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Design, synthesis, and evaluation of dioxane-based antiviral agents targeted against the Sindbis virus capsid protein.

Dioxane-based antiviral agents targeted to the hydrophobic binding pocket of Sindbis virus capsid protein were designed by computer graphics molecular modeling and synthesized. Virus production using SIN-IRES-Luc and capsid assembly were monitored to evaluate antiviral activity. A compound with a three-carbon linker chain connecting two dioxane moieties inhibited virus production by 50% at a concentration of 40 microM, while (R)-hydroxymethyldioxane inhibited virus production by 50% at a concentration of 1 microM. Both compounds were not cytotoxic in uninfected BHK cells at concentrations of 1mM.

Animals↗

Expression and subcellular targeting of canine parvovirus capsid proteins in baculovirus-transduced NLFK cells.

A mammalian baculovirus delivery system was developed to study targeting in Norden Laboratories feline kidney (NLFK) cells of the capsid proteins of canine parvovirus (CPV), VP1 and VP2, or corresponding counterparts fused to EGFP. VP1 and VP2, when expressed alone, both had equal nuclear and cytoplasmic distribution. However, assembled form of VP2 had a predominantly cytoplasmic localization. When VP1 and VP2 were simultaneously present in cells, their nuclear localization increased. Thus, confocal immunofluorescence analysis of cells transduced with the different baculovirus constructs or combinations thereof in the absence or presence of infecting CPV revealed that the VP1 protein is a prerequisite for efficient targeting of VP2 to the nucleus. The baculovirus vectors were functional and the genes of interest efficiently introduced to this CPV susceptible mammalian cell line. Thus, we show evidence that the system could be utilized to study targeting of the CPV capsid proteins.

Animals↗

Protection against Atlantic halibut nodavirus in turbot is induced by recombinant capsid protein vaccination but not following DNA vaccination.

Fish nodaviruses (betanodaviruses) are small, non-enveloped icosahedral single-stranded positive-sense RNA viruses that can cause viral encephalopathy and retinopathy (VER) in a number of cultured marine teleost species, including Atlantic halibut (Hippoglossus hippoglossus). A recombinant protein vaccine and a DNA vaccine were produced, based on the same capsid-encoding region of the Atlantic halibut nodavirus (AHNV) genome, and tested for protection in juvenile turbot (Scophthalmus maximus). Vaccine efficacy was demonstrated in the fish vaccinated with recombinant capsid protein but not in the DNA-vaccinated fish, despite the fact that in vivo expression of the DNA vaccine-encoded antigen was confirmed by RNA in situ hybridisation and immunohistochemistry. Combined DNA and recombinant vaccine administration did not improve the effect of the latter. Surprisingly, fish vaccinated with 50 microg recombinant protein demonstrated a threefold lower survival rate than the two groups that received 10 microg recombinant protein. Neither the recombinant protein vaccine nor the DNA vaccine induced anti-viral antibodies 9 weeks after immunisation, while antibodies reactive with the recombinant protein were detectable mainly in fish vaccinated with 50 microg recombinant protein. The study also demonstrates evidence of viral replication inside the myocytes of intramuscularly challenged fish.

Animals↗

In vitro expression, monoclonal antibody and bioactivity for capsid protein of porcine circovirus type II without nuclear localization signal.

We expressed firstly the Capsid protein gene defecting the nuclear localization signal (NLS) of Porcine circovirus type II (PCV2) in Escherichia coli as a fusion protein with glutathione S-transferase (rGST-dCap protein). The purified rGST-dCap protein and the recombinant NLS-defected Cap protein of PCV2 (rdCap protein) from the purified rGST-dCap protein reacted specifically with swine antiserum to PCV2. Furthermore, the obtained monoclonal antibodies (mAbs) to rdCap protein were shown to bind to PCV2 particles replicated in PK15 cell and capsid protein (Cap protein) of PCV2 expressed in PK15 cells, respectively. mAbs to rdCap protein also revealed the neutralizing ability to PCV2 particles. These results demonstrated that rGST-dCap protein expressed in E. coli was folded correctly or at least partly, and mAbs to rdCap protein possessed the binding epitopes of PCV2 particles whereas mAbs 4C4 and 3F6 to rdCap protein remained the neutralization epitope of PCV2 particle, showing a possibility of neutralizing mAb to rdCap protein as an immnuotherapeutic agent and a potential of rGST-dCap protein as a vaccine antigen or serodiagnostic reagent.

Animals↗

Epidemic spread of recombinant noroviruses with four capsid types in Hungary.

BACKGROUND: Noroviruses are common pathogens in gastro-enteritis outbreaks in humans worldwide. Noroviruses are genetically diverse group of viruses with multiple genogroups (GG) and genotypes. More recently, naturally occurring recombinant noroviruses were described. These viruses had a distinct polymerase gene sequence (designated GGIIb/Hilversum) and were disseminated through waterborne and food-borne transmission in Europe. OBJECTIVES: Our aim was to characterize these emerging recombinant noroviruses causing outbreaks of gastro-enteritis in Hungary. STUDY DESIGN: From January 2001 to May 2004, samples containing "GGIIb/Hilversum polymerase" (GGIIb-pol) were selected for analysis of the viral capsid region (ORF2) by reverse transcription-polymerase chain reaction (RT-PCR) and sequencing. RESULTS: Thirty-four (14.4%) of 236 confirmed norovirus outbreaks were caused by the variant lineage with the GGIIb-pol. Four different recombinants were detected with capsids of Hu/NLV/GGII/Mexico/1989 (n=9, 43%), Hu/NLV/GGII/Snow Mountain/1976 (n=6, 28%), Hu/NLV/GGII/Hawaii/1971 (n=4, 19%) and Hu/NLV/GGII/Lordsdale/1993 (n=1, 5%). CONCLUSIONS: In Hungary, emerging recombinant noroviruses became the second most common norovirus variants-next to GGII-4/Lordsdale virus-causing epidemics of gastroenteritis in the last 4 years.

Caliciviridae Infections↗

Crystal structure of an empty capsid of turnip yellow mosaic virus.

Empty capsids (artificial top component) of turnip yellow mosaic virus were co-crystallized with an encapsidation initiator RNA hairpin. No clear density was observed for the RNA, but there were clear differences in the conformation of a loop of the coat protein at the opening of the pentameric capsomer (formed by five A-subunits) protruding from the capsid, compared to the corresponding loop in the intact virus. Further differences were found at the N terminus of the A-subunit. These differences have implications for the mechanism of decapsidation of the virus, required for infection.

Capsid↗

Cooperative reorganization of a 420 subunit virus capsid.

The complex protein capsids of many viruses exhibit dramatic reorganizations at critical stages in their life-cycle. Here, time-resolved solution X-ray scattering was used to study a dynamic, large-scale conformational maturation of the 420 subunit, 13 MDa, icosahedrally symmetric HK97 bacteriophage capsid. Isoscattering points in the time-resolved scattering patterns and singular value decomposition revealed that the expansion occurs as a cooperative, two-state reaction. The analysis demonstrates that the population shift from Prohead-II to Expansion Intermediate I, EI-I (60 A larger than Prohead-II) occurs in minutes, but does not reveal the time required for individual transitions that occur stochastically. Any intermediate forms that may be traversed during this conversion are unstable and do not constitute an appreciable population of the ensemble of particles. In an energetic landscape view, particles must undergo an energy barrier-crossing event in order to successfully convert from Prohead-II to EI-I. This implies that the particles "hop" over the energy barrier stochastically as they individually attain an expansion-active state. Interestingly, systematic deviations from single-exponential kinetics were observed for the population shift. This may indicate that in undergoing the irreversible conversion from Prohead-II to EI-I, particles are subject to a complex energy landscape that links the initial and final particle forms.

Capsid Proteins↗

Distinguishing reversible from irreversible virus capsid assembly.

Capsids of spherical viruses may be constructed from hundreds or thousands of copies of the major capsid protein(s). These assembly reactions are poorly understood. Here we consider the predicted behavior for assembly where the component reactions have weak association energy and are reversible and compare them to essentially irreversible reactions. The comparisons are based on mass action calculations and the behavior predicted from kinetic simulations where assembly is described as a cascade of low order reactions. Reversible reactions are characterized by a pseudo-critical concentration, whereas irreversible reactions consume all free subunits. Irreversible reactions are more susceptible to kinetic traps comprised of numerous small intermediates. In the case where only the ultimate step is irreversible, very low concentrations of intermediates slow the completion of the reaction so that overall it closely matches the predictions for the reversible reactions that make up the majority of the cascade. Data in the literature strongly support the hypothesis that most viruses are held together by many weak interactions.

Capsid↗

IFN-gamma production and astrocyte recognition by autoreactive T cells induced by Theiler's virus infection: role of viral strains and capsid proteins.

From mice infected with the DA strain of Theiler's murine encephalomyelitis virus (TMEV), CD8+ cytotoxic T lymphocytes (CTLs) could be detected after stimulation with TMEV infected antigen presenting cells (APCs). These CTLs killed not only TMEV infected but also uninfected syngeneic cells. Killing was associated with interferon (IFN)-gamma production in ELISPOT assays. The CTLs were efficiently induced by vaccinia virus encoding DA virus capsid proteins, but not by APCs infected with the GDVII strain of TMEV. The CTLs produced IFN-gamma in response to TMEV infected, but not uninfected, astrocytes. The CTLs could be maintained in the presence of interleukin (IL)-2. We hypothesized that, in DA virus infection, CD8+ CTLs specific for viral capsid protein could recognize self protein on oligodendrocytes by molecular mimicry, leading to demyelination.

Animals↗

Chemiluminescence-based electrophoretic mobility shift assay of RNA-protein interactions: application to binding of viral capsid proteins to RNA.

A chemiluminescence electrophoretic mobility shift assay was introduced for the study of RNA-protein interactions that include association of genomic RNA and viral capsid proteins. Binding of the capsid protein of Venezuelan equine encephalitis virus (VEEV) to several types of RNA was used as a model system to test the application of the method. The effects of RNA secondary structures and the significance of electrostatic interaction on binding were identified. This method may have wide application to the study of RNA-protein interactions.

Capsid Proteins↗

Ovine progressive pneumonia virus capsid is B-cell immunodominant using Western blot analysis: a comparison of sensitivity between Western blot analysis and immunoprecipitation.

A Western blot assay was developed and analyzed against the comparable standard, immunoprecipitation of (35)[S]-methionine/cysteine-labeled ovine progressive pneumonia virus (OPPV) proteins, for its ability to detect anti-OPPV antibodies using endpoint titers. Western blot analysis is 12-fold more sensitive in detecting endpoint anti-capsid antibody titers than IP, and the capsid is the B-cell immunodominant OPPV protein when utilizing Western blot analysis. Since the surface envelope glycoprotein is the B-cell immunodominant OPPV protein when utilizing immunoprecipitation, this suggests immunoprecipitation and Western blot analysis measure different types of antibody that are more specific for conformational and linear OPPV protein epitopes, respectively.

Animals↗

Bacteriophage T4 nanoparticle capsid surface SOC and HOC bipartite display with enhanced classical swine fever virus immunogenicity: a powerful immunological approach.

The phage T4 HOC, SOC bipartite display system is attractive for the expression of cDNA and display of peptides or proteins at high copy numbers on the phage capsid surface. Until recently, using T4 phage vector to display large foreign molecular immunogens resulted only from either an SOC or HOC single site. In this report, the main advantages of the phage T4 system over other display technologies are substantiated by using the phage T4 SOC, HOC dual site display vector T4-Zh(-) to express: (1) on the SOC site, the classical swine fever virus (CSFV) major antigenic determinant cluster mE2 (123 amino acid, aa) through gene fusion to the SOC gene C-terminus of T4 genome, and (2) on the HOC site, full-length CSFV primary antigen E2 (371 aa) through a co-transformed plasmid, hence leading to a simultaneous display of both proteins on the T4 capsid surface. The immunogenicities of these constructs were measured by ID-ELISA, dot-ELISA, Western blotting, and immunogenic response in mice including humoral and cellular immunity tests. The immunological efficiencies both in vitro and in mice of phage T4 with both single site and dual site displays, as well as conventional Escherichia coli plasmid expression, were evaluated. The animal immune response data showed that the antibody titers elicited by the T4 phage-CSFV recombinants were significantly higher than those obtained by E. coli plasmid expression, and the unpurified double site display T4 phage particles were around two times higher than either single site display or plasmid expression while being at lower phage concentrations than the single site phages. The immunogens were effective in the absence of eukaryotic protein modifications. Therefore, the phage T4 dual site display emerges as a powerful method with an enhanced immune response in animals for research and development of immunological products.

Animals↗

A directed evolution approach to select for novel Adeno-associated virus capsids on an HIV-1 producer T cell line.

A directed evolution approach was used to select for Adeno-associated virus (AAV) capsids that would exhibit more tropism toward an HIV-1 producer T cell line with the long-term goal of developing improved gene transfer vectors. A library of AAV variants was used to infect H9 T cells previously infected or uninfected by HIV-1 followed by AAV amplification with wild-type adenovirus. Six rounds of biological selection were performed, including negative selection and diversification after round three. The H9 T cells were successfully infected with all three wild-type viruses (AAV, adenovirus, and HIV-1). Four AAV cap mutants best representing the small number of variants emerging after six rounds of selection were chosen for further study. These mutant capsids were used to package an AAV vector and subsequently used to infect H9 cells that were previously infected or uninfected by HIV-1. A quantitative polymerase chain reaction assay was performed to measure cell-associated AAV genomes. Two of the four cap mutants showed a significant increase in the amount of cell-associated genomes as compared to wild-type AAV2. This study shows that directed evolution can be performed successfully to select for mutants with improved tropism for a T cell line in the presence of HIV-1.

Capsid↗

An avian influenza vaccine for humans targeting the polymerase B2 protein inside the capsid instead of hemagglutinin or neuramidase on the virus surface.

Vaccines for avian influenza typically are aimed at hemagglutinin or neuramidase on the outside of the virus capsid. A major problem with such an approach is that the genes coding for these proteins have a very rapid mutation rate, forcing commercial producers to wait for mutations to occur before developing effective new versions of standard vaccines. However, a recent study has revealed that the 1918 flu virus, like the H5N1 avian flu virus, has an E627K mutation in its polymerase B2 component, which is located inside the virus capsid. Other research has indicated that this mutation strongly influences the virulence of H5N1. It seems reasonable to believe that the constancy, over more than 80 years, of the E627K mutation could be exploited to begin developing a vaccine now, rather than waiting for new mutations. Consequently, a publicly available database at the National Center for Biotechnology Information (NCBI) website, and the SYFPEITHI online computer algorithm, were used to generate a hypothesis about a peptide-based vaccine targeted at the E627K mutation in PB2 of the avian influenza virus. It was found that the peptide sequence, DTVQIIKLL, present in the PB2 protein of the H5N1 virus, would be expected to bind to HLA-A26 restricted immune system cell surface receptors. Hence, the bound peptide might be capable of stimulating protection from cytotoxic T lymphocytes. Should the present hypothesis be confirmed in laboratory studies, and an effective vaccine developed for individuals expressing the HLA-A26 receptor; further research would be indicated. This research would be aimed at determining whether molecular modifications to the DTVQIIKLL peptide could make it effective with other members of the HLA-A1 supertype to which HLA-A26 belongs. In addition to allowing vaccine development to begin now, this peptide-based approach would have the advantage of avoiding the use of dangerous, live, avian influenza virus during mass production.

Capsid Proteins↗

Recombinant expression of Garlic virus C (GARV-C) capsid protein in insect cells and its potential for the production of specific antibodies.

Garlic cultivars in Brazil are infected by a complex of viruses and for some virus species, such as the allexivirus, purification of the virions is sometimes cumbersume. To overcome this problem, recombinant expression of viral proteins in heterologous systems is an alternative method for producing antibodies. The capsid gene from Garlic virus C (GarV-C), an Allexivirus, was inserted into the genome of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) generating the recombinant virus vSynGarV-C. The recombinant protein expression was confirmed by SDS-PAGE and western-blot of extracts from recombinant virus infected insect cells, where a protein band of approximately 32KDa was observed only in extracts from recombinant infected cells. This protein corresponded to the predicted size of the capsid protein of the GarV-C. A rabbit polyclonal antibody was raised against this protein, shown to be specific for the GarV-C protein in western-blot and dot-Elisa, however with a low titer.

Animals↗

Complete genome characterization, phylogenetic analysis, and capsid P2 variation of a goose astrovirus genotype 2 isolate from Guizhou, China.

Goose astrovirus genotype 2 (GAstV-2) is associated with gout and renal disease in goslings, but its occurrence in Guizhou Province remains poorly documented. We isolated a GAstV-2 strain, designated GZJP2024, from goslings with visceral gout on a farm in Jinping County, Guizhou Province, China. PCR detected GAstV-2 but not goose parvovirus, goose reovirus, Tembusu virus, fowl adenovirus, or goose astrovirus genotype 1. Serial passage in goose embryos produced mortality and hemorrhagic lesions during the third passage. Whole-genome sequencing yielded a 7,251-nt genome containing three overlapping open reading frames (ORF1a, ORF1b, and ORF2). Sequence identity and phylogenetic analyses assigned GZJP2024 to the GAstV-2 lineage. ORF1b was the most conserved coding region, whereas ORF2 was more variable. Comparison with consensus sequences from representative GAstV-2 strains identified five amino acid substitutions in ORF1a and ten in ORF2. Four ORF2 substitutions (E456D, L540Q, S608T, and A614T) occurred in the capsid P2 domain and overlapped or neighbored predicted B-cell epitope-rich regions. Template-based mapping placed E456D, S608T, and A614T on exposed regions of a spike-like capsid structure. These findings document a GAstV-2 isolate from a gout-affected goose farm in Guizhou and provide sequence data for future regional surveillance.

Capsid P2↗

X-ray structure of RLI, an essential twin cassette ABC ATPase involved in ribosome biogenesis and HIV capsid assembly.

The ABC ATPase RNase-L inhibitor (RLI) emerges as a key enzyme in ribosome biogenesis, formation of translation preinitiation complexes, and assembly of HIV capsids. To help reveal the structural mechanism of RLI, we determined the Mg2+-ADP bound crystal structure of the twin cassette ATPase of P. furiosus RLI at 1.9 A resolution and analyzed functional motifs in yeast in vivo. RLI shows similarities but also differences to known ABC enzyme structures. Twin nucleotide binding domains (NBD1 and NBD2) are arranged to form two composite active sites in their interface cleft, indicating they undergo the ATP-driven clamp-like motion of the NBDs of ABC transporters. An unusual "hinge" domain along the NBD1:NBD2 interface provides a frame for association and possibly ATP-driven conformational changes of the NBDs. Our results establish a first structural basis for ABC domain heterodimers and suggest that RLI may act as mechanochemical enzyme in ribosome and HIV capsid biogenesis.

Adenosine Triphosphatases↗

Features of reovirus outer capsid protein mu1 revealed by electron cryomicroscopy and image reconstruction of the virion at 7.0 Angstrom resolution.

Reovirus is a useful model for addressing the molecular basis of membrane penetration by one of the larger nonenveloped animal viruses. We now report the structure of the reovirus virion at approximately 7.0 A resolution as obtained by electron cryomicroscopy and three-dimensional image reconstruction. Several features of the myristoylated outer capsid protein mu1, not seen in a previous X-ray crystal structure of the mu1-sigma3 heterohexamer, are evident in the virion. These features appear to be important for stabilizing the outer capsid, regulating the conformational changes in mu1 that accompany perforation of target membranes, and contributing directly to membrane penetration during cell entry.

Algorithms↗