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At least 793 records · Page 44Linked to original sources
PubMed1993

Capsid localization of the bacteriophage P4 Psu protein.

In addition to its polarity-suppressing activity, the Psu protein of bacteriophage P4 also serves to stabilize the capsid against heat treatment and binds externally to the phage capsid. However, the heat stability is lost upon purification of the virus, indicating a loss of Psu protein from the capsid. By using three-dimensional reconstruction from cryo-electron micrographs of P4 psu1 amber mutants lacking Psu, and of P4 virions, which have been saturated with Psu protein to regain heat stability, we have determined the position of this protein on the virus surface. Our results are consistent with the hypothesis that the function of Psu is to stabilize the hexameric capsomer assembly.

Capsid↗
PubMed1994

HIV-2 viral protein X association with the GAG p27 capsid protein.

VPX is a 16 kDa accessory protein expressed in cells infected with HIV-2 and most SIV strains and is packaged into virus particles. In order to define the requirements for incorporation of VPX into virions, VPX and HIV-2 GAG-POL were expressed independently from a vaccinia virus-based transient expression system. Under these conditions, VPX was exported from transfected cells only when coexpressed with the HIV-2 GAG-POL plasmid. A 27 kDa protein coprecipitating with VPX was found to have an identical electrophoretic mobility as the GAG capsid protein, and reacted with an anti-GAG antiserum. Coexpression of VPX and GAG-POL resulted in virus-like particles containing both proteins, as determined by sucrose gradient analyses. Expression of VPX and HIV-2 GAG without POL gave similar results. VPX association with HIV-2 GAG p27 capsid protein was specific, since no association was found with the HIV-1 GAG p25/p24 capsid protein.

Animals↗
PubMed1995

Human papillomavirus type 16 capsid proteins produced from recombinant Semliki Forest virus assemble into virus-like particles.

Several neutralizing sites of the human papillomavirus (HPV) capsid are known to be critically dependent on the conformation of the capsid. However, efficient production of HPV16 capsids in mammalian cells has been difficult, possibly because the HPV genome contains negative regulatory elements. To circumvent these problems, we cloned the HPV16 L1 and L2 genes from a healthy HPV16-infected woman into a Semliki Forest virus based expression vector (P. Liljeström and H. Garoff, Biotechnology 9, 1356-1361, 1991). Recombinant HPV16 L1- or L2-producing Semliki Forest virus was generated and used for infection of mammalian cells. The HPV16 L1 and L2 proteins were efficiently expressed and the majority of the L1 protein self assembled into virus-like particles (VLPs). Coexpression of L1 and L2 resulted in incorporation of L2 into the VLPs. The particles had a density of approximately 1.3 g/ml as determined by density gradient centrifugation. Transmission electron microscopy revealed that the particles had a morphology similar to native virions. The HPV16 VLPs produced by the Semliki Forest virus expression system may be useful as a conformationally correctly assembled target for studies of HPV attachment, assembly, serology, or vaccination.

Animals↗
PubMed2000

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

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.

Animals↗
PubMed2002

Yeast coexpression of human papillomavirus types 6 and 16 capsid proteins.

The L1 and L2 capsid proteins of animal and human papillomaviruses (HPVs) can self-assemble into virus-like particles (VLPs) that closely resemble native virions. The use of different animal models shows that VLPs can be very efficient at inducing a protective immune response. However, studies with infectious HPV virions and VLPs of different HPV types indicate that the immune response is predominantly type-specific. We have generated a diploid yeast strain that coexpresses the L1 and L2 capsid proteins of both HPV-6b and HPV-16, and we have purified fully assembled VLPs banding in a cesium chloride gradient at the expected density of 1.29-1.3 mg/ml. Experimental evidence strongly indicated that the four proteins coassembled into VLPs. Western blot analysis, using anti-HPV-6 and anti-HPV-16 L1-specific monoclonal antibodies and type-specific L2 antisera, demonstrated that all four proteins copurified. Most importantly, immunoprecipitation experiments, carried out using type-specific anti-L1 monoclonals and either total yeast cell extracts or purified VLPs, confirmed the interaction and the formation of covalent disulfide bonds between the two L1 proteins. Finally, HPV-6/16 VLPs administered to mice induced conformational antibodies against both L1 protein types. These results suggest that coexpression of different capsid proteins may provide new tools for the induction of antibodies directed against multiple HPV types.

Animals↗
PubMed1998

Characterizing sequence variation in the VP1 capsid proteins of foot and mouth disease virus (serotype 0) with respect to virion structure.

The VP1 capsid protein of foot and mouth disease virus (FMDV) is highly polymorphic and contains several of the major immunogenic sites important to effective antibody neutralization and subsequent viral clearance by the immune system. Whether this high level of polymorphism is of adaptive value to the virus remains unknown. In this study we examined sequence data from a set of 55 isolates in order to establish the nature of selective pressures acting on this gene. Using the known molecular structure of VP1, the rates and ratios of different types of nonsynonymous and synonymous changes were compared between different parts of the protein. All parts of the protein are subject to purifying selection, but this is greatest amongst those amino acid residues within beta-strands and is significantly reduced at residues exposed on the capsid surface, which include those residues demonstrated by previous mutational analyses to permit the virus to escape from monoclonal antibody binding. The ratios of nonsynonymous substitution resulting in various forms of physicochemically radical and conserved amino acid change were shown to be largely equal throughout these different parts of the protein. There was a consistently higher level of nonsynonymous and charge radical sites in those regions of the gene coding for residues exposed on the outer surface of the capsid and a marked difference in the use of amino acids between surface and nonsurface regions of the protein. However, the analysis is consistent with the hypothesis that the observed sequence variation arises where it is least likely to be disruptive to the higher-order structure of the protein and is not necessarily due to positive Darwinian selection.

Amino Acid Sequence↗
PubMed1997

The minor outer capsid protein P2 of rice gall dwarf virus has a primary structure conserved with, yet is chemically dissimilar to, rice dwarf virus P2, a protein associated with virus infectivity.

The nucleotide sequence of the genome segment 2 (S2) of rice gall dwarf virus (RGDV), a phytoreovirus, when compared with the amino acid sequence of a component protein of the virus, showed that S2 potentially encoded a 127K minor outer capsid protein. This 127K protein designated as P2 and the 127K minor outer capsid protein (also termed P2) of rice dwarf virus (RDV) are similar in size, located in the outer capsid, and have well-conserved predicted polypeptide sequences, suggesting similar functions. Infectivity to insect vector cell monolayers of RGDV was maintained and the P2 protein was retained irrespective of carbon tetrachloride (CCl4) treatment. This is in contrast to the infectivity of RDV which is removed along with P2 protein following CCl4 treatment. RGDV with P2 was acquired by vector insects and transmitted to host plants, although RDV lacking P2 could not be transmitted to plants as previously published. These results imply that RDV and RGDV require P2 proteins for virus infectivity to vector insects.

Amino Acid Sequence↗
PubMed1988

Roles of operator and non-operator RNA sequences in bacteriophage R17 capsid assembly.

In order to understand the role of sequences other than the translational operator on bacteriophage R17 assembly, in vitro capsid assembly was studied with R17 coat protein and a variety of RNAs. For a series of RNA oligomers of the same chain length, sequences that bind coat protein dimer with a lower affinity require higher concentrations of RNA and protein for assembly. Among a series of non-specific RNA molecules of differing lengths, lower protein and RNA concentrations are required for assembly of capsids containing longer RNAs. For RNA molecules of any length, the presence of a single high-affinity translational operator sequence lowered the concentration requirements for capsid assembly. However, the advantage for encapsidation provided by the operator sequence is small for large RNA molecules. The experiments indicate that in the overall assembly process the interaction of coat protein with non-specific sequences is at least as important as its interaction with the specific translational operator sequence. In light of the data, a mechanism of achieving selective packaging of the R17 genomic RNA in vivo is discussed.

Bacteriophages↗
PubMed1984

Electron microscopic studies on the connections between capsomers in the adenovirus capsid.

Electron microscopic observation of negatively stained virions suggests that the interhexonal spaces within the adenovirus capsid are bridged by interhexonal connective elements. Similar connections are also detected between pentons and the five peripentonal hexons. Examinations of electron micrographs by Markham's rotational integration technique reveals the presence of a regular network of the connective elements between the capsomers in the whole capsid. The length of these connections corresponds to about one-third of the diameter of the hexons. The connections consist of two approximately parallel elements, thus every hexon is joined to its six nearest neighbours by 12 connections. A possible mutual orientation pattern of the connective elements in the whole capsid is presented on a tentative virus model.

Adenoviridae↗
PubMed1991

Outer capsid glycoprotein vp7 is recognized by cross-reactive, rotavirus-specific, cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTLs) generated in mice orally inoculated with rotaviruses lyse target cells infected with different rotavirus serotypes (cross-reactive CTLs). Using vaccinia virus recombinants expressing individual rotavirus proteins from two different rotavirus serotypes, we found that cross-reactive CTLs recognize target cells expressing outer capsid protein vp7 better than those expressing outer capsid protein vp4 or inner capsid protein vp6. These findings may be relevant to vaccine strategies which include immunization with reassortant rotaviruses or viral or bacterial vectors expressing individual rotavirus proteins. The region or regions of vp7 which are antigenically conserved among different rotavirus serotypes and recognized by cross-reactive CTLs remain to be determined.

Animals↗
PubMed1995

A synthetic parvovirus B19 capsid protein can replace viral antigen in antibody-capture enzyme immunoassays.

To establish a renewable source of parvovirus B19 antigens for diagnostic tests, gene sequences for the viral capsid proteins, VP1 and VP2, were cloned into baculovirus expression vectors and the recombinant viruses used to infect Sf9 insect cells. Cell lysates examined by immunoblotting demonstrated reactive proteins corresponding to the expected sizes of native VP1 (83 kDa) and VP2 (58 kDa). The VP2 protein was produced efficiently in quantity and self-assembled into empty capsids as shown by density equilibration in a CsCl step gradient. The VP2 protein was purified and used as an antigen in antibody-capture enzyme immunoassays for the detection of B19 IgG and IgM antibodies. Compared to a standard antibody-capture EIA based on whole viral antigen, the VP2-EIA gave a sensitivity of 100% and specificity of 97% in detection of B19 IgM in 138 patients suspected of B19 infection. No IgM-positive specimens were missed. IgG detection yielded a sensitivity of 100% and specificity of 96% in the same population. Recombinant VP2 capsid proteins expressed in baculovirus-infected insect cells can substitute for serum-derived B19 virus in standard antibody-capture EIA for the detection of B19 IgG and IgM with comparable results.

Adult↗
PubMed2006

IgG antibodies to HPV16, 52, 58 and 6 L1-capsids and spontaneous regression of cervical intraepithelial neoplasia.

To identify the predictive markers for spontaneous regression of cervical intraepithelial neoplasia (CIN), we examined whether IgG antibody responses to common human papillomavirus (HPV) L1-capsids correlate with CIN regression. In a cohort study, a total of 116 Japanese women with CIN grade I/II were tested for cervical HPV DNA and serum IgG antibodies to HPV16/52/58/6 L1-capsids. Our data suggest that baseline IgG reactivities to HPV L1-capsids do not serve as a predictive marker of CIN regression, in contrast to histological CIN grades and HPV DNA status.

Antibodies, Viral↗
PubMed2005

Putative autocleavage of reovirus mu1 protein in concert with outer-capsid disassembly and activation for membrane permeabilization.

Capsid proteins of several different families of non-enveloped animal viruses with single-stranded RNA genomes undergo autocatalytic cleavage (autocleavage) as a maturation step in assembly. Similarly, the 76 kDa major outer-capsid protein mu1 of mammalian orthoreoviruses (reoviruses), which are non-enveloped and have double-stranded RNA genomes, undergoes putative autocleavage between residues 42 and 43, yielding N-terminal N-myristoylated fragment mu1N and C-terminal fragment mu1C. Cleavage at this site allows release of mu1N, which is thought to be critical for penetration of the host-cell membrane during cell entry. Most previous studies have suggested that cleavage at the mu1N/mu1C junction precedes addition to the outer capsid during virion assembly, such that only a small number of the mu1 subunits in mature virions remain uncleaved at that site (approximately 5%). In this study, we varied the conditions for disruption of virions before running the proteins on denaturing gels and in several circumstances recovered much higher levels of uncleaved mu1 (up to approximately 60%). Elements of the disruption conditions that allowed greater recovery of uncleaved protein were increased pH, absence of reducing agent, and decreased temperature. These same elements allowed comparably higher levels of the mu1delta protein, in which cleavage at the mu1N/delta junction has not occurred, to be recovered from particle uncoating intermediates in which mu1 had been previously cleaved by chymotrypsin in a distinct protease-sensitive region near residue 580. The capacity to recover higher levels of mu1delta following disruption of these particles for electrophoresis was lost, however, in concert with a series of structural changes that activate the particles for membrane permeabilization, suggesting that the putative autocleavage is itself one of these changes.

Capsid↗
PubMed2006

VP4 protein from human rhinovirus 14 is released by pressure and locked in the capsid by the antiviral compound WIN.

Rhinoviruses are the major causative agents of the common cold in humans. Here, we studied the stability of human rhinovirus type 14 (HRV14) under conditions of high hydrostatic pressure, low temperature, and urea in the absence and presence of an antiviral drug. Capsid dissociation and changes in the protein conformation were monitored by fluorescence spectroscopy, light scattering, circular dichroism, gel filtration chromatography, mass spectrometry and infectivity assays. The data show that high pressure induces the dissociation of HRV14 and that this process is inhibited by WIN 52084. MALDI-TOF mass spectrometry experiments demonstrate that VP4, the most internal viral protein, is released from the capsid by pressure treatment. This release of VP4 is concomitant with loss of infectivity. Our studies also show that at least one antiviral effect of the WIN drugs involves the locking of VP4 inside the capsid by blocking the dynamics associated with cell attachment.

Antiviral Agents↗
PubMed2004

A tiling approach to virus capsid assembly explaining a structural puzzle in virology.

A novel approach for the description of the protein stoichiometry of viral capsids, that is the protein shells protecting the viral genome, is introduced based on tiling theory. This approach generalizes Caspar-Klug theory of quasi-equivalence to account also for non-quasi-equivalent subunit arrangements in icosahedral virus capsids that have been observed experimentally but are not covered by the Caspar-Klug approach. In particular, the new approach is used to explain the structure of polyoma virus, Simian Virus 40 and L-A virus capsids, which are considered structural puzzles in view of Caspar-Klug theory.

Capsid↗
PubMed2005

Crosslinking in viral capsids via tiling theory.

A vital part of a virus is its protein shell, called the viral capsid, that encapsulates and hence protects the viral genome. It has been shown in Twarock [2004. A tiling approach to vius capsids assembly explaining a structural puzzle in virology. J. Theor. Biol. 226, 477-482] that the surface structures of viruses with icosahedrally symmetric capsids can be modelled in terms of tilings that encode the locations of the protein subunits. This theory is extended here to multi-level tilings in order to model crosslinking structures. The new framework is demonstrated for the case of bacteriophage HK97, and it is shown, how the theory can be used in general to decide if crosslinking, and what type of crosslinking, is compatible from a mathematical point of view with the geometrical surface structure of a virus.

Bacteriophages↗
PubMed2007

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

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.

Animals↗
PubMed2006

Consideration of the three-dimensional structure of core shells (capsids) in spherical retroviruses.

The problem of three-dimensional organization of retroviral cores has been a matter of interest for the past 30 years. The general opinion in favor of icosahedral symmetry based on electron microscopy observations was questioned when cryo-electron microscopy failed to provide convincing evidence in its favor. More recent studies by cryo-electron microscopy, X-ray crystallography and in vitro assembly of the CA domain of Human immuno deficiency virus (HIV), Murine leukemia virus (MuLV) and Rous sarcoma virus (RSV) threw new light on the organization of retroviral cores. In this communication we report how we produced a three-dimensional (3D) model of MuLV core using data from CA assembly on a lipid film [Ganser, B.K., Cheng, A., Sundquist, W.I., Yeager, M., 2003. Three-dimensional structure of the M-MuLV CA protein on a lipid monolayer: a general model for retroviral capsid assembly. EMBO J. 22, 2886-2892]. The resulting structure revealed that the molecular organization of the core shell is specific and the presence of a 5,3,2 rotational symmetry of the 3D model provides support for icosahedral shape of MuLV cores. The model made it possible to determine the diameter of the cores and calculate the number of CA copies as well as the molecular mass of a core of specific diameter. Thus MuLV cores 68 (or 81.6) nm in diameter consist of 1500 (or 2160) copies of CA. About 12% of molecules from fullerene-like Gag shells versus 71% of molecules of closely packed (core-like). Gag shells were not incorporated into the core shells (capsids). Our 3D models received support from X-ray data of MuLV CA NTD domain published by Mortuza et al. [Mortuza, G., Haire, L.F., Stevens, A., Smerdon, S.J., Stoye, J.P., Taylor, I.A., 2004. High resolution structure of a retroviral capsid hexameric amino-terminal domain. Nature 431, 481-485].

Animals↗