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At least 811 records · Page 45Linked to original sources
PubMed2005

Conservation of the capsid structure in tailed dsDNA bacteriophages: the pseudoatomic structure of phi29.

Bacteriophage phi29 is one of the smallest and simplest known dsDNA phages, making it amenable to structural investigations. The three-dimensional structure of a fiberless, isometric variant has been determined to 7.9 A resolution by cryo-electron microscopy (cryo-EM), allowing the identification of alpha helices and beta sheets. Their arrangement indicates that the folds of the phi29 and bacteriophage HK97 capsid proteins are similar except for an additional immunoglobulin-like domain of the phi29 protein. An atomic model that incorporates these two domains fits well into the cryo-EM density of the T = 3, fiberless isometric phi29 particle, and cryo-EM structures of fibered isometric and fiberless prolate prohead phi29 particles at resolutions of 8.7 A and 12.7 A, respectively. Thus, phi29 joins the growing number of phages that utilize the HK97 capsid structure, suggesting that this protein fold may be as prevalent in capsids of dsDNA phages as the jelly roll fold is in eukaryotic viruses.

Bacillus Phages↗
PubMed2000

Production and purification of SV40 major capsid protein (VP1) in Escherichia coli strains deficient for the GroELS chaperone machine.

Production of the major capsid protein of SV40, VP1, is of great interest for the study on capsid assembly in vitro. Production of soluble His6-VP1 in Escherichia coli strains deficient in the GroELS chaperone machine was substantially higher than in the wild-type strain. The His6-VP1 produced in a groEL mutant strain was readily purified. The protein was able to form higher-order structures as evidenced by analysis of the soluble fraction by gel filtration, by sedimentation in sucrose gradient, and by electron microscopy. We propose the use of groE mutants for the production of the major capsid protein of SV40 and perhaps also other papovaviruses.

Capsid↗
PubMed2000

Nucleotide sequence of the gene encoding the major capsid protein of herpesvirus of turkeys.

The gene encoding the major capsid protein (MCP) VP5 of herpesvirus of turkeys (HVT) was identified and sequenced. It has a single open reading frame of 4236 nucleotides encoding 1412 aa protein. The gene is flanked by VP23 and UL20 sequences and is localized in the unique long region (UL) within the BamHI-B fragment. Comparison of amino acid homology has shown its clear position among the alpha-herpesviruses rather than beta- or gamma-herpesviruses. The VP5 is expressed from polycistronic mRNA together with the UL20 and the VP23 genes. The 7,2 kb RNA transcript is lacking any promoter elements or polyA signal in intergenomic regions between VP5 and UL20 or VP5 and VP23 genes, respectively. Multiple alignment of known major capsid protein sequences of all herpesvirus groups revealed presence of seven highly homologous clusters suggesting-that the corresponding protein domains might play an important role in folding of MCP and assembly of herpesvirus capsid.

Amino Acid Sequence↗
PubMed2006

An in vitro fluorescence screen to identify antivirals that disrupt hepatitis B virus capsid assembly.

Virus assembly has not been routinely targeted in the development of antiviral drugs, in part because of the lack of tractable methods for screening in vitro. We have developed an in vitro assay of hepatitis B virus (HBV) capsid assembly, based on fluorescence quenching of dye-labeled capsid protein, for testing potential inhibitors. This assay is adaptable to high-throughput screening and can identify small-molecule inhibitors of virus assembly that prevent, inappropriately accelerate and/or misdirect capsid formation to yield aberrant particles. An in vitro primary screen has the advantage of identifying promising lead compounds affecting assembly without the requirement that they be taken up by cells in culture and be nontoxic. Our approach may facilitate the identification of antivirals targeting viruses other than HBV, such as avian influenza and HIV.

Antiviral Agents↗
PubMed2015

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

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.

Animals↗
PubMed2005

Identification of structural domains involved in astrovirus capsid biology.

Coat proteins of non-enveloped, icosahedral viruses must perform a variety of functions during their life cycle such as assembly of the coat protein subunits into a closed shell, specific encapsidation of the viral nucleic acid, maturation of the capsid, interaction with host receptors, and disassembly to deliver the genetic information into the newly infected cell. A thorough understanding of the multiple capsid properties at the molecular level is required in order to identify potential targets for antiviral therapy and the prevention of viral disease. The system we have chosen for study is the astrovirus, a family of icosahedral, single-stranded RNA viruses that cause disease in mammals and birds. Very little is known about what regions of the coat protein contribute to the diverse capsid functions. This review will present novel structural predictions for the coat protein sequence of different astrovirus family members. Based on these predictions, we hypothesize that the assembly and RNA packaging functions of the astrovirus coat protein constitutes an individual domain distinct from the determinants required for receptor binding and internalization. Information derived from these structural predictions will serve as an important tool in designing experiments to understand astrovirus biology.

Capsid↗
PubMed2006

Dynamic monitoring of oncolytic adenovirus in vivo by genetic capsid labeling.

BACKGROUND: Conditionally replicative adenoviruses represent a promising strategy to address the limited efficacy and safety issues associated with conventional cancer treatment. Despite rapid translation into human clinical trials and demonstrated safety, the fundamental properties of oncolytic adenovirus replication and spread and host-vector interactions in vivo have not been completely evaluated. METHODS: We developed a noninvasive dynamic monitoring system to detect adenovirus replication. We constructed capsid-labeled E1/E3-deleted and wild-type adenoviruses (Ad-wt) by fusing the minor capsid protein IX with red fluorescent proteins mRFP1 and tdimer2(12), resulting in Ad-IX-mRFP1, Ad-IX-tdimer2(12), and Ad-wt-IX-mRFP1. Virus DNA replication, encapsidation, cytopathic effect, thermostability, and binding to primary receptor (coxsackie adenovirus receptor) were analyzed using real-time quantitative polymerase chain reaction, cell viability (MTS) assay, and fluorescence microscopy. Athymic mice (n = 4) carrying xenograft tumors that were derived from A549 lung adenocarcinoma cells were intratumorally inoculated with Ad-wt-IX-mRFP1, and adenovirus replication was dynamically monitored with a fluorescence noninvasive imaging system. Correlations between fluorescence signal intensity and viral DNA synthesis and replication were calculated using Pearson's correlation coefficient (r). RESULTS: The red fluorescence label had little effect on viral DNA replication, encapsidation, cytopathic effect, thermostability, and coxsackie adenovirus receptor binding. The fluorescent signal correlated with viral DNA synthesis and infectious progeny production both in vitro and in vivo (in A549 cells, r = .99 and r = .65; in tumors, r = .93 and r = .92, respectively). The replication efficiency of Ad-wt-IX-mRFP1 in vivo was variable, and replication and viral spreading and persistence were limited, consistent with clinical observations. CONCLUSIONS: Genetic capsid labeling provides a promising approach for the dynamic assessment of oncolytic adenovirus function in vivo.

Adenocarcinoma↗
PubMed1982

Immunochemical studies of polioviruses: identification of immunoreactive virus capsid polypeptides.

Investigation of the immunological reactions with individual poliovirus capsid polypeptides of antisera and monoclonal antibodies raised against poliovirus type 3 antigens are described. Virus polypeptides were separated by electrophoresis, transferred electrophoretically to nitrocellulose sheets and treated with antibody preparations. Antibody binding specifically to the virus polypeptides was then detected by application of 125I-labelled anti-immunoglobulin followed by autoradiography. The technique readily enabled the identification of the polypeptides recognized by the antibody. Antibodies present in polyclonal, type-specific neutralizing sera to poliovirus type 3 bound to the two largest capsid polypeptides (VP1 and VP2) of the homotypic poliovirus, and also to the VP1 of poliovirus type 1 and type 2. There was no obvious difference between the antibody binding patterns obtained with neutralizing and non-neutralizing antisera or between C-specific and D-specific antisera. VP1 appeared to be the immunodominant virus polypeptide. Among monoclonal antibodies specific for the C antigen of poliovirus type 3, a proportion reacted homotypically with the VP1 of poliovirus type 3. Other monoclonal antibodies of C antigen or D antigen specificity, or which reacted both with D and C antigens, some of which had potent virus-neutralizing activity, failed to give demonstrable binding reactions. The non-correlation of neutralization and immunoblot reactivity suggests that sequence determinants alone do not mediate virus neutralization which may depend on antigenic determinants specified by complex conformational arrangements of the virus capsid proteins.

Antibodies, Monoclonal↗
PubMed1991

Nucleotide sequences of genome segments S8, encoding a capsid protein, and S10, encoding a 36K protein, of rice gall dwarf virus.

The nucleotide sequences of DNAs complementary to the eighth (S8) and the tenth (S10) largest of the 12 genome segments of rice gall dwarf virus (RGDV) were determined. The S8 and S10 segments consist of 1578 and 1198 nucleotides, each with a single open reading frame extending for 1278 nucleotides from nucleotide 21, and 960 nucleotides from nucleotide 22, respectively. S8 encodes a polypeptide of 426 amino acids with an Mr of 47419. The amino acid sequences of several peptide fragments of the major outer capsid protein reported as 45K were contained in the predicted polypeptide. This protein, renamed the 47K protein, showed high homology with the outer capsid proteins of rice dwarf virus (RDV) and wound tumour virus (WTV); there was 56, 52 and 48% amino acid sequence identity between RGDV and WTV, RGDV and RDV, and RDV and WTV, respectively. S10 had the coding potential for a polypeptide of 320 amino acids with an Mr of 36,095 (36K protein), which exhibits 32% and 35% amino acid sequence identity with the predicted translation product of RDV S9 and the P9 capsid protein encoded by WTV S11, respectively. The conserved terminal sequences 5' GG...GAU 3' which are present in all genome segments of WTV and RDV so far analysed, and in S9 of RGDV, were also found in RGDV S8 and S10. This conserved sequence together with the segment-specific inverted repeats found in the terminal sequence of RGDV S8 and S10 are thus characteristic structures common to all three phytoreoviruses. The nucleotide sequence of the region surrounding the inverted repeats was more similar between RGDV and WTV than between RGDV and RDV.

Amino Acid Sequence↗
PubMed1992

Characterization of the genes encoding two of the major capsid proteins of epizootic haemorrhagic disease virus indicates a close genetic relationship to bluetongue virus.

The sequences of the genes of two of the major capsid proteins of epizootic haemorrhagic disease virus serotype 1 (EHDV-1, Orbivirus genus, Reoviridae) have been determined by analyses of cDNA clones representing the L2 and S7 RNA segments. The EHDV-1 S7 RNA segment, which encodes the VP7 core protein, is 1162 nucleotides in length and has the capacity to encode 349 amino acids (M(r) 38,243). The EHDV-1 L2 RNA segment, which encodes the outer capsid VP2 protein (M(r) 113,249) is 2968 nucleotides in length and has an open reading frame of 971 codons. The potential secondary structure of the EHDV-1 S7 mRNA species, in particular that of the terminal regions, is comparable to those of the corresponding segments of bluetongue virus (BTV) and African horse sickness virus (AHSV); the EHDV-1 L2 mRNA species has a secondary structure similar to that of the L2 mRNA of BTV. The EHDV-1 VP2 and VP7 proteins, as well as those of the other two major structural proteins of EHDV published previously (the inner core VP3 protein and the second outer capsid, VP5), are closely related to the corresponding proteins of BTV. The EHDV and BTV VP7 sequences are more distantly related to the sequence of the AHSV VP7 protein published recently.

Amino Acid Sequence↗
PubMed1999

Two determinants in the capsid of a persistent type 3 poliovirus exert different effects on mutant virus uncoating.

Mutant polioviruses (PV) have been previously found to be capable of establishing persistent infections in HEp-2c cells. Together, two amino acid substitutions in the viral capsid of a type 3 poliovirus (PV-3), at positions VP213 and VP1(290), are sufficient to confer the persistent phenotype to a normally lytic virus. When susceptible cells are infected, the double mutant T7L+2L(13)1N(290) undergoes unique conformational changes in the capsid, modifying its sedimentation coefficient from 160S to 147S. In the present study, we have further investigated mutant PV decapsidation and, in particular, the effect of each determinant independently. Our results indicate that the novel 147S form was also generated by a mutant carrying only the determinant 1N290. This form was not produced as a result of inherent capsid instability and it was generated only upon specific PV-host cell interactions. The second viral determinant, 2L13, also modified receptor-induced conformational changes, although differently from 1N290.

Capsid↗
PubMed1999

Only the non-glycosylated fraction of hepatitis E virus capsid (open reading frame 2) protein is stable in mammalian cells.

Hepatitis E virus (HEV) is a non-enveloped, positive-strand RNA virus, with the genome encoding three open reading frames (ORFs) of which ORF 2 directs translation of the capsid protein, PORF2. Following pulse-labelling and cell fractionation of PORF2 expressed in mammalian cells using the Semliki Forest virus replicon, the capsid protein was detected as three major species of 78 (PORF2), 82 and 86 kDa, with P82 and P86 being N-glycosylated (gPORF2 and ggPORF2, respectively). Although gPORF2 and ggPORF2 species represented 79% of total PORF2 after 20 min metabolic labelling and were largely membrane-associated, the glycosylated PORF2 species were much less stable than non-glycosylated PORF2, which was present in the cytosol and represented the major product accumulated in the cell. In the absence of detectable surface expression or export of PORF2, this suggests that glycosylated ORF 2 proteins may not be intermediates in HEV capsid assembly.

Animals↗
PubMed2005

Mechanical properties of viral capsids.

Viruses are known to tolerate wide ranges of pH and salt conditions and to withstand internal pressures as high as 100 atmospheres . In this paper we investigate the mechanical properties of viral capsids, calling explicit attention to the inhomogeneity of the shells that is inherent to their discrete and polyhedral nature. We calculate the distribution of stress in these capsids and analyze their response to isotropic internal pressure (arising, for instance, from genome confinement and/or osmotic activity). We compare our results with appropriate generalizations of classical (i.e., continuum) elasticity theory. We also examine competing mechanisms for viral shell failure, e.g., in-plane crack formation vs radial bursting. The biological consequences of the special stabilities and stress distributions of viral capsids are also discussed.

Biomechanical Phenomena↗
PubMed2005

Capsid structure and dynamics of a human rhinovirus probed by hydrogen exchange mass spectrometry.

Viral capsids are dynamic protein assemblies surrounding viral genomes. Despite the high-resolution structures determined by X-ray crystallography and cryo-electron microscopy, their in-solution structure and dynamics can be probed by hydrogen exchange. We report here using hydrogen exchange combined with protein enzymatic fragmentation and mass spectrometry to determine the capsid structure and dynamics of a human rhinovirus, HRV14. Capsid proteins (VP1-4) were labeled with deuterium by incubating intact virus in D(2)O buffer at neutral pH. The labeled proteins were digested by immobilized pepsin to give peptides analyzed by capillary reverse-phase HPLC coupled with nano-electrospray mass spectrometry. Deuterium levels incorporated at amide linkages in peptic fragments were measured for different exchange times from 12 sec to 30 h to assess the amide hydrogen exchange rates along each of the four protein backbones. Exchange results generally agree with the crystal structure of VP1-4,with extended, flexible terminal and surface-loop regions in fast exchange and folded helical and sheet structures in slow exchange. In addition, three alpha-helices, one from each of VP1-3, exhibited very slow exchange, indicating high stability of the protomeric interface. The beta-strands at VP3 N terminus also had very slow exchange, suggesting stable pentamer contacts. It was noted, however, that the interface around the fivefold axis had fast and intermediate exchange, indicating relatively more flexibility. Even faster exchange rates were found in the N terminus of VP1 and most segments of VP4, suggesting high flexibilities, which may correspond to their potential roles in virus uncoating.

Capsid↗
PubMed2000

Expression of recombinant capsid proteins of chitta virus, a genogroup II Norwalk virus, and development of an ELISA to detect the viral antigen.

The second open reading frame (ORF2) gene of the Chitta virus (CHV) was cloned to construct a recombinant baculovirus. The CHV ORF2 is predicted to encode a capsid protein of 535 amino acids (aa). CHV showed a high aa identity in the capsid region with genogroup II Norwalk virus (NV) (65-85%), but a low aa identity with genogroup I NV (44-46%). Phylogenetic analysis of the ORF2 gene demonstrated that CHV is genetically closely related to the Hawaii virus included in genogroup II NV. The recombinant capsid protein of CHV (rCHV) self-assembled to form empty virus-like particles (VLPs) when expressed in insect cells with the recombinant baculovirus. An enzyme-linked immunosorbent assay (ELISA) based on antisera to rCHV was developed to detect CHV antigen in stools. The antigen ELISA appeared to be highly specific to both rCHV and CHV-like strains. In addition, combined use of antigen ELISAs using antibodies against two antigenically distinct recombinant VLPs, the recombinant Chiba virus (rCV) and recombinant Seto virus (rSEV), enabled us to determine the genetic as well as antigenic relationship among these three viruses.

Animals↗
PubMed2000

Characterization of monoclonal antibodies generated against Norwalk virus GII capsid protein expressed in Escherichia coli.

The Norwalk virus (NV) causes outbreaks of acute non-bacterial gastroenteritis in humans. The virus capsid is composed of a single 60 kDa protein. The capsid protein of NV36 (genogroup II, Mexico virus type) was expressed in an Escherichia coli system and ten monoclonal antibodies (MAbs) were generated against it. The reactivity of these MAbs was characterized using enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) analysis towards 20 overlapping fragments of the NV36 capsid protein expressed in E. coli. All of the MAbs recognized sequential (continuous) epitopes on the three antigenic regions. Six of the 10 MAbs recognized fragment 2 (equivalent residues 31-70), three MAbs recognized fragment 13 (residues 361-403) and one MAb recognized fragment 7 (residues 181-220), suggesting that the N-terminal domain (residues 1-220) may contain more antigenic epitopes than the C-terminal domain (residues 210-548). Furthermore, two MAbs (1B4 and 1F6) reacted in WB with three purified NV strains (genogroup II) derived from patients' stool samples. It was also found that genogroup I recombinant NV96-908 (genogroup I, KY89 type) could be detected as sensitively as recombinant NV36 (genogroup II) by ELISA with a set of the MAbs produced here.

Amino Acid Sequence↗
PubMed1999

X-ray crystallographic structure of the Norwalk virus capsid.

Norwalk virus, a noncultivatable human calicivirus, is the major cause of epidemic gastroenteritis in humans. The first x-ray structure of a calicivirus capsid, which consists of 180 copies of a single protein, has been determined by phase extension from a low-resolution electron microscopy structure. The capsid protein has a protruding (P) domain connected by a flexible hinge to a shell (S) domain that has a classical eight-stranded beta-sandwich motif. The structure of the P domain is unlike that of any other viral protein with a subdomain exhibiting a fold similar to that of the second domain in the eukaryotic translation elongation factor-Tu. This subdomain, located at the exterior of the capsid, has the largest sequence variation among Norwalk-like human caliciviruses and is likely to contain the determinants of strain specificity and cell binding.

Amino Acid Sequence↗
PubMed2000

Seeing the herpesvirus capsid at 8.5 A.

Human herpesviruses are large and structurally complex viruses that cause a variety of diseases. The three-dimensional structure of the herpesvirus capsid has been determined at 8.5 angstrom resolution by electron cryomicroscopy. More than 30 putative alpha helices were identified in the four proteins that make up the 0.2 billion-dalton shell. Some of these helices are located at domains that undergo conformational changes during capsid assembly and DNA packaging. The unique spatial arrangement of the heterotrimer at the local threefold positions accounts for the asymmetric interactions with adjacent capsid components and the unusual co-dependent folding of its subunits.

Capsid↗