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At least 919 records · Page 51Linked to original sources
PubMed2005

Characterization of the major capsid proteins of myxoma virus particles using MALDI-TOF mass spectrometry.

The protein composition of poxvirus particles remains uncertain because of their large size and structural complexity. This has complicated the characterization of even well-studied Orthopoxviruses, like vaccinia virus, and little or nothing is known about the capsid composition of viruses belonging to other poxvirus genera. This paper describes methods that address this problem and have been used to identify 17 different Leporipoxvirus capsid proteins. Myxoma virus particles were purified using sucrose and Nicodenz gradient centrifugation and subfractionated into membrane and core fractions by thiol and detergent treatment. These materials were further fractionated using reverse-phase chromatography and SDS-PAGE and the resulting proteins identified by mass spectroscopy. Most of the myxoma proteins identified in this manner were homologs of either vaccinia virus structural proteins (F17R, L4R, J1R, H3L, A3L, A10L, A27L, and A45R) or virion-associated enzymes (I7L, H4L, D11L, A7L, and A22R). However, the myxoma homolog of the vaccinia P4a/A10L protein (M099L) differs from P4a protein in being proteolytically cleaved only once. M095L and M151R were also detected in core fractions. M095L and M151R are homologs of vaccinia A6L and B13R proteins, respectively, and poxvirus proteins not previously known to be capsid components. M093L, a protein of unknown function and having no certain Orthopoxvirus homolog associates with membrane fractions. These studies illustrate the conservation of Chordopoxvirion architecture and the methods that can be used to elucidate the proteins comprising these structures.

Amino Acid Sequence↗
PubMed2004

Is RNA-laden p24 capsid protein a vector for HIV?

The consensus HIV infection pathway, although well supported by abundant experimental evidence, fails to account for infection of cells (e.g., neurons) that lack CD4 and/or coreceptors. We present the hypothesis that p24 capsid protein laden with RNA may be an alternative virulent agent. A novel mechanism of HIV infection may involve binding of the capsid with membrane receptors, followed by internalization of the capsid-receptor complex. This new pathway suggests novel strategies for antiviral pharmacotherapy.

Capsid Proteins↗
PubMed2005

Mammalian SCAN domain dimer is a domain-swapped homolog of the HIV capsid C-terminal domain.

Retroviral assembly is driven by multiple interactions mediated by the Gag polyprotein, the main structural component of the forming viral shell. Critical determinants of Gag oligomerization are contained within the C-terminal domain (CTD) of the capsid protein, which also harbors a conserved sequence motif, the major homology region (MHR), in the otherwise highly variable Gag. An unexpected clue about the MHR function in retroviral assembly emerges from the structure of the zinc finger-associated SCAN domain we describe here. The SCAN dimer adopts a fold almost identical to that of the retroviral capsid CTD but uses an entirely different dimerization interface caused by swapping the MHR-like element between the monomers. Mutations in retroviral capsid proteins and functional data suggest that a SCAN-like MHR-swapped CTD dimer forms during immature particle assembly. In the SCAN-like dimer, the MHR contributes the major part of the large intertwined dimer interface explaining its functional significance.

Amino Acid Sequence↗
PubMed2006

Stability and dynamics of virus capsids described by coarse-grained modeling.

We report a study of the structural dynamics of viral capsids, simulated on a microsecond timescale, by employing a coarse-graining molecular dynamics method. The method was calibrated against an all-atom simulation of one complete virus. Among the studied capsids, some collapsed rapidly, while others were found to be stable. Interlocking between coat proteins is found to be a key factor determining the stability of the capsids.

Biochemistry↗
PubMed2006

Human CD8+ cytotoxic T cell responses to adenovirus capsid proteins.

