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PubMed2005

AAV hybrid serotypes: improved vectors for gene delivery.

In recent years, significant efforts have been made on studying and engineering adeno-associated virus (AAV) capsid, in order to increase efficiency in targeting specific cell types that are non-permissive to wild type (wt) viruses and to improve efficacy in infecting only the cell type of interest. With our previous knowledge of the viral properties of the naturally occurring serotypes and the elucidation of their capsid structures, we can now generate capsid mutants, or hybrid serotypes, by various methods and strategies. In this review, we summarize the studies performed on AAV retargeting, and categorize the available hybrid serotypes to date, based on the type of modification: 1) transcapsidation, 2) adsorption of bi-specific antibody to capsid surface, 3) mosaic capsid, and 4) chimeric capsid. Not only these hybrid serotypes could achieve high efficiency of gene delivery to a specific targeted cell type, which can be better-tailored for a particular clinical application, but also serve as a tool for studying AAV biology such as receptor binding, trafficking and genome delivery into the nucleus.

Animals↗
PubMed1996

Two-dimensional gel analysis of [35S]methionine labelled and phosphorylated proteins present in virions and light particles of herpes simplex virus type 1, and detection of potentially new structural proteins.

Cells infected with herpes simplex virus (HSV) synthesize both infectious viruses and non-infectious light particles (L-particles). The latter contain the envelope and tegument components of the virions, but lack virus capsid and DNA. Electrophoresis in SDS-polyacrylamide gels (SDS-PAGE) has been used extensively for analysis of structural proteins in virions and L-particles. Two-dimensional (2-D) gel electrophoresis, however has a markedly higher resolution, and in the present work we have used this technique to study both [35S]methionine labelled and phosphorylated structural proteins in virions and L-particles. Proteins were assigned to the tegument or the envelope by the analysis of L-particles. Localization of structural proteins was also determined by stepwise solubilization in the presence of the neutral detergent NP-40 and NaCl, and by isolation of capsids from nuclei of infected cells. Different steps in posttranslational modification can be detected by 2-D gel electrophoresis such that a single polypeptide may appear as several spots. This was most clearly observed for some of the HSV-encoded glycoproteins which were shown to exist in multiple forms in the virion. Some polypeptides apparently not identified previously were either capsid associated, or localized in the tegument or envelope. The degrees of phosphorylation in L-particles and virions are almost identical for some proteins, but markedly different for others. Thus, glycoprotein E of HSV-1 is for the first time shown to be phosphorylated, and most heavily so in virions. The IE VMW)110 protein represents a group of proteins which are more phosphorylated in L-particles than in virions. Attempts are made to correlate the proteins detected by 2-D analysis with those previously separated by SDS-PAGE.

Animals↗
PubMed1990

Transport and assembly of gag proteins into Moloney murine leukemia virus.

We have studied the process of Moloney murine leukemia virus (M-MuLV) assembly by characterization of core (gag) protein mutants and analysis of wild-type (wt) gag proteins produced by cells in the presence of the ionophore monensin. Our genetic studies involved examination of linker insertion mutants of a Gag-beta-galactosidase (Gag-beta-gal) fusion protein, GBG2051, which is incorporated into virus particles when expressed in the presence of wt viral proteins. Analysis indicated that the amino-terminal two-thirds of the gag matrix domain is essential for targeting of proteins to the plasma membrane; mutant proteins localized to the cytoplasm or were trapped on intracellular membranes. Mutations through most of the coding region of the gag capsid domain generated proteins which were released from cells in membrane vesicles but not in virions. In contrast, linker insertions into p12gag or carboxy-terminal portions of the matrix or capsid coding regions did not affect assembly of fusion proteins into virus particles. Monensin, which blocks vesicular transport, inhibited gag protein intracellular transport and release from cells. Our results suggest that a significant proportion of M-MuLV myristylated gag proteins travel via vesicles to the cell surface. Specific matrix protein polypeptide regions and myristic acid modification are both necessary for appropriate gag protein transport, while capsid protein interactions appear to mediate the final phase of virion formation.

Acylation↗
PubMed2004

Fluorescently labeled adenovirus with pIX-EGFP for vector detection.

Adenoviruses are extensively studied in terms of their use as gene therapy vectors and pathogenesis. These vectors have been targeted on both transcriptional and transductional levels to achieve cell-specific gene delivery. Current detection strategies, including reporter gene expression, viral component detection, and vector labeling with fluorophores, have been applied to analyze adenoviral vectors; however, these methods are inadequate for assessing transductional targeting. As an alternative to conventional vector detection techniques, we developed a specific genetic labeling system whereby an adenoviral vector incorporates a fusion between capsid protein IX and EGFP. DNA packaging and thermostability were marginally hampered by the modification while DNA replication, cytopathic effect, and CAR-dependent binding were not affected. The fluorescent label was associated with the virus capsid and conferred a fluorescent property useful in detecting adenoviral particles in flow cytometry, tracking, and tissue sections. We believe our genetic adenovirus labeling system has important implications for vector development, detecting adenovirus vectors in targeting schemes, and studying adenovirus biology. In addition, this technique has potential utility for dynamic monitoring of adenovirus replication and spread.

Adenovirus E1 Proteins↗
PubMed2007

Solubilization and stabilization of bacteriophage MS2 in organic solvents.

Several techniques were examined for the solubilization of bacteriophage MS2 in organic solvents. Direct extraction of the MS2 from an aqueous phase into isooctane containing 2 mM AOT, a proven approach for the organic solubilization of many proteins, was not successful. However, predried samples of MS2 were solubilized through the direct addition of organic solvents containing 500 mM AOT. As an alternative procedure, reverse micelles containing aqueous solutions of MS2 were prepared in isooctane using AOT, dehydrated through solvent evaporation and azeotropic drying, and resolubilized in a solvent of choice. The structure and microenvironment of organic-solubilized MS2 were investigated by UV absorbance, the fluorescence emission of an attached solvatochromatic dye, tