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At least 109 records · Page 6Linked to original sources
PubMed1995

Assembly of foot-and-mouth disease virus empty capsids synthesized by a vaccinia virus expression system.

cDNA cassettes encoding the foot-and-mouth disease virus (FMDV) structural protein precursor (P1-2A) together with the 3C protease, which cleave this molecule to 1AB, 1C and 1D, were constructed. These cassettes were introduced into vaccinia virus (VV) transfer vectors. Attempts to isolate recombinant VVs constitutively expressing these cassettes were unsuccessful. However, when the P1-2A-3C cassette was placed under the control of the bacteriophage T7 promoter, stable VV/FMDV recombinants were isolated. Co-infection with recombinant VV vTF7-3 (which expresses T7 RNA polymerase) led to the production of correctly processed FMDV capsid proteins. Analysis by sucrose gradient centrifugation showed that material which co-sedimented with natural empty capsid particles (70S) was formed. Electron microscopy revealed empty capsid-like particles with diameters of about 30 nm. Studies using monoclonal antibodies specific for conformational epitopes indicated that the antigenicity of the synthetic particles was similar to whole virions and natural empty capsid particles. Surprisingly, merely the modification of a single amino acid residue within the myristoylation consensus sequence at the N terminus of P1-2A allowed the isolation of a recombinant VV which constitutively expressed the correctly processed proteins. However, the capsid proteins expressed from this mutant cassette failed to assemble into 70S empty particles.

Aphthovirus↗
PubMed2004

Fiber-mosaic adenovirus as a novel approach to design genetically modified adenoviral vectors.

Genetic modification of the adenovirus (Ad) capsid is one of the successful strategies to achieve viral retargeting. However, it has been widely recognized that structural constraints imposed by viral proteins limit the number and nature of incorporated targeting ligands and often hamper viral propagation. To address this issue, we propose a genetic fiber-mosaic virus (having two distinct fibers in one viral particle) as a means to facilitate fiber modifications. Fiber-mosaic virus having tandem fibers: a wild type (wt) fiber and second adjunctive fiber, will utilize natural viral entry for the conventional propagation of the vectors whereas, adjunctive fiber will serve multiple potential purposes such as targeting, purification, or imaging of viral particles via genetic incorporation of the corresponding functional moieties. We generated the mosaic adenovirus vector encoding two fibers: wild-type and adjunctive fiber--Fiber-Fibritin (FF) and confirmed incorporation of FF in the mosaic viral particles. We investigated binding specificity of the mosaic virus and the possible interference of the two fibers during virus life cycle. Fiber-mosaic Ad attained new binding properties provided by the second fiber, while preserving the binding ability attributed to the wt fiber. Our results suggest that the two fibers being presented and structurally separated on the viral particle may also function separately as binding counterparts for virus attachment. Therefore, the mosaic setting will allow more flexibility in Ad retargeting approaches.

Adenoviridae↗
PubMed2001

Gene therapy: recombinant adeno-associated virus vectors.

Gene transfer using recombinant adeno-associated virus (rAAV) vectors shows great promise for human gene therapy. The broad host range, low level of immune response, and longevity of gene expression observed with these vectors in numerous disease paradigms has enabled the initiation of a number of clinical trials using this gene delivery system. This review presents an overview of the current developments in the field of AAV-mediated gene delivery. Such developments include the establishment of new production methods allowing the generation of high titer preparations, improved purification methods, the use of alternative AAV serotypes, and the generation of trans-splicing rAAV genomes. Together, these developments have improved results interpretation, host range, and the coding capacity of rAAV vectors. Furthermore, the recent identification of regions within the viral capsid that are amenable to modification has begun to address the issue of direct rAAV vector targeting, which could potentially allow targeted gene delivery to specific cell populations. The versatility shown by this vector has enabled new diseases to be realistically considered for therapeutic intervention and considerably broadened the scope of gene therapy.

Dependovirus↗
PubMed1997

A fish encephalitis virus that differs from other nodaviruses by its capsid protein processing.

RNA2, the short segment of the genome of Dicenthrarchus labrax encephalitis virus (DIEV), a fish nodavirus causing seabass encephalitis, was cloned. Sequence analysis revealed that DIEV RNA2 contains a single open reading frame (ORF), which carries the catalytic D-75 residue but lacks the site for autocatalytic proteolysis, the process yielding the two capsid proteins of insect nodaviruses. Nevertheless, SDS-PAGE analysis of mature virions revealed a 43-45 kDa protein doublet. In order to determine the mechanism of synthesis of the two capsid proteins in DIEV, wild type and mutagenized forms of RNA2 were expressed in cell-free translation extracts and in transfected cells. Results showed that, despite the presence of the catalytic D-75 residue, the DIEV capsid protein doublet did not result from the assembly-dependent autocatalytic cleavage of a protein precursor. Moreover, our data show that, although suggested by sequence analysis, the DIEV capsid protein doublet results from neither an alternative initiation codon usage nor from a--1 ribosomal frameshift. Results of cell-free translation experiments demonstrate that the capsid protein doublet neither results of the proteolytic cleavage of a precursor nor of a degradation process. Kinetics of capsid protein synthesis in cell-free translation programmed with RNA2 revealed, instead, that the two capsid proteins are cosynthesized. Together these data strongly suggest that the DIEV capsid protein doublet results from cotranslational modification(s) of the ORF-encoded protein.

Amino Acid Sequence↗
PubMed2000

Mass spectrometry in viral proteomics.

Mass spectrometry is a valuable tool in structural and functional viral proteomics, where it has been used to identify viral capsid proteins, viral mutants, and posttranslational modifications. Further, mass-based approaches combined with time-resolved proteolysis (mass mapping) have revealed the dynamic nature of viral particles in solution; this method is contributing to an understanding of the dynamic domains of the viral capsid which may have significant value in developing new approaches for viral inactivation. As a result of these experiments, and by comparison with complementary data from X-ray crystallography, a new dimension to viral protein structure and function is emerging.

Mass Spectrometry↗
PubMed1981

Differences in the subpopulations of the structural proteins of polyoma virions and capsids: biological functions of the multiple VP1 species.

The structural proteins of polyoma virions and capsids were analyzed by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polyoma virion VP1 was found to be composed of six distinct species which had pI's between pH 6.75 and 5.75. Polyoma capsid VP1 was found to contain four species with pI's between pH 6.60 and 5.75. The different forms of virion and capsid VP1 appeared to be generated by modifications (phosphorylation and acetylation) of the initial translation product. The most basic of the virion VP1 species (pI, pH 6.75) was absent in capsids and was found to be exclusively associated with the viral nucleoprotein complex. Three of the virion VP1 species and three of the capsid VP1 species were found in capsomere preparations enriched for hexon subunits. Two VP1 species were specifically immune precipitated from virions with hemagglutination-inhibiting antibodies. These two VP1 species were common to both virions and capsids. Polyoma virions, but not capsids, possessed a single VP1 species which was immune precipitated with neutralizing antibodies. Both virion and capsid VP2 were found to have pI's of approximately pH 5.50. Virion VP3 had a pI of approximately pH 7.00, whereas capsid VP3 had a pI of approximately pH 6.50.

Acetylation↗
PubMed2005

Glycosylation of beet western yellows virus proteins is implicated in the aphid transmission of the virus.

Beet western yellows virus relies on the aphid M. persicae for its transmission in a persistent and circulative mode. To be transmitted, the virus must cross the midgut and the accessory salivary gland epithelial barriers by a transcytosis mechanism where vector receptors interact with virions. The aphid and the peptidic viral determinants implicated in this interaction mechanism have been studied. In this paper, we report that the coat and the readthrough proteins that constitute the capsid of this virus are glycosylated. Modification of the glucidic core of these structural viral proteins by oxidation with sodium metaperiodate or deglycosylation with N-glycosidase F or alpha-D-galactosidase abrogates the aphid transmission of the virus. Aphid transmission could also be inhibited by lectins directed against alpha-D-galactose when aphids were allowed to acquire virus on artificial membranes. These results suggest that the glucidic cores of the capsid proteins of beet western yellows virus contain alpha-D-galactose residues that are implicated in virus-aphid interaction and promote aphid transmission of the virus.

Amino Acid Sequence↗
PubMed1965

REOVIRUSES. II. STRUCTURE AND COMPOSITION OF THE VIRION.

Mayor, Heather D. (Baylor University College of Medicine, Houston, Tex.), Richard M. Jamison, Liane E. Jordan, and M. Van Mitchell. Reoviruses. II. Structure and composition of the virion. J. Bacteriol. 89:1548-1556. 1965.-Reovirus type 1 has been grown in green monkey kidney cells and harvested 24 hr after infection ("early" virus) and 96 hr after infection ("late" virus). A number of biological parameters have been determined on purified preparations of both "early" and "late" harvests of reovirus. There were no significant differences in values obtained for the molecular weight, RNA content, and buoyant density of virions prepared from early or late harvests. The size of the capsids and their morphology were also identical. Late harvests of reovirus were particularly rich in empty viral capsids (density, 1.28 in cesium chloride), and a significant number of empty inner capsid shells were routinely found. These shells could be prepared readily by controlled digestion of complete virus particles with trypsin. The inner shell appears to be composed of subunits packed with icosahedral symmetry to form a 45-mmu foundation on which the outer 92-subunit capsid is assembled. The inner shell is somewhat reminiscent in size and morphology of the capsid of papovaviruses. The fact that it can exist as a discrete entity has prompted us to propose some modifications to the current models for the reovirus capsid.

Animals↗
PubMed2004

Mimicking live flavivirus immunization with a noninfectious RNA vaccine.

Flaviviruses are human pathogens of world-wide medical importance. They have recently received much additional attention because of their spread to new regions (such as West Nile virus to North America), highlighting their potential as newly emerging disease agents. Using tick-borne encephalitis virus, we have developed and evaluated in mice a new genetic vaccine based on self-replicating but noninfectious RNA. This RNA contains all of the necessary genetic information for establishing its replication machinery in the host cell, thus mimicking a natural infection. However, genetic modifications in the region encoding the capsid protein simultaneously prevent the assembly of infectious virus particles and promote the secretion of noninfectious subviral particles that elicit neutralizing antibodies. These characteristics demonstrate that a new generation of flavivirus vaccines can be designed that stimulate the same spectrum of innate and specific immune responses as a live vaccine but have the safety features of an inactivated vaccine.

Animals↗
PubMed2003

Structural and phylogenetic analysis of adenovirus hexons by use of high-resolution x-ray crystallographic, molecular modeling, and sequence-based methods.

A major impediment to the use of adenovirus as a gene therapy vector and for vaccine applications is the host immune response to adenovirus hexon-the major protein component of the icosahedral capsid. A solution may lie in novel vectors with modified or chimeric hexons designed to evade the immune response. To facilitate this approach, we have distinguished the portion of hexon that all serotypes have in common from the hypervariable regions that are responsible for capsid diversity and type-specific immunogenicity. The common hexon core-conserved because it forms the viral capsid-sets boundaries to the regions where modifications can be made to produce nonnative hexons. The core has been defined from the large and diverse set of known hexon sequences by an accurate alignment based on the newly refined crystal structures of human adenovirus types 2 (Ad2) and Ad5 hexon. Comparison of the two hexon models, which are the most accurate so far, reveals that over 90% of the residues in each have three-dimensional positions that closely match. Structures for more distant hexons were predicted by building molecular models of human Ad4, chimpanzee adenovirus (AdC68), and fowl adenovirus 1 (FAV1 or CELO). The five structures were then used to guide the alignment of the 40 full-length (>900 residues) hexon sequences in public databases. Distance- and parsimony-based phylogenetic trees are consistent and reveal evolutionary relationships between adenovirus types that parallel those of their animal hosts. The combination of crystallography, molecular modeling, and phylogenetic analysis defines a conserved molecular core that can serve as the armature for the directed design of novel hexons.

Adenoviruses, Human↗
PubMed2004

Cell-selective viral gene delivery vectors for the vasculature.

Clinical gene therapy for cardiovascular disease remains achievable. To date, however, preclinical studies and clinical trials have highlighted shortfalls in viral gene delivery to vascular cells. These include poor efficiency, poor target tissue selectivity, the presence of pre-existing neutralizing antibodies and immunogenicity generated by the host to vectors such as adenovirus. These important issues require careful consideration when applying viral vectors for gene therapy. Each delivery vector requires precise optimization and tailoring for each disease application since parameters relating to vector : tissue exposure time, route of delivery and target cell type vary considerably. Optimization can be achieved through modification of the structure of the virus capsid proteins and expression cassette to generate vectors that are highly selective and efficient for target cell binding and entry as well as instilling transcriptional control and/or longevity on transgene expression. This ultimately will improve the efficacy and toxicity profiles of gene delivery vectors and has become a very important area in gene therapy. Here, we review recent advances in the targeting of viral gene delivery vectors to the vasculature.

Adenoviridae↗
PubMed2004

Assembly protein precursor (pUL80.5 homolog) of simian cytomegalovirus is phosphorylated at a glycogen synthase kinase 3 site and its downstream "priming" site: phosphorylation affects interactions of protein with itself and with major capsid protein.

Capsid assembly among the herpes-group viruses is coordinated by two related scaffolding proteins. In cytomegalovirus (CMV), the main scaffolding constituent is called the assembly protein precursor (pAP). Like its homologs in other herpesviruses, pAP is modified by proteolytic cleavage and phosphorylation. Cleavage is essential for capsid maturation and production of infectious virus, but the role of phosphorylation is undetermined. As a first step in evaluating the significance of this modification, we have identified the specific sites of phosphorylation in the simian CMV pAP. Two were established previously to be adjacent serines (Ser156 and Ser157) in a casein kinase II consensus sequence. The remaining two, identified here as Thr231 and Ser235, are within consensus sequences for glycogen synthase kinase 3 (GSK-3) and mitogen-activated protein kinase, respectively. Consistent with Thr231 being a GSK-3 substrate, its phosphorylation required a downstream "priming" phosphate (i.e., Ser235) and was reduced by a GSK-3-specific inhibitor. Phosphorylation of Ser235 converts pAP to an electrophoretically slower-mobility isoform, pAP*; subsequent phosphorylation of pAP* at Thr231 converts pAP* to a still-slower isoform, pAP**. The mobility shift to pAP* was mimicked by substituting an acidic amino acid for either Thr231 or Ser235, but the shift to pAP** required that both positions be phosphorylated. Glu did not substitute for pSer235 in promoting phosphorylation of Thr231. We suggest that phosphorylation of Thr231 and Ser235 causes charge-driven conformational changes in pAP, and we demonstrate that preventing these modifications alters interactions of pAP with itself and with major capsid protein, suggesting a functional significance.

Amino Acid Sequence↗
PubMed1999

Insertion of an RGD motif into the HI loop of adenovirus fiber protein alters the distribution of transgene expression of the systemically administered vector.

Adenoviral vectors are attractive gene delivery vehicles, but their in vivo utility is reduced by lack of cell-specific infection. Tropism modification of the virion by genetic manipulation of capsid proteins is an attractive strategy to achieve targeted transduction. However, no genetic targeting strategies have yet been shown to modify the distribution of transgene expression following systemic administration of vector. This is an essential requirement if such approaches are to form a basis for further vector develop- ment. In this report we present data showing that insertion of a RGD motif into the HI loop of the adenoviral fiber knob results in a significant change in transgene expression profile following intravenous administration. The key finding that a motif in the HI loop is available for cellular interaction when administered systemically means that such modifications can be rationally considered as a foundation upon which further genetic modifications can be superimposed for targeted systemic gene therapy.

Adenoviridae↗
PubMed2002

Engineering of adenovirus vectors containing heterologous peptide sequences in the C terminus of capsid protein IX.

The utility of the present generation of adenovirus (Ad) vectors for gene therapy applications could be improved by restricting native viral tropism to selected cell types. In order to achieve modification of Ad tropism, we proposed to exploit a minor component of viral capsid, protein IX (pIX), for genetic incorporation of targeting ligands. Based on the proposed structure of pIX, we hypothesized that its C terminus could be used as a site for incorporation of heterologous peptide sequences. We engineered recombinant Ad vectors containing modified pIX carrying a carboxy-terminal Flag epitope along with a heparan sulfate binding motif consisting of either eight consecutive lysines or a polylysine sequence. Using an anti-Flag antibody, we have shown that modified pIXs are incorporated into virions and display Flag-containing C-terminal sequences on the capsid surface. In addition, both lysine octapeptide and polylysine ligands were accessible for binding to heparin-coated beads. In contrast to virus bearing lysine octapeptide, Ad vector displaying a polylysine was capable of recognizing cellular heparan sulfate receptors. We have demonstrated that incorporation of a polylysine motif into the pIX ectodomain results in a significant augmentation of Ad fiber knob-independent infection of CAR-deficient cell types. Our data suggest that the pIX ectodomain can serve as an alternative to the fiber knob, penton base, and hexon proteins for incorporation of targeting ligands for the purpose of Ad tropism modification.

Adenoviruses, Human↗
PubMed2002

The phosphorylated form of the ORF3 protein of hepatitis E virus interacts with its non-glycosylated form of the major capsid protein, ORF2.

Hepatitis E virus (HEV) is a human RNA virus containing three open reading frames. Of these, ORF1 encodes the viral nonstructural polyprotein; ORF2 encodes the major capsid protein, which exists in a glycosylated and non-glycosylated form; and ORF3 codes for a phosphoprotein of undefined function. Using fluorescence-based colocalization, yeast two-hybrid experiments, transiently transfected COS-1 cell co-immunoprecipitation, and cell-free coupled transcription-translation techniques, we have shown that the ORF3 protein interacts with the ORF2 protein. The domains involved in this ORF2-ORF3 association have been identified and mapped. Our deletion analysis showed that a 25-amino acid region (residues 57-81) of the ORF3 protein is required for this interaction. Using a Mexican HEV isolate, site-directed mutagenesis of ORF3, and a phosphatase digestion assay, we showed that the ORF2-ORF3 interaction is dependent upon the phosphorylation at Ser(80) of ORF3. Finally, using COS-1 cell immunoprecipitation experiments, we found that the phosphorylated ORF3 protein preferentially interacts with the non-glycosylated ORF2 protein. These findings were confirmed using tunicamycin inhibition, point mutants, and deletion mutants expressing only non-glycosylated ORF2. ORF3 maps in the structural region of the HEV genome and now interacts with the major capsid protein, ORF2, in a post-translational modification-dependent manner. Such an interaction of ORF2 with ORF3 suggests a possible well regulated role for ORF3 in HEV structural assembly.

Amino Acid Sequence↗
PubMed2004

Spacers increase the accessibility of peptide ligands linked to the carboxyl terminus of adenovirus minor capsid protein IX.

The efficiency and specificity of gene transfer with human adenovirus (hAd)-derived gene transfer vectors would be improved if the native viral tropism could be modified. Here, we demonstrate that the minor capsid protein IX (pIX), which is present in 240 copies in the Ad capsid, can be exploited as an anchor for heterologous polypeptides. Protein IX-deleted hAd5 vectors were propagated in hAd5 helper cells expressing pIX variants, with heterologous carboxyl-terminal extensions of up to 113 amino acids in length. The extensions evaluated consist of alpha-helical spacers up to 75 A in length and to which peptide ligands were fused. The pIX variants were efficiently incorporated into the capsids of Ad particles. On intact particles, the MYC-tagged-pIX molecules were readily accessible to anti-MYC antibodies, as demonstrated by electron microscopic analyses of immunogold-labeled virus particles. The labeling efficiency improved with increasing spacer length, suggesting that the spacers lift and expose the ligand at the capsid surface. Furthermore, we found that the addition of an integrin-binding RGD motif to the pIX markedly stimulated the transduction of coxsackievirus group B and hAd receptor-deficient endothelioma cells, demonstrating the utility of pIX modification in gene transfer. Our data demonstrate that the minor capsid protein IX can be used as an anchor for the addition of polypeptide ligands to Ad particles.

Adenoviridae↗
PubMed1999

Molecular characterization of a ranavirus isolated from largemouth bass Micropterus salmoides.

An iridovirus, isolated from largemouth bass Micropterus salmoides following a die-off among adult fish and provisionally designated largemouth bass virus (LMBV), was characterized by analysis of viral protein synthesis in infected cells, viral DNA restriction fragment length polymorphisms (RFLP), and sequence determination of the major capsid protein and viral DNA methyltransferase genes. All 3 approaches yielded results consistent with the suggestion that LMBV was a member of the genus Ranavirus. Moreover, LMBV was nearly identical to 2 isolates from Southeast Asia which had been previously detected in imported ornamental fish. It remains to be determined whether infection of largemouth bass resulted from exposure to an imported virus, or whether the presence of similar viruses in southeast Asia and the southeastern United States indicates that iridovirus species are not geographically limited as suggested earlier, but rather globally distributed.

Amino Acid Sequence↗