Adenoviruses (Ads) cause fatal disease in allogeneic stem cell transplant recipients, but there is no established therapy. Ad-specific CD8+ T cells were detected in PBMC from healthy adults at a mean frequency of 77 per 10(5) CD8+ T cells (range 8-260) by interferon-gamma ELISPOT and cytokine flow cytometry assays. CD8+ T cell lines from 7 of 7 donors exhibited MHC-class-I-restricted killing of targets expressing the capsid protein hexon. In contrast, cytotoxicity against the capsid proteins fiber and penton base was weaker or not detected. Two HLA-A2-restricted hexon epitopes and one HLA-B-restricted epitope were identified, all of which are adjacent to or overlap an HLA-DP4-restricted epitope in the highly conserved C-terminus. Thus, hexon is the immunodominant T cell target among capsid proteins and contains multiple C-terminal epitopes conserved among serotypes. These data support evaluation of donor lymphocyte infusions for treatment of Ad disease post-transplant.

Adenoviridae↗
PubMed2004

Plasmid-based generation of recombinant coxsackievirus B3 particles carrying capsid gene replacement replicons.

Recombinant infectious coxsackievirus B3 (CVB3) particles were generated by packaging of modified viral genomes in which the capsid coding P1-region was replaced by an EGFP-luciferase reporter gene. Efficient packaging of the recombinant genome was achieved by a novel method based on cotransfection of a plasmid encoding the subgenomic viral replicon together with two alternative helper plasmids carrying expression cassettes of the CVB3 capsid proteins, and a T7 RNA polymerase expression plasmid. Transcription of a reporter gene and expression of capsid proteins were achieved in a single step, eliminating the need of a helper virus. Recombinant viral stocks were used to infect human embryonal cardiomyocytes (hCMC) and other cell types, and luciferase activity was measured at different timepoints after infection. Neither progeny virus nor wildtype CVB3 was produced upon infection of target cells, facilitating analyses of infected cells without viral spread. The presence of an IRES sequence upstream of the P1 open reading frame in the helper plasmids was indispensable for the generation of recombinant particles, as no packaging was observed using helper plasmids without this feature. Luciferase data obtained by transfection of reporter plasmids with and without upstream 5'-NTR sequences suggests that the CVB3 IRES facilitates translation in T7 RNA polymerase-dependent gene transcription, both in presence and absence of viral replication.

Capsid↗
PubMed2003

Osmotic shock and the strength of viral capsids.

Osmotic shock is a familiar means for rupturing viral capsids and exposing their genomes intact. The necessary conditions for providing this shock involve incubation in high-concentration salt solutions, and lower permeability of the capsids to salt ions than to water molecules. We discuss here how values of the capsid strength can be inferred from calculations of the osmotic pressure differences associated with measured values of the critical concentration of incubation solution.

Capsid↗
PubMed2000

Packaging of up to 240 subunits of a 17 kDa nuclease into the interior of recombinant hepatitis B virus capsids.

The icosahedral nucleocapsid of hepatitis B virus (HBV) consists of multiple subunits of a single 183 amino acids (aa) core protein encasing the viral genome. However, recombinant core protein alone also forms capsid-like particles. We have recently shown that a 238 aa protein centrally inserted into the core protein can be displayed on the particle surface. Here we demonstrate that replacement of the C-terminal basic domain by the 17 kDa Staphylococcus aureus nuclease also yields particles but that in these the foreign domains are located in the interior. The packaged nuclease is enzymatically active, and the chimeric protein forms mosaic particles with the wild-type core protein. Hence the HBV capsid is useful as a molecular platform which, dependent on the fusion site, allows foreign protein domains to either be packaged into or be exposed on the exterior of the particle. These results are of relevance for the use of the HBV capsid as a vaccine carrier, and as a target for antiviral therapy.

Amino Acid Sequence↗
PubMed2002

Conformational stability of dimeric and monomeric forms of the C-terminal domain of human immunodeficiency virus-1 capsid protein.

The unfolding equilibrium of the C-terminal domain of human immunodeficiency virus-1 (HIV-1) capsid protein has been analyzed by circular dichroism and fluorescence spectroscopy. The results for the dimeric, natural domain are consistent with a three-state model (N(2)<-->2I<-->2U). The dimer (N(2)) dissociates and partially unfolds in a coupled cooperative process, into a monomeric intermediate (I) of very low conformational stability. This intermediate, which is the only significantly populated form at low (1 microM) protein concentrations, fully preserves the secondary structure but has lost part of the tertiary (intramonomer) interactions found in the dimer. In a second transition, the intermediate cooperatively unfolds into denatured monomer (U). The latter process is the equivalent of a two-state unfolding transition observed for a monomeric domain in which Trp184 at the dimer interface had been truncated to Ala. A highly conserved, disulfide-bonded cysteine, but not the disulfide bond itself, and three conserved residues within the major homology region of the retroviral capsid are important for the conformational stability of the monomer. All these residues are involved also in the association process, despite being located far away from the dimerization interface. It is proposed that dimerization of the C-terminal domain of the HIV-1 capsid protein involves induced-fit recognition, and the conformational reorganization also improves substantially the low intrinsic stability of each monomeric half.

Amino Acid Substitution↗
PubMed2003

Visualization by cryo-electron microscopy of genomic RNA that binds to the protein capsid inside bacteriophage MS2.

The icosahedrally symmetrized structure of bacteriophage MS2 as determined by cryo-electron microscopy (EM) reveals the presence of genomic RNA that attaches to coat-protein dimers. Earlier X-ray diffraction studies revealed similar interactions between the unique operator hairpin of the MS2 genomic RNA and the coat-protein dimer. This observation leads us to conclude that not only the operator, but also many other RNA sequences in the genome of MS2, are able to bind to the coat-protein dimer. A substantial number of potential coat-protein-dimer binding sites are present in the genome of MS2 that can account for the observed RNA densities in the EM map. Moreover, it appears that these stem-loop structures are able to bind in a similar fashion to the coat protein dimer as the wild-type operator hairpin. The EM map also shows additional density between the potential operator-binding sites, linking the RNA stem-loops together to form an icosahedral network around the 3 and 5-fold axes. This RNA network is bound to the inside of the MS2 capsid and probably influences both capsid stability and formation, supporting the idea that capsid formation and RNA packaging are intimately linked to each other.

Capsid Proteins↗
PubMed2003

Morphological variants of Sindbis virus produced by a mutation in the capsid protein.

Sindbis virus is a complex aggregate of RNA, protein and lipid. The virus is organized as two nested T = 4 icosahedral protein shells between which is sandwiched a lipid bilayer. The virus RNA resides within the inner protein shell. The inner protein shell is attached to the outer protein shell through contacts to proteins in the outer shell, which penetrate the lipid bilayer. The data presented in the following manuscript show that mutations in the capsid protein can result in the assembly of the virus structural proteins into icosahedra of different triangulation numbers. The triangulation numbers calculated, for these morphological variants, follow the sequence T = 4, 9, 16, 25 and 36. All fall into the class P = 1 of icosadeltahedra as was predicted by. The data support their hypothesis that families of icosahedra would be developed by altering the distance between the points of insertion of the five-fold axis. This capsid protein defect also results in the incorporation of much of the capsid protein, into large cytoplasmic aggregates of protein and RNA. These observations support models suggesting that the geometry of a pre-formed nucleocapsid organizes the assembly of the virus membrane proteins into a structure of identical configuration and argues against models suggesting that assembly of the membrane glycoproteins directs the assembly of the nucleocapsid.

Animals↗
PubMed2003

Digestion pattern of reovirus outer capsid protein sigma3 determined by mass spectrometry.

Reovirus is an enteric virus comprising eight structural proteins that form a double-layered capsid. During reovirus entry into cells, the outermost capsid layer (composed of proteins sigma3 and mu1C) is proteolytically processed to generate an infectious subviral particle (ISVP) that is subsequently uncoated to produce the transcriptionally active core particle. Kinetic studies suggest that protein sigma3 is rapidly removed from virus particles and then protein mu1C is cleaved. Initial cleavage of mu1C has been well described and generates an amino (N)-terminal delta peptide and a carboxyl (C)-terminal phi peptide. However, cleavage and removal of sigma3 is an extremely rapid event that has not been well defined. We have treated purified reovirus serotype 1 Lang virions with a variety of endoproteases. Time-course digestions with chymotrypsin, Glu-C, pepsin, and trypsin resulted in the initial generation of two peptides that were resolved in SDS-PAGE and analyzed by in-gel tryptic digestion and MALDI-Qq-TOFMS. Most tested proteases cut sigma3 within a "hypersensitive" region between amino acids 217 and 238. In addition, to gain a better understanding of the sequence of subsequent proteolytic events that result in generation of reovirus subviral particles, time-course digestions of purified particles were performed under physiologic salt conditions and released peptide fragments ranging from 500 to 5000 Da were directly analyzed by MALDI-Qq-TOFMS. Trypsin digestion initially released a peptide that corresponded to the C-terminus of mu1C, followed by a peptide that corresponded to amino acids 214-236 of the sigma3 protein. Other regions of mu1C were not observed until protein sigma3 was completely digested. Similar experiments with Glu-C indicated the hypersensitive region of sigma3 was cut first when virions were treated at pH values of 4.5 or 7.4, but treatment of virions with pepsin at pH 3.0 released different sigma3 peptides, suggesting acid-induced conformational changes in this outer capsid protein. These studies also revealed that the N-terminus of sigma3 is acetylated.

Amino Acid Sequence↗
PubMed2003

Deleterious effect of peptide insertions in a permissive site of the AAV2 capsid.

A permissive site for insertion of heterologous peptide sequences has been identified in the capsid proteins of AAV2. While attempting to use this site for insertion of a nuclear localization sequence, we have observed a drastic reduction in the yield of DNA-containing particles. ELISA analysis showed that capsid assembly was modestly affected, whereas genome encapsidation was more profoundly altered, a phenomenon we did not observed when a RGD peptide was inserted at the same location. Furthermore, the NLS viruses displayed poor transduction efficiency on HeLa and 293 cells. Altogether, these results indicate that the nature of the peptide inserted at position 587 in the capsid may have important consequences on both particle formation and infectivity.

Capsid↗
PubMed2003

Recombinant adenovirus co-expressing capsid proteins of two serotypes of foot-and-mouth disease virus (FMDV): in vitro characterization and induction of neutralizing antibodies against FMDV in swine.

Human adenovirus type 5 (Ad5) has been evaluated as a novel gene delivery vector for the development of live-viral vaccines for foot-and-mouth disease (FMD). In this study, we constructed an Ad5 vector co-expressing the capsid precursor proteins, P1, of FMD virus (FMDV) field strains A24 Cruzeiro and O1 Campos and examined the neutralizing antibody responses in swine after inoculation with the vector. To construct the Ad5 vector, a bicistronic expression cassette containing a cytomegalovirus promoter, the P1 coding sequence of FMDV A24, the internal ribosomal entry site (IRES) of FMDV A12, the P1 coding sequence of FMDV O1 Campos and the coding region of A12 3C protease was inserted into the E1 region of an E1/E3-deleted Ad5. The recombinant adenovirus, Ad5A24+O1, was generated by transfection of 293 cells with full-length pAd5A24+O1 recombinant plasmid DNA. The recombinant Ad5 co-expressed P1 of both A24 and O1 in infected 293 cells and P1 of both serotypes was processed to produce VP0, VP3, and VP1. We further demonstrated the formation of capsid protein complexes by co-precipitation of VP0, VP3, and VP1 with monoclonal antibodies against viral capsid proteins. Swine inoculated with Ad5A24+O1 generated neutralizing antibodies against both A24 and O1. However, the overall neutralizing antibody response was considerably lower than that induced by a commercial FMD vaccine or a monovalent Ad5-A24 vaccine.

Adenoviruses, Human↗
PubMed1998

Mutational analysis of discontinuous epitopes of foot-and-mouth disease virus using an unprocessed capsid protomer precursor.

An unprocessed capsid precursor (P1) of foot-and-mouth disease virus (FMDV) has been expressed in mammalian cells to study discontinuous epitopes involved in viral neutralization. Amino acid replacements found in virus-escape mutants were engineered in the P1 precursor by site-directed mutagenesis of the plasmid. In all cases the replacements abolished recognition of unprocessed P1 by the relevant monoclonal antibodies (MAbs), paralleling the effects of the corresponding substitutions in neutralization of infectious FMDV. Five capsid surface residues within the same discontinuous antigenic area that were never found replaced in escape mutants were also engineered in P1. None of the substitutions affected antibody recognition, suggesting that these residues were not directly involved in the interaction with the antibodies tested. The results validate site-directed mutagenesis of constructs encoding capsid precursors as an approach to probe the structure of viral discontinuous epitopes not amenable to analysis with synthetic peptides.

Amino Acid Sequence↗
PubMed2001

Immunological analyses of human papillomavirus capsids.

Recombinant human papillomavirus (HPV) virus-like particles (VLPs) are promising vaccine candidates for controlling anogenital HPV disease. Questions remain, however, concerning the extent of capsid antigenic similarity between closely related virus genotypes. To investigate this issue, we produced VLPs and corresponding polyclonal immune sera from several anogenital HPV types, and examined these reagents in enzyme-linked immunosorbent assays (ELISAs) and in cross-neutralization studies. Despite varying degrees of L1 genetic sequence relatedness, VLPs of each type examined induced high-titer serum polyclonal antibody responses that were entirely genotype-specific. In an in vitro infectivity assay, only cognate VLP antisera were able to neutralize pseudovirions of HPV-16, HPV-18 and HPV-33, with two exceptions: HPV-31 and HPV-45 VLP post-immune sera demonstrated low levels of neutralizing activity against pseudovirions of HPV-33 and HPV-18, respectively. In other experiments, epitopes shared between closely related types were found to be less immunogenic than, and antigenically distinct from, primary type-specific B-cell determinants of the viral capsid. In addition, results from epitope blocking experiments suggested a close correlation between primary type-specific capsid antigenic sites and virion neutralization. These findings support the view that papillomavirus genotypes denote unique viral serotypes, and suggest that a successful vaccine for these viruses will likely require the inclusion of VLPs of each serotype for which protection is desired.

Absorption↗
PubMed1999

Sequence variation within the capsid protein of Australian isolates of feline calicivirus.

The capsid protein of Australian feline calicivirus (FCV) isolates is demonstrably different from the prototype strain F9. Five Australian isolates of FCV, dating from 1970 to 1989, were analysed by western blotting and immunoprecipitation. Varying reactivity to a panel of F9 specific monoclonal antibodies (MAbs) was observed. DNA sequencing of RT-PCR generated clones supported the observation of variation between capsid proteins. Predicted amino acid sequences varied by 11 to 17.5% across the whole capsid when compared to the published F9 sequence. Differences in amino acid sequence were most apparent in previously described hypervariable regions (C and E). Within hypervariable region E differences of 22 to 34% were observed compared to F9. The observed lack of reactivity to F9 MAbs correlated with amino acid changes within previously characterized binding sites within region E.

Amino Acid Sequence↗
PubMedDate not supplied

Processing of the minor capsid protein of the cauliflower mosaic virus requires a cysteine proteinase.

The major capsid protein of the cauliflower mosaic virus (CaMV) is processed in vivo. The viral aspartic proteinase that catalyses this maturation has been characterized previously and is coded by the CaMV gene V. This virus has a second capsid protein, a minor component, encoded by gene III. This protein, P3, is also processed at its C-terminus in vivo. To determine whether P3 is matured by the CaMV proteinase P5, we expressed, in Saccharomyces cerevisiae, P3, P5 and a fusion protein P7-P4, containing potential sites of cleavage. P5 was found to be involved in maturation of P7-P4 but did not cleave P3. The latter result was confirmed by experiments carried out with an in vitro translation system (the reticulocyte lysate) and with preparations of replication complexes purified from infected plants. Moreover, [N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leu cyl]-amido(4-guanido)butane, a specific inhibitor of cysteine proteinases, inhibited the maturation of P3, suggesting that the two CaMV capsid proteins are not processed by the same proteolytic event.

Capsid↗