differentiation of these viruses from each other and from ESV/ECV and FV3/GV. These simple and rapid tests to distinguish important ranaviruses from the regions of Europe, Australia and America will help regulatory authorities assess the need for disease control responses in the event of occurrence of ranavirus infection in aquaculture species. PMID:12030764

  3. Synthetic antibodies and peptides recognizing progressive multifocal leukoencephalopathy-specific point mutations in polyomavirus JC capsid viral protein 1.

    PubMed

    Chen, Gang; Gorelik, Leonid; Simon, Kenneth J; Pavlenco, Alevtina; Cheung, Anne; Brickelmaier, Margot; Chen, Ling Ling; Jin, Ping; Weinreb, Paul H; Sidhu, Sachdev S

    2015-01-01

    Polyomavirus JC (JCV) is the causative agent of progressive multifocal leukoencephalopathy (PML), a rare and frequently fatal brain disease that afflicts a small fraction of the immune-compromised population, including those affected by AIDS and transplantation recipients on immunosuppressive drug therapy. Currently there is no specific therapy for PML. The major capsid viral protein 1 (VP1) involved in binding to sialic acid cell receptors is believed to be a key player in pathogenesis. PML-specific mutations in JCV VP1 sequences present at the binding pocket of sialic acid cell receptors, such as L55F and S269F, abolish sialic acid recognition and might favor PML onset. Early diagnosis of these PML-specific mutations may help identify patients at high risk of PML, thus reducing the risks associated with immunosuppressive therapy. As a first step in the development of such early diagnostic tools, we report identification and characterization of affinity reagents that specifically recognize PML-specific mutations in VP1 variants using phage display technology. We first identified 2 peptides targeting wild type VP1 with moderate specificity. Fine-tuning via selection of biased libraries designed based on 2 parental peptides yielded peptides with different, yet still moderate, bindinspecificities. In contrast, we had great success in identifying synthetic antibodies that recognize one of the PML-specific mutations (L55F) with high specificity from the phage-displayed libraries. These peptides and synthetic antibodies represent potential candidates for developing tailored immune-based assays for PML risk stratification in addition to complementing affinity reagents currently available for the study of PML and JCV. PMID:25879139

  4. Protective cell-mediated immunity by DNA vaccination against Papillomavirus L1 capsid protein in the Cottontail Rabbit Papillomavirus model.

    PubMed

    Hu, Jiafen; Cladel, Nancy M; Budgeon, Lynn R; Reed, Cynthia A; Pickel, Martin D; Christensen, Neil D

    2006-01-01

    Papillomavirus major capsid protein L1 has successfully stimulated protective immunity against virus infection by induction of neutralizing antibodies in animal models and in clinical trials. However, the potential impact of L1-induced protective cell-mediated immune (CMI) responses is difficult to measure in vivo because of the coincidence of anti-L1 antibody. In this study, we tested the hypothesis that L1 could activate CMI, using the Cottontail Rabbit Papillomavirus (CRPV)-rabbit model. A unique property of this model is that infections can be initiated with viral DNA, thus bypassing all contributions to protection via neutralizing anti-L1 antibody. DNA vaccines containing either CRPV L1, or subfragments of L1 (amino-terminal two-thirds of L1 [L1N] and the carboxylterminal two-thirds of L1 [L1C]), were delivered intracutaneously into rabbits, using a gene gun. After three booster immunizations, the rabbits were challenged with several viral DNA constructs: wild-type CRPV, CRPV L1ATGko (an L1 ATG knockout mutation), and CRPV-ROPV hybrid (CRPV with a replacement L1 from Rabbit Oral Papillomavirus). Challenge of L1 DNA-vaccinated rabbits with wild-type CRPV resulted in significantly fewer papillomas when compared with challenge with CRPV L1ATGko DNA. Significantly smaller papillomas were found in CRPV L1-, L1N-, and L1C-vaccinated rabbits. In addition, rabbits vaccinated with either L1 or L1N grew significantly fewer and smaller papillomas when challenged with CRPV-ROPV hybrid DNA. Therefore, CRPV L1 DNA vaccination induced CMI responses to CRPV DNA infections that can contribute to protective immunity. Cross-protective immunity against CRPV L1 and ROPV L1 was elicited in these CRPV L1- and subfragment-vaccinated rabbits. PMID:16987067

  5. Construction and immunogenicity of a recombinant pseudorabies virus co-expressing porcine circovirus type 2 capsid protein and interleukin 18.

    PubMed

    Zheng, Lan-lan; Guo, Xiao-qing; Zhu, Qian-lei; Chao, An-jun; Fu, Peng-fei; Wei, Zhan-yong; Wang, Shu-juan; Chen, Hong-ying; Cui, Bao-an

    2015-04-01

    A novel recombinant pseudorabies virus (PRV) expressing porcine circovirus type 2 (PCV2) capsid protein and IL-18 was constructed. The PCV2 open reading frame 2 (ORF2) and porcine IL-18 genes were amplified by PCR and then inserted into the PRV transfer vector (pG) to produce a recombinant plasmid (pGO18). Plasmid pGO18 was transfected into porcine kidney cell (PK15) pre-infected with PRV HB98 vaccine strain to generate a recombinant virus. The recombinant virus PRV-ORF2-IL18 was purified by green fluorescent plaque purification and the inserts were confirmed by PCR, enzyme digestion, sequencing, and Western blot. The humoral and cellular responses induced by the recombinant virus were assessed in mice. Mice (n=10) were immunized with PRV-ORF2-IL18, PRV-ORF2, PRV HB98, or inactivated PCV2. PRV-ORF2-IL18 elicited high titers of ELISA and serum neutralizing antibodies and strong cell-mediated immune responses in mice as indicated by anti-PCV2 ELISA, PRV-neutralizing assay, PCV2 specific lymphocyte proliferation assay, CD3(+), CD4(+) and CD8(+) T lymphocytes analysis, respectively. And PRV-ORF2-IL18 immunization protected mice against a lethal challenge of a virulent PRV Fa strain and significantly reduced the amount of PCV2 viremia. These results suggest an adjuvant effect of IL-18 on cellular immune responses. The recombinant virus might be an attractive candidate vaccine for preventing PCV2 and PRV infections in pigs. PMID:25701744

  6. Interaction between Simian Virus 40 Major Capsid Protein VP1 and Cell Surface Ganglioside GM1 Triggers Vacuole Formation

    PubMed Central

    Luo, Yong; Motamedi, Nasim; Magaldi, Thomas G.; Gee, Gretchen V.; Atwood, Walter J.

    2016-01-01

    ABSTRACT Simian virus 40 (SV40), a polyomavirus that has served as an important model to understand many aspects of biology, induces dramatic cytoplasmic vacuolization late during productive infection of monkey host cells. Although this activity led to the discovery of the virus in 1960, the mechanism of vacuolization is still not known. Pentamers of the major SV40 capsid protein VP1 bind to the ganglioside GM1, which serves as the cellular receptor for the virus. In this report, we show that binding of VP1 to cell surface GM1 plays a key role in SV40 infection-induced vacuolization. We previously showed that SV40 VP1 mutants defective for GM1 binding fail to induce vacuolization, even though they replicate efficiently. Here, we show that interfering with GM1-VP1 binding by knockdown of GM1 after infection is established abrogates vacuolization by wild-type SV40. Vacuole formation during permissive infection requires efficient virus release, and conditioned medium harvested late during SV40 infection rapidly induces vacuoles in a VP1- and GM1-dependent fashion. Furthermore, vacuolization can also be induced by a nonreplicating SV40 pseudovirus in a GM1-dependent manner, and a mutation in BK pseudovirus VP1 that generates GM1 binding confers vacuole-inducing activity. Vacuolization can also be triggered by purified pentamers of wild-type SV40 VP1, but not by GM1 binding-defective pentamers or by intracellular expression of VP1. These results demonstrate that SV40 infection-induced vacuolization is caused by the binding of released progeny viruses to GM1, thereby identifying the molecular trigger for the activity that led to the discovery of SV40. PMID:27006465

  7. Nonicosahedral pathways for capsid expansion

    NASA Astrophysics Data System (ADS)

    Cermelli, Paolo; Indelicato, Giuliana; Twarock, Reidun

    2013-09-01

    For a significant number of viruses a structural transition of the protein container that encapsulates the viral genome forms an important part of the life cycle and is a prerequisite for the particle becoming infectious. Despite many recent efforts the mechanism of this process is still not fully understood, and a complete characterization of the expansion pathways is still lacking. We present here a coarse-grained model that captures the essential features of the expansion process and allows us to investigate the conditions under which a viral capsid becomes unstable. Based on this model we demonstrate that the structural transitions in icosahedral viral capsids are likely to occur through a low-symmetry cascade of local expansion events spreading in a wavelike manner over the capsid surface.

  8. A novel fusion protein domain III-capsid from dengue-2, in a highly aggregated form, induces a functional immune response and protection in mice

    SciTech Connect

    Valdes, Iris; Bernardo, Lidice; Pavon, Alekis; Guzman, Maria G.

    2009-11-25

    Based on the immunogenicity of domain III from the Envelope protein of dengue virus as well as the proven protective capacity of the capsid antigen, we have designed a novel domain III-capsid chimeric protein with the goal of obtaining a molecule potentially able to induce both humoral and cell-mediated immunity (CMI). After expression of the recombinant gene in Escherichia coli, the domain III moiety retained its antigenicity as evaluated with anti-dengue sera. In order to explore alternatives for modulating the immunogenicity of the protein, it was mixed with oligodeoxynucleotides in order to obtain particulated aggregates and then immunologically evaluated in mice in comparison with non-aggregated controls. Although the humoral immune response induced by both forms of the protein was equivalent, the aggregated variant resulted in a much stronger CMI as measured by in vitro IFN-gamma secretion and protection experiments, mediated by CD4{sup +} and CD8{sup +} cells. The present work provides additional evidence in support for a crucial role of CMI in protection against dengue virus and describes a novel vaccine candidate against the disease based on a recombinant protein that can stimulate both arms of the acquired immune system.

  9. Delivering Transgenic DNA Exceeding the Carrying Capacity of AAV Vectors

    PubMed Central

    Hirsch, Matthew L.; Wolf, Sonya J.; Samulski, R.J.

    2016-01-01

    Gene delivery using recombinant adeno-associated virus (rAAV) has emerged to the forefront demonstrating safe and effective phenotypic correction of diverse diseases including hemophilia B and Leber’s congenital amaurosis. In addition to rAAV’s high efficiency of transduction and the capacity for long-term transgene expression, the safety profile of rAAV remains unsoiled in humans with no deleterious vector-related consequences observed thus far. Despite these favorable attributes, rAAV vectors have a major disadvantage preventing widespread therapeutic applications; as the AAV capsid is the smallest described to date, it cannot package “large” genomes. Currently, the packaging capacity of rAAV has yet to be definitively defined but is approximately 5 kb, which has served as a limitation for large gene transfer. There are two main approaches that have been developed to overcome this limitation, split AAV vectors, and fragment AAV (fAAV) genome reassembly (Hirsch et al., Mol Ther 18(1):6–8, 2010). Split rAAV vector applications were developed based upon the finding that rAAV genomes naturally concatemerize in the cell post-transduction and are substrates for enhanced homologous recombination (HR) (Hirsch et al., Mol Ther 18(1):6–8, 2010; Duan et al., J Virol 73(1):161–169, 1999; Duan et al., J Virol 72(11):8568–8577, 1998; Duan et al., Mol Ther 4(4):383–391, 2001; Halbert et al., Nat Biotechnol 20(7):697–701, 2002). This method involves “splitting” the large transgene into two separate vectors and upon co-transduction, intracellular large gene reconstruction via vector genome concatemerization occurs via HR or nonhomologous end joining (NHEJ). Within the split rAAV approaches there currently exist three strategies: overlapping, trans-splicing, and hybrid trans-splicing (Duan et al., Mol Ther 4(4):383–391, 2001; Halbert et al., Nat Biotechnol 20(7):697–701, 2002; Ghosh et al., Mol Ther 16(1):124–130, 2008; Ghosh et al., Mol Ther 15

  10. Local Administration of AAV-BDNF to Subventricular Zone Induces Functional Recovery in Stroke Rats

    PubMed Central

    Yu, Seong-Jin; Tseng, Kuan-Yin; Shen, Hui; Harvey, Brandon K.; Airavaara, Mikko; Wang, Yun

    2013-01-01

    Migration of new neuroprogenitor cells (NPCs) from the subventricular zone (SVZ) plays an important role in neurorepair after injury. Previous studies have shown that brain derived neurotrophic factor (BDNF) enhances the migration of NPCs from SVZ explants in neonatal mice in vitro. The purpose of this study was to identify the role of BDNF in SVZ cells using AAV-BDNF in an animal model of stroke. BDNF protein production after AAV‐BDNF infection was verified in primary neuronal culture. AAV-BDNF or AAV-RFP was injected into the left SVZ region of adult rats at 14 days prior to right middle cerebral artery occlusion (MCAo). SVZ tissues were collected from the brain and placed in Metrigel cultures 1 day after MCAo. Treatment with AAV-BDNF significantly increased the migration of SVZ cells in the stroke brain in vitro. In another set of animals, AAV-GFP was co-injected with AAV-BDNF or AAV-RFP to label cells in left SVZ prior to right MCAo. Local administration of AAV-BDNF significantly enhanced recovery of locomotor function and migration of GFP-positive cells from the SVZ toward the lesioned hemisphere in stroke rats. Our data suggest that focal administration of AAV-BDNF to the SVZ increases behavioral recovery post stroke, possibly through the enhancement of migration of cells from SVZ in stroke animals. Regional manipulation of BDNF expression through AAV may be a novel approach for neurorepair in stroke brains. PMID:24312581

  11. Magnetic Resonance Imaging Revealed Splenic Targeting of Canine Parvovirus Capsid Protein VP2

    NASA Astrophysics Data System (ADS)

    Ma, Yufei; Wang, Haiming; Yan, Dan; Wei, Yanquan; Cao, Yuhua; Yi, Peiwei; Zhang, Hailu; Deng, Zongwu; Dai, Jianwu; Liu, Xiangtao; Luo, Jianxun; Zhang, Zhijun; Sun, Shiqi; Guo, Huichen

    2016-03-01

    Canine parvovirus (CPV) is a highly contagious infectious virus, whose infectious mechanism remains unclear because of acute gastroenteritis and the lack of an efficient tool to visualize the virus in real time during virology research. In this study, we developed an iron oxide nanoparticle supported by graphene quantum dots (GQD), namely, FeGQD. In this composite material, GQD acts as a stabilizer; thus, vacancies are retained on the surface for further physical adsorption of the CPV VP2 protein. The FeGQD@VP2 nanocomposite product showed largely enhanced colloidal stability in comparison with bare FeGQD, as well as negligible toxicity both in vitro and in vivo. The composite displayed high uptake into transferrin receptor (TfR) positive cells, which are distinguishable from FeGQD or TfR negative cells. In addition, the composite developed a significant accumulation in spleen rather than in liver, where bare FeGQD or most iron oxide nanoparticles gather. As these evident targeting abilities of FeGQD@VP2 strongly suggested, the biological activity of CPV VP2 was retained in our study, and its biological functions might correspond to CPV when the rare splenic targeting ability is considered. This approach can be applied to numerous other biomedical studies that require a simple yet efficient approach to track proteins in vivo while retaining biological function and may facilitate virus-related research.

  12. Magnetic Resonance Imaging Revealed Splenic Targeting of Canine Parvovirus Capsid Protein VP2

    PubMed Central

    Ma, Yufei; Wang, Haiming; Yan, Dan; Wei, Yanquan; Cao, Yuhua; Yi, Peiwei; Zhang, Hailu; Deng, Zongwu; Dai, Jianwu; Liu, Xiangtao; Luo, Jianxun; Zhang, Zhijun; Sun, Shiqi; Guo, Huichen

    2016-01-01

    Canine parvovirus (CPV) is a highly contagious infectious virus, whose infectious mechanism remains unclear because of acute gastroenteritis and the lack of an efficient tool to visualize the virus in real time during virology research. In this study, we developed an iron oxide nanoparticle supported by graphene quantum dots (GQD), namely, FeGQD. In this composite material, GQD acts as a stabilizer; thus, vacancies are retained on the surface for further physical adsorption of the CPV VP2 protein. The FeGQD@VP2 nanocomposite product showed largely enhanced colloidal stability in comparison with bare FeGQD, as well as negligible toxicity both in vitro and in vivo. The composite displayed high uptake into transferrin receptor (TfR) positive cells, which are distinguishable from FeGQD or TfR negative cells. In addition, the composite developed a significant accumulation in spleen rather than in liver, where bare FeGQD or most iron oxide nanoparticles gather. As these evident targeting abilities of FeGQD@VP2 strongly suggested, the biological activity of CPV VP2 was retained in our study, and its biological functions might correspond to CPV when the rare splenic targeting ability is considered. This approach can be applied to numerous other biomedical studies that require a simple yet efficient approach to track proteins in vivo while retaining biological function and may facilitate virus-related research. PMID:26996514

  13. Magnetic Resonance Imaging Revealed Splenic Targeting of Canine Parvovirus Capsid Protein VP2.

    PubMed

    Ma, Yufei; Wang, Haiming; Yan, Dan; Wei, Yanquan; Cao, Yuhua; Yi, Peiwei; Zhang, Hailu; Deng, Zongwu; Dai, Jianwu; Liu, Xiangtao; Luo, Jianxun; Zhang, Zhijun; Sun, Shiqi; Guo, Huichen

    2016-01-01

    Canine parvovirus (CPV) is a highly contagious infectious virus, whose infectious mechanism remains unclear because of acute gastroenteritis and the lack of an efficient tool to visualize the virus in real time during virology research. In this study, we developed an iron oxide nanoparticle supported by graphene quantum dots (GQD), namely, FeGQD. In this composite material, GQD acts as a stabilizer; thus, vacancies are retained on the surface for further physical adsorption of the CPV VP2 protein. The FeGQD@VP2 nanocomposite product showed largely enhanced colloidal stability in comparison with bare FeGQD, as well as negligible toxicity both in vitro and in vivo. The composite displayed high uptake into transferrin receptor (TfR) positive cells, which are distinguishable from FeGQD or TfR negative cells. In addition, the composite developed a significant accumulation in spleen rather than in liver, where bare FeGQD or most iron oxide nanoparticles gather. As these evident targeting abilities of FeGQD@VP2 strongly suggested, the biological activity of CPV VP2 was retained in our study, and its biological functions might correspond to CPV when the rare splenic targeting ability is considered. This approach can be applied to numerous other biomedical studies that require a simple yet efficient approach to track proteins in vivo while retaining biological function and may facilitate virus-related research. PMID:26996514

  14. Cell culture adaptation mutations in foot-and-mouth disease virus serotype A capsid proteins: implications for receptor interactions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this study we describe the adaptive changes fixed on the capsid of several foot-and-mouth disease virus serotype A strains during propagation in cell monolayers. Viruses passaged extensively in three cell lines (BHK-21, LFBK and IB-RS-2), consistently gained several positively charged amino acids...

  15. Structural and Functional Analysis of Murine Polyomavirus Capsid Proteins Establish the Determinants of Ligand Recognition and Pathogenicity.

    PubMed

    Buch, Michael H C; Liaci, A Manuel; O'Hara, Samantha D; Garcea, Robert L; Neu, Ursula; Stehle, Thilo

    2015-10-01

    Murine polyomavirus (MuPyV) causes tumors of various origins in newborn mice and hamsters. Infection is initiated by attachment of the virus to ganglioside receptors at the cell surface. Single amino acid exchanges in the receptor-binding pocket of the major capsid protein VP1 are known to drastically alter tumorigenicity and spread in closely related MuPyV strains. The virus represents a rare example of differential receptor recognition directly influencing viral pathogenicity, although the factors underlying these differences remain unclear. We performed structural and functional analyses of three MuPyV strains with strikingly different pathogenicities: the low-tumorigenicity strain RA, the high-pathogenicity strain PTA, and the rapidly growing, lethal laboratory isolate strain LID. Using ganglioside deficient mouse embryo fibroblasts, we show that addition of specific gangliosides restores infectability for all strains, and we uncover a complex relationship between virus attachment and infection. We identify a new infectious ganglioside receptor that carries an additional linear [α-2,8]-linked sialic acid. Crystal structures of all three strains complexed with representative oligosaccharides from the three main pathways of ganglioside biosynthesis provide the molecular basis of receptor recognition. All strains bind to a range of sialylated glycans featuring the central [α-2,3]-linked sialic acid present in the established receptors GD1a and GT1b, but the presence of additional sialic acids modulates binding. An extra [α-2,8]-linked sialic acid engages a protein pocket that is conserved among the three strains, while another, [α-2,6]-linked branching sialic acid lies near the strain-defining amino acids but can be accommodated by all strains. By comparing electron density of the oligosaccharides within the binding pockets at various concentrations, we show that the [α-2,8]-linked sialic acid increases the strength of binding. Moreover, the amino acid

  16. Structural and Functional Analysis of Murine Polyomavirus Capsid Proteins Establish the Determinants of Ligand Recognition and Pathogenicity

    PubMed Central

    O’Hara, Samantha D.; Garcea, Robert L.; Neu, Ursula; Stehle, Thilo

    2015-01-01

    Murine polyomavirus (MuPyV) causes tumors of various origins in newborn mice and hamsters. Infection is initiated by attachment of the virus to ganglioside receptors at the cell surface. Single amino acid exchanges in the receptor-binding pocket of the major capsid protein VP1 are known to drastically alter tumorigenicity and spread in closely related MuPyV strains. The virus represents a rare example of differential receptor recognition directly influencing viral pathogenicity, although the factors underlying these differences remain unclear. We performed structural and functional analyses of three MuPyV strains with strikingly different pathogenicities: the low-tumorigenicity strain RA, the high-pathogenicity strain PTA, and the rapidly growing, lethal laboratory isolate strain LID. Using ganglioside deficient mouse embryo fibroblasts, we show that addition of specific gangliosides restores infectability for all strains, and we uncover a complex relationship between virus attachment and infection. We identify a new infectious ganglioside receptor that carries an additional linear [α-2,8]-linked sialic acid. Crystal structures of all three strains complexed with representative oligosaccharides from the three main pathways of ganglioside biosynthesis provide the molecular basis of receptor recognition. All strains bind to a range of sialylated glycans featuring the central [α-2,3]-linked sialic acid present in the established receptors GD1a and GT1b, but the presence of additional sialic acids modulates binding. An extra [α-2,8]-linked sialic acid engages a protein pocket that is conserved among the three strains, while another, [α-2,6]-linked branching sialic acid lies near the strain-defining amino acids but can be accommodated by all strains. By comparing electron density of the oligosaccharides within the binding pockets at various concentrations, we show that the [α-2,8]-linked sialic acid increases the strength of binding. Moreover, the amino acid

  17. Modified Indirect Porcine Circovirus (PCV) Type 2-Based and Recombinant Capsid Protein (ORF2)-Based Enzyme-Linked Immunosorbent Assays for Detection of Antibodies to PCV

    PubMed Central

    Nawagitgul, Porntippa; Harms, Perry A.; Morozov, Igor; Thacker, Brad J.; Sorden, Steven D.; Lekcharoensuk, Chalermpol; Paul, Prem S.

    2002-01-01

    Postweaning multisystemic wasting syndrome of swine associated with porcine circovirus (PCV) is a recently reported and economically important disease. Simple and reliable diagnostic methods are needed for detecting antibodies to PCV type 2 (PCV2) for monitoring of PCV infection. Here, we report the development of two modified indirect enzyme-linked immunosorbent assays (ELISAs): a PCV2 ELISA based on cell-culture-propagated PCV2 and an ORF2 ELISA based on recombinant major capsid protein. PCV2 and ORF2 ELISA detected antibodies to PCV2 and the capsid protein, respectively, in sera from pigs experimentally infected with PCV2 as early as 14 and 21 days postinoculation (dpi). The kinetics of the antibody response to PCV2 and the major capsid protein were similar. Repeatability tests revealed that the coefficients of variation of positive sera within and between runs for both assays were less than 30%. To validate the assays, PCV2 and ORF2 ELISAs were performed with 783 serum samples of young and adult pigs collected from different herds in the Midwestern United States and compared with an indirect immunofluorescent assay (IIF). Six out of 60 samples collected from nursery and growing pigs in 1987 were positive by both ELISA and IIF. Compared with IIF, the diagnostic sensitivity, specificity, and accuracy of PCV2 and ORF2 ELISAs were similar (>90%). The tests showed no cross-reactivity with antibodies to porcine parvovirus and porcine reproductive and respiratory syndrome virus. There was good agreement between the two ELISAs and between the ELISAs and IIF. The availability of the two ELISAs should accelerate our understanding of the host immune response to PCV2 and facilitate the development of prevention and control strategies by elucidating the ecology of PCV2 within swine populations. PMID:11777826

  18. A Kinase Chaperones Hepatitis B Virus Capsid Assembly and Captures Capsid Dynamics in vitro

    PubMed Central

    Chen, Chao; Wang, Joseph Che-Yen; Zlotnick, Adam

    2011-01-01

    The C-terminal domain (CTD) of Hepatitis B virus (HBV) core protein is involved in regulating multiple stages of the HBV lifecycle. CTD phosphorylation correlates with pregenomic-RNA encapsidation during capsid assembly, reverse transcription, and viral transport, although the mechanisms remain unknown. In vitro, purified HBV core protein (Cp183) binds any RNA and assembles aggressively, independent of phosphorylation, to form empty and RNA-filled capsids. We hypothesize that there must be a chaperone that binds the CTD to prevent self-assembly and nonspecific RNA packaging. Here, we show that HBV capsid assembly is stalled by the Serine Arginine protein kinase (SRPK) binding to the CTD, and reactivated by subsequent phosphorylation. Using the SRPK to probe capsids, solution and structural studies showed that SRPK bound to capsid, though the CTD is sequestered on the capsid interior. This result indicates transient CTD externalization and suggests that capsid dynamics could be crucial for directing HBV intracellular trafficking. Our studies illustrate the stochastic nature of virus capsids and demonstrate the appropriation of a host protein by a virus for a non-canonical function. PMID:22114561

  19. Antigenic composition and immunoreactivity differences between HEV recombinant capsid proteins generated from different genotypes.

    PubMed

    Behloul, Nouredine; Wen, Jiyue; Dai, Xing; Dong, Chen; Meng, Jihong

    2015-08-01

    Appreciable variability has been observed in hepatitis E virus (HEV) serological diagnostics. Four recombinant proteins (p166s) were generated from position 452 to 617 aa of ORF2 of different HEV genotypes and used in an indirect ELISA to detect anti-HEV IgMs and IgGs in serially diluted sera of patients infected with different HEV genotypes (genotype 1, n=15; genotype 3, n=12; genotype 4, n=17). To evaluate the differences at a conformational level, 3D-structure models of p166s were predicted, and different bioinformatics tools were used to analyze the antigenic composition. With both anti-HEV IgMs and IgGs antibodies, there was a considerable variability between the four antigens immunoreactivities. In silico results revealed the region 483-533 aa with the highest antigenic potential and contains six key aa at positions 488, 489, 512, 533, 483 and 530. This immunoreactivity variation could affect diagnosis results and seroprevalence estimations and the identification in silico of a region highly antigenic would guide the development of efficient serological assays and epitope-based vaccines. PMID:26122075

  20. High yield production of pigeon circovirus capsid protein in the E. coli by evaluating the key parameters needed for protein expression

    PubMed Central

    2014-01-01

    Background Pigeon circovirus (PiCV) is considered to be a viral agent central to the development of young pigeon disease syndrome (YPDS). The Cap protein, a structural protein encoded by the cap (or C1) gene of PiCV, has been shown to be responsible for not only capsid assembly, but also has been used as antigen for detecting antibody when the host is infected with PiCV. The antigenic characteristics of the Cap protein potentially may allow the development of a detection kit that could be applied to control PiCV infection. However, poor expression and poor protein solubility have hampered the production of recombinant Cap protein in the bacteria. This study was undertaken to develop the optimal expression of recombinant full-length Cap protein of PiCV using an E. coli expression system. Results The PiCV cap gene was cloned and fused with different fusion partners including a His-tag, a GST-tag (glutathioine-S-transferase tag) and a Trx-His-tag (thioredoxin-His tag). The resulting constructs were then expressed after transformation into a number of different E. coli strains; these then had their protein expression evaluated. The expression of the recombinant Cap protein in E. coli was significantly increased when Cap protein was fused with either a GST-tag or a Trx-His tag rather than a His-tag. After various rare amino acid codons presented in the Cap protein were optimized to give the sequence rCapopt, the expression level of the GST-rCapopt in E. coli BL21(DE3) was further increased to a significant degree. The highest protein expression level of GST-rCapopt obtained was 394.27 ± 26.1 mg/L per liter using the E. coli strain BL21(DE3)-pLysS. Moreover, approximately 74.5% of the expressed GST-rCapopt was in soluble form, which is higher than the soluble Trx-His-rCapopt expressed using the BL21(DE3)-pLysS strain. After purification using a GST affinity column combined with ion-exchange chromatography, the purified recombinant GST-rCapopt protein was found to

  1. Production of a recombinant capsid protein VP1 from a newly described polyomavirus (RacPyV) for downstream use in virus characterization.

    PubMed

    Church, Molly E; Dela Cruz, Florante N; Kim, Kevin; Persiani, Michele; Woods, Leslie W; Pesavento, Patricia A; Woolard, Kevin D

    2016-06-01

    Here we describe the methods for production of a recombinant viral capsid protein and subsequent use in an indirect enzyme linked immunosorbent assay (ELISA), and for use in production of a rabbit polyclonal antibody. These reagents were utilized in development and optimization of an ELISA, which established the extent of exposure of free ranging raccoons to a newly described polyomavirus (RacPyV) [1]. Production of a polyclonal antibody has allowed for further characterization of RacPyV, including immunohistochemistry and immunocytochemistry techniques, in order to answer questions about pathogenesis of this virus. PMID:26955649

  2. Production of a recombinant capsid protein VP1 from a newly described polyomavirus (RacPyV) for downstream use in virus characterization

    PubMed Central

    Church, Molly E.; Dela Cruz, Florante N.; Kim, Kevin; Persiani, Michele; Woods, Leslie W.; Pesavento, Patricia A.; Woolard, Kevin D.

    2016-01-01

    Here we describe the methods for production of a recombinant viral capsid protein and subsequent use in an indirect enzyme linked immunosorbent assay (ELISA), and for use in production of a rabbit polyclonal antibody. These reagents were utilized in development and optimization of an ELISA, which established the extent of exposure of free ranging raccoons to a newly described polyomavirus (RacPyV) [1]. Production of a polyclonal antibody has allowed for further characterization of RacPyV, including immunohistochemistry and immunocytochemistry techniques, in order to answer questions about pathogenesis of this virus. PMID:26955649

  3. Inner Limiting Membrane Barriers to AAV-mediated Retinal Transduction From the Vitreous

    PubMed Central

    Dalkara, Deniz; Kolstad, Kathleen D; Caporale, Natalia; Visel, Meike; Klimczak, Ryan R; Schaffer, David V; Flannery, John G

    2009-01-01

    Adeno-associated viral gene therapy has shown great promise in treating retinal disorders, with three promising clinical trials in progress. Numerous adeno-associated virus (AAV) serotypes can infect various cells of the retina when administered subretinally, but the retinal detachment accompanying this injection induces changes that negatively impact the microenvironment and survival of retinal neurons. Intravitreal administration could circumvent this problem, but only AAV2 can infect retinal cells from the vitreous, and transduction is limited to the inner retina. We therefore sought to investigate and reduce barriers to transduction from the vitreous. We fluorescently labeled several AAV serotype capsids and followed their retinal distribution after intravitreal injection. AAV2, 8, and 9 accumulate at the vitreoretinal junction. AAV1 and 5 show no accumulation, indicating a lack of appropriate receptors at the inner limiting membrane (ILM). Importantly, mild digestion of the ILM with a nonspecific protease enabled substantially enhanced transduction of multiple retinal cell types from the vitreous, with AAV5 mediating particularly remarkable expression in all retinal layers. This protease treatment has no effect on retinal function as shown by electroretinogram (ERG) and visual cortex cell population responses. These findings may help avoid limitations, risks, and damage associated with subretinal injections currently necessary for clinical gene therapy. PMID:19672248

  4. Structure of N-linked oligosaccharides attached to chlorovirus PBCV-1 major capsid protein reveals unusual class of complex N-glycans.

    PubMed

    De Castro, Cristina; Molinaro, Antonio; Piacente, Francesco; Gurnon, James R; Sturiale, Luisa; Palmigiano, Angelo; Lanzetta, Rosa; Parrilli, Michelangelo; Garozzo, Domenico; Tonetti, Michela G; Van Etten, James L

    2013-08-20

    The major capsid protein Vp54 from the prototype chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1) contains four Asn-linked glycans. The structure of the four N-linked oligosaccharides and the type of substitution at each glycosylation site was determined by chemical, spectroscopic, and spectrometric analyses. Vp54 glycosylation is unusual in many ways, including: (i) unlike most viruses, PBCV-1 encodes most, if not all, of the machinery to glycosylate its major capsid protein; (ii) the glycans are attached to the protein by a β-glucose linkage; (iii) the Asn-linked glycans are not located in a typical N-X-(T/S) consensus site; and (iv) the process probably occurs in the cytoplasm. The four glycoforms share a common core structure, and the differences are related to the nonstoichiometric presence of two monosaccharides. The most abundant glycoform consists of nine neutral monosaccharide residues, organized in a highly branched fashion. Among the most distinctive features of the glycoforms are (i) a dimethylated rhamnose as the capping residue of the main chain, (ii) a hyperbranched fucose unit, and (iii) two rhamnose residues with opposite absolute configurations. These glycoforms differ from what has been reported so far in the three domains of life. Considering that chloroviruses and other members of the family Phycodnaviridae may have a long evolutionary history, we suggest that the chlorovirus glycosylation pathway is ancient, possibly existing before the development of the endoplasmic reticulum and Golgi pathway, and involves still unexplored mechanisms. PMID:23918378

  5. Structure of N-linked oligosaccharides attached to chlorovirus PBCV-1 major capsid protein reveals unusual class of complex N-glycans

    PubMed Central

    De Castro, Cristina; Molinaro, Antonio; Piacente, Francesco; Gurnon, James R.; Sturiale, Luisa; Palmigiano, Angelo; Lanzetta, Rosa; Parrilli, Michelangelo; Garozzo, Domenico; Tonetti, Michela G.; Van Etten, James L.

    2013-01-01

    The major capsid protein Vp54 from the prototype chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1) contains four Asn-linked glycans. The structure of the four N-linked oligosaccharides and the type of substitution at each glycosylation site was determined by chemical, spectroscopic, and spectrometric analyses. Vp54 glycosylation is unusual in many ways, including: (i) unlike most viruses, PBCV-1 encodes most, if not all, of the machinery to glycosylate its major capsid protein; (ii) the glycans are attached to the protein by a β-glucose linkage; (iii) the Asn-linked glycans are not located in a typical N-X-(T/S) consensus site; and (iv) the process probably occurs in the cytoplasm. The four glycoforms share a common core structure, and the differences are related to the nonstoichiometric presence of two monosaccharides. The most abundant glycoform consists of nine neutral monosaccharide residues, organized in a highly branched fashion. Among the most distinctive features of the glycoforms are (i) a dimethylated rhamnose as the capping residue of the main chain, (ii) a hyperbranched fucose unit, and (iii) two rhamnose residues with opposite absolute configurations. These glycoforms differ from what has been reported so far in the three domains of life. Considering that chloroviruses and other members of the family Phycodnaviridae may have a long evolutionary history, we suggest that the chlorovirus glycosylation pathway is ancient, possibly existing before the development of the endoplasmic reticulum and Golgi pathway, and involves still unexplored mechanisms. PMID:23918378

  6. IRES-driven Expression of the Capsid Protein of the Venezuelan Equine Encephalitis Virus TC-83 Vaccine Strain Increases Its Attenuation and Safety

    PubMed Central

    Guerbois, Mathilde; Volkova, Eugenia; Forrester, Naomi L.; Rossi, Shannan L.; Frolov, Ilya; Weaver, Scott C.

    2013-01-01

    The live-attenuated TC-83 strain is the only licensed veterinary vaccine available to protect equids against Venezuelan equine encephalitis virus (VEEV) and to protect humans indirectly by preventing equine amplification. However, TC-83 is reactogenic due to its reliance on only two attenuating point mutations and has infected mosquitoes following equine vaccination. To increase its stability and safety, a recombinant TC-83 was previously engineered by placing the expression of the viral structural proteins under the control of the Internal Ribosome Entry Site (IRES) of encephalomyocarditis virus (EMCV), which drives translation inefficiently in insect cells. However, this vaccine candidate was poorly immunogenic. Here we describe a second generation of the recombinant TC-83 in which the subgenomic promoter is maintained and only the capsid protein gene is translated from the IRES. This VEEV/IRES/C vaccine candidate did not infect mosquitoes, was stable in its attenuation phenotype after serial murine passages, and was more attenuated in newborn mice but still as protective as TC-83 against VEEV challenge. Thus, by using the IRES to modulate TC-83 capsid protein expression, we generated a vaccine candidate that combines efficient immunogenicity and efficacy with lower virulence and a reduced potential for spread in nature. PMID:23675542

  7. Alphavirus capsid proteins self-assemble into core-like particles in insect cells: A promising platform for nanoparticle vaccine development.

    PubMed

    Hikke, Mia C; Geertsema, Corinne; Wu, Vincen; Metz, Stefan W; van Lent, Jan W; Vlak, Just M; Pijlman, Gorben P

    2016-02-01

    The mosquito-borne chikungunya virus (CHIKV) causes arthritic diseases in humans, whereas the aquatic salmonid alphavirus (SAV) is associated with high mortality in aquaculture of salmon and trout. Using modern biotechnological approaches, promising vaccine candidates based upon highly immunogenic, enveloped virus-like particles (eVLPs) have been developed. However, the eVLP structure (core, lipid membrane, surface glycoproteins) is more complex than that of non-enveloped, protein-only VLPs, which are structurally and morphologically 'simple'. In order to develop an alternative to alphavirus eVLPs, in this paper we engineered recombinant baculovirus vectors to produce high levels of alphavirus core-like particles (CLPs) in insect cells by expression of the CHIKV and SAV capsid proteins. The CLPs localize in dense nuclear bodies within the infected cell nucleus and are purified through a rapid and scalable protocol involving cell lysis, sonication and low-speed centrifugation steps. Furthermore, an immunogenic epitope from the alphavirus E2 glycoprotein can be successfully fused to the N-terminus of the capsid protein without disrupting the CLP self-assembling properties. We propose that immunogenic epitope-tagged alphavirus CLPs produced in insect cells present a simple and perhaps more stable alternative to alphavirus eVLPs. PMID:26287127

  8. [Expression of cDNA of the Gene for the Capsid Protein VP2 of German Cockroach Densovirus in the Transgenic Strain of Drosophila melanogaster].

    PubMed

    Kozlov, E N; Martynova, E U; Roshina, N V; Karakozova, M V; Mukha, D V

    2016-04-01

    Transgenic strains of Drosophila melanogaster capable of expressing a cDNA fragment corresponding to open reading frame (ORF) of the gene for the German cockroach densonucleosis virus capsid protein VP2 (ORF VP2) in specific tissues and at a certain stage of development depending on the type of chosen driver strains (GAL-UAS system) were obtained. The ORF VP2 transcription was examined at the imago stage after crossing the obtained transgenic Drosophila with the driver line expressing the inducer protein (GAL4) under control of actin promoter (the ORF VP2 expression is induced in all tissues of the first-generation Drosophila). It was demonstrated that the greater part of transcribed foreign RNA was represented by three spliced variants in which RNA fragments either between nucleotides 137 and 353 or between nucleotides 609 and 1925 were excised; the third spliced variant was represented by RNA lacking both introns. Using the next-generation sequencing (NGS) technique, the proportion of unspliced form relative to spliced variants of the analyzed RNA was assessed. It was shown that the ratio of unspliced form to the identified spliced variants of the analyzed RNA was approximately 1:6. It is suggested that splicing of viral RNA foreign to Drosophila can be a sort of defense mechanism preventing the large-scale production of the capsid protein, potentially hazardous to the host organism. PMID:27529987

  9. AAV9 supports wide-scale transduction of the CNS and TDP-43 disease modeling in adult rats

    PubMed Central

    Jackson, Kasey L; Dayton, Robert D; Klein, Ronald L

    2015-01-01

    AAV9 has emerged as an efficient adeno-associated virus (AAV) serotype for gene transfer to the central nervous system. We have used this technique to study aspects of amyotrophic lateral sclerosis (ALS) by administering AAV encoding the ALS-related gene transactive response DNA binding protein of 43 kDa (TDP-43) to neonatal rats. However, inducing the expression in adult subjects would be preferable to mimic the adult onset of symptoms in ALS. We expressed either green fluorescent protein (GFP) or TDP-43 in adult rats after an intravenous (i.v.) route of administration to attempt wide-scale transduction of the spinal cord for disease modeling. In order to optimize the gene transfer, we made comparisons of efficiency by age, gender, and across several AAV serotypes (AAV1, AAV8, AAV9, and AAV10). The data indicate more efficient neuronal transduction in neonates, with little evidence of glial transduction at either age, no gender-related differences in transduction, and that AAV9 was efficient in adults relative to the other serotypes tested. Based on these data, AAV9 TDP-43 was expressed at three vector doses in adult female rats yielding highly consistent, dose-dependent motor deficits. AAV9 can be delivered i.v. to adult rats to achieve consistent pathophysiological changes and a relevant adult-onset system for disease modeling. PMID:26445725

  10. Immune Response to Recombinant Capsid Proteins of Adenovirus in Humans: Antifiber and Anti-Penton Base Antibodies Have a Synergistic Effect on Neutralizing Activity

    PubMed Central

    Gahéry-Ségard, Hanne; Farace, Françoise; Godfrin, Dominique; Gaston, Jesintha; Lengagne, Renée; Tursz, Thomas; Boulanger, Pierre; Guillet, Jean-Gérard

    1998-01-01

    Replication-deficient adenovirus used in humans for gene therapy induces a strong immune response to the vector, resulting in transient recombinant protein expression and the blocking of gene transfer upon a second administration. Therefore, in this study we examined in detail the capsid-specific humoral immune response in sera of patients with lung cancer who had been given one dose of a replication-defective adenovirus. We analyzed the immune response to the three major components of the viral capsid, hexon (Hx), penton base (Pb), and fiber (Fi). A longitudinal study of the humoral response assayed on adenovirus particle-coated enzyme-linked immunosorbent assay plates showed that patients had preexisting immunity to adenovirus prior to the administration of adenovirus–β-gal. The level of the response increased in three patients after adenovirus administration and remained at a maximum after three months. One patient had a strong immune response to adenovirus prior to treatment, and this response was unaffected by adenovirus administration. Sera collected from the patients were assayed for recognition of each individual viral capsid protein to determine more precisely the molecular basis of the humoral immune response. Clear differences existed in the humoral response to the three major components of the viral capsid in serum from humans. Sequential appearance of these antibodies was observed: anti-Fi antibodies appeared first, followed by anti-Pb antibodies and then by anti-Hx antibodies. Moreover, anti-Fi antibodies preferentially recognized the native trimeric form of Fi protein, suggesting that they recognized conformational epitopes. Our results showed that sera with no neutralizing activity contained only anti-Fi antibodies. In contrast, neutralizing activity was only obtained with sera containing anti-Fi and anti-Pb antibodies. More importantly, we showed that anti-native Fi and anti-Pb antibodies had a synergistic effect on neutralization. The

  11. An unexpected twist in viral capsid maturation

    SciTech Connect

    Gertsman, Ilya; Gan, Lu; Guttman, Miklos; Lee, Kelly; Speir, Jeffrey A.; Duda, Robert L.; Hendrix, Roger W.; Komives, Elizabeth A.; Johnson, John E.

    2009-04-14

    Lambda-like double-stranded (ds) DNA bacteriophage undergo massive conformational changes in their capsid shell during the packaging of their viral genomes. Capsid shells are complex organizations of hundreds of protein subunits that assemble into intricate quaternary complexes that ultimately are able to withstand over 50 atm of pressure during genome packaging. The extensive integration between subunits in capsids requires the formation of an intermediate complex, termed a procapsid, from which individual subunits can undergo the necessary refolding and structural rearrangements needed to transition to the more stable capsid. Although various mature capsids have been characterized at atomic resolution, no such procapsid structure is available for a dsDNA virus or bacteriophage. Here we present a procapsid X-ray structure at 3.65 {angstrom} resolution, termed prohead II, of the lambda-like bacteriophage HK97, the mature capsid structure of which was previously solved to 3.44 {angstrom}. A comparison of the two largely different capsid forms has unveiled an unprecedented expansion mechanism that describes the transition. Crystallographic and hydrogen/deuterium exchange data presented here demonstrate that the subunit tertiary structures are significantly different between the two states, with twisting and bending motions occurring in both helical and -sheet regions. We also identified subunit interactions at each three-fold axis of the capsid that are maintained throughout maturation. The interactions sustain capsid integrity during subunit refolding and provide a fixed hinge from which subunits undergo rotational and translational motions during maturation. Previously published calorimetric data of a closely related bacteriophage, P22, showed that capsid maturation was an exothermic process that resulted in a release of 90 kJ mol{sup -1} of energy. We propose that the major tertiary changes presented in this study reveal a structural basis for an exothermic

  12. An Unexpected Twist in Viral Capsid Maturation

    PubMed Central

    Gertsman, Ilya; Gan, Lu; Guttman, Miklos; Lee, Kelly; Speir, Jeffrey A.; Duda, Robert L.; Hendrix, Roger W.; Komives, Elizabeth A.; Johnson, John E.

    2009-01-01

    Lambda-like dsDNA bacteriophage undergo massive conformational changes in their capsid shell during the packaging of their viral genomes. Capsid shells are complex organizations of hundreds of protein subunits that assemble into intricate quaternary complexes that ultimately are able to withstand over 50 atm. of pressure during genome packaging1. The extensive integration between subunits in capsids is unlikely to form in a single assembly step, therefore requiring formation of an intermediate complex, termed a procapsid, from which individual subunits can undergo the necessary refolding and structural rearrangements needed to transition to the more stable capsid. Though various mature capsids have been characterized at atomic resolution, no such procapsid structure is available for a dsDNA virus or bacteriophage that undergoes large scale conformational changes. We present a procapsid x-ray structure at 3.65Å resolution, termed Prohead II, of the lambda like bacteriophage HK97, whose mature capsid structure was previously solved to 3.44 Å2. A comparison of the two largely different capsid forms has unveiled an unprecedented expansion mechanism that describes the transition. Crystallographic and Hydrogen/Deuterium exchange data presented here demonstrates that the subunit tertiary structures are significantly different between the two states, with twisting and bending motions occurring in both helical and β-sheet regions. We have also discovered conserved subunit interactions at each 3-fold of the virus capsid, from which capsid subunits maintain their integrity during refolding, facilitating the rotational and translational motions of maturation. Calormetric data of a closely related bacteriophage, P22, showed that capsid maturation was an exothermic process that resulted in a release of 90KJ/mol of energy3. We propose the major tertiary changes presented in this study reveal a structural basis for an exothermic maturation process likely present in many ds

  13. Viral capsid assembly as a model for protein aggregation diseases: Active processes catalyzed by cellular assembly machines comprising novel drug targets.

    PubMed

    Marreiros, Rita; Müller-Schiffmann, Andreas; Bader, Verian; Selvarajah, Suganya; Dey, Debendranath; Lingappa, Vishwanath R; Korth, Carsten

    2015-09-01

    Viruses can be conceptualized as self-replicating multiprotein assemblies, containing coding nucleic acids. Viruses have evolved to exploit host cellular components including enzymes to ensure their replicative life cycle. New findings indicate that also viral capsid proteins recruit host factors to accelerate their assembly. These assembly machines are RNA-containing multiprotein complexes whose composition is governed by allosteric sites. In the event of viral infection, the assembly machines are recruited to support the virus over the host and are modified to achieve that goal. Stress granules and processing bodies may represent collections of such assembly machines, readily visible by microscopy but biochemically labile and difficult to isolate by fractionation. We hypothesize that the assembly of protein multimers such as encountered in neurodegenerative or other protein conformational diseases, is also catalyzed by assembly machines. In the case of viral infection, the assembly machines have been modified by the virus to meet the virus' need for rapid capsid assembly rather than host homeostasis. In the case of the neurodegenerative diseases, it is the monomers and/or low n oligomers of the so-called aggregated proteins that are substrates of assembly machines. Examples for substrates are amyloid β peptide (Aβ) and tau in Alzheimer's disease, α-synuclein in Parkinson's disease, prions in the prion diseases, Disrupted-in-schizophrenia 1 (DISC1) in subsets of chronic mental illnesses, and others. A likely continuum between virus capsid assembly and cell-to-cell transmissibility of aggregated proteins is remarkable. Protein aggregation diseases may represent dysfunction and dysregulation of these assembly machines analogous to the aberrations induced by viral infection in which cellular homeostasis is pathologically reprogrammed. In this view, as for viral infection, reset of assembly machines to normal homeostasis should be the goal of protein aggregation

  14. Large-Scale Functional Purification of Recombinant HIV-1 Capsid

    PubMed Central

    Jin, Debi; Wong, Melanie; Leavitt, Stephanie; Brendza, Katherine M.; Liu, Xiaohong; Sakowicz, Roman

    2013-01-01

    During human immunodeficiency virus type-1 (HIV-1) virion maturation, capsid proteins undergo a major rearrangement to form a conical core that protects the viral nucleoprotein complexes. Mutations in the capsid sequence that alter the stability of the capsid core are deleterious to viral infectivity and replication. Recently, capsid assembly has become an attractive target for the development of a new generation of anti-retroviral agents. Drug screening efforts and subsequent structural and mechanistic studies require gram quantities of active, homogeneous and pure protein. Conventional means of laboratory purification of Escherichia coli expressed recombinant capsid protein rely on column chromatography steps that are not amenable to large-scale production. Here we present a function-based purification of wild-type and quadruple mutant capsid proteins, which relies on the inherent propensity of capsid protein to polymerize and depolymerize. This method does not require the packing of sizable chromatography columns and can generate double-digit gram quantities of functionally and biochemically well-behaved proteins with greater than 98% purity. We have used the purified capsid protein to characterize two known assembly inhibitors in our in-house developed polymerization assay and to measure their binding affinities. Our capsid purification procedure provides a robust method for purifying large quantities of a key protein in the HIV-1 life cycle, facilitating identification of the next generation anti-HIV agents. PMID:23472130

  15. Large-scale functional purification of recombinant HIV-1 capsid.

    PubMed

    Hung, Magdeleine; Niedziela-Majka, Anita; Jin, Debi; Wong, Melanie; Leavitt, Stephanie; Brendza, Katherine M; Liu, Xiaohong; Sakowicz, Roman

    2013-01-01

    During human immunodeficiency virus type-1 (HIV-1) virion maturation, capsid proteins undergo a major rearrangement to form a conical core that protects the viral nucleoprotein complexes. Mutations in the capsid sequence that alter the stability of the capsid core are deleterious to viral infectivity and replication. Recently, capsid assembly has become an attractive target for the development of a new generation of anti-retroviral agents. Drug screening efforts and subsequent structural and mechanistic studies require gram quantities of active, homogeneous and pure protein. Conventional means of laboratory purification of Escherichia coli expressed recombinant capsid protein rely on column chromatography steps that are not amenable to large-scale production. Here we present a function-based purification of wild-type and quadruple mutant capsid proteins, which relies on the inherent propensity of capsid protein to polymerize and depolymerize. This method does not require the packing of sizable chromatography columns and can generate double-digit gram quantities of functionally and biochemically well-behaved proteins with greater than 98% purity. We have used the purified capsid protein to characterize two known assembly inhibitors in our in-house developed polymerization assay and to measure their binding affinities. Our capsid purification procedure provides a robust method for purifying large quantities of a key protein in the HIV-1 life cycle, facilitating identification of the next generation anti-HIV agents. PMID:23472130

  16. Magic Angle Spinning NMR Reveals Sequence-Dependent Structural Plasticity, Dynamics, and the Spacer Peptide 1 Conformation in HIV-1 Capsid Protein Assemblies

    SciTech Connect

    Han, Yun; Hou, Guangjin; Suiter, Christopher L.; Ahn, Jinwoo; Byeon, In-Ja L.; Lipton, Andrew S.; Burton, Sarah D.; Hung, Ivan; Gorkov, Peter L.; Gan, Zhehong; Brey, William W.; Rice, David M.; Gronenborn, Angela M.; Polenova, Tatyana E.

    2013-11-27

    Maturation of HIV-1 virus into an infectious virion requires cleavage of the Gag polyprotein into its constituent domains and formation of a conical capsid core that encloses viral RNA and a small complement of proteins for replication. The final step of this process is the cleavage of the SP1 peptide from the CA-SP1 maturation intermediate, which triggers the condensation of the CA protein into a conical capsid. The mechanism of this step, including the conformation of the SP1 peptide in CA-SP1, is under intense debate. In this report, we examine the tubular assemblies of CA and the CA-SP1 maturation intermediate using Magic Angle Spinning NMR spectroscopy. At the magnetic fields of 19.9 T and above, tubular CA and CA-SP1 assemblies yield outstanding-quality 2D and 3D MAS NMR spectra, which are amenable to resonance assignments and detailed structural characterization. Dipolar- and scalar-based correlation experiments unequivocally indicate that SP1 peptide is in a random coil conformation and mobile in the assembled CA-SP1. Analysis of two sequence variants reveals that remarkably, the conformation of SP1 tail, of the functionally important CypA loop, and of the loop preceding helix 8 are sequence dependent and modulated by the residue variations at distal sites. These findings challenge the role of SP1 as a conformational switch in the maturation process and establish sequence-dependent conformational plasticity in CA.

  17. Genetic Variation of Capsid Protein VP7 in Genotype G4 Human Rotavirus Strains: Simultaneous Emergence and Spread of Different Lineages in Argentina

    PubMed Central

    Bok, Karin; Matson, David O.; Gomez, Jorge A.

    2002-01-01

    Rotavirus is the most-common cause of severe diarrhea in young children. Complete rotavirus characterization includes determination of the antigenic type of the two outer capsid proteins, VP7 and VP4, designated G and P types, respectively. During a nationwide rotavirus surveillance study, genotype G4 frequency increased during the second year. To evaluate further the mechanism of emergence and the relationship among G4 strains, the genetic diversity of VP7 capsid protein in these samples was studied in detail. Overall nucleotide sequence divergence ranged from less than 0.1 to 19.5%, a higher divergence than that observed for other rotavirus G types (0.1 to 9%). Sequences were classified into two major lineages (designated I and II) based on their nucleotide distances. The most heterogeneous lineage was further subdivided into four sublineages (designated Ia to Id). Most Argentine sequences were of sublineages Ib and Ic, which were confirmed to be independent sequence clusters by parsimony analysis. This study describes different lineages and sublineages within G4 strains and shows that Argentine strains are distantly related to reference strain ST3. The appearance of at least two G4 genotype (sub)lineages during 1998 demonstrates that the increased frequency of these strains was due to the synchronized emergence of different groups of strains. PMID:12037057

  18. Vaccination of horses with a recombinant modified vaccinia Ankara virus (MVA) expressing African horse sickness (AHS) virus major capsid protein VP2 provides complete clinical protection against challenge

    PubMed Central

    Alberca, Berta; Bachanek-Bankowska, Katarzyna; Cabana, Marta; Calvo-Pinilla, Eva; Viaplana, Elisenda; Frost, Lorraine; Gubbins, Simon; Urniza, Alicia; Mertens, Peter; Castillo-Olivares, Javier

    2014-01-01

    African horse sickness virus (AHSV) is an arthropod-borne pathogen that infects all species of equidae and causes high mortality in horses. Previously, a recombinant modified vaccinia Ankara (MVA) virus expressing the protein VP2 of AHSV serotype 4 was shown to induce virus neutralising antibodies in horses and protected interferon alpha receptor gene knock-out mice (IFNAR −/−) against virulent AHSV challenge. This study builds on the previous work, examining the protective efficacy of MVA-VP2 vaccination in the natural host of AHSV infection. A study group of 4 horses was vaccinated twice with a recombinant MVA virus expressing the major capsid protein (VP2) of AHSV serotype 9. Vaccinated animals and a control group of unvaccinated horses were then challenged with a virulent strain of AHSV-9. The vaccinated animals were completely protected against clinical disease and also against viraemia as measured by standard end-point dilution assays. In contrast, all control horses presented viraemia after challenge and succumbed to the infection. These results demonstrate the potential of recombinant MVA viruses expressing the outer capsid VP2 of AHSV as a protective vaccine against AHSV infection in the field. PMID:24837765

  19. Structural and mechanistic basis of anti-termination of Rho-dependent transcription termination by bacteriophage P4 capsid protein Psu

    PubMed Central

    Ranjan, Amitabh; Sharma, Savita; Banerjee, Ramanuj; Sen, Udayaditya; Sen, Ranjan

    2013-01-01

    The conserved bacterial transcription terminator, Rho, is a potent target for bactericidal agents. Psu, a bacteriophage P4 capsid protein, is capable of inducing anti-termination to the Rho-dependent transcription termination. Knowledge of structural and mechanistic basis of this anti-termination is required to design peptide-inhibitor(s) of Rho from Psu. Using suppressor genetics, cross-linking, protein foot-printing and FRET analyses, we describe a conserved disordered structure, encompassing 139–153 amino acids of Rho, as the primary docking site for Psu. Also a neighbouring helical structure, comprising 347–354 amino acids, lining its central channel, plays a supportive role in the Rho–Psu complex formation. Based on the crystal structure of Psu, its conformation in the capsid of the P4 phage, and its interacting regions on Rho, we build an energy-minimized structural model of the Rho:Psu complex. In this model, a V-shaped dimer of Psu interacts with the two diagonally opposite subunits of a hexameric Rho, enabling Psu to form a ‘lid’ on the central channel of the latter. We show that this configuration of Psu makes the central channel of Rho inaccessible, and it causes a mechanical impediment to its translocase activity. PMID:23703205

  20. The advent of AAV9 expands applications for brain and spinal cord gene delivery

    PubMed Central

    Dayton, Robert D; Wang, David B; Klein, Ronald L

    2012-01-01

    Introduction Straightforward studies compared adeno-associated virus (AAV) serotypes to determine the most appropriate one for robust expression in the CNS. AAV9 was efficient when directly injected into the brain, but more surprisingly, AAV9 produced global expression in the brain and spinal cord after a peripheral, systemic route of administration to neonatal mice. Areas covered Topics include AAV9 gene delivery from intraparenchymal, intravenous, intrathecal and intrauterine routes of administration, and related preclinical studies and disease models. Systemic AAV9 gene transfer yields remarkably consistent neuronal expression, though only in early development. AAV9 is versatile to study neuropathological proteins: microtubule-associated protein tau and transactive response DNA-binding protein 43 kDa (TDP-43). Expert opinion AAV9 will be more widely used based on current data, although other natural serotypes and recombineered vectors may also support or improve upon wide-scale expression. A peripheral-to-central gene delivery that can affect the entire CNS without having to inject the CNS is promising for basic functional experiments, and potentially for gene therapy. Systemic or intra-cerebrospinal fluid routes of AAV9 administration should be considered for spinal muscular atrophy, lysosomal storage diseases and amyotrophic lateral sclerosis, if more neuronal expression can be achieved in adults, or if glial expression can be exploited. PMID:22519910

  1. Therapeutic efficacy of an oncolytic adenovirus containing RGD ligand in minor capsid protein IX and Fiber, Δ24DoubleRGD, in an ovarian cancer model

    PubMed Central

    Gamble, Lena J.; Ugai, Hideyo; Wang, Minghui; Borovjagin, Anton V.; Matthews, Qiana L.

    2013-01-01

    Ovarian cancer is the leading cause of gynecological disease death despite advances in medicine. Therefore, novel strategies are required for ovarian cancer therapy. Conditionally replicative adenoviruses (CRAds), genetically modified as anti-cancer therapeutics, are one of the most attractive candidate agents for cancer therapy. However, a paucity of coxsackie B virus and adenovirus receptor (CAR) expression on the surface of ovarian cancer cells has impeded treatment of ovarian cancer using this approach. This study sought to engineer a CRAd with enhanced oncolytic ability in ovarian cancer cells, “Δ24DoubleRGD.” Δ24DoubleRGD carries an arginine-glycine-aspartate (RGD) motif incorporated into both fiber and capsid protein IX (pIX) and its oncolytic efficacy was evaluated in ovarian cancer. In vitro analysis of cell viability showed that infection of ovarian cancer cells with Δ24DoubleRGD leads to increased cell killing relative to the control CRAds. Data from this study suggested that not only an increase in number of RGD motifs on the CRAd capsid, but also a change in the repertoir of targeted integrins could lead to enhanced oncolytic potency of Δ24DoubleRGD in ovarian cancer cells in vitro. In an intraperitoneal model of ovarian cancer, mice injected with Δ24DoubleRGD showed, however, a similar survival rate as mice treated with control CRAds. PMID:23998042

  2. A porcine circovirus-2 mutant isolated in Brazil contains low-frequency substitutions in regions of immunoprotective epitopes in the capsid protein.

    PubMed

    Salgado, Rafael Locatelli; Vidigal, Pedro Marcus Pereira; Gonzaga, Natalia F; de Souza, Luiz F L; Polêto, Marcelo D; Onofre, Thiago Souza; Eller, Monique R; Pereira, Carlos Eduardo Real; Fietto, Juliana L R; Bressan, Gustavo C; Guedes, Roberto M C; Almeida, Márcia R; Silva Júnior, Abelardo

    2015-11-01

    Porcine circovirus-2 (PCV2) is the etiologic agent of several diseases in pigs, including multi-systemic wasting syndrome (PMWS). In this work, a new mutant PCV2b was isolated from PMWS-affected pigs on a Brazilian farm. Its genome showed high sequence similarity (>99% identity) to those from a group of emerging mutants isolated from cases of PMWS outbreaks in vaccinated pigs in China, the USA and South Korea. Here, we show that these isolates share a combination of low-frequency substitutions (single amino acid polymorphisms with a frequency of ≤25%) in the viral capsid protein, mainly in regions of immunoprotective epitopes, and an additional lysine residue at position 234. These isolates were phylogenetically grouped in the PCV2b clade, reinforcing the idea of the emergence of a new group of mutants PCV2b associated with outbreaks worldwide. The identification of these polymorphisms in the viral capsid highlights the importance of considering these isolates for the development of more-effective vaccines. PMID:26271152

  3. Evolution of the capsid protein genes of foot-and-mouth disease virus: antigenic variation without accumulation of amino acid substitutions over six decades.

    PubMed Central

    Martínez, M A; Dopazo, J; Hernández, J; Mateu, M G; Sobrino, F; Domingo, E; Knowles, N J

    1992-01-01

    The genetic diversification of foot-and-mouth disease virus (FMDV) of serotype C over a 6-decade period was studied by comparing nucleotide sequences of the capsid protein-coding regions of viruses isolated in Europe, South America, and The Philippines. Phylogenetic trees were derived for VP1 and P1 (VP1, VP2, VP3, and VP4) RNAs by using the least-squares method. Confidence intervals of the derived phylogeny (significance levels of nodes and standard deviations of branch lengths) were placed by application of the bootstrap resampling method. These procedures defined six highly significant major evolutionary lineages and a complex network of sublines for the isolates from South America. In contrast, European isolates are considerably more homogeneous, probably because of the vaccine origin of several of them. The phylogenetic analysis suggests that FMDV CGC Ger/26 (one of the earliest FMDV isolates available) belonged to an evolutionary line which is now apparently extinct. Attempts to date the origin (ancestor) of the FMDVs analyzed met with considerable uncertainty, mainly owing to the stasis noted in European viruses. Remarkably, the evolution of the capsid genes of FMDV was essentially associated with linear accumulation of silent mutations but continuous accumulation of amino acid substitutions was not observed. Thus, the antigenic variation attained by FMDV type C over 6 decades was due to fluctuations among limited combinations of amino acid residues without net accumulation of amino acid replacements over time. PMID:1316467

  4. Analysis of SAT Type Foot-And-Mouth Disease Virus Capsid Proteins and the Identification of Putative Amino Acid Residues Affecting Virus Stability

    PubMed Central

    Maree, Francois F.; Blignaut, Belinda; de Beer, Tjaart A. P.; Rieder, Elizabeth

    2013-01-01

    Foot-and-mouth disease virus (FMDV) initiates infection by adhering to integrin receptors on target cells, followed by cell entry and disassembly of the virion through acidification within endosomes. Mild heating of the virions also leads to irreversible dissociation into pentamers, a characteristic linked to reduced vaccine efficacy. In this study, the structural stability of intra- and inter-serotype chimeric SAT2 and SAT3 virus particles to various conditions including low pH, mild temperatures or high ionic strength, was compared. Our results demonstrated that while both the SAT2 and SAT3 infectious capsids displayed different sensitivities in a series of low pH buffers, their stability profiles were comparable at high temperatures or high ionic strength conditions. Recombinant vSAT2 and intra-serotype chimeric viruses were used to map the amino acid differences in the capsid proteins of viruses with disparate low pH stabilities. Four His residues at the inter-pentamer interface were identified that change protonation states at pH 6.0. Of these, the H145 of VP3 appears to be involved in interactions with A141 in VP3 and K63 in VP2, and may be involved in orientating H142 of VP3 for interaction at the inter-pentamer interfaces. PMID:23717387

  5. Analysis of SAT type foot-and-mouth disease virus capsid proteins and the identification of putative amino acid residues affecting virus stability.

    PubMed

    Maree, Francois F; Blignaut, Belinda; de Beer, Tjaart A P; Rieder, Elizabeth

    2013-01-01

    Foot-and-mouth disease virus (FMDV) initiates infection by adhering to integrin receptors on target cells, followed by cell entry and disassembly of the virion through acidification within endosomes. Mild heating of the virions also leads to irreversible dissociation into pentamers, a characteristic linked to reduced vaccine efficacy. In this study, the structural stability of intra- and inter-serotype chimeric SAT2 and SAT3 virus particles to various conditions including low pH, mild temperatures or high ionic strength, was compared. Our results demonstrated that while both the SAT2 and SAT3 infectious capsids displayed different sensitivities in a series of low pH buffers, their stability profiles were comparable at high temperatures or high ionic strength conditions. Recombinant vSAT2 and intra-serotype chimeric viruses were used to map the amino acid differences in the capsid proteins of viruses with disparate low pH stabilities. Four His residues at the inter-pentamer interface were identified that change protonation states at pH 6.0. Of these, the H145 of VP3 appears to be involved in interactions with A141 in VP3 and K63 in VP2, and may be involved in orientating H142 of VP3 for interaction at the inter-pentamer interfaces. PMID:23717387

  6. Crystallization and X-ray analysis of the T = 4 particle of hepatitis B capsid protein with an N-terminal extension

    SciTech Connect

    Tan, Wen Siang; McNae, Iain W.; Ho, Kok Lian; Walkinshaw, Malcolm D.

    2007-08-01

    Hepatitis B virus capsids have significant potential as carriers for immunogenic peptides. The crystal structure of the T = 4 particle of hepatitis B core protein containing an N-terminal extension reveals that the fusion peptide is exposed on the exterior of the particle. Hepatitis B core (HBc) particles have been extensively exploited as carriers for foreign immunological epitopes in the development of multicomponent vaccines and diagnostic reagents. Crystals of the T = 4 HBc particle were grown in PEG 20 000, ammonium sulfate and various types of alcohols. A temperature jump from 277 or 283 to 290 K was found to enhance crystal growth. A crystal grown using MPD as a cryoprotectant diffracted X-rays to 7.7 Å resolution and data were collected to 99.6% completeness at 8.9 Å. The crystal belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 352.3, b = 465.5, c = 645.0 Å. The electron-density map reveals a protrusion that is consistent with the N-terminus extending out from the surface of the capsid. The structure presented here supports the idea that N-terminal insertions can be exploited in the development of diagnostic reagents, multicomponent vaccines and delivery vehicles into mammalian cells.

  7. Sulfolobus Spindle-Shaped Virus 1 Contains Glycosylated Capsid Proteins, a Cellular Chromatin Protein, and Host-Derived Lipids

    PubMed Central

    Quemin, Emmanuelle R. J.; Pietilä, Maija K.; Oksanen, Hanna M.; Forterre, Patrick; Rijpstra, W. Irene C.; Schouten, Stefan; Bamford, Dennis H.; Prangishvili, David

    2015-01-01

    ABSTRACT Geothermal and hypersaline environments are rich in virus-like particles, among which spindle-shaped morphotypes dominate. Currently, viruses with spindle- or lemon-shaped virions are exclusive to Archaea and belong to two distinct viral families. The larger of the two families, the Fuselloviridae, comprises tail-less, spindle-shaped viruses, which infect hosts from phylogenetically distant archaeal lineages. Sulfolobus spindle-shaped virus 1 (SSV1) is the best known member of the family and was one of the first hyperthermophilic archaeal viruses to be isolated. SSV1 is an attractive model for understanding virus-host interactions in Archaea; however, the constituents and architecture of SSV1 particles remain only partially characterized. Here, we have conducted an extensive biochemical characterization of highly purified SSV1 virions and identified four virus-encoded structural proteins, VP1 to VP4, as well as one DNA-binding protein of cellular origin. The virion proteins VP1, VP3, and VP4 undergo posttranslational modification by glycosylation, seemingly at multiple sites. VP1 is also proteolytically processed. In addition to the viral DNA-binding protein VP2, we show that viral particles contain the Sulfolobus solfataricus chromatin protein Sso7d. Finally, we provide evidence indicating that SSV1 virions contain glycerol dibiphytanyl glycerol tetraether (GDGT) lipids, resolving a long-standing debate on the presence of lipids within SSV1 virions. A comparison of the contents of lipids isolated from the virus and its host cell suggests that GDGTs are acquired by the virus in a selective manner from the host cytoplasmic membrane, likely during progeny egress. IMPORTANCE Although spindle-shaped viruses represent one of the most prominent viral groups in Archaea, structural data on their virion constituents and architecture still are scarce. The comprehensive biochemical characterization of the hyperthermophilic virus SSV1 presented here brings novel and

  8. All-Atom Molecular Dynamics of Virus Capsids as Drug Targets.

    PubMed

    Perilla, Juan R; Hadden, Jodi A; Goh, Boon Chong; Mayne, Christopher G; Schulten, Klaus

    2016-05-19

    Virus capsids are protein shells that package the viral genome. Although their morphology and biological functions can vary markedly, capsids often play critical roles in regulating viral infection pathways. A detailed knowledge of virus capsids, including their dynamic structure, interactions with cellular factors, and the specific roles that they play in the replication cycle, is imperative for the development of antiviral therapeutics. The following Perspective introduces an emerging area of computational biology that focuses on the dynamics of virus capsids and capsid-protein assemblies, with particular emphasis on the effects of small-molecule drug binding on capsid structure, stability, and allosteric pathways. When performed at chemical detail, molecular dynamics simulations can reveal subtle changes in virus capsids induced by drug molecules a fraction of their size. Here, the current challenges of performing all-atom capsid-drug simulations are discussed, along with an outlook on the applicability of virus capsid simulations to reveal novel drug targets. PMID:27128262

  9. Neutralizing Antibodies Against Adeno-Associated Viral Capsids in Patients with mut Methylmalonic Acidemia.

    PubMed

    Harrington, Elizabeth A; Sloan, Jennifer L; Manoli, Irini; Chandler, Randy J; Schneider, Mark; McGuire, Peter J; Calcedo, Roberto; Wilson, James M; Venditti, Charles P

    2016-05-01

    Isolated methylmalonic acidemia (MMA), a group of autosomal recessive inborn errors of metabolism, is most commonly caused by complete (mut(0)) or partial (mut(-)) deficiency of the enzyme methylmalonyl-CoA mutase (MUT). The severe metabolic instability and increased mortality experienced by many affected individuals, especially those with mut(0) MMA, has led centers to use elective liver transplantation as a treatment for these patients. We have previously demonstrated the efficacy of systemic adeno-associated viral (AAV) gene delivery as a treatment for MMA in a murine model and therefore sought to survey AAV antibody titers against serotypes 2, 8, and 9 in a group of well-characterized MMA patients, accrued via a dedicated natural history study ( clinicaltrials.gov ID: NCT00078078). Plasma samples provided by 42 patients (8 mut(-) and 34 mut(0); 10 had received organ transplantation), who ranged in age between 2 and 31 years, were analyzed to examine AAV2 (n = 35), AAV8 (n = 41), and AAV9 (n = 42) antibody titers. In total, the seroprevalence of antibodies against AAV2, AAV8, or AAV9 was 20%, 22%, and 24%, respectively. We observed a lower-than-expected seropositivity rate (titers ≥1:20) in the pediatric MMA patients (2-18 years) for both AAV2 (p < 0.05) and AAV8 (p < 0.01) neutralizing antibodies (NAbs) compared with historical controls. Those with positive NAb titers were typically older than 18 years (p < 0.05 all serotypes) or had received solid organ transplantation (p < 0.01 AAV8, AAV9). The mut(0) patients who had not been transplanted (n = 24)-that is, the subset with the greatest need for improved treatments-represented the seronegative majority, with 21 out of 24 patients lacking Abs against all AAV capsids tested. The unexpected lack of NAbs against AAV in this patient population has encouraging implications for systemic gene delivery as a treatment for mut MMA. PMID:26790480

  10. Widespread AAV1- and AAV2-mediated transgene expression in the nonhuman primate brain: implications for Huntington’s disease

    PubMed Central

    Hadaczek, Piotr; Stanek, Lisa; Ciesielska, Agnieszka; Sudhakar, Vivek; Samaranch, Lluis; Pivirotto, Philip; Bringas, John; O’Riordan, Catherine; Mastis, Bryan; San Sebastian, Waldy; Forsayeth, John; Cheng, Seng H; Bankiewicz, Krystof S; Shihabuddin, Lamya S

    2016-01-01

    Huntington’s disease (HD) is caused by a toxic gain-of-function associated with the expression of the mutant huntingtin (htt) protein. Therefore, the use of RNA interference to inhibit Htt expression could represent a disease-modifying therapy. The potential of two recombinant adeno-associated viral vectors (AAV), AAV1 and AAV2, to transduce the cortico-striatal tissues that are predominantly affected in HD was explored. Green fluorescent protein was used as a reporter in each vector to show that both serotypes were broadly distributed in medium spiny neurons in the striatum and cortico-striatal neurons after infusion into the putamen and caudate nucleus of nonhuman primates (NHP), with AAV1-directed expression being slightly more robust than AAV2-driven expression. This study suggests that both serotypes are capable of targeting neurons that degenerate in HD, and it sets the stage for the advanced preclinical evaluation of an RNAi-based therapy for this disease. PMID:27408903

  11. Epitope Mapping of the Major Capsid Protein of Type 2 Porcine Circovirus (PCV2) by Using Chimeric PCV1 and PCV2

    PubMed Central

    Lekcharoensuk, Porntippa; Morozov, Igor; Paul, Prem S.; Thangthumniyom, Nattarat; Wajjawalku, Worawidh; Meng, X. J.

    2004-01-01

    Type 2 porcine circovirus (PCV2) is associated with postweaning multisystemic wasting syndrome in pigs, whereas the genetically related type 1 PCV (PCV1) is nonpathogenic. In this study, seven monoclonal antibodies (MAbs) against PCV2-ORF2 capsid protein were generated, biologically characterized, and subsequently used to map the antigenic sites of PCV2 capsid protein by using infectious PCV DNA clones containing PCV1/PCV2-ORF2 chimeras. The PCV1/PCV2-ORF2 chimeras were constructed by serial deletions of PCV2-ORF2 and replacement with the corresponding sequences of the PCV1-ORF2. The reactivities of chimeric PCV1/PCV2 clones in transfected PK-15 cells with the seven MAbs were detected by an immunofluorescence assay (IFA). The chimera (r140) with a deletion of 47 amino acids at the N terminus of PCV2-ORF2 reacted strongly to all seven MAbs. Expanding the deletion of PCV2-ORF2 from residues 47 to 57 (r175) abolished the recognition of MAb 3B7, 3C11, 4A10, 6H2, or 8F6 to the chimera. Further deletion of PCV2-ORF2 to 62 residues disrupted the binding of this chimera to all seven MAbs. IFA reactivities with all MAbs were absent when residues 165 to 233 at the C terminus of PCV2-ORF2 was replaced with that of PCV1-ORF2. Extending the sequence of PCV2-ORF2 from residues 165 (r464) to 185 (r526), 200 (r588), or 224 (r652) restored the ability of the three chimeras to react with MAbs 3C11, 6H2, 9H7, and 12G3 but not with 8F6, 3B7, or 4A10. When the four amino acids at the C terminus of r588 were replaced with that of PCV2-ORF2, the resulting chimera (r588F) reacted with all seven MAbs. The results from this study suggest that these seven MAbs recognized at least five different but overlapping conformational epitopes within residues 47 to 63 and 165 to 200 and the last four amino acids at the C terminus of the PCV2 capsid protein. PMID:15254185

  12. Analysis of self-association of West Nile virus capsid protein and the crucial role played by Trp 69 in homodimerization.

    PubMed

    Bhuvanakantham, Raghavan; Ng, Mah-Lee

    2005-04-01

    The understanding of capsid (C) protein interactions with itself would provide important data on how the core is organized in flaviviruses during assembly. In this study, West Nile (WN) virus C protein was shown to form homodimers using yeast two-hybrid analysis in conjunction with mammalian two-hybrid and in vivo co-immunoprecipitation assays. To delineate the region on the C protein which mediates C-C dimerization, truncation studies were carried out. The results obtained clearly showed that the internal hydrophobic segment flanked by helix I and helix III of WN virus C protein is essential for the self-association of C protein. The crucial role played by Trp 69 in stabilizing the self-association of C protein was also demonstrated by mutating Trp to Gly/Arg/Phe. Substitution of the Trp residue with Gly/Arg abolished the dimerization, whereas substitution with Phe decreased the self-association significantly. The results of this study pinpoint a critical residue in the C protein that potentially plays a role in stabilizing the homotypic interaction. PMID:15721300

  13. Live cell imaging of interactions between replicase and capsid protein of Brome mosaic virus using Bimolecular Fluorescence Complementation: Implications for replication and genome packaging

    SciTech Connect

    Chaturvedi, Sonali; Rao, A.L.N.

    2014-09-15

    In Brome mosaic virus, it was hypothesized that a physical interaction between viral replicase and capsid protein (CP) is obligatory to confer genome packaging specificity. Here we tested this hypothesis by employing Bimolecular Fluorescent Complementation (BiFC) as a tool for evaluating protein–protein interactions in living cells. The efficacy of BiFC was validated by a known interaction between replicase protein 1a (p1a) and protein 2a (p2a) at the endoplasmic reticulum (ER) site of viral replication. Additionally, co-expression in planta of a bona fide pair of interacting protein partners of p1a and p2a had resulted in the assembly of a functional replicase. Subsequent BiFC assays in conjunction with mCherry labeled ER as a fluorescent cellular marker revealed that CP physically interacts with p2a, but not p1a, and this CP:p2a interaction occurs at the cytoplasmic phase of the ER. The significance of the CP:p2a interaction in BMV replication and genome packaging is discussed. - Highlights: • YFP fusion proteins of BMV p1a and p2a are biologically active. • Self-interaction was observed for p1a, p2a and CP. • CP interacts with p2a but not p1a. • Majority of reconstituted YFP resulting from bona fide fusion protein partners localized on ER.

  14. Cyclophilins Facilitate Dissociation of the Human Papillomavirus Type 16 Capsid Protein L1 from the L2/DNA Complex following Virus Entry

    PubMed Central

    Bienkowska-Haba, Malgorzata; Williams, Carlyn; Kim, Seong Man; Garcea, Robert L.

    2012-01-01

    Human papillomaviruses (HPV) are composed of the major and minor capsid proteins, L1 and L2, that encapsidate a chromatinized, circular double-stranded DNA genome. At the outset of infection, the interaction of HPV type 16 (HPV16) (pseudo)virions with heparan sulfate proteoglycans triggers a conformational change in L2 that is facilitated by the host cell chaperone cyclophilin B (CyPB). This conformational change results in exposure of the L2 N terminus, which is required for infectious internalization. Following internalization, L2 facilitates egress of the viral genome from acidified endosomes, and the L2/DNA complex accumulates at PML nuclear bodies. We recently described a mutant virus that bypasses the requirement for cell surface CyPB but remains sensitive to cyclosporine for infection, indicating an additional role for CyP following endocytic uptake of virions. We now report that the L1 protein dissociates from the L2/DNA complex following infectious internalization. Inhibition and small interfering RNA (siRNA)-mediated knockdown of CyPs blocked dissociation of L1 from the L2/DNA complex. In vitro, purified CyPs facilitated the dissociation of L1 pentamers from recombinant HPV11 L1/L2 complexes in a pH-dependent manner. Furthermore, CyPs released L1 capsomeres from partially disassembled HPV16 pseudovirions at slightly acidic pH. Taken together, these data suggest that CyPs mediate the dissociation of HPV L1 and L2 capsid proteins following acidification of endocytic vesicles. PMID:22761365

  15. AAV Vectors for Cardiac Gene Transfer: Experimental Tools and Clinical Opportunities

    PubMed Central

    Pacak, Christina A; Byrne, Barry J

    2011-01-01

    Since the first demonstration of in vivo gene transfer into myocardium there have been a series of advancements that have driven the evolution of cardiac gene delivery from an experimental tool into a therapy currently at the threshold of becoming a viable clinical option. Innovative methods have been established to address practical challenges related to tissue-type specificity, choice of delivery vehicle, potency of the delivered material, and delivery route. Most importantly for therapeutic purposes, these strategies are being thoroughly tested to ensure safety of the delivery system and the delivered genetic material. This review focuses on the development of recombinant adeno-associated virus (rAAV) as one of the most valuable cardiac gene transfer agents available today. Various forms of rAAV have been used to deliver “pre-event” cardiac protection and to temper the severity of hypertrophy, cardiac ischemia, or infarct size. Adeno-associated virus (AAV) vectors have also been functional delivery tools for cardiac gene expression knockdown studies and successfully improving the cardiac aspects of several metabolic and neuromuscular diseases. Viral capsid manipulations along with the development of tissue-specific and regulated promoters have greatly increased the utility of rAAV-mediated gene transfer. Important clinical studies are currently underway to evaluate AAV-based cardiac gene delivery in humans. PMID:21792180

  16. Development of type-specific and cross-reactive serological probes for the minor capsid protein of human papillomavirus type 33.

    PubMed Central

    Volpers, C; Sapp, M; Komly, C A; Richalet-Secordel, P; Streeck, R E

    1993-01-01

    Human papillomavirus type 33 (HPV33) is associated with malignant tumors of the cervix. In an attempt to develop immunological probes for HPV33 infections, antisera against various bacterial fusion proteins carrying sequences of the minor capsid protein encoded by L2 were raised in animals. Antigenic determinants on the HPV33 L2 protein were identified by using truncated fusion proteins and were classified as type specific or cross-reactive with respect to HPV1, -8, -11, -16, and -18. Cross-reactive epitopes map to amino acids 98 to 107 or to amino acids 102 to 112 and 107 to 117, respectively, depending on the fusion protein used for immunization. Antibodies directed toward these epitopes detect L2 proteins of HPV11, -16, and -18, but not of HPV1 and -8, in Western immunoblots and enzyme-linked immunosorbent assays. HPV33 L2 amino acids 82 to 94 and 117 to 130 induce type-specific antibodies, with the major response directed to amino acids 117 to 130. By using a synthetic peptide corresponding to L2 amino acids 117 to 130, high-titered, type-specific antisera were obtained. These antisera should be useful as immunological probes for HPV33 infection. Images PMID:8383218

  17. Anti-clarin-1 AAV-delivered ribozyme induced apoptosis in the mouse cochlea.

    PubMed

    Aarnisalo, A A; Pietola, L; Joensuu, J; Isosomppi, J; Aarnisalo, P; Dinculescu, A; Lewin, A S; Flannery, J; Hauswirth, W W; Sankila, E-M; Jero, J

    2007-08-01

    Usher syndrome type 3 is caused by mutations in the USH3A gene, which encodes the protein clarin-1. Clarin-1 is a member of the tetraspanin superfamily (TM4SF) of transmembrane proteins, expressed in the organ of Corti and spiral ganglion cells of the mouse ear. We have examined whether the AAV-mediated anti-clarin ribozyme delivery causes apoptotic cell death in vivo in the organ of Corti. We used an AAV-2 vector delivered hammerhead ribozyme, AAV-CBA-Rz, which specifically recognizes and cleaves wild type mouse clarin-1 mRNA. Cochleae of CD-1 mice were injected either with 1mul of the AAV-CBA-Rz, or control AAV vectors containing the green fluorescent protein (GFP) marker gene (AAV-CBA-GFP). Additional controls were performed with saline only. At one-week and one-month post-injection, the animals were sacrificed and the cochleae were studied by histology and fluorescence imaging. Mice injected with AAV-CBA-GFP displayed GFP reporter expression of varying fluorescence intensity throughout the length of the cochlea in the outer and inner hair cells and stria vascularis, and to a lesser extent, in vestibular epithelial cells. GFP expression was not detectable in the spiral ganglion. The pro-apoptotic effect of AAV-CBA-delivered anti-clarin-1 ribozymes was evaluated by TUNEL-staining. We observed in the AAV-CBA-Rz, AAV-CBA-GFP and saline control groups apoptotic nuclei in the outer and inner hair cells and in the stria vascularis one week after the microinjection. The vestibular epithelium was also observed to contain apoptotic cells. No TUNEL-positive spiral ganglion neurons were detected. After one-month post-injection, the AAV-CBA-Rz-injected group had significantly more apoptotic outer and inner hair cells and cells of the stria vascularis than the AAV-CBA-GFP group. In this study, we demonstrate that AAV-CBA mediated clarin-1 ribozyme may induce apoptosis of the cochlear hair cells and cells of the stria vascularis. Surprisingly, we did not observe apoptosis in

  18. Self-assembly of Ni-NTA-modified β-annulus peptides into artificial viral capsids and encapsulation of His-tagged proteins.

    PubMed

    Matsuura, Kazunori; Nakamura, Tomohiro; Watanabe, Kenta; Noguchi, Takanori; Minamihata, Kosuke; Kamiya, Noriho; Kimizuka, Nobuo

    2016-08-16

    β-Annulus peptides bearing Cys at the N-terminal from tomato bushy stunt virus were synthesised using a standard Fmoc-protected solid-phase method, and the peptide was modified with Ni-NTA at the N-terminal. The Ni-NTA-modified β-annulus peptide self-assembled into virus-like nanocapsules of approximately 40 nm in diameter. The critical aggregation concentration of these nanocapsules in 10 mM Tris-HCl buffer (pH 7.3) at 25 °C was 0.053 μM, which is 470 times lower than that of unmodified β-annulus peptides. Moreover, size exclusion chromatography of the peptide assembly indicated encapsulation of His-tagged green fluorescent protein in the Ni-NTA-modified artificial viral capsid. PMID:27386944

  19. Cloning of the Major Capsid Protein (MCP) of Grouper Iridovirus of Taiwan (TGIV) and Preliminary Evaluation of a Recombinant MCP Vaccine against TGIV.

    PubMed

    Liu, Hsin-I; Chiou, Pinwen Peter; Gong, Hong-Yi; Chou, Hsin-Yiu

    2015-01-01

    Fish iridoviruses cause systemic diseases with high mortality in various species of wild and farm-raised fish, resulting in severe economic losses. In 1998, we isolated a new epizootic iridovirus in cultured grouper (Epinephelus sp.) in Taiwan, thus named as grouper iridovirus of Taiwan (TGIV). We report here the cloning of TGIV major capsid protein (MCP). Phylogenetic analysis of the iridoviral MCPs confirmed the classification of TGIV into the Megalocytivirus genus. Recombinant TGIV MCP and GIV MCP were then generated to produce polyclonal antibodies. Western blot analysis revealed that the two antisera were species-specific, indicating no common epitope shared by the MCPs of the two viruses. We further assayed the potency of a subunit vaccine containing recombinant TGIV MCP. The vaccine effectively protected grouper from TGIV infection. The result demonstrated that MCP is a suitable antigen for anti-TGIV vaccines. PMID:26633384

  20. Cloning of the Major Capsid Protein (MCP) of Grouper Iridovirus of Taiwan (TGIV) and Preliminary Evaluation of a Recombinant MCP Vaccine against TGIV

    PubMed Central

    Liu, Hsin-I; Chiou, Pinwen Peter; Gong, Hong-Yi; Chou, Hsin-Yiu

    2015-01-01

    Fish iridoviruses cause systemic diseases with high mortality in various species of wild and farm-raised fish, resulting in severe economic losses. In 1998, we isolated a new epizootic iridovirus in cultured grouper (Epinephelus sp.) in Taiwan, thus named as grouper iridovirus of Taiwan (TGIV). We report here the cloning of TGIV major capsid protein (MCP). Phylogenetic analysis of the iridoviral MCPs confirmed the classification of TGIV into the Megalocytivirus genus. Recombinant TGIV MCP and GIV MCP were then generated to produce polyclonal antibodies. Western blot analysis revealed that the two antisera were species-specific, indicating no common epitope shared by the MCPs of the two viruses. We further assayed the potency of a subunit vaccine containing recombinant TGIV MCP. The vaccine effectively protected grouper from TGIV infection. The result demonstrated that MCP is a suitable antigen for anti-TGIV vaccines. PMID:26633384

  1. Identification and characterization of the herpes simplex virus type 2 gene encoding the essential capsid protein ICP32/VP19c.

    PubMed Central

    Yei, S P; Chowdhury, S I; Bhat, B M; Conley, A J; Wold, W S; Batterson, W

    1990-01-01

    We describe the characterization of the herpes simplex virus type 2 (HSV-2) gene encoding infected cell protein 32 (ICP32) and virion protein 19c (VP19c). We also demonstrate that the HSV-1 UL38/ORF.553 open reading frame (ORF), which has been shown to specify a viral protein essential for capsid formation (B. Pertuiset, M. Boccara, J. Cebrian, N. Berthelot, S. Chousterman, F. Puvian-Dutilleul, J. Sisman, and P. Sheldrick, J. Virol. 63: 2169-2179, 1989), must encode the cognate HSV type 1 (HSV-1) ICP32/VP19c protein. The region of the HSV-2 genome deduced to contain the gene specifying ICP32/VP19c was isolated and subcloned, and the nucleotide sequence of 2,158 base pairs of HSV-2 DNA mapping immediately upstream of the gene encoding the large subunit of the viral ribonucleotide reductase was determined. This region of the HSV-2 genome contains a large ORF capable of encoding two related 50,538- and 49,472-molecular-weight polypeptides. Direct evidence that this ORF encodes HSV-2 ICP32/VP19c was provided by immunoblotting experiments that utilized antisera directed against synthetic oligopeptides corresponding to internal portions of the predicted polypeptides encoded by the HSV-2 ORF or antisera directed against a TrpE/HSV-2 ORF fusion protein. The type-common immunoreactivity of the two antisera and comparison of the primary amino acid sequences of the predicted products of the HSV-2 ORF and the equivalent genomic region of HSV-1 provided evidence that the HSV-1 UL38 ORF encodes the HSV-1 ICP32/VP19c. Analysis of the expression of the HSV-1 and HSV-2 ICP32/VP19c cognate proteins indicated that there may be differences in their modes of synthesis. Comparison of the predicted structure of the HSV-2 ICP32/VP19c protein with the structures of related proteins encoded by other herpes viruses suggested that the internal capsid architecture of the herpes family of viruses varies substantially. Images PMID:2154597

  2. O-GlcNAcylation of the Plum pox virus capsid protein catalyzed by SECRET AGENT: characterization of O-GlcNAc sites by electron transfer dissociation mass spectrometry.

    PubMed

    Kim, Young-Cheon; Udeshi, Namrata D; Balsbaugh, Jeremy L; Shabanowitz, Jeffrey; Hunt, Donald F; Olszewski, Neil E

    2011-03-01

    The capsid protein of Plum pox virus (PPV-CP) is modified with O-linked β-N-acetylglucosamine (O-GlcNAc). In Arabidopsis thaliana this modification is made by an O-GlcNAc transferase named SECRET AGENT (SEC). Modification of PPV-CP by SEC is hypothesized to have a direct role in the infection process, because virus titer and rate of spread are reduced in SEC mutants. Previous studies used deletion mapping and site-directed mutagenesis to identify four O-GlcNAc sites on the capsid protein that are modified by Escherichia coli-expressed SEC. The infection process was not affected when two of these sites were mutated suggesting that O-GlcNAcylation of these sites does not have a significant role in the infection process or that a subset of the modifications is sufficient. Since it is possible that the mutational mapping approach missed or incorrectly identified O-GlcNAc sites, the modifications produced by E. coli-expressed SEC were characterized using mass spectrometry. O-GlcNAcylated peptides were enzymatically tagged with galactose, the products were enriched on immobilized Ricinus communis agglutinin I and sequenced by electron transfer dissociation (ETD) mass spectrometry. Five O-GlcNAc sites on PPV-CP were identified. Two of these sites were not identified in by the previous mutational mapping. In addition, one site previously predicted by mutation mapping was not detected, but modification of this site was not supported when the mutation mapping was repeated. This study suggests that mapping modification sites by ETD mass spectrometry is more comprehensive and accurate than mutational mapping. PMID:20676902

  3. Baculovirus as a PRRSV and PCV2 bivalent vaccine vector: baculovirus virions displaying simultaneously GP5 glycoprotein of PRRSV and capsid protein of PCV2.

    PubMed

    Xu, Xin-Gang; Wang, Zhi-Sheng; Zhang, Qi; Li, Zhao-Cai; Ding, Li; Li, Wei; Wu, Hung-Yi; Chang, Ching-Dong; Lee, Long-Huw; Tong, De-Wen; Liu, Hung-Jen

    2012-02-01

    The GP5 glycoprotein of PRRSV is the main target for inducing neutralizing antibodies and protective immunity in the natural host. The capsid (Cap) protein is the major immunogenic protein and associated with the production of PCV2-specific neutralizing antibodies. In the present study, one genetic recombinant baculovirus BacSC-Dual-GP5-Cap was constructed. This virus displays simultaneously histidine-tagged GP5 and Cap proteins with the baculovirus glycoprotein gp64 TM and CTD on the virion surface as well as the surface of the virus-infected cells. After infection, the GP5 and Cap proteins were expressed and anchored simultaneously on the plasma membrane of Sf-9 cells, as revealed by Western blot and confocal microscopy. This report demonstrated first that both GP5 and Cap proteins were displayed successfully on the viral surface, revealed by immunogold electron microscopy. Vaccination of swine with recombinant baculovirus BacSC-Dual-GP5-Cap elicited significantly higher GP5 and Cap ELISA antibody titers in swine than the control groups. Virus neutralization test also showed that serum from the BacSC-Dual-GP5-Cap treated swine had significant levels of virus neutralization titers. Lymphocyte proliferation responses could be induced in swine immunized with BacSC-Dual-GP5-Cap than the control groups. These findings demonstrate that the BacSC-Dual-GP5-Cap bivalent subunit vaccine can be a potential vaccine against PRRSV and PCV2 infections. PMID:22172969

  4. Homologous recombination is required for AAV-mediated gene targeting

    PubMed Central

    Vasileva, Ana; Linden, R. Michael; Jessberger, Rolf

    2006-01-01

    High frequencies of gene targeting can be achieved by infection of mammalian cells with recombinant adeno-associated virus (rAAV) vectors [D. W. Russell and R. K. Hirata (1998) Nature Genet., 18, 325–330; D. W. Russell and R. K. Hirata (2000) J. Virol., 74, 4612–4620; R. Hirata et al. (2002) Nat. Biotechnol., 20, 735–738], but the mechanism of targeting is unclear and random integration often occurs in parallel. We assessed the role of specific DNA repair and recombination pathways in rAAV gene targeting by measuring correction of a mutated enhanced green fluorescent protein (EGFP) gene in cells where homologous recombination (HR) or non-homologous end-joining (NHEJ) had been suppressed by RNAi. EGFP-negative cells were transduced with rAAV vectors carrying a different inactivating deletion in the EGFP, and in parallel with rAAV vectors carrying red fluorescent protein (RFP). Expression of RFP accounted for viral transduction efficiency and long-term random integration. Approximately 0.02% of the infected GFP-negative cells were stably converted to GFP positive cells. Silencing of the essential NHEJ component DNA-PK had no significant effect on the frequency of targeting at any time point examined. Silencing of the SNF2/SWI2 family members RAD54L or RAD54B, which are important for HR, reduced the rate of stable rAAV gene targeting ∼5-fold. Further, partial silencing of the Rad51 paralogue XRCC3 completely abolished stable long-term EGFP expression. These results show that rAAV gene targeting requires the Rad51/Rad54 pathway of HR. PMID:16822856

  5. Dynamic Models for Templated Viral Capsid Assembly

    NASA Astrophysics Data System (ADS)

    Hagan, Michael

    2008-03-01

    The replication of many viruses with single-stranded genomes requires the simultaneous assembly of an ordered protein shell, or capsid, and encapsidation of the genome. In this talk, I will present coarse-grained computational and theoretical models that describe the assembly of viral capsid proteins around interior cores, such as polymers and rigid spheres. These models are motivated by two recently developed experimental model systems in which viral proteins dynamically encapsidate inorganic nanoparticles and polyelectrolytes. Model predictions suggest that some forms of cooperative interactions between subunits and cores can dramatically enhance rates and robustness of assembly, as compared to the spontaneous assembly of subunits into empty capsids. For large core-subunit interactions, subunits adsorb onto a core en masse in a disordered manner, and then undergo a cooperative rearrangement into an ordered capsid structure. These assembly pathways are unlike any seen for empty capsids formation. While model predictions suggest that cooperative interactions between disparate assembling components can overcome some limitations of spontaneous assembly, the complexity of multicomponent assembly introduces new forms of kinetic traps that can frustrate assembly, and hence introduces new limitations. These findings have implications for a mechanism in which viruses use interactions between proteins and genomic molecules to promote and control assembly, and thereby control the replication process.

  6. Modeling HIV-1 viral capsid nucleation by dynamical systems.

    PubMed

    Sadre-Marandi, Farrah; Liu, Yuewu; Liu, Jiangguo; Tavener, Simon; Zou, Xiufen

    2015-12-01

    There are two stages generally recognized in the viral capsid assembly: nucleation and elongation. This paper focuses on the nucleation stage and develops mathematical models for HIV-1 viral capsid nucleation based on six-species dynamical systems. The Particle Swarm Optimization (PSO) algorithm is used for parameter fitting to estimate the association and dissociation rates from biological experiment data. Numerical simulations of capsid protein (CA) multimer concentrations demonstrate a good agreement with experimental data. Sensitivity and elasticity analysis of CA multimer concentrations with respect to the association and dissociation rates further reveals the importance of CA trimer-of- dimers in the nucleation stage of viral capsid self- assembly. PMID:26596714

  7. Tyrosine triple mutated AAV2-BDNF gene therapy in a rat model of transient IOP elevation

    PubMed Central

    Igarashi, Tsutomu; Kobayashi, Maika; Kameya, Shuhei; Fujimoto, Chiaki; Nakamoto, Kenji; Takahashi, Hisatomo; Igarashi, Toru; Miyake, Noriko; Iijima, Osamu; Hirai, Yukihiko; Shimada, Takashi; Okada, Takashi; Takahashi, Hiroshi

    2016-01-01

    Purpose We examined the neuroprotective effects of exogenous brain-derived neurotrophic factor (BDNF), which provides protection to retinal ganglion cells (RGCs) in rodents, in a model of transient intraocular pressure (IOP) elevation using a mutant (triple Y-F) self-complementary adeno-associated virus type 2 vector encoding BDNF (tm-scAAV2-BDNF). Methods The tm-scAAV2-BDNF or control vector encoding green fluorescent protein (GFP; tm-scAAV2-GFP) was intravitreally administered to rats, which were then divided into four groups: control, ischemia/reperfusion (I/R) injury only, I/R injury with tm-scAAV2-GFP, and tm-scAAV2-BDNF. I/R injury was then induced by transiently increasing IOP, after which the rats were euthanized to measure the inner retinal thickness and cell counts in the RGC layer. Results Intravitreous injection of tm-scAAV2-BDNF resulted in high levels of BDNF expression in the neural retina. Histological analysis showed that the inner retinal thickness and cell numbers in the RGC layer were preserved after transient IOP elevation in eyes treated with tm-scAAV2-BDNF but not in the other I/R groups. Significantly reduced glial fibrillary acidic protein (GFAP) immunostaining after I/R injury in the rats that received tm-scAAV2-BDNF indicated reduced retinal stress, and electroretinogram (ERG) analysis confirmed preservation of retinal function in the tm-scAAV2-BDNF group. Conclusions These results demonstrate the feasibility and effectiveness of neuroprotective gene therapy using tm-scAAV2-BDNF to protect the inner retina from transiently high intraocular pressure. An in vivo gene therapeutic approach to the clinical management of retinal diseases in conditions such as glaucoma, retinal artery occlusion, hypertensive retinopathy, and diabetic retinopathy thus appears feasible. PMID:27440998

  8. Prediction of stability changes upon mutation in an icosahedral capsid

    PubMed Central

    Hickman, Samuel J.

    2015-01-01

    ABSTRACT Identifying the contributions to thermodynamic stability of capsids is of fundamental and practical importance. Here we use simulation to assess how mutations affect the stability of lumazine synthase from the hyperthermophile Aquifex aeolicus, a T = 1 icosahedral capsid; in the simulations the icosahedral symmetry of the capsid is preserved by simulating a single pentamer and imposing crystal symmetry, in effect simulating an infinite cubic lattice of icosahedral capsids. The stability is assessed by estimating the free energy of association using an empirical method previously proposed to identify biological units in crystal structures. We investigate the effect on capsid formation of seven mutations, for which it has been experimentally assessed whether they disrupt capsid formation or not. With one exception, our approach predicts the effect of the mutations on the capsid stability. The method allows the identification of interaction networks, which drive capsid assembly, and highlights the plasticity of the interfaces between subunits in the capsid. Proteins 2015; 83:1733–1741. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc PMID:26178267

  9. High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development

    PubMed Central

    2011-01-01

    Background Chicken anemia virus (CAV), the causative agent chicken anemia, is the only member of the genus Gyrovirus of the Circoviridae family. CAV is an immune suppressive virus and causes anemia, lymph organ atrophy and immunodeficiency. The production and biochemical characterization of VP1 protein and its use in a subunit vaccine or as part of a diagnostic kit would be useful to CAV infection prevention. Results Significantly increased expression of the recombinant full-length VP1 capsid protein from chicken anemia virus was demonstrated using an E. coli expression system. The VP1 gene was cloned into various different expression vectors and then these were expressed in a number of different E. coli strains. The expression of CAV VP1 in E. coli was significantly increased when VP1 was fused with GST protein rather than a His-tag. By optimizing the various rare amino acid codons within the N-terminus of the VP1 protein, the expression level of the VP1 protein in E. coli BL21(DE3)-pLysS was further increased significantly. The highest protein expression level obtained was 17.5 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 2 h. After purification by GST affinity chromatography, the purified full-length VP1 protein produced in this way was demonstrated to have good antigenicity and was able to be recognized by CAV-positive chicken serum in an ELISA assay. Conclusions Purified recombinant VP1 protein with the gene's codons optimized in the N-terminal region has potential as chimeric protein that, when expressed in E. coli, may be useful in the future for the development of subunit vaccines and diagnostic tests. PMID:21781331

  10. Characterization and epitope mapping of monoclonal antibodies raised against rat hepatitis E virus capsid protein: An evaluation of their neutralizing activity in a cell culture system.

    PubMed

    Kobayashi, Tominari; Takahashi, Masaharu; Tanggis; Mulyanto; Jirintai, Suljid; Nagashima, Shigeo; Nishizawa, Tsutomu; Okamoto, Hiroaki

    2016-07-01

    Hepatitis E virus (HEV) is the causative agent of acute hepatitis. Rat HEV is a recently discovered virus related to, but distinct from, human HEV. Since laboratory rats can be reproducibly infected with rat HEV and a cell culture system has been established for rat HEV, this virus may be used as a surrogate virus for human HEV, enabling studies on virus replication and mechanism of infection. However, monoclonal antibodies (MAbs) against rat HEV capsid (ORF2) protein are not available. In this study, 12 murine MAbs were generated against a recombinant ORF2 protein of rat HEV (rRatHEV-ORF2: amino acids 101-644) and were classified into at least six distinct groups by epitope mapping and a cross-reactivity analysis with human HEV ORF2 proteins. Two non-cross-reactive MAbs recognizing the protruding (P) domain detected both non-denatured and denatured rRatHEV-ORF2 protein and efficiently captured cell culture-produced rat HEV particles that had been treated with deoxycholate and trypsin, but not those without prior treatment. In addition, these two MAbs were able to efficiently neutralize replication of cell culture-generated rat HEV particles without lipid membranes (but not those with lipid membranes) in a cell culture system, similar to human HEV. PMID:26992654

  11. Development of an IP-Free Biotechnology Platform for Constitutive Production of HPV16 L1 Capsid Protein Using the Pichia pastoris PGK1 Promoter

    PubMed Central

    Mariz, F. C.; Coimbra, E. C.; Jesus, A. L. S.; Nascimento, L. M.; Torres, F. A. G.; Freitas, A. C.

    2015-01-01

    The human papillomavirus (HPV) L1 major capsid protein, which forms the basis of the currently available vaccines against cervical cancer, self-assembles into virus-like particles (VLPs) when expressed heterologously. We report the development of a biotechnology platform for HPV16 L1 protein expression based on the constitutive PGK1 promoter (PPGK1) from the methylotrophic yeast Pichia pastoris. The L1 gene was cloned under regulation of PPGK1 into pPGKΔ3 expression vector to achieve intracellular expression. In parallel, secretion of the L1 protein was obtained through the use of an alternative vector called pPGKΔ3α, in which a codon optimized α-factor signal sequence was inserted. We devised a work-flow based on the detection of the L1 protein by dot blot, colony blot, and western blot to classify the positive clones. Finally, intracellular HPV VLPs assembly was demonstrated for the first time in yeast cells. This study opens up perspectives for the establishment of an innovative platform for the production of HPV VLPs or other viral antigens for vaccination purposes, based on constitutive expression in P. pastoris. PMID:26090426

  12. Capsid protease domain as a tool for assessing protein-domain folding during organelle import of nascent polypeptides in living cells.

    PubMed

    Kang, Kongsoo; Takahara, Michiyo; Sakaue, Haruka; Sakaguchi, Masao

    2016-05-01

    Various proteins synthesized by ribosomes are imported into specific organelles. To elucidate the behavior of protein domains during import, we developed a folding probe, in which the capsid protease (CP) domain of the Semliki Forest virus was connected to enhanced green fluorescent protein (EGFP). The probe was fused to appropriate N-terminal organelle-targeting signal sequences and expressed in cultured cells. When the entire CP-domain was present in the cytosol, it became folded and cleaved off the following EGFP-domain. Once cleaved, EGFP stability was not affected by upstream sequences. Based on EGFP localization, we estimated the extent of CP-domain folding in the cytosolic space. When fused to mitochondrial hydrophobic multispanning membrane protein ABCB10, more than half of the EGFP remained in the cytoplasm, whereas most of the CP-portion was in the mitochondrial fraction. When fused to the endoplasmic reticulum (ER) signal, the cleaved EGFP was observed only in the ER fraction, confirming that the CP-domain cannot fold on the cytoplasmic side during cotranslational ER translocation. Thus, import of the ABCB10 molecule was not as tightly coupled with chain elongation as ER translocation. Use of this probe to quantitatively examine stop-translocation at the ER translocon in living cells revealed that positively charged residues on the translocating nascent chain stall at the ER translocon. PMID:26711239

  13. Development of an IP-Free Biotechnology Platform for Constitutive Production of HPV16 L1 Capsid Protein Using the Pichia pastoris PGK1 Promoter.

    PubMed

    Mariz, F C; Coimbra, E C; Jesus, A L S; Nascimento, L M; Torres, F A G; Freitas, A C

    2015-01-01

    The human papillomavirus (HPV) L1 major capsid protein, which forms the basis of the currently available vaccines against cervical cancer, self-assembles into virus-like particles (VLPs) when expressed heterologously. We report the development of a biotechnology platform for HPV16 L1 protein expression based on the constitutive PGK1 promoter (PPGK1) from the methylotrophic yeast Pichia pastoris. The L1 gene was cloned under regulation of PPGK1 into pPGKΔ3 expression vector to achieve intracellular expression. In parallel, secretion of the L1 protein was obtained through the use of an alternative vector called pPGKΔ3α, in which a codon optimized α-factor signal sequence was inserted. We devised a work-flow based on the detection of the L1 protein by dot blot, colony blot, and western blot to classify the positive clones. Finally, intracellular HPV VLPs assembly was demonstrated for the first time in yeast cells. This study opens up perspectives for the establishment of an innovative platform for the production of HPV VLPs or other viral antigens for vaccination purposes, based on constitutive expression in P. pastoris. PMID:26090426

  14. Structure of the HIV-1 Full-Length Capsid Protein in a Conformationally Trapped Unassembled State Induced by Small-Molecule Binding

    SciTech Connect

    Du, Shoucheng; Betts, Laurie; Yang, Ruifeng; Shi, Haibin; Concel, Jason; Ahn, Jinwoo; Aiken, Christopher; Zhang, Peijun; Yeh, Joanne I.

    2012-11-26

    The capsid (CA) protein plays crucial roles in HIV infection and replication, essential to viral maturation. The absence of high-resolution structural data on unassembled CA hinders the development of antivirals effective in inhibiting assembly. Unlike enzymes that have targetable, functional substrate-binding sites, the CA does not have a known site that affects catalytic or other innate activity, which can be more readily targeted in drug development efforts. We report the crystal structure of the HIV-1 CA, revealing the domain organization in the context of the wild-type full-length (FL) unassembled CA. The FL CA adopts an antiparallel dimer configuration, exhibiting a domain organization sterically incompatible with capsid assembly. A small compound, generated in situ during crystallization, is bound tightly at a hinge site ('H site'), indicating that binding at this interdomain region stabilizes the ADP conformation. Electron microscopy studies on nascent crystals reveal both dimeric and hexameric lattices coexisting within a single condition, in agreement with the interconvertibility of oligomeric forms and supporting the feasibility of promoting assembly-incompetent dimeric states. Solution characterization in the presence of the H-site ligand shows predominantly unassembled dimeric CA, even under conditions that promote assembly. Our structure elucidation of the HIV-1 FL CA and characterization of a potential allosteric binding site provides three-dimensional views of an assembly-defective conformation, a state targeted in, and thus directly relevant to, inhibitor development. Based on our findings, we propose an unprecedented means of preventing CA assembly, by 'conformationally trapping' CA in assembly-incompetent conformational states induced by H-site binding.

  15. Production and purification of polymerization-competent HIV-1 capsid protein p24 (CA) in NiCo21(DE3) Escherichia coli

    PubMed Central

    2013-01-01

    Background HIV genome is packaged and organized in a conical capsid, which is made up of ~1,500 copies of the viral capsid protein p24 (CA). Being a primary structural component and due to its critical roles in both late and early stages of the HIV replication cycle, CA has attracted increased interest as a drug discovery target in recent years. Drug discovery studies require large amounts of highly pure and biologically active protein. It is therefore desirable to establish a simple and reproducible process for efficient production of HIV-1 CA. Result In this work, 6-His-tagged wild type CA from HIV-1 (NL4.3) was expressed in rare tRNA-supplemented NiCo21(DE3) Escherichia coli, and its production was studied in shake flask culture condition of expression. Influences of various key cultivation parameters were examined to identify optimal conditions for HIV-1 CA production. It was found that a culture temperature of 22°C and induction with 0.05 mM IPTG at the early stage of growth were ideal, leading to a maximum biomass yield when grown in Super broth supplemented with 1% glucose. With optimized culture conditions, a final biomass concentration of ~27.7 g L-1 (based on optical density) was obtained in 12 hours post-induction, leading to a yield of about ~170 mg L-1 HIV-1 CA. A two-step purification strategy (chitin beads + IMAC) was employed, which efficiently removed metal affinity resin-binding bacterial proteins that contaminate recombinant His-tagged protein preparation, and resulted in highly pure HIV-1 CA. The purified protein was capable of polymerization when tested in an in vitro polymerization assay. Conclusions By using this optimized expression and purification procedure, milligram amounts of highly pure and polymerization-competent recombinant HIV-1 CA can be produced at the lab-scale and thus used for further biochemical studies. PMID:24304876

  16. Lytic Replication of Kaposi's Sarcoma-Associated Herpesvirus Results in the Formation of Multiple Capsid Species: Isolation and Molecular Characterization of A, B, and C Capsids from a Gammaherpesvirus

    PubMed Central

    Nealon, K.; Newcomb, W. W.; Pray, T. R.; Craik, C. S.; Brown, J. C.; Kedes, D. H.

    2001-01-01

    Despite the discovery of Epstein-Barr virus more than 35 years ago, a thorough understanding of gammaherpesvirus capsid composition and structure has remained elusive. We approached this problem by purifying capsids from Kaposi's sarcoma-associated herpesvirus (KSHV), the only other known human gammaherpesvirus. The results from our biochemical and imaging analyses demonstrate that KSHV capsids possess a typical herpesvirus icosahedral capsid shell composed of four structural proteins. The hexameric and pentameric capsomers are composed of the major capsid protein (MCP) encoded by open reading frame 25. The heterotrimeric complexes, forming the capsid floor between the hexons and pentons, are each composed of one molecule of ORF62 and two molecules of ORF26. Each of these proteins has significant amino acid sequence homology to capsid proteins in alpha- and betaherpesviruses. In contrast, the fourth protein, ORF65, lacks significant sequence homology to its structural counterparts from the other subfamilies. Nevertheless, this small, basic, and highly antigenic protein decorates the surface of the capsids, as does, for example, the even smaller basic capsid protein VP26 of herpes simplex virus type 1. We have also found that, as with the alpha- and betaherpesviruses, lytic replication of KSHV leads to the formation of at least three capsid species, A, B, and C, with masses of approximately 200, 230, and 300 MDa, respectively. A capsids are empty, B capsids contain an inner array of a fifth structural protein, ORF17.5, and C capsids contain the viral genome. PMID:11222712

  17. Chondroitin sulfate N-acetylgalactosaminyltransferase-2 contributes to the replication of infectious bursal disease virus via interaction with the capsid protein VP2.

    PubMed

    Zhang, Lizhou; Ren, Xiangang; Chen, Yuming; Gao, Yulong; Wang, Nian; Lu, Zhen; Gao, Li; Qin, Liting; Wang, Yongqiang; Gao, Honglei; Li, Kai; Jiang, Lili; Cui, Hongyu; Liu, Changjun; Zhang, Yanping; Qi, Xiaole; Wang, Xiaomei

    2015-03-01

    Infectious bursal disease virus (IBDV) is a birnavirus that causes a highly contagious immunosuppressive disease in young chickens. The capsid protein VP2 of IBDV plays multiple roles in its life cycle. To more comprehensively understand the functions of VP2 involved in the communication between virus and host, we used yeast two-hybrid screening to identify the cellular factors that interact with this protein. We found that chondroitin sulfate N-acetylgalactosaminyltransferase-2 (CSGalNAcT2), a typical type II transmembrane protein located in Golgi apparatus, could interact with VP2, and we confirmed this interaction by co-immunoprecipitation and confocal laser scanning microscopy assays. Additionally, up-regulation of CSGalNAcT2 during IBDV infection was observed. Overexpression and siRNA-mediated knockdown of CSGalNAcT2 assays suggested that CSGalNAcT2 promoted IBDV replication. Moreover, this enhancing effect of CSGalNAcT2 could be inhibited by Brefeldin A, which is a Golgi-disturbing agent. This indicated that the integrity of Golgi apparatus structure was involved in the function of CSGalNAcT2. Taken together, we concluded that CSGalNAcT2, located in the Golgi apparatus, contributed to the replication of IBDV via interaction with VP2. PMID:25807054

  18. Chondroitin Sulfate N-acetylgalactosaminyltransferase-2 Contributes to the Replication of Infectious Bursal Disease Virus via Interaction with the Capsid Protein VP2

    PubMed Central

    Zhang, Lizhou; Ren, Xiangang; Chen, Yuming; Gao, Yulong; Wang, Nian; Lu, Zhen; Gao, Li; Qin, Liting; Wang, Yongqiang; Gao, Honglei; Li, Kai; Jiang, Lili; Cui, Hongyu; Liu, Changjun; Zhang, Yanping; Qi, Xiaole; Wang, Xiaomei

    2015-01-01

    Infectious bursal disease virus (IBDV) is a birnavirus that causes a highly contagious immunosuppressive disease in young chickens. The capsid protein VP2 of IBDV plays multiple roles in its life cycle. To more comprehensively understand the functions of VP2 involved in the communication between virus and host, we used yeast two-hybrid screening to identify the cellular factors that interact with this protein. We found that chondroitin sulfate N-acetylgalactosaminyltransferase-2 (CSGalNAcT2), a typical type II transmembrane protein located in Golgi apparatus, could interact with VP2, and we confirmed this interaction by co-immunoprecipitation and confocal laser scanning microscopy assays. Additionally, up-regulation of CSGalNAcT2 during IBDV infection was observed. Overexpression and siRNA-mediated knockdown of CSGalNAcT2 assays suggested that CSGalNAcT2 promoted IBDV replication. Moreover, this enhancing effect of CSGalNAcT2 could be inhibited by Brefeldin A, which is a Golgi-disturbing agent. This indicated that the integrity of Golgi apparatus structure was involved in the function of CSGalNAcT2. Taken together, we concluded that CSGalNAcT2, located in the Golgi apparatus, contributed to the replication of IBDV via interaction with VP2. PMID:25807054

  19. Diversity of in-vivo assembled HIV-1 capsids

    NASA Astrophysics Data System (ADS)

    Lee, Se Il; Nguyen, Toan

    2008-03-01

    Understanding the capsid assembly process of Human Immunodeficiency Virus (HIV), the causative agent of Acute Immuno Deficiency Syndrom (AIDS), is very important because of recent intense interest in capsid-oriented viral therapy. The unique conical shapes of mature HIV-1 capsid have drawn significant interests in the biological community and started to attract attention from the physics community. Previous studies showed that in a free assembly process, the HIV-1 conical shape is not thermodynamically stable. However, if the volume of the capsid is constrained during assembly and the capsid protein shell has high spontaneous curvature, the conical shape is stable. In this work, we focus on in-vivo HIV-1 capsid assembly. For this case, the viral envelope membrane present during assembly imposes constraint on the length of the capsid. We use an elastic continuum shell theory to approximate the energies of various HIV-1 capsid shapes (spherical, cylindrical and conical). We show that for certain range of viral membrane diameter, the conical and cylindrical shapes are both thermodynamically stable. This result is supported by experimental observation that in-vivo assembled HIV-1 capsids are very heterogeneous in shapes and sizes. Numerical calculation is also performed to improve theoretical approximation.

  20. The semipermeability of simple spherical virus capsids.

    PubMed

    Durham, A C; Witz, J; Bancroft, J B

    1984-02-01

    Hydrogen-ion titration curves are reported for tomato bushy stunt virus, two strains of cowpea chlorotic mottle virus, and turnip crinkle virus, with particular attention to the hysteresis loops associated with the swelling and contraction of virions. There appears to be an archetypal shape of hysteresis loops, which is shared by viruses in several groups, suggestive of many intermediate states in the swelling of any one particle. In contrast, eggplant mosaic virus behaves as if its protein capsid is impermeable to small ions in mild conditions; its cation-binding sites were revealed by treatment with high concentrations of salt or urea, or at raised temperatures. Putting these observations together with the fact that a spherical virus capsid is a closed, holey, charged surface leads to a theory of titration hysteresis: its key feature is that the protein capsids of simple viruses are inherently semipermeable, with many of the ion-handling properties usually attributed only to complex lipid membranes. PMID:18639804

  1. Induction of robust immunity response in mice by dual-expression-system-based recombinant baculovirus expressing the capsid protein of porcine circovirus type 2

    PubMed Central

    2013-01-01

    Background Porcine circovirus type 2 (PCV2) is associated with post-weaning multisystemic wasting syndrome (PMWS), an emerging swine disease that causes progressive weight loss, dyspnea, tachypnea, anemia, jaundice, and diarrhea in piglets. Although baculovirus is an enveloped virus that infects insects in nature, it has emerged as a vaccine vector, and we used it to develop a novel candidate vaccine for a preventive or therapeutic strategy to control PCV2 infections. Methods Immunoblotting analysis of recombinant baculovirus and immunofluorescent staining of baculovirus-infected cells were followed using anti-ORF2 monoclonal antibodies. The BALB/c mice were immunized intramuscularly with this baculovirus. The titers of antibodies were mensurated with a Cap-protein-specific enzyme-linked immunosorbent assay (ELISA) and a serum neutralization assay. The IFN-γ response in splenocytes harvested from immunized mice was measured by ELISA. Student's t-test was used to compare immune responses of different groups. Results In this study, we successfully constructed a dual-expression-system-based recombinant baculovirus BV-GD-ORF2, which can display the PCV2 capsid (Cap) protein and VSV-G protein on the viral envelope and also expressing Cap protein on transduced mammalian cells, thereby functioning as both a subunit and a DNA vaccine. After infection, the Cap protein was expressed and displayed on the viral surface, as demonstrated with an indirect fluorescence assay and immunoblotting. The vaccination of mice with recombinant baculovirus BV-GD-ORF2 successfully induced robust Cap-protein-specific humoral and cellular immune responses. Conclusions Our findings collectively demonstrate that the recombinant baculovirus BV-GD-ORF2 is a potential vaccine against PCV2 infections. PMID:24161107

  2. Inhibition of iridovirus protein synthesis and virus replication by antisense morpholino oligonucleotides targeted to the major capsid protein, the 18 kDa immediate-early protein, and a viral homolog of RNA polymerase II

    SciTech Connect

    Sample, Robert; Bryan, Locke; Long, Scott; Majji, Sai; Hoskins, Glenn; Sinning, Allan; Olivier, Jake; Chinchar, V. Gregory . E-mail: vchinchar@microbio.umsmed.edu

    2007-02-20

    Frog virus 3 (FV3) is a large DNA virus that encodes {approx} 100 proteins. Although the general features of FV3 replication are known, the specific roles that most viral proteins play in the virus life cycle have not yet been elucidated. To address the question of viral gene function, antisense morpholino oligonucleotides (asMOs) were used to transiently knock-down expression of specific viral genes and thus infer their role in virus replication. We designed asMOs directed against the major capsid protein (MCP), an 18 kDa immediate-early protein (18K) that was thought to be a viral regulatory protein, and the viral homologue of the largest subunit of RNA polymerase II (vPol-II{alpha}). All three asMOs successfully inhibited translation of the targeted protein, and two of the three asMOs resulted in marked phenotypic changes. Knock-down of the MCP resulted in a marked reduction in viral titer without a corresponding drop in the synthesis of other late viral proteins. Transmission electron microscopy (TEM) showed that in cells treated with the anti-MCP MO assembly sites were devoid of viral particles and contained numerous aberrant structures. In contrast, inhibition of 18K synthesis did not block virion formation, suggesting that the 18K protein was not essential for replication of FV3 in fathead minnow (FHM) cells. Finally, consistent with the view that late viral gene expression is catalyzed by a virus-encoded or virus-modified Pol-II-like protein, knock-down of vPol-II{alpha} triggered a global decline in late gene expression and virus yields without affecting the synthesis of early viral genes. Collectively, these results demonstrate the utility of using asMOs to elucidate the function of FV3 proteins.

  3. Molecular characterization of mouse-virulent poliovirus type 1 Mahoney mutants: involvement of residues of polypeptides VP1 and VP2 located on the inner surface of the capsid protein shell.

    PubMed Central

    Couderc, T; Hogle, J; Le Blay, H; Horaud, F; Blondel, B

    1993-01-01

    Most poliovirus (PV) strains, including PV PV-1/Mahoney, are unable to cause paralysis in mice. Determinants for restriction of PV-1/Mahoney in mice have been identified by manipulating PV-1 cDNA and located on the viral capsid protein VP1. These determinants consist of a highly exposed amino acid sequence on the capsid surface corresponding to the B-C loop (M. Murray, J. Bradley, X. Yang, E. Wimmer, E. Moss, and V. Racaniello, Science 241:213-215, 1988; A. Martin, C. Wychowski, T. Couderc, R. Crainic, J. Hogle, and M. Girard, EMBO J. 7:2839-2847, 1988) and of residues belonging to the N-terminal sequence located on the inner surface of the protein shell (E. Moss and V. Racaniello, EMBO J. 10:1067-1074, 1991). Using an in vivo approach, we isolated two mouse-neurovirulent PV-1 mutants in the mouse central nervous system after a single passage of PV-1/Mahoney inoculated by the intracerebral route. Both mutants were subjected to two additional passages in mice, plaque purified, and subsequently characterized. The two cloned mutants, Mah-NK13 and Mah-NL32, retained phenotypic characteristics of the parental PV-1/Mahoney, including epitope map, heat lability, and temperature sensitivity. Mah-NK13 exhibited slightly smaller plaques than did the parental virus. The nucleotide sequences of the mutant genomes were determined, and mutations were identified. Mutations were independently introduced into the parental PV-1/Mahoney genome by single-site mutagenesis. Mutated PV-1/Mahoney viruses were then tested for their neurovirulence in mice. A single amino acid substitution in the capsid proteins VP1 (Thr-22-->Ile) and VP2 (Ser-31-->Thr) identified in the Mah-NK13 and Mah-NL32 genomes, respectively, conferred the mouse-virulent phenotype to the mouse-avirulent PV-1/Mahoney. Ile-22 in VP1 was responsible for the small-plaque phenotype of Mah-NK13. Both mutations arose during the first passage in the mouse central nervous system. We thus identified a new mouse adaptation

  4. Tilable nature of virus capsids and the role of topological constraints in natural capsid design

    NASA Astrophysics Data System (ADS)

    Mannige, Ranjan V.; Brooks, Charles L., III

    2008-05-01

    Virus capsids are highly specific assemblies that are formed from a large number of often chemically identical capsid subunits. In the present paper we ask to what extent these structures can be viewed as mathematically tilable objects using a single two-dimensional tile. We find that spherical viruses from a large number of families—eight out of the twelve studied—qualitatively possess properties that allow their representation as two-dimensional monohedral tilings of a bound surface, where each tile represents a subunit. This we did by characterizing the extent to which individual spherical capsids display subunit-subunit (1) holes, (2) overlaps, and (3) gross structural variability. All capsids with T numbers greater than 1 from the Protein Data Bank, with homogeneous protein composition, were used in the study. These monohedral tilings, called canonical capsids due to their platonic (mathematical) form, offer a mathematical segue into the structural and dynamical understanding of not one, but a large number of virus capsids. From our data, it appears as though one may only break the long-standing rules of quasiequivalence by the introduction of subunit-subunit structural variability, holes, and gross overlaps into the shell. To explore the utility of canonical capsids in understanding structural aspects of such assemblies, we used graph theory and discrete geometry to enumerate the types of shapes that the tiles (and hence the subunits) must possess. We show that topology restricts the shape of the face to a limited number of five-sided prototiles, one of which is the “bisected trapezoid” that is a platonic representation of the most ubiquitous capsid subunit shape seen in nature (the trapezoidal jelly-roll motif). This motif is found in a majority of seemingly unrelated virus families that share little to no host, size, or amino acid sequence similarity. This suggests that topological constraints may exhibit dominant roles in the natural design of

  5. Mucopolysaccharidosis IIIB confers enhanced neonatal intracranial transduction by AAV8 but not by 5, 9 or rh10

    PubMed Central

    Gilkes, J A; Bloom, M D; Heldermon, C D

    2016-01-01

    Sanfilippo syndrome type B (mucopolysaccharidosis IIIB, MPS IIIB) is a lysosomal storage disease resulting from deficiency of N-acetyl-glucosaminidase (NAGLU) activity. To determine the possible therapeutic utility of recombinant adeno-associated virus (rAAV) in early gene therapy-based interventions, we performed a comprehensive assessment of transduction and biodistribution profiles of four central nervous system (CNS) administered rAAV serotypes, -5, -8, -9 and -rh10. To simulate optimal earliest treatment of the disease, each rAAV serotype was injected into the CNS of neonatal MPS IIIB and control animals. We observed marked differences in biodistribution and transduction profiles between the serotypes and this differed in MPS IIIB compared with healthy control mice. Overall, in control mice, all serotypes performed comparably, although some differences were observed in certain focal areas. In MPS IIIB mice, AAV8 was more efficient than AAV5, -9 and -rh10 for gene delivery to most structures analyzed, including the cerebral cortex, hippocampus and thalamus. Noteworthy, the pattern of biodistribution within the CNS varied by serotype and genotype. Interestingly, AAV8 also produced the highest green fluorescent protein intensity levels compared with any other serotype and demonstrated improved transduction in NAGLU compared with control brains. Importantly, we also show leakage of AAV8, -9 and -rh10, but not AAV5, from CNS parenchyma to systemic organs. Overall, our data suggest that AAV8 represents the best therapeutic gene transfer vector for early intervention in MPS IIIB. PMID:26674264

  6. Mucopolysaccharidosis IIIB confers enhanced neonatal intracranial transduction by AAV8 but not by 5, 9 or rh10.

    PubMed

    Gilkes, J A; Bloom, M D; Heldermon, C D

    2016-03-01

    Sanfilippo syndrome type B (mucopolysaccharidosis IIIB, MPS IIIB) is a lysosomal storage disease resulting from deficiency of N-acetyl-glucosaminidase (NAGLU) activity. To determine the possible therapeutic utility of recombinant adeno-associated virus (rAAV) in early gene therapy-based interventions, we performed a comprehensive assessment of transduction and biodistribution profiles of four central nervous system (CNS) administered rAAV serotypes, -5, -8, -9 and -rh10. To simulate optimal earliest treatment of the disease, each rAAV serotype was injected into the CNS of neonatal MPS IIIB and control animals. We observed marked differences in biodistribution and transduction profiles between the serotypes and this differed in MPS IIIB compared with healthy control mice. Overall, in control mice, all serotypes performed comparably, although some differences were observed in certain focal areas. In MPS IIIB mice, AAV8 was more efficient than AAV5, -9 and -rh10 for gene delivery to most structures analyzed, including the cerebral cortex, hippocampus and thalamus. Noteworthy, the pattern of biodistribution within the CNS varied by serotype and genotype. Interestingly, AAV8 also produced the highest green fluorescent protein intensity levels compared with any other serotype and demonstrated improved transduction in NAGLU compared with control brains. Importantly, we also show leakage of AAV8, -9 and -rh10, but not AAV5, from CNS parenchyma to systemic organs. Overall, our data suggest that AAV8 represents the best therapeutic gene transfer vector for early intervention in MPS IIIB. PMID:26674264

  7. A Cell Internalizing Antibody Targeting Capsid Protein (p24) Inhibits the Replication of HIV-1 in T Cells Lines and PBMCs: A Proof of Concept Study

    PubMed Central

    Ali, Syed A.; Teow, Sin-Yeang; Omar, Tasyriq Che; Khoo, Alan Soo-Beng; Choon, Tan Soo; Yusoff, Narazah Mohd

    2016-01-01

    There remains a need for newer therapeutic approaches to combat HIV/AIDS. Viral capsid protein p24 plays important roles in HIV pathogenesis. Peptides and small molecule inhibitors targeting p24 have shown to inhibit virus replication in treated cell. High specificity and biological stability of monoclonal antibodies (mAbs) make them an attractive contender for in vivo treatments. However, mAbs do not enter into cells, thus are restricted to target surface molecules. This also makes targeting intracellular HIV-1 p24 a challenge. A mAb specific to p24 that can internalize into the HIV-infected cells is hypothesized to inhibit the virus replication. We selected a mAb that has previously shown to inhibit p24 polymerization in an in vitro assay and chemically conjugated it with cell penetrating peptides (CPP) to generate cell internalizing anti-p24 mAbs. Out of 8 CPPs tested, κFGF-MTS -conjugated mAbs internalized T cells most efficiently. At nontoxic concentration, the κFGF-MTS-anti-p24-mAbs reduced the HIV-1 replication up to 73 and 49% in T-lymphocyte and PBMCs respectively. Marked inhibition of HIV-1 replication in relevant cells by κFGF-MTS-anti-p24-mAbs represents a viable strategy to target HIV proteins present inside the cells. PMID:26741963

  8. Cleavage of the HPV16 Minor Capsid Protein L2 during Virion Morphogenesis Ablates the Requirement for Cellular Furin during De Novo Infection

    PubMed Central

    Cruz, Linda; Biryukov, Jennifer; Conway, Michael J.; Meyers, Craig

    2015-01-01

    Infections by high-risk human papillomaviruses (HPV) are the causative agents for the development of cervical cancer. As with other non-enveloped viruses, HPVs are taken up by the cell through endocytosis following primary attachment to the host cell. Through studies using recombinant pseudovirus particles (PsV), many host cellular proteins have been implicated in the process. The proprotein convertase furin has been demonstrated to cleave the minor capsid protein, L2, post-attachment to host cells and is required for infectious entry by HPV16 PsV. In contrast, using biochemical inhibition by a furin inhibitor and furin-negative cells, we show that tissue-derived HPV16 native virus (NV) initiates infection independent of cellular furin. We show that HPV16 L2 is cleaved during virion morphogenesis in differentiated tissue. In addition, HPV45 is also not dependent on cellular furin, but two other alpha papillomaviruses, HPV18 and HPV31, are dependent on the activity of cellular furin for infection. PMID:26569287

  9. A Cell Internalizing Antibody Targeting Capsid Protein (p24) Inhibits the Replication of HIV-1 in T Cells Lines and PBMCs: A Proof of Concept Study.

    PubMed

    Ali, Syed A; Teow, Sin-Yeang; Omar, Tasyriq Che; Khoo, Alan Soo-Beng; Choon, Tan Soo; Yusoff, Narazah Mohd

    2016-01-01

    There remains a need for newer therapeutic approaches to combat HIV/AIDS. Viral capsid protein p24 plays important roles in HIV pathogenesis. Peptides and small molecule inhibitors targeting p24 have shown to inhibit virus replication in treated cell. High specificity and biological stability of monoclonal antibodies (mAbs) make them an attractive contender for in vivo treatments. However, mAbs do not enter into cells, thus are restricted to target surface molecules. This also makes targeting intracellular HIV-1 p24 a challenge. A mAb specific to p24 that can internalize into the HIV-infected cells is hypothesized to inhibit the virus replication. We selected a mAb that has previously shown to inhibit p24 polymerization in an in vitro assay and chemically conjugated it with cell penetrating peptides (CPP) to generate cell internalizing anti-p24 mAbs. Out of 8 CPPs tested, κFGF-MTS -conjugated mAbs internalized T cells most efficiently. At nontoxic concentration, the κFGF-MTS-anti-p24-mAbs reduced the HIV-1 replication up to 73 and 49% in T-lymphocyte and PBMCs respectively. Marked inhibition of HIV-1 replication in relevant cells by κFGF-MTS-anti-p24-mAbs represents a viable strategy to target HIV proteins present inside the cells. PMID:26741963

  10. Development of two Trichoplusia ni larvae-derived ELISAs for the detection of antibodies against replicase and capsid proteins of porcine circovirus type 2 in domestic pigs.

    PubMed

    Pérez-Martín, Eva; Grau-Roma, Llorenç; Argilaguet, Jordi M; Nofrarías, Miquel; Escribano, José M; Gómez-Sebastián, Silvia; Segalés, Joaquim; Rodríguez, Fernando

    2008-12-01

    The main aim of the present study was to describe new methods for the identification of antibodies against the PCV2 capsid (Cap) and replicase (Rep) proteins in pig sera. Specifically, two new indirect enzyme-linked immunosorbent assays (ELISA) were developed based on recombinant PCV2 Cap (rCap) and Rep/Rep' (rRep) proteins expressed in baculovirus and produced in Trichoplusia ni insect larvae. Both assays were validated by testing serum samples in a longitudinal study of 107 animals with different clinico-pathological features of PCV2 infection: pigs with postweaning multisystemic wasting syndrome (PMWS), wasted pigs without a diagnosis of PMWS and healthy animals. Longitudinal antibody profiles indicated that healthy animals had significantly higher anti-Cap and anti-Rep antibody levels than the rest of the animal groups at 11 weeks of age. Moreover, PMWS affected pigs could be distinguished from the rest of the pig groups by their lower anti-Rep antibody levels at 11 weeks of age and at necropsy. The results demonstrate the potential of these two ELISAs for large-scale serological studies. This study represents the first longitudinal study of the induction of anti-Cap and anti-Rep antibodies in farms affected by PMWS, from 1 week of age until the occurrence of disease. PMID:18773923

  11. Self-assembly of virus-like particles of canine parvovirus capsid protein expressed from Escherichia coli and application as virus-like particle vaccine.

    PubMed

    Xu, Jin; Guo, Hui-Chen; Wei, Yan-Quan; Dong, Hu; Han, Shi-Chong; Ao, Da; Sun, De-Hui; Wang, Hai-Ming; Cao, Sui-Zhong; Sun, Shi-Qi

    2014-04-01

    Canine parvovirus disease is an acute infectious disease caused by canine parvovirus (CPV). Current commercial vaccines are mainly attenuated and inactivated; as such, problems concerning safety may occur. To resolve this problem, researchers developed virus-like particles (VLPs) as biological nanoparticles resembling natural virions and showing high bio-safety. This property allows the use of VLPs for vaccine development and mechanism studies of viral infections. Tissue-specific drug delivery also employs VLPs as biological nanomaterials. Therefore, VLPs derived from CPV have a great potential in medicine and diagnostics. In this study, small ubiquitin-like modifier (SUMO) fusion motif was utilized to express a whole, naturalVP2 protein of CPV in Escherichia coli. After the cleavage of the fusion motif, the CPV VP2 protein has self-assembled into VLPs. The VLPs had a size and shape that resembled the authentic virus capsid. However, the self-assembly efficiency of VLPs can be affected by different pH levels and ionic strengths. The mice vaccinated subcutaneously with CPV VLPs and CPV-specific immune responses were compared with those immunized with the natural virus. This result showed that VLPs can effectively induce anti-CPV specific antibody and lymphocyte proliferation as a whole virus. This result further suggested that the antigen epitope of CPV was correctly present on VLPs, thereby showing the potential application of a VLP-based CPV vaccine. PMID:24413974

  12. The red clover necrotic mosaic virus capsid protein N-terminal amino acids possess specific RNA binding activity and are required for stable virion assembly.

    PubMed

    Park, Sang-Ho; Sit, Tim L; Kim, Kook-Hyung; Lommel, Steven A

    2013-09-01

    The red clover necrotic mosaic virus (RCNMV) bipartite RNA genome is packaged into two virion populations containing either RNA-1 and RNA-2 or multiple copies of RNA-2 only. To understand this distinctive packaging scheme, we investigated the RNA-binding properties of the RCNMV capsid protein (CP). Maltose binding protein-CP fusions exhibited the highest binding affinities for RNA probes containing the RNA-2 trans-activator or the 3' non-coding region from RNA-1. Other viral and non-viral RNA probes displayed CP binding but to a much lower degree. Deletion of the highly basic N-terminal 50 residues abolished CP binding to viral RNA transcripts. In planta studies of select CP deletion mutants within this N-terminal region revealed that it was indispensable for stable virion formation and the region spanning CP residues 5-15 is required for systemic movement. Thus, the N-terminal region of the CP is involved in both producing two virion populations due to its RNA binding properties and virion stability. PMID:23747688

  13. Self-assembly of virus-like particles of rabbit hemorrhagic disease virus capsid protein expressed in Escherichia coli and their immunogenicity in rabbits.

    PubMed

    Guo, Huimin; Zhu, Jie; Tan, Yonggui; Li, Chuanfeng; Chen, Zongyan; Sun, Shiqi; Liu, Guangqing

    2016-07-01

    In this study, virus-like particles (VLPs) derived from rabbit hemorrhagic disease virus (RHDV) were evaluated for the development of a vaccine against RHDV infection. The VP60 gene was cloned and inserted into a pSMK expression vector containing a small ubiquitin-like modifier (SUMO) tag that can promote the soluble expression of heterologous proteins in Escherichia coli cells. After expression and purification of His-SUMO-VP60 and cleavage of the SUMO tag, we found that the RHDV VP60 protein had self-assembled into VLPs with a similar shape and smaller size compared with authentic RHDV capsid. Next, the antigenicity and immunogenicity of the VLPs were examined. The results showed that RHDV-specific responses were clearly induced in rabbits and that all rabbits in the VLP group survived while those in the negative control group died within 72 h post-infection. These results suggest that VLP-based RHDV could be a promising RHDV vaccine candidate. PMID:27118636

  14. Assessment of recombinant beak and feather disease virus capsid protein as a vaccine for psittacine beak and feather disease.

    PubMed

    Bonne, Nicolai; Shearer, Patrick; Sharp, Margaret; Clark, Phillip; Raidal, Shane

    2009-03-01

    Beak and feather disease virus (BFDV) is a significant pathogen of wild Australasian and African psittacine birds. We assessed the immunogenicity of recombinant BFDV capsid (recBFDVcap) to protect against the development of psittacine beak and feather disease (PBFD). Long-billed corellas (Cacatua tenuirostris) (n=13) received (by injection) 1 ml vaccine containing 10 microg recBFDVcap on day 0 and 0.4 ml vaccine containing 66.8 microg recBFDVcap on day 11. All vaccinated corellas and five non-vaccinated control corellas were given 0.4 ml BFDV suspension [titre=log(2) 12 haemagglutination units (HAU) 50 microl(-1)] intramuscularly and 0.1 ml orally 16 days after booster vaccination. Blood was collected during the vaccination period and blood and feathers were collected after BFDV administration. Testing of blood samples included BFDV DNA detection by PCR and quantitative PCR (qPCR) as well as antibody detection by haemagglutination inhibition (HI) and on feather samples, BFDV DNA and antigen was detected by haemagglutination (HA) and qPCR. Four of 97 blood samples collected from vaccinated birds after virus challenge tested positive by PCR, whereas 17 of 35 samples taken from non-vaccinated control corellas tested positive. Vaccinated birds did not develop feather lesions, had only transient PCR-detectable viraemia and had no evidence of persistent infection 270 days post-challenge using PCR, histopathology and immunohistochemistry. Non-vaccinated control corellas developed transient feather lesions and had PCR, HI and HA test results consistent with PBFD. They were BFDV PCR-positive for up to 41 days post-challenge and qPCR demonstrated reduced virus replication in vaccinated birds compared with non-vaccinated control birds. PMID:19218209

  15. Virus-like particles from Escherichia Coli-derived untagged papaya ringspot virus capsid protein purified by immobilized metal affinity chromatography enhance the antibody response against a soluble antigen.

    PubMed

    Guerrero-Rodríguez, Jesús; Manuel-Cabrera, Carlos Alberto; Palomino-Hermosillo, Y Apatzingan; Delgado-Guzmán, Paola Guadalupe; Escoto-Delgadillo, Martha; Silva-Rosales, Laura; Herrera-Rodríguez, Sara Elisa; Sánchez-Hernández, Carla; Gutiérrez-Ortega, Abel

    2014-12-01

    There is a growing interest in using virus-like particles (VLPs) as scaffolds for the presentation of antigens of choice to the immune system. In this work, VLPs from papaya ringspot virus capsid protein expressed in Escherichia coli were evaluated as enhancers of antibody response against a soluble antigen. Interestingly, although the capsid protein lacks a histidine tag, its purification by immobilized metal affinity chromatography was achieved. The formation of VLPs was demonstrated by electron microscopy for the first time for this capsid protein. VLPs were enriched by polyethylene glycol precipitation. Additionally, these VLPs were chemically coupled to green fluorescent protein in order to evaluate them as antigen carriers; however, bioconjugate instability was observed. Nonetheless, the adjuvant effect of these VLPs on BALB/c mice was evaluated, using GFP as antigen, resulting in a significant increase in anti-GFP IgG response, particularly, IgG1 class, demonstrating that the VLPs enhance the immune response against the antigen chosen in this study. PMID:25119647

  16. Whole-Chain Tick Saliva Proteins Presented on Hepatitis B Virus Capsid-Like Particles Induce High-Titered Antibodies with Neutralizing Potential

    PubMed Central

    Kolb, Philipp; Wallich, Reinhard; Nassal, Michael

    2015-01-01

    Ticks are vectors for various, including pathogenic, microbes. Tick saliva contains multiple anti-host defense factors that enable ticks their bloodmeals yet also facilitate microbe transmission. Lyme disease-causing borreliae profit specifically from the broadly conserved tick histamine release factor (tHRF), and from cysteine-rich glycoproteins represented by Salp15 from Ixodes scapularis and Iric-1 from Ixodes ricinus ticks which they recruit to their outer surface protein C (OspC). Hence these tick proteins are attractive targets for anti-tick vaccines that simultaneously impair borrelia transmission. Main obstacles are the tick proteins´ immunosuppressive activities, and for Salp15 orthologs, the lack of efficient recombinant expression systems. Here, we exploited the immune-enhancing properties of hepatitis B virus core protein (HBc) derived capsid-like particles (CLPs) to generate, in E. coli, nanoparticulate vaccines presenting tHRF and, as surrogates for the barely soluble wild-type proteins, cysteine-free Salp15 and Iric-1 variants. The latter CLPs were exclusively accessible in the less sterically constrained SplitCore system. Mice immunized with tHRF CLPs mounted a strong anti-tHRF antibody response. CLPs presenting cysteine-free Salp15 and Iric-1 induced antibodies to wild-type, including glycosylated, Salp15 and Iric-1. The broadly distributed epitopes included the OspC interaction sites. In vitro, the anti-Salp15 antibodies interfered with OspC binding and enhanced human complement-mediated killing of Salp15 decorated borreliae. A mixture of all three CLPs induced high titered antibodies against all three targets, suggesting the feasibility of combination vaccines. These data warrant in vivo validation of the new candidate vaccines´ protective potential against tick infestation and Borrelia transmission. PMID:26352137

  17. Long-range effects of tag sequence on marginally stabilized structure in HIV-1 p24 capsid protein monitored using NMR.

    PubMed

    Okazaki, Honoka; Kaneko, Chie; Hirahara, Miyuki; Watanabe, Satoru; Tochio, Naoya; Kigawa, Takanori; Nishimura, Chiaki

    2014-09-01

    N-terminal domain of HIV-1 p24 capsid protein is a globular fold composed of seven helices and two β-strands with a flexible structure including the α4-5 loop and both N- and C-terminal ends. However, the protein shows a high tendency (48%) for an intrinsically disordered structure based on the PONDR VL-XT prediction from the primary sequence. To assess the possibility of marginally stabilized structure under physiological conditions, the N-terminal domain of p24 was destabilized by the addition of an artificial flexible tag to either N- or C-terminal ends, and it was analyzed using T1, T2, hetero-nuclear NOE, and amide-proton exchange experiments. When the C-terminal tag (12 residues) was attached, the regions of the α3-4 loop and helix 6 as well as the α4-5 loop attained the flexible structures. Furthermore, in the protein containing the N-terminal tag (27 residues), helix 4 in addition to the above-mentioned area including α3-4 and α4-5 loops as well as helix 6 exhibited highly disordered structures. Thus, the long-range effects of the existence of tag sequence was observed in the stepwise manner of the appearance of disordered structures (step 1: α4-5 loop, step 2: α3-4 loop and helix 6, and step 3: helix 4). Furthermore, the disordered regions in tagged proteins were consistent with the PONDR VL-XT disordered prediction. The dynamic structure located in the middle part (α3-4 loop to helix 6) of the protein shown in this study may be related to the assembly of the viral particle. PMID:24960591

  18. Coarse-grained simulation reveals key features of HIV-1 capsid self-assembly

    NASA Astrophysics Data System (ADS)

    Grime, John M. A.; Dama, James F.; Ganser-Pornillos, Barbie K.; Woodward, Cora L.; Jensen, Grant J.; Yeager, Mark; Voth, Gregory A.

    2016-05-01

    The maturation of HIV-1 viral particles is essential for viral infectivity. During maturation, many copies of the capsid protein (CA) self-assemble into a capsid shell to enclose the viral RNA. The mechanistic details of the initiation and early stages of capsid assembly remain to be delineated. We present coarse-grained simulations of capsid assembly under various conditions, considering not only capsid lattice self-assembly but also the potential disassembly of capsid upon delivery to the cytoplasm of a target cell. The effects of CA concentration, molecular crowding, and the conformational variability of CA are described, with results indicating that capsid nucleation and growth is a multi-stage process requiring well-defined metastable intermediates. Generation of the mature capsid lattice is sensitive to local conditions, with relatively subtle changes in CA concentration and molecular crowding influencing self-assembly and the ensemble of structural morphologies.

  19. Coarse-grained simulation reveals key features of HIV-1 capsid self-assembly.

    PubMed

    Grime, John M A; Dama, James F; Ganser-Pornillos, Barbie K; Woodward, Cora L; Jensen, Grant J; Yeager, Mark; Voth, Gregory A

    2016-01-01

    The maturation of HIV-1 viral particles is essential for viral infectivity. During maturation, many copies of the capsid protein (CA) self-assemble into a capsid shell to enclose the viral RNA. The mechanistic details of the initiation and early stages of capsid assembly remain to be delineated. We present coarse-grained simulations of capsid assembly under various conditions, considering not only capsid lattice self-assembly but also the potential disassembly of capsid upon delivery to the cytoplasm of a target cell. The effects of CA concentration, molecular crowding, and the conformational variability of CA are described, with results indicating that capsid nucleation and growth is a multi-stage process requiring well-defined metastable intermediates. Generation of the mature capsid lattice is sensitive to local conditions, with relatively subtle changes in CA concentration and molecular crowding influencing self-assembly and the ensemble of structural morphologies. PMID:27174390

  20. Coarse-grained simulation reveals key features of HIV-1 capsid self-assembly

    PubMed Central

    Grime, John M. A.; Dama, James F.; Ganser-Pornillos, Barbie K.; Woodward, Cora L.; Jensen, Grant J.; Yeager, Mark; Voth, Gregory A.

    2016-01-01

    The maturation of HIV-1 viral particles is essential for viral infectivity. During maturation, many copies of the capsid protein (CA) self-assemble into a capsid shell to enclose the viral RNA. The mechanistic details of the initiation and early stages of capsid assembly remain to be delineated. We present coarse-grained simulations of capsid assembly under various conditions, considering not only capsid lattice self-assembly but also the potential disassembly of capsid upon delivery to the cytoplasm of a target cell. The effects of CA concentration, molecular crowding, and the conformational variability of CA are described, with results indicating that capsid nucleation and growth is a multi-stage process requiring well-defined metastable intermediates. Generation of the mature capsid lattice is sensitive to local conditions, with relatively subtle changes in CA concentration and molecular crowding influencing self-assembly and the ensemble of structural morphologies. PMID:27174390

  1. Quantitative comparison of expression with adeno-associated virus (AAV-2) brain-specific gene cassettes.

    PubMed

    Xu, R; Janson, C G; Mastakov, M; Lawlor, P; Young, D; Mouravlev, A; Fitzsimons, H; Choi, K L; Ma, H; Dragunow, M; Leone, P; Chen, Q; Dicker, B; During, M J

    2001-09-01

    This study compared a range of mammalian CNS expression cassettes in recombinant adeno-associated virus (AAV-2) vectors using strong endogenous promoter sequences, with or without a strong post-regulatory element and polyadenylation signal. Changes in these elements led to transgene expression varying by over three orders of magnitude. In experiments conducted in primary cell culture and in >100 stereotactically injected rats, we observed highly efficient and stable (>15 months) gene expression in neurons and limited expression in glia; the highest expression occurred with endogenous, nonviral promoters such as neuron-specific enolase and beta-actin. The packaging size of AAV-2 was maximized at 5.7 kb without impairing gene expression, as judged by direct comparison with a number of smaller AAV-2 constructs. The genomic insert size and titer were confirmed by Southern blot and quantitative PCR, and infectivity was tested by particle titer using ELISA with a conformation-dependent epitope that requires the full intact capsid. A packaging and purification protocol we describe allows for high-titer, high-capacity AAV-2 vectors that can transduce over 2 x 10(5) neurons in vivo per microliter of vector, using the strongest expression cassette. PMID:11571569

  2. Membrane-mediated interaction between retroviral capsids

    NASA Astrophysics Data System (ADS)

    Zhang, Rui; Nguyen, Toan

    2012-02-01

    A retrovirus is an RNA virus that is replicated through a unique strategy of reverse transcription. Unlike regular enveloped viruses which are assembled inside the host cells, the assembly of retroviral capsids happens right on the cell membrane. During the assembly process, the partially formed capsids deform the membrane, giving rise to an elastic energy. When two such partial capsids approach each other, this elastic energy changes. Or in other words, the two partial capsids interact with each other via the membrane. This membrane mediated interaction between partial capsids plays an important role in the kinetics of the assembly process. In this work, this membrane mediated interaction is calculated both analytically and numerically. It is worth noting that the diferential equation determining the membrane shape in general nonlinear and cannot be solved analytically,except in the linear region of small deformations. And it is exactly the nonlinear regime that is important for the assembly kinetics of retroviruses as it provides a large energy barrier. The theory developed here is applicable to more generic cases of membrane mediated interactions between two membrane-embedded proteins.

  3. Identification and Characterization of Nuclear and Nucleolar Localization Signals in the Adeno-Associated Virus Serotype 2 Assembly-Activating Protein

    PubMed Central

    Earley, Lauriel F.; Kawano, Yasuhiro; Adachi, Kei; Sun, Xiao-Xin; Dai, Mu-Shui

    2014-01-01

    ABSTRACT Assembly-activating protein (AAP) of adeno-associated virus serotype 2 (AAV2) is a nucleolar-localizing protein that plays a critical role in transporting the viral capsid VP3 protein to the nucleolus for assembly. Here, we identify and characterize AAV2 AAP (AAP2) nuclear (NLS) and nucleolar (NoLS) localization signals near the carboxy-terminal region of AAP2 (amino acid positions 144 to 184) (AAP2144–184). This region contains five basic-amino-acid-rich (BR) clusters, KSKRSRR (AAP2BR1), RRR (AAP2BR2), RFR (AAP2BR3), RSTSSR (AAP2BR4), and RRIK (AAP2BR5), from the amino terminus to the carboxy terminus. We created 30 AAP2BR mutants by arginine/lysine-to-alanine mutagenesis or deletion of AAP2BRs and 8 and 1 green fluorescent protein (GFP)-AAP2BR and β-galactosidase–AAP2BR fusion proteins, respectively, and analyzed their intracellular localization in HeLa cells by immunofluorescence microscopy. The results showed that AAP2144–184 has redundant multipartite NLSs and that any combinations of 4 AAP2BRs, but not 3 or less, can constitute a functional NLS-NoLS; AAP2BR1 and AAP2BR2 play the most influential role for nuclear localization, but either one of the two AAP2BRs is dispensable if all 4 of the other AAP2BRs are present, resulting in 3 different, overlapping NLS motifs; and the NoLS is shared redundantly among the five AAP2BRs and functions in a context-dependent manner. AAP2BR mutations not only resulted in aberrant intracellular localization, but also attenuated AAP2 protein expression to various degrees, and both of these abnormalities have a significant negative impact on capsid production. Thus, this study reveals the organization of the intermingling NLSs and NoLSs in AAP2 and provides insights into their functional roles in capsid assembly. IMPORTANCE Adeno-associated virus (AAV) has become a popular and successful vector for in vivo gene therapy; however, its biology has yet to be fully understood. In this regard, the recent discovery of

  4. The use of additive and subtractive approaches to examine the nuclear localization sequence of the polyomavirus major capsid protein VP1

    NASA Technical Reports Server (NTRS)

    Chang, D.; Haynes, J. I. 2nd; Brady, J. N.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    A nuclear localization signal (NLS) has been identified in the N-terminal (Ala1-Pro-Lys-Arg-Lys-Ser-Gly-Val-Ser-Lys-Cys11) amino acid sequence of the polyomavirus major capsid protein VP1. The importance of this amino acid sequence for nuclear transport of VP1 protein was demonstrated by a genetic "subtractive" study using the constructs pSG5VP1 (full-length VP1) and pSG5 delta 5'VP1 (truncated VP1, lacking amino acids Ala1-Cys11). These constructs were used to transfect COS-7 cells, and expression and intracellular localization of the VP1 protein was visualized by indirect immunofluorescence. These studies revealed that the full-length VP1 was expressed and localized in the nucleus, while the truncated VP1 protein was localized in the cytoplasm and not transported to the nucleus. These findings were substantiated by an "additive" approach using FITC-labeled conjugates of synthetic peptides homologous to the NLS of VP1 cross-linked to bovine serum albumin or immunoglobulin G. Both conjugates localized in the nucleus after microinjection into the cytoplasm of 3T6 cells. The importance of individual amino acids found in the basic sequence (Lys3-Arg-Lys5) of the NLS was also investigated. This was accomplished by synthesizing three additional peptides in which lysine-3 was substituted with threonine, arginine-4 was substituted with threonine, or lysine-5 was substituted with threonine. It was found that lysine-3 was crucial for nuclear transport, since substitution of this amino acid with threonine prevented nuclear localization of the microinjected, FITC-labeled conjugate.

  5. Mechanical oscillations of a viral capsid

    NASA Astrophysics Data System (ADS)

    Benson, Daryn; Sankey, Otto; Dykeman, Eric

    2010-03-01

    Viruses are sub-microscopic infectious agents that infect almost every living creature on Earth. They are unable to grow or reproduce outside of a host cell and are therefore parasitic in nature. A virus' internal genetic material is protected by an external protein coat (capsid). We developed a theoretical model which uses the interaction of light with a viral capsid to create large amplitude motions within the capsid. This work displays the results of the model on the tobacco mosaic virus (TMV) with attached RNA genome. The development of this model was motivated by the experimental work of Tsen et. al. [1] who used ultra-short laser pulses to inactivate viruses. [1] K-T. Tsen et al., J. of Physics -- Cond. Mat. 19, 472201 (2007).

  6. N-terminal basic amino acid residues of Beet black scorch virus capsid protein play a critical role in virion assembly and systemic movement

    PubMed Central

    2013-01-01

    Background Beet black scorch virus (BBSV) is a small single-stranded, positive-sense RNA plant virus belonging to the genus Necrovirus, family Tombusviridae. Its capsid protein (CP) contains a 13 amino acid long basic region at the N-terminus, rich in arginine and lysine residues, which is thought to interact with viral RNA to initiate virion assembly. Results In the current study, a series of BBSV mutants containing amino acid substitutions as well as deletions within the N-terminal region were generated and examined for their effects on viral RNA replication, virion assembly, and long distance spread in protoplasts and whole host plants of BBSV. The RNA-binding activities of the mutated CPs were also evaluated in vitro. These experiments allowed us to identify two key basic amino acid residues in this region that are responsible for initiating virus assembly through RNA-binding. Proper assembly of BBSV particles is in turn needed for efficient viral systemic movement. Conclusions We have identified two basic amino acid residues near the N-terminus of the BBSV CP that bind viral RNA with high affinity to initiate virion assembly. We further provide evidence showing that systemic spread of BBSV in infected plants requires intact virions. This study represents the first in-depth investigation of the role of basic amino acid residues within the N-terminus of a necroviral CP. PMID:23786675

  7. Development of an in process control filtration-assisted chemiluminometric immunoassay to quantify foot and mouth disease virus (FMDV) non-capsid proteins in vaccine-antigen batches.

    PubMed

    Capozzo, Alejandra Victoria; Martínez, Manuel Rosendo; Schielen, Wilhelmus Joseph Gerardus

    2010-09-14

    In many countries, foot and mouth disease (FMD) is controlled by vaccination and surveillance against non-capsid proteins (NCP); therefore vaccines are required not to induce antibodies against NCP. Vaccine purity is evaluated by repeated inoculation of naïve cattle, an expensive and time consuming protocol that raises several animal welfare concerns. We have developed an in process control filtration-assisted chemiluminometric immunoassay (FAL-ELISA), to detect and quantify NCP in vaccine-antigen batches regardless of its volume and composition. Samples are filtered through PVDF-filter microplates pre-coated with a monoclonal antibody against NCP. Filtration removes all unbound components in the sample and captured NCP are detected by anti-NCP conjugate followed by incubation with the substrate, luminol/peroxide. Analytical detection limit was 2 ng for purified NCP and 4 ng for vaccine-antigen batches spiked with NCP, which makes this assay sensitive enough to be applied to purity control of FMD vaccines. Vaccine components did not interfere with the antibody and substrate reactions in the assay. FAL-ELISA is an alternative for the in vivo tests, observing the objective to Replace, Reduce and Refine the use of animals for quality control of immunobiologicals. PMID:20685600

  8. Identification of the major capsid protein of erythrocytic necrosis virus (ENV) and development of quantitative real-time PCR assays for quantification of ENV DNA.

    PubMed

    Purcell, Maureen K; Pearman-Gillman, Schuyler; Thompson, Rachel L; Gregg, Jacob L; Hart, Lucas M; Winton, James R; Emmenegger, Eveline J; Hershberger, Paul K

    2016-07-01

    Viral erythrocytic necrosis (VEN) is a disease of marine and anadromous fish that is caused by the erythrocytic necrosis virus (ENV), which was recently identified as a novel member of family Iridoviridae by next-generation sequencing. Phylogenetic analysis of the ENV DNA polymerase grouped ENV with other erythrocytic iridoviruses from snakes and lizards. In the present study, we identified the gene encoding the ENV major capsid protein (MCP) and developed a quantitative real-time PCR (qPCR) assay targeting this gene. Phylogenetic analysis of the MCP gene sequence supported the conclusion that ENV does not group with any of the currently described iridovirus genera. Because there is no information regarding genetic variation of the MCP gene across the reported host and geographic range for ENV, we also developed a second qPCR assay for a more conserved ATPase-like gene region. The MCP and ATPase qPCR assays demonstrated good analytical and diagnostic sensitivity and specificity based on samples from laboratory challenges of Pacific herring Clupea pallasii The qPCR assays had similar diagnostic sensitivity and specificity as light microscopy of stained blood smears for the presence of intraerythrocytic inclusion bodies. However, the qPCR assays may detect viral DNA early in infection prior to the formation of inclusion bodies. Both qPCR assays appear suitable for viral surveillance or as a confirmatory test for ENV in Pacific herring from the Salish Sea. PMID:27154315

  9. Functional analysis of the N-terminal basic motif of a eukaryotic satellite RNA virus capsid protein in replication and packaging.

    PubMed

    Sivanandam, Venkatesh; Mathews, Deborah; Garmann, Rees; Erdemci-Tandogan, Gonca; Zandi, Roya; Rao, A L N

    2016-01-01

    Efficient replication and assembly of virus particles are integral to the establishment of infection. In addition to the primary role of the capsid protein (CP) in encapsidating the RNA progeny, experimental evidence on positive sense single-stranded RNA viruses suggests that the CP also regulates RNA synthesis. Here, we demonstrate that replication of Satellite tobacco mosaic virus (STMV) is controlled by the cooperative interaction between STMV CP and the helper virus (HV) Tobacco mosaic virus (TMV) replicase. We identified that the STMV CP-HV replicase interaction requires a positively charged residue at the third position (3R) in the N-terminal 13 amino acid (aa) motif. Far-Northwestern blotting showed that STMV CP promotes binding between HV-replicase and STMV RNA. An STMV CP variant having an arginine to alanine substitution at position 3 in the N-terminal 13aa motif abolished replicase-CP binding. The N-terminal 13aa motif of the CP bearing alanine substitutions for positively charged residues located at positions 5, 7, 10 and 11 are defective in packaging full-length STMV, but can package a truncated STMV RNA lacking the 3' terminal 150 nt region. These findings provide insights into the mechanism underlying the regulation of STMV replication and packaging. PMID:27193742

  10. Functional analysis of the N-terminal basic motif of a eukaryotic satellite RNA virus capsid protein in replication and packaging

    PubMed Central

    Sivanandam, Venkatesh; Mathews, Deborah; Garmann, Rees; Erdemci-Tandogan, Gonca; Zandi, Roya; Rao, A. L. N.

    2016-01-01

    Efficient replication and assembly of virus particles are integral to the establishment of infection. In addition to the primary role of the capsid protein (CP) in encapsidating the RNA progeny, experimental evidence on positive sense single-stranded RNA viruses suggests that the CP also regulates RNA synthesis. Here, we demonstrate that replication of Satellite tobacco mosaic virus (STMV) is controlled by the cooperative interaction between STMV CP and the helper virus (HV) Tobacco mosaic virus (TMV) replicase. We identified that the STMV CP-HV replicase interaction requires a positively charged residue at the third position (3R) in the N-terminal 13 amino acid (aa) motif. Far-Northwestern blotting showed that STMV CP promotes binding between HV-replicase and STMV RNA. An STMV CP variant having an arginine to alanine substitution at position 3 in the N-terminal 13aa motif abolished replicase-CP binding. The N-terminal 13aa motif of the CP bearing alanine substitutions for positively charged residues located at positions 5, 7, 10 and 11 are defective in packaging full-length STMV, but can package a truncated STMV RNA lacking the 3′ terminal 150 nt region. These findings provide insights into the mechanism underlying the regulation of STMV replication and packaging. PMID:27193742

  11. Unveiling of the Diversity of Prasinoviruses (Phycodnaviridae) in Marine Samples by Using High-Throughput Sequencing Analyses of PCR-Amplified DNA Polymerase and Major Capsid Protein Genes

    PubMed Central

    Clerissi, Camille; Ogata, Hiroyuki; Hingamp, Pascal; Poulain, Julie; Desdevises, Yves

    2014-01-01

    Viruses strongly influence the ecology and evolution of their eukaryotic hosts in the marine environment, but little is known about their diversity and distribution. Prasinoviruses infect an abundant and widespread class of phytoplankton, the Mamiellophyceae, and thereby exert a specific and important role in microbial ecosystems. However, molecular tools to specifically identify this viral genus in environmental samples are still lacking. We developed two primer sets, designed for use with polymerase chain reactions and 454 pyrosequencing technologies, to target two conserved genes, encoding the DNA polymerase (PolB gene) and the major capsid protein (MCP gene). While only one copy of the PolB gene is present in Prasinovirus genomes, there are at least seven paralogs for MCP, the copy we named number 6 being shared with other eukaryotic alga-infecting viruses. Primer sets for PolB and MCP6 were thus designed and tested on 6 samples from the Tara Oceans project. The results suggest that the MCP6 amplicons show greater richness but that PolB gave a wider coverage of Prasinovirus diversity. As a consequence, we recommend use of the PolB primer set, which will certainly reveal exciting new insights about the diversity and distribution of prasinoviruses at the community scale. PMID:24632251

  12. Identification of the major capsid protein of erythrocytic necrosis virus (ENV) and development of quantitative real-time PCR assays for quantification of ENV DNA

    USGS Publications Warehouse

    Purcell, Maureen K.; Pearman-Gillman, Schuyler; Thompson, Rachel L.; Gregg, Jacob L.; Hart, Lucas M.; Winton, James R.; Emmenegger, Eveline J.; Hershberger, Paul K.

    2016-01-01

    Viral erythrocytic necrosis (VEN) is a disease of marine and anadromous fish that is caused by the erythrocytic necrosis virus (ENV), which was recently identified as a novel member of family Iridoviridae by next-generation sequencing. Phylogenetic analysis of the ENV DNA polymerase grouped ENV with other erythrocytic iridoviruses from snakes and lizards. In the present study, we identified the gene encoding the ENV major capsid protein (MCP) and developed a quantitative real-time PCR (qPCR) assay targeting this gene. Phylogenetic analysis of the MCP gene sequence supported the conclusion that ENV does not group with any of the currently described iridovirus genera. Because there is no information regarding genetic variation of the MCP gene across the reported host and geographic range for ENV, we also developed a second qPCR assay for a more conserved ATPase-like gene region. The MCP and ATPase qPCR assays demonstrated good analytical and diagnostic sensitivity and specificity based on samples from laboratory challenges of Pacific herring Clupea pallasii. The qPCR assays had similar diagnostic sensitivity and specificity as light microscopy of stained blood smears for the presence of intraerythrocytic inclusion bodies. However, the qPCR assays may detect viral DNA early in infection prior to the formation of inclusion bodies. Both qPCR assays appear suitable for viral surveillance or as a confirmatory test for ENV in Pacific herring from the Salish Sea.

  13. The capsid protein of Turnip crinkle virus overcomes two separate defense barriers to facilitate systemic movement of the virus in Arabidopsis.

    PubMed

    Cao, Mingxia; Ye, Xiaohong; Willie, Kristen; Lin, Junyan; Zhang, Xiuchun; Redinbaugh, Margaret G; Simon, Anne E; Morris, T Jack; Qu, Feng

    2010-08-01

    The capsid protein (CP) of Turnip crinkle virus (TCV) is a multifunctional protein needed for virus assembly, suppression of RNA silencing-based antiviral defense, and long-distance movement in infected plants. In this report, we have examined genetic requirements for the different functions of TCV CP and evaluated the interdependence of these functions. A series of TCV mutants containing alterations in the CP coding region were generated. These alterations range from single-amino-acid substitutions and domain truncations to knockouts of CP translation. The latter category also contained two constructs in which the CP coding region was replaced by either the cDNA of a silencing suppressor of a different virus or that of green fluorescent protein. These mutants were used to infect Arabidopsis plants with diminished antiviral silencing capability (dcl2 dcl3 dcl4 plants). There was a strong correlation between the ability of mutants to reach systemic leaves and the silencing suppressor activity of mutant CP. Virus particles were not essential for entry of the viral genome into vascular bundles in the inoculated leaves in the absence of antiviral silencing. However, virus particles were necessary for egress of the viral genome from the vasculature of systemic leaves. Our experiments demonstrate that TCV CP not only allows the viral genome to access the systemic movement channel through silencing suppression but also ensures its smooth egress by way of assembled virus particles. These results illustrate that efficient long-distance movement of TCV requires both functions afforded by the CP. PMID:20504923

  14. High cerebrospinal fluid levels of interleukin-10 attained by AAV in dogs.

    PubMed

    Pleticha, J; Malkmus, S A; Heilmann, L F; Veesart, S L; Rezek, R; Xu, Q; Yaksh, T L; Beutler, A S

    2015-02-01

    Intrathecal (IT) gene transfer using adeno-associated virus (AAV) may be clinically promising as a treatment for chronic pain if it can produce sufficiently high levels of a transgene product in the cerebrospinal fluid (CSF). Although this strategy was developed in rodents, no studies investigating CSF levels of an analgesic or antiallodynic protein delivered by IT AAV have been performed in large animals. Interleukin-10 (IL-10) is an antiallodynic cytokine for which target therapeutic levels have been established in rats. The present study tested IT AAV8 encoding either human IL-10 (hIL-10) or enhanced green fluorescent protein (EGFP) in a dog model of IT drug delivery. AAV8/hIL-10 at a dose of 3.5 × 10(12) genome copies induced high hIL-10 levels in the CSF, exceeding the target concentration previously found to be antiallodynic in rodents by >1000-fold. AAV8/EGFP targeted the primary sensory and motor neurons and the meninges. hIL-10, a xenogeneic protein in dogs, induced anti-hIL-10 antibodies detectable in the CSF and serum of dogs. The high hIL-10 levels demonstrate the efficacy of AAV for delivery of secreted transgenes into the IT space of large animals, suggesting a strong case for further development toward clinical testing. PMID:25354684

  15. Multivalent Viral Capsids with Internal Cargo for Fibrin Imaging

    PubMed Central

    Obermeyer, Allie C.; Capehart, Stacy L.; Jarman, John B.; Francis, Matthew B.

    2014-01-01

    Thrombosis is the cause of many cardiovascular syndromes and is a significant contributor to life-threatening diseases, such as myocardial infarction and stroke. Thrombus targeted imaging agents have the capability to provide molecular information about pathological clots, potentially improving detection, risk stratification, and therapy of thrombosis-related diseases. Nanocarriers are a promising platform for the development of molecular imaging agents as they can be modified to have external targeting ligands and internal functional cargo. In this work, we report the synthesis and use of chemically functionalized bacteriophage MS2 capsids as biomolecule-based nanoparticles for fibrin imaging. The capsids were modified using an oxidative coupling reaction, conjugating ∼90 copies of a fibrin targeting peptide to the exterior of each protein shell. The ability of the multivalent, targeted capsids to bind fibrin was first demonstrated by determining the impact on thrombin-mediated clot formation. The modified capsids out-performed the free peptides and were shown to inhibit clot formation at effective concentrations over ten-fold lower than the monomeric peptide alone. The installation of near-infrared fluorophores on the interior surface of the capsids enabled optical detection of binding to fibrin clots. The targeted capsids bound to fibrin, exhibiting higher signal-to-background than control, non-targeted MS2-based nanoagents. The in vitro assessment of the capsids suggests that fibrin-targeted MS2 capsids could be used as delivery agents to thrombi for diagnostic or therapeutic applications. PMID:24960118

  16. All-Atom Molecular Dynamics of Virus Capsids as Drug Targets

    PubMed Central

    2016-01-01

    Virus capsids are protein shells that package the viral genome. Although their morphology and biological functions can vary markedly, capsids often play critical roles in regulating viral infection pathways. A detailed knowledge of virus capsids, including their dynamic structure, interactions with cellular factors, and the specific roles that they play in the replication cycle, is imperative for the development of antiviral therapeutics. The following Perspective introduces an emerging area of computational biology that focuses on the dynamics of virus capsids and capsid–protein assemblies, with particular emphasis on the effects of small-molecule drug binding on capsid structure, stability, and allosteric pathways. When performed at chemical detail, molecular dynamics simulations can reveal subtle changes in virus capsids induced by drug molecules a fraction of their size. Here, the current challenges of performing all-atom capsid–drug simulations are discussed, along with an outlook on the applicability of virus capsid simulations to reveal novel drug targets. PMID:27128262

  17. All-atom molecular dynamics calculation study of entire poliovirus empty capsids in solution

    SciTech Connect

    Andoh, Y.; Yoshii, N.; Yamada, A.; Kojima, H.; Mizutani, K.; Okazaki, S.; Fujimoto, K.; Nakagawa, A.; Nomoto, A.

    2014-10-28

    Small viruses that belong, for example, to the Picornaviridae, such as poliovirus and foot-and-mouth disease virus, consist simply of capsid proteins and a single-stranded RNA (ssRNA) genome. The capsids are quite stable in solution to protect the genome from the environment. Here, based on long-time and large-scale 6.5 × 10{sup 6} all-atom molecular dynamics calculations for the Mahoney strain of poliovirus, we show microscopic properties of the viral capsids at a molecular level. First, we found equilibrium rapid exchange of water molecules across the capsid. The exchange rate is so high that all water molecules inside the capsid (about 200 000) can leave the capsid and be replaced by water molecules from the outside in about 25 μs. This explains the capsid's tolerance to high pressures and deactivation by exsiccation. In contrast, the capsid did not exchange ions, at least within the present simulation time of 200 ns. This implies that the capsid can function, in principle, as a semipermeable membrane. We also found that, similar to the xylem of trees, the pressure of the solution inside the capsid without the genome was negative. This is caused by coulombic interaction of the solution inside the capsid with the capsid excess charges. The negative pressure may be compensated by positive osmotic pressure by the solution-soluble ssRNA and the counter ions introduced into it.

  18. All-atom molecular dynamics calculation study of entire poliovirus empty capsids in solution

    NASA Astrophysics Data System (ADS)

    Andoh, Y.; Yoshii, N.; Yamada, A.; Fujimoto, K.; Kojima, H.; Mizutani, K.; Nakagawa, A.; Nomoto, A.; Okazaki, S.

    2014-10-01

    Small viruses that belong, for example, to the Picornaviridae, such as poliovirus and foot-and-mouth disease virus, consist simply of capsid proteins and a single-stranded RNA (ssRNA) genome. The capsids are quite stable in solution to protect the genome from the environment. Here, based on long-time and large-scale 6.5 × 106 all-atom molecular dynamics calculations for the Mahoney strain of poliovirus, we show microscopic properties of the viral capsids at a molecular level. First, we found equilibrium rapid exchange of water molecules across the capsid. The exchange rate is so high that all water molecules inside the capsid (about 200 000) can leave the capsid and be replaced by water molecules from the outside in about 25 μs. This explains the capsid's tolerance to high pressures and deactivation by exsiccation. In contrast, the capsid did not exchange ions, at least within the present simulation time of 200 ns. This implies that the capsid can function, in principle, as a semipermeable membrane. We also found that, similar to the xylem of trees, the pressure of the solution inside the capsid without the genome was negative. This is caused by coulombic interaction of the solution inside the capsid with the capsid excess charges. The negative pressure may be compensated by positive osmotic pressure by the solution-soluble ssRNA and the counter ions introduced into it.

  19. Detection of Foot-and-mouth Disease Virus RNA and Capsid Protein in Lymphoid Tissues of Convalescent Pigs Does Not Indicate Existence of a Carrier State.

    PubMed

    Stenfeldt, C; Pacheco, J M; Smoliga, G R; Bishop, E; Pauszek, S J; Hartwig, E J; Rodriguez, L L; Arzt, J

    2016-04-01

    A systematic study was performed to investigate the potential of pigs to establish and maintain persistent foot-and-mouth disease virus (FMDV) infection. Infectious virus could not be recovered from sera, oral, nasal or oropharyngeal fluids obtained after resolution of clinical infection with any of five FMDV strains within serotypes A, O and Asia-1. Furthermore, there was no isolation of live virus from tissue samples harvested at 28-100 days post-infection from convalescent pigs recovered from clinical or subclinical FMD. Despite lack of detection of infectious FMDV, there was a high prevalence of FMDV RNA detection in lymph nodes draining lesion sites harvested at 35 days post-infection, with the most frequent detection recorded in popliteal lymph nodes (positive detection in 88% of samples obtained from non-vaccinated pigs). Likewise, at 35 dpi, FMDV capsid antigen was localized within follicles of draining lymph nodes, but without concurrent detection of FMDV non-structural protein. There was a marked decline in the detection of FMDV RNA and antigen in tissue samples by 60 dpi, and no antigen or viral RNA could be detected in samples obtained at 100 dpi. The data presented herein provide the most extensive investigation of FMDV persistence in pigs. The overall conclusion is that domestic pigs are unlikely to be competent long-term carriers of infectious FMDV; however, transient persistence of FMDV protein and RNA in lymphoid tissues is common following clinical or subclinical infection. PMID:24943477

  20. Computational and molecular tools for scalable rAAV-mediated genome editing.

    PubMed

    Stoimenov, Ivaylo; Ali, Muhammad Akhtar; Pandzic, Tatjana; Sjöblom, Tobias

    2015-03-11

    The rapid discovery of potential driver mutations through large-scale mutational analyses of human cancers generates a need to characterize their cellular phenotypes. Among the techniques for genome editing, recombinant adeno-associated virus (rAAV)-mediated gene targeting is suited for knock-in of single nucleotide substitutions and to a lesser degree for gene knock-outs. However, the generation of gene targeting constructs and the targeting process is time-consuming and labor-intense. To facilitate rAAV-mediated gene targeting, we developed the first software and complementary automation-friendly vector tools to generate optimized targeting constructs for editing human protein encoding genes. By computational approaches, rAAV constructs for editing ~71% of bases in protein-coding exons were designed. Similarly, ~81% of genes were predicted to be targetable by rAAV-mediated knock-out. A Gateway-based cloning system for facile generation of rAAV constructs suitable for robotic automation was developed and used in successful generation of targeting constructs. Together, these tools enable automated rAAV targeting construct design, generation as well as enrichment and expansion of targeted cells with desired integrations. PMID:25488813

  1. HIV-1 Capsid Stabilization Assay.

    PubMed

    Fricke, Thomas; Diaz-Griffero, Felipe

    2016-01-01

    The stability of the HIV-1 core in the cytoplasm is crucial for productive HIV-1 infection. Mutations that stabilize or destabilize the core showed defects in HIV-1 reverse transcription and infection. We developed a novel and simple assay to measure stability of in vitro-assembled HIV-1 CA-NC complexes. This assay allowed us to demonstrate that cytosolic extracts strongly stabilize the HIV-1 core (Fricke et al., J Virol 87:10587-10597, 2013). By using our novel assay, one can measure the ability of different drugs to modulate the stability of in vitro-assembled HIV-1 CA-NC complexes, such as PF74, CAP-1, IXN-053, cyclosporine A, Bi2, and the peptide CAI. We also found that purified CPSF6 (1-321) protein stabilizes in vitro-assembled HIV-1 CA-NC complexes (Fricke et al., J Virol 87:10587-10597, 2013). Here we describe in detail the use of this capsid stability assay. We believe that our assay can be a powerful tool to assess HIV-1 capsid stability in vitro. PMID:26714703

  2. Enhancing Transgene Expression from Recombinant AAV8 Vectors in Different Tissues Using Woodchuck Hepatitis Virus Post-Transcriptional Regulatory Element

    PubMed Central

    Wang, Lizheng; Wang, Zixuan; Zhang, Fangfang; Zhu, Rui; Bi, Jinpeng; Wu, Jiaxin; Zhang, Haihong; Wu, Hui; Kong, Wei; Yu, Bin; Yu, Xianghui

    2016-01-01

    Adeno-associated virus (AAV) vectors have been utilized extensively in gene therapy and gene function studies, as strong transgene expression is a prerequisite for positive outcomes. AAV8 was reported as the most efficient AAV serotype for transduction of the liver, brain and muscle compared with other serotypes. However, AAV8-mediated transduction of human hepatocytes is rather poor with approximately 20-fold lower efficiency compared with that of mouse hepatocytes. Therefore, we applied the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) to enhance AAV8-mediated transgene expression driven by a combination promoter (CAG promoter) with a CMV-IE enhancer and chicken beta-actin promoter for a more efficient viral vector. Transgene expression from recombinant AAV8 (rAAV8) vectors harboring a red fluorescent protein (RFP) reporter gene with or without WPRE were evaluated in vitro and in vivo. The results demonstrated that WPRE improved AAV8-mediated RFP expression in different cell lines with clear increases of transgene expression in the liver, brain or muscle of animals. The findings of this study will help to substantially reduce the quantity of viral particles that must be injected in order to reach a therapeutic level of transgene expression in gene therapy. Consequently, such dose reductions may lessen the potential risks associated with high doses of viral vectors. PMID:27076785

  3. Membrane Binding Properties and Terminal Residues of the Mature Hepatitis C Virus Capsid Protein in Insect Cells

    PubMed Central

    Ogino, Tomoaki; Fukuda, Hiroyuki; Imajoh-Ohmi, Shinobu; Kohara, Michinori; Nomoto, Akio

    2004-01-01

    The immature core protein (p23, residues 1 to 191) of hepatitis C virus undergoes posttranslational modifications including intramembranous proteolysis within its C-terminal signal sequence by signal peptide peptidase to generate the mature form (p21). In this study, we analyzed the cleavage site and other amino acid modifications that occur on the core protein. To produce the posttranslationally modified core protein, we used a baculovirus-insect cell expression model system. As previously reported, p23 is processed to form p21 in insect as well as in mammalian cells. p21 was found to be associated with the cytoplasmic membrane, and its significant portion behaved as an integral membrane protein. The protein was purified from the membrane by a simple and unique procedure on the basis of its membrane-binding properties and solubility in detergents. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of purified p21 showed that the average molecular mass (m/z 19,307) of its single-charged ion differs by m/z 1,457 from that calculated for p23. To determine the posttranslational modifications, tryptic p21 peptides were analyzed by MALDI-TOF MS. We found three peptides that did not match the theoretically derived peptides of p23. Analysis of these peptides by MALDI-TOF tandem MS revealed that they correspond to N-terminal peptides (residues 2 to 9 and 2 to 10) starting with α-N-acetylserine and C-terminal peptide (residues 150 to 177) ending with phenylalanine. These results suggest that the mature core protein (molecular mass of 19,306 Da) includes residues 2 to 177 and that its N terminus is blocked with an acetyl group. PMID:15479818

  4. Development and characterization of a potential diagnostic monoclonal antibody against capsid protein VP1 of the chicken anemia virus

    PubMed Central

    Lien, Yi-Yang; Huang, Chi-Hung; Sun, Fang-Chun; Sheu, Shyang-Chwen; Lu, Tsung-Chi; Lee, Meng-Shiunn; Hsueh, Shu-Chin; Chen, Hsi-Jien

    2012-01-01

    Chicken anemia virus (CAV) is an important viral pathogen that causes anemia and severe immunodeficiency syndrome in chickens worldwide. In this study, a potential diagnostic monoclonal antibody against the CAV VP1 protein was developed which can precisely recognize the CAV antigen for diagnostic and virus recovery purposes. The VP1 gene of CAV encoding the N-terminus-deleted VP1 protein, VP1Nd129, was cloned into an Escherichia (E.) coli expression vector. After isopropyl-β-D-thiogalactopyronoside induction, VP1Nd129 protein was shown to be successfully expressed in the E. coli. By performing an enzyme-linked immunoabsorbent assay using two coating antigens, purified VP1Nd129 and CAV-infected liver tissue lysate, E3 monoclonal antibody (mAb) was found to have higher reactivity against VP1 protein than the other positive clones according to the result of limiting dilution method from 64 clones. Using immunohistochemistry, the presence of the VP1-specific mAb, E3, was confirmed using CAV-infected liver and thymus tissues as positive-infected samples. Additionally, CAV particle purification was also performed using an immunoaffinity column containing E3 mAb. The monoclonal E3 mAb developed in this study will not only be very useful for detecting CAV infection and performing histopathology studies of infected chickens, but may also be used to purify CAV particles in the future. PMID:22437539

  5. Production of Monoclonal Antibodies against the Major Capsid Protein of the Lactococcus Bacteriophage ul36 and Development of an Enzyme-Linked Immunosorbent Assay for Direct Phage Detection in Whey and Milk

    PubMed Central

    Moineau, Sylvain; Bernier, Denis; Jobin, Marie; Hébert, Jacques; Klaenhammer, Todd R.; Pandian, Sithian

    1993-01-01

    The only major structural protein (35 kDa) of the lactococcal small isometric-headed bacteriophage ul36, a member of the P335 species, was isolated from a preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Monoclonal antibodies (MAbs) were raised against the denatured 35-kDa protein. Six MAbs were selected and characterized. Western blots (immunoblots) showed that all MAbs recognized the 35 kDa but also a 45 kDa that is in lower concentration in the phage structure. Binding inhibition assays identified five families of MAbs that recognized nonoverlapping epitopes of the 35- and 45-kDa proteins. Immunoelectron microscopy showed that these two proteins are localized within the phage head, therefore indicating that the 35 kDa is a major capsid protein of ul36 and that the 45 kDa is a minor capsid protein. With two MAbs, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed for direct detection of lactococcal phages in whey and milk samples. Whey and milk components, however, interfered with the conduct of the assay. Partial denaturation of milk samples by heat treatment in the presence of SDS and β-mercaptoethanol removed the masking effect and increased the sensitivity of the assay by 100-fold. With the method used here, 107 PFU/ml were detected by the ELISA within 2 h without any steps to enrich or isolate bacteriophages. Images PMID:16348980

  6. Involvement of the major capsid protein and two early-expressed phage genes in the activity of the lactococcal abortive infection mechanism AbiT.

    PubMed

    Labrie, Simon J; Tremblay, Denise M; Moisan, Maxim; Villion, Manuela; Magadán, Alfonso H; Campanacci, Valérie; Cambillau, Christian; Moineau, Sylvain

    2012-10-01

    The dairy industry uses the mesophilic, Gram-positive, lactic acid bacterium (LAB) Lactococcus lactis to produce an array of fermented milk products. Milk fermentation processes are susceptible to contamination by virulent phages, but a plethora of phage control strategies are available. One of the most efficient is to use LAB strains carrying phage resistance systems such as abortive infection (Abi) mechanisms. Yet, the mode of action of most Abi systems remains poorly documented. Here, we shed further light on the antiviral activity of the lactococcal AbiT system. Twenty-eight AbiT-resistant phage mutants derived from the wild-type AbiT-sensitive lactococcal phages p2, bIL170, and P008 were isolated and characterized. Comparative genomic analyses identified three different genes that were mutated in these virulent AbiT-insensitive phage derivatives: e14 (bIL170 [e14(bIL170)]), orf41 (P008 [orf41(P008)]), and orf6 (p2 [orf6(p2)] and P008 [orf6(P008)]). The genes e14(bIL170) and orf41(P008) are part of the early-expressed genomic region, but bioinformatic analyses did not identify their putative function. orf6 is found in the phage morphogenesis module. Antibodies were raised against purified recombinant ORF6, and immunoelectron microscopy revealed that it is the major capsid protein (MCP). Coexpression in L. lactis of ORF6(p2) and ORF5(p2), a protease, led to the formation of procapsids. To our knowledge, AbiT is the first Abi system involving distinct phage genes. PMID:22820334

  7. Identification of regions of the Beet mild curly top virus (family Geminiviridae) capsid protein involved in systemic infection, virion formation and leafhopper transmission.

    PubMed

    Soto, Maria J; Chen, Li-Fang; Seo, Young-Su; Gilbertson, Robert L

    2005-10-25

    Plant viruses in the genus Curtovirus (family Geminiviridae) are vectored by the beet leafhopper (Circulifer tenellus) and cause curly top disease in a wide range of dicotyledonous plants. An infectious clone of an isolate of Beet mild curly top virus (BMCTV-[W4]), associated with an outbreak of curly top in pepper and tomato crops, was characterized and used to investigate the role of the capsid protein (CP) in viral biology and pathogenesis. Frameshift mutations were introduced into the overlapping CP and V2 genes, and a series of CP alanine scanning mutations were generated. All mutants replicated in tobacco protoplasts or systemically infected plants, consistent with these gene products not being required for viral DNA replication. The CP frameshift mutant and most C-terminal alanine scanning mutants did not systemically infect Nicotiana benthamiana plants or form detectable virions, and were not leafhopper-transmitted. In contrast, most N-terminal alanine scanning mutants systemically infected N. benthamiana and induced disease symptoms, formed virions and were leafhopper-transmissible; thus, these substitution mutations did not significantly alter the functional properties of this region. One N-terminal mutant (CP49-51) systemically infected N. benthamiana, but did not form detectable virions; whereas another (CP25-28) systemically infected N. benthamiana and formed virions, but was not insect-transmissible. These mutants may reveal regions involved in virus movement through the plant and/or leafhopper vector. Together, these results indicate an important role for virions in systemic infection (long-distance movement) and insect transmission, and strongly suggest that virions are the form in which BMCTV moves, long distance, in the phloem. PMID:16085227

  8. Co-expression of Ubiquitin gene and capsid protein gene enhances the potency of DNA immunization of PCV2 in mice

    PubMed Central

    2011-01-01

    A recombinant plasmid that co-expressed ubiquitin and porcine circovirus type 2 (PCV2) virus capsid protein (Cap), denoted as pc-Ub-Cap, and a plasmid encoding PCV2 virus Cap alone, denoted as pc-Cap, were transfected into 293T cells. Indirect immunofluorescence (IIF) and confocal microscopy were performed to measure the cellular expression of Cap. Three groups of mice were then vaccinated once every three weeks for a total of three doses with pc-Ub-Cap, pc-Cap or the empty vector pCAGGS, followed by challenging all mice intraperitoneally with 0.5 mL 106.5 TCID50/mL PCV2. To characterize the protective immune response against PCV2 infection in mice, assays of antibody titer (including different IgG isotypes), flow cytometric analysis (FCM), lymphocyte proliferation, cytokine production and viremia were evaluated. The results showed that pc-Ub-Cap and pc-Cap were efficiently expressed in 293T cells. However, pc-Ub-Cap-vaccinated animals had a significantly higher level of Cap-specific antibody and induced a stronger Th1 type cellular immune response than did pc-Cap-vaccinated animals, suggesting that ubiquitin conjugation improved both the cellular and humoral immune responses. Additionally, viral replication in blood was lower in the pc-Ub-Cap-vaccinated group than in the pc-Cap and empty vector groups, suggesting that the protective immunity induced by pc-Ub-Cap is superior to that induced by pc-Cap. PMID:21624113

  9. A pseudotype baculovirus expressing the capsid protein of foot-and-mouth disease virus and a T-Cell immunogen shows enhanced immunogenicity in mice

    PubMed Central

    2011-01-01

    Background Foot-and-mouth disease (FMD) is a highly contagious disease of livestock which causes severe economic loss in cloven-hoofed animals. Vaccination is still a major strategy in developing countries to control FMD. Currently, inactivated vaccine of FMDV has been used in many countries with limited success and safety concerns. Development of a novel effective vaccine is must. Methods In the present study, two recombinant pseudotype baculoviruses, one expressing the capsid of foot-and-mouth disease virus (FMDV) under the control of a cytomegalovirus immediate early enhancer/promoter (CMV-IE), and the other the caspid plus a T-cell immunogen coding region under a CAG promoter were constructed, and their expression was characterized in mammalian cells. In addition, their immunogenicity in a mouse model was investigated. The humoral and cell-mediated immune responses induced by pseudotype baculovirus were compared with those of inactivated vaccine. Results Indirect immunofluorescence assay (IFA) and indirect sandwich-ELISA (IS-ELISA) showed both recombinant baculoviruses (with or without T-cell epitopes) were transduced efficiently and expressed target proteins in BHK-21 cells. In mice, intramuscular inoculation of recombinants with 1 × 109 or 1 × 1010 PFU/mouse induced the production of FMDV-specific neutralizing antibodies and gamma interferon (IFN-γ). Furthermore, recombinant baculovirus with T-cell epitopes had better immunogenicity than the recombinant without T-cell epitopes as demonstrated by significantly enhanced IFN-γ production (P < 0.01) and higher neutralizing antibody titer (P < 0.05). Although the inactivated vaccine produced the highest titer of neutralizing antibodies, a lower IFN-γ expression was observed compared to the two recombinant pseudotype baculoviruses. Conclusions These results indicate that pseudotype baculovirus-mediated gene delivery could be a alternative strategy to develop a new generation of vaccines against FMDV infection

  10. [Enzyme immunoassay for detection of porcine circovirus type 2, by using the recombinant capsid protein ORF-2].

    PubMed

    Shkaeva, M A; Bogdanova, V S; Tsibezov, V V; Gibadulin, R A; Musienko, M I; Alekseev, K P; Grebennikova, T V; Verkhovskiĭ, O A; Zaberezhnyĭ, A D; Aliper, T I

    2006-01-01

    Recombinant antigen ORF2 from porcine circovirus type 2 (PCV-2) was produced, by using the baculovirus expression system, with histidine tags to allow purification by metal-chelate affinity chromatography. The purity of the protein was verified by polyacrylamide gel electrophoresis; and its immunospecificity was confirmed by the immunoblotting test using reference PCV-2-positive and PCV-2-negative porcine sera and monoclonal antibodies. The protein was used as an antigen to develop an indirect enzyme immunoassay (EIA) of PCV-2 antibodies. EIA was shown to have a high sensitivity and specificity as compared with indirect immunofluorescence test. Porcine serum samples from 15 pig-breeding farms of the Russian Federation were studied. Seropositive samples were found in all age pig groups in all the farms, The number of seropositive animals was shown to be directly related to its age. PMID:17087066

  11. In vitro expression and development of indirect ELISA for Capsid protein of duck circovirus without nuclear localization signal

    PubMed Central

    Lu, Yanyan; Jia, Renyong; Zhang, Zhilong; Wang, Mingshu; Xu, Yu; Zhu, Dekang; Chen, Shun; Liu, Mafeng; Yin, Zhongqiong; Chen, Xiaoyue; Cheng, Anchun

    2014-01-01

    Duck circovirus was a newly discovered pathogen that causing ducks immunosuppression in recent years, but it can not been cultured in vitro that limited its depth study. In the present study, the Cap gene that defect the nuclear localization signal (NLS) of DuCV was amplified and connected to the express vector pET-32a (+), to express the recombinant Cap protein in the bacterium Escherichia coli Rosetta. The recombinant Cap protein was purified and an indirect ELISA method was developed based on the recombinant Cap protein. The results showed that the truncated Cap gene was 567 bp and cloned into pET-32a (+) vector successfully. The recombinant Cap protein was expressed as inclusion bodies. The results of optimization for indirect ELISA revealed that the optimal antigen and serum dilutions were selected to be 4 μg/well and 1:40, respectively; the coating condition was 37°C for 1 h and 4°C overnight; the blocking time in 1% BSA was 1 h at 37°C and the second antibody dilution was 1:800, the cut-off value was 0.352. Known anti-sera samples of other duck common pathogens were tested by the developed ELISA and the results showed it was specific for DuCV anti-sera detection. The sensitivity of indirect ELISA reached 1:2560, and the coefficient of variation between intra-assay and inter-assay were less than 10%. Compared the PCR and indirect ELISA methods, the positive compliance rate was 95.6% for detected 59 duck samples. PMID:25197365

  12. Statistical mechanical models of virus capsid assembly

    NASA Astrophysics Data System (ADS)

    Hicks, Stephen Daniel

    Viruses have become an increasingly popular subject of physics investigation, particularly in the last decade. Advances in imaging of virus capsids---the protective protein shells---in a wide variety of stages of assembly have encouraged physical assembly models at a similarly wide variety of scales, while the apparent simplicity of the capsid system---typically, many identical units assembling spontaneously into an icosahedrally symmetric (rather than amorphous) shell---makes the problem particularly interesting. We take a look at the existing physical assembly models in light of the question of how a particular assembly target can be consistently achieved in the presence of so many possible incorrect results. This review leads us to pose our own model of fully irreversible virus assembly, which we study in depth using a large ensemble of simulated assembled capsids, generated under a variety of capsid shell elastic parameters. While this irreversible model (predictably) did not yield consistently symmetric results, we do glean some insight into the effect of elasticity on growth, as well as an understanding of common failure modes. In particular, we found that (i) capsid size depends strongly on the spontaneous curvature and weakly on the ratio of bending to stretching elastic stiffnesses, (ii) the probability of successful capsid completion decays exponentially with capsid size, and (iii) the degree of localization of Gaussian curvature depends heavily on the ratio of elastic stiffnesses. We then go on to consider more thoroughly the nature of the ensemble of symmetric and almost-symmetric capsids---ultimately computing a phase diagram of minimum-energy capsids as a function of the two above-mentioned elastic parameters---and also look at a number of modifications we can make to our irreversible model, finally putting forth a rather different type of model potentially appropriate for understanding immature HIV assembly, and concluding with a fit of this new

  13. Assembly of Epstein-Barr Virus Capsid in Promyelocytic Leukemia Nuclear Bodies

    PubMed Central

    Wang, Wen-Hung; Kuo, Chung-Wen; Chang, Li-Kwan; Hung, Chen-Chia; Chang, Tzu-Hsuan

    2015-01-01

    ABSTRACT The Epstein-Barr virus (EBV) capsid contains a major capsid protein, VCA; two minor capsid proteins, BDLF1 and BORF1; and a small capsid protein, BFRF3. During the lytic cycle, these capsid proteins are synthesized and imported into the host nucleus for capsid assembly. This study finds that EBV capsid proteins colocalize with promyelocytic leukemia (PML) nuclear bodies (NBs) in P3HR1 cells during the viral lytic cycle, appearing as nuclear speckles under a confocal laser scanning microscope. In a glutathione S-transferase pulldown study, we show that BORF1 interacts with PML-NBs in vitro. BORF1 also colocalizes with PML-NBs in EBV-negative Akata cells after transfection and is responsible for bringing VCA and the VCA-BFRF3 complex from the cytoplasm to PML-NBs in the nucleus. Furthermore, BDLF1 is dispersed throughout the cell when expressed alone but colocalizes with PML-NBs when BORF1 is also present in the cell. In addition, this study finds that knockdown of PML expression by short hairpin RNA does not influence the intracellular levels of capsid proteins but reduces the number of viral particles produced by P3HR1 cells. Together, these results demonstrate that BORF1 plays a critical role in bringing capsid proteins to PML-NBs, which may likely be the assembly sites of EBV capsids. The mechanisms elucidated in this study are critical to understanding the process of EBV capsid assembly. IMPORTANCE Capsid assembly is an important event during the Epstein-Barr virus (EBV) lytic cycle, as this process is required for the production of virions. In this study, confocal microscopy revealed that the EBV capsid protein BORF1 interacts with promyelocytic leukemia (PML) nuclear bodies (NBs) in the host nucleus and is responsible for transporting the other EBV capsid proteins, including VCA, BDLF1, and BFRF3, to these subnuclear locations prior to initiation of capsid assembly. This study also found that knockdown of PML expression by short hairpin RNA

  14. Kinetics versus Thermodynamics in Virus Capsid Polymorphism.

    PubMed

    Moerman, Pepijn; van der Schoot, Paul; Kegel, Willem

    2016-07-01

    Virus coat proteins spontaneously self-assemble into empty shells in aqueous solution under the appropriate physicochemical conditions, driven by an interaction free energy per bond on the order of 2-5 times the thermal energy kBT. For this seemingly modest interaction strength, each protein building block nonetheless gains a very large binding free energy, between 10 and 20 kBT. Because of this, there is debate about whether the assembly process is reversible or irreversible. Here we discuss capsid polymorphism observed in in vitro experiments from the perspective of nucleation theory and of the thermodynamics of mass action. We specifically consider the potential contribution of a curvature free energy term to the effective interaction potential between the proteins. From these models, we propose experiments that may conclusively reveal whether virus capsid assembly into a mixture of polymorphs is a reversible or an irreversible process. PMID:27027925

  15. Functional label-free assays for characterizing the in vitro mechanism of action of small molecule modulators of capsid assembly.

    PubMed

    Lad, Latesh; Clancy, Sheila; Koditek, David; Wong, Melanie H; Jin, Debi; Niedziela-Majka, Anita; Papalia, Giuseppe A; Hung, Magdeleine; Yant, Stephen; Somoza, John R; Hu, Eric; Chou, Chienhung; Tse, Winston; Halcomb, Randall; Sakowicz, Roman; Pagratis, Nikos

    2015-04-01

    HIV capsid protein is an important target for antiviral drug design. High-throughput screening campaigns have identified two classes of compounds (PF74 and BI64) that directly target HIV capsid, resulting in antiviral activity against HIV-1 and HIV-2 laboratory strains. Using recombinant proteins, we developed a suite of label-free assays to mechanistically understand how these compounds modulate capsid activity. PF74 preferentially binds to the preassembled hexameric capsid form and prevents disruption of higher-order capsid structures by stabilizing capsid intersubunit interactions. BI64 binds only the monomeric capsid and locks the protein in the assembly incompetent monomeric form by disrupting capsid intersubunit interactions. We also used these assays to characterize the interaction between capsid and the host protein cleavage and polyadenylation specific factor 6 (CPSF6). Consistent with recently published results, our assays revealed CPSF6 activates capsid polymerization and preferentially binds to the preassembled hexameric capsid form similar to the small molecule compound, PF74. Furthermore, these label-free assays provide a robust method for facilitating the identification of a different class of small molecule modulators of capsid function. PMID:25774576

  16. Comprehensive Small RNA-Seq of Adeno-Associated Virus (AAV)-Infected Human Cells Detects Patterns of Novel, Non-Coding AAV RNAs in the Absence of Cellular miRNA Regulation.

    PubMed

    Stutika, Catrin; Mietzsch, Mario; Gogol-Döring, Andreas; Weger, Stefan; Sohn, Madlen; Chen, Wei; Heilbronn, Regine

    2016-01-01

    Most DNA viruses express small regulatory RNAs, which interfere with viral or cellular gene expression. For adeno-associated virus (AAV), a small ssDNA virus with a complex biphasic life cycle miRNAs or other small regulatory RNAs have not yet been described. This is the first comprehensive Illumina-based RNA-Seq analysis of small RNAs expressed by AAV alone or upon co-infection with helper adenovirus or HSV. Several hotspots of AAV-specific small RNAs were detected mostly close to or within the AAV-ITR and apparently transcribed from the newly identified anti-p5 promoter. An additional small RNA hotspot was located downstream of the p40 promoter, from where transcription of non-coding RNAs associated with the inhibition of adenovirus replication were recently described. Parallel detection of known Ad and HSV miRNAs indirectly validated the newly identified small AAV RNA species. The predominant small RNAs were analyzed on Northern blots and by human argonaute protein-mediated co-immunoprecipitation. None of the small AAV RNAs showed characteristics of bona fide miRNAs, but characteristics of alternative RNA processing indicative of differentially regulated AAV promoter-associated small RNAs. Furthermore, the AAV-induced regulation of cellular miRNA levels was analyzed at different time points post infection. In contrast to other virus groups AAV infection had virtually no effect on the expression of cellular miRNA, which underscores the long-established concept that wild-type AAV infection is apathogenic. PMID:27611072

  17. Overexpression of recombinant infectious bursal disease virus (IBDV) capsid protein VP2 in the middle silk gland of transgenic silkworm.

    PubMed

    Xu, Hanfu; Yuan, Lin; Wang, Feng; Wang, Yuancheng; Wang, Riyuan; Song, Chunnuan; Xia, Qingyou; Zhao, Ping

    2014-10-01

    Infectious bursal disease virus (IBDV) is the causative agent of a highly contagious disease affecting young chickens and causes serious economic losses to the poultry industry worldwide. Development of subunit vaccine using its major caspid protein, VP2, is one of the promising strategies to protect against IBDV. This study aim to test the feasibility of using silkworm to produce recombinant VP2 protein (rVP2) derived from a very virulent strain of IBDV (vvIBDV). A total of 16 transgenic silkworm lines harboring a codon-optimized VP2 gene driven by the sericin1 promoter were generated and analyzed. The results showed that the rVP2 was synthesized in the middle silk gland of all lines and secreted into their cocoons. The content of rVP2 in the cocoon of each line was ranged from 0.07 to 16.10 % of the total soluble proteins. The rVP2 was purified from 30 g cocoon powders with a yield of 3.33 mg and a purity >90 %. Further analysis indicated that the rVP2 was able to tolerate high temperatures up to 80 °C, and exhibited specific immunogenic activity in mice. To our knowledge, this is the first report of overexpressing rVP2 in the middle silk gland of transgenic silkworm, which demonstrates the capability of silkworm as an efficient tool to produce recombinant immunogens for use in new vaccines against animal diseases. PMID:25106848

  18. Genome Sequence of Bluetongue Virus Type 2 from India: Evidence for Reassortment between Outer Capsid Protein Genes

    PubMed Central

    Maan, Narender S.; Belaganahalli, Manjunatha N.; Kumar, Aman; Batra, Kanisht; Rao, Pavuluri Panduranga; Hemadri, Divakar; Reddy, Yella Narasimha; Putty, Kalyani; Krishnajyothi, Yadlapati; Reddy, G. Hanmanth; Singh, Karam Pal; Hegde, Nagendra R.; Nomikou, Kyriaki; Sreenivasulu, Daggupati

    2015-01-01

    Southern Indian isolate IND1994/01 of bluetongue virus serotype 2 (BTV-2), from the Orbivirus Reference Collection at the Pirbright Institute (http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/btv-2.htm#IND1994/01), was sequenced. Its genome segment 6 (Seg-6) [encoding VP5(OCP2)] is identical to that of the Indian BTV-1 isolate (IND2003/05), while Seg-5 and Seg-9 are closely related to isolates from South Africa and the United States, respectively. PMID:25858823

  19. Genome Sequence of Bluetongue Virus Type 2 from India: Evidence for Reassortment between Outer Capsid Protein Genes.

    PubMed

    Maan, Sushila; Maan, Narender S; Belaganahalli, Manjunatha N; Kumar, Aman; Batra, Kanisht; Rao, Pavuluri Panduranga; Hemadri, Divakar; Reddy, Yella Narasimha; Putty, Kalyani; Krishnajyothi, Yadlapati; Reddy, G Hanmanth; Singh, Karam Pal; Hegde, Nagendra R; Nomikou, Kyriaki; Sreenivasulu, Daggupati; Mertens, Peter P C

    2015-01-01

    Southern Indian isolate IND1994/01 of bluetongue virus serotype 2 (BTV-2), from the Orbivirus Reference Collection at the Pirbright Institute (http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/btv-2.htm#IND1994/01), was sequenced. Its genome segment 6 (Seg-6) [encoding VP5(OCP2)] is identical to that of the Indian BTV-1 isolate (IND2003/05), while Seg-5 and Seg-9 are closely related to isolates from South Africa and the United States, respectively. PMID:25858823

  20. Capsid modification strategies for detargeting adenoviral vectors.

    PubMed

    Parker, Alan L; Bradshaw, Angela C; Alba, Raul; Nicklin, Stuart A; Baker, Andrew H

    2014-01-01

    Adenoviral vectors hold immense potential for a wide variety of gene therapy based applications; however, their efficacy and toxicity is dictated by "off target" interactions that preclude cell specific targeting to sites of disease. A number of "off target" interactions have been described in the literature that occur between the three major capsid proteins (hexon, penton, and fiber) and components of the circulatory system, including cells such as erythrocytes, white blood cells, and platelets, as well as circulatory proteins including complement proteins, coagulation factors, von Willebrand Factor, p-selectin as well as neutralizing antibodies. Thus, to improve efficacious targeting to sites of disease and limit nonspecific uptake of virus to non-target tissues, specifically the liver and the spleen, it is necessary to develop suitable strategies for genetically modifying the capsid proteins to preclude these interactions. To this end we have developed versatile systems based on homologous recombination for modification of each of the major capsid proteins, which are described herein. PMID:24132476

  1. Molecular characterization of Indian isolate of peanut mottle virus and immunodiagnosis using bacterial expressed core capsid protein.

    PubMed

    Soumya, K; Yogita, M; Prasanthi, Y; Anitha, K; Kishor, P B Kavi; Jain, R K; Mandal, Bikash

    2014-01-01

    Peanut mottle virus (PeMoV), a seed borne potyvirus was recorded in India in 1978, however the virus was not characterized at molecular level. In the present study, an isolate of PeMoV infecting peanut in southern India was characterized based on host reactions and coat protein (CP) gene sequence, which revealed that the Indian isolate was very close to a peanut isolate reported from Israel and distinct from pea isolate reported from USA. The core region of CP gene that contained majority of the predicted epitopes was successfully expressed (1.75 mg/l) in Escherichia coli as a 22 kDa protein. A high titer polyclonal antibody (PAb) to the expressed core CP was produced, which efficiently detected PeMoV. The antiserum was useful in specific detection of PeMoV as it showed negligible cross reactivity with the other potyviruses e.g., peanut stripe virus, potato virus Y, papaya ringspot virus and onion yellow dwarf virus. The PAb was validated in ELISA using 1,169 field and greenhouse samples of peanut which showed 1.85-26.3 % incidence of PeMoV in peanut seed multiplication field during 2011-2012. This is the first report of immunodiagnosis of PeMoV with a PAb to recombinant core CP of PeMoV. PMID:25674600

  2. Scaffold expulsion and genome packaging trigger stabilization of herpes simplex virus capsids

    PubMed Central

    Roos, Wouter H.; Radtke, Kerstin; Kniesmeijer, Edward; Geertsema, Hylkje; Sodeik, Beate; Wuite, Gijs J. L.

    2009-01-01

    Herpes simplex virus type 1 (HSV1) capsids undergo extensive structural changes during maturation and DNA packaging. As a result, they become more stable and competent for nuclear egress. To further elucidate this stabilization process, we used biochemical and nanoindentation approaches to analyze the structural and mechanical properties of scaffold-containing (B), empty (A), and DNA-containing (C) nuclear capsids. Atomic force microscopy experiments revealed that A and C capsids were mechanically indistinguishable, indicating that the presence of DNA does not account for changes in mechanical properties during capsid maturation. Despite having the same rigidity, the scaffold-containing B capsids broke at significantly lower forces than A and C capsids. An extraction of pentons with guanidine hydrochloride (GuHCl) increased the flexibility of all capsids. Surprisingly, the breaking forces of the modified A and C capsids dropped to similar values as those of the GuHCl-treated B capsids, indicating that mechanical reinforcement occurs at the vertices. Nonetheless, it also showed that HSV1 capsids possess a remarkable structural integrity that was preserved after removal of pentons. We suggest that HSV1 capsids are stabilized after removal of the scaffold proteins, and that this stabilization is triggered by the packaging of DNA, but independent of the actual presence of DNA. PMID:19487681

  3. Scaffold expulsion and genome packaging trigger stabilization of herpes simplex virus capsids.

    PubMed

    Roos, Wouter H; Radtke, Kerstin; Kniesmeijer, Edward; Geertsema, Hylkje; Sodeik, Beate; Wuite, Gijs J L

    2009-06-16

    Herpes simplex virus type 1 (HSV1) capsids undergo extensive structural changes during maturation and DNA packaging. As a result, they become more stable and competent for nuclear egress. To further elucidate this stabilization process, we used biochemical and nanoindentation approaches to analyze the structural and mechanical properties of scaffold-containing (B), empty (A), and DNA-containing (C) nuclear capsids. Atomic force microscopy experiments revealed that A and C capsids were mechanically indistinguishable, indicating that the presence of DNA does not account for changes in mechanical properties during capsid maturation. Despite having the same rigidity, the scaffold-containing B capsids broke at significantly lower forces than A and C capsids. An extraction of pentons with guanidine hydrochloride (GuHCl) increased the flexibility of all capsids. Surprisingly, the breaking forces of the modified A and C capsids dropped to similar values as those of the GuHCl-treated B capsids, indicating that mechanical reinforcement occurs at the vertices. Nonetheless, it also showed that HSV1 capsids possess a remarkable structural integrity that was preserved after removal of pentons. We suggest that HSV1 capsids are stabilized after removal of the scaffold proteins, and that this stabilization is triggered by the packaging of DNA, but independent of the actual presence of DNA. PMID:19487681

  4. Periodic Table of Virus Capsids: Implications for Natural Selection and Design

    PubMed Central

    Mannige, Ranjan V.; Brooks, Charles L.

    2010-01-01

    Background For survival, most natural viruses depend upon the existence of spherical capsids: protective shells of various sizes composed of protein subunits. So far, general evolutionary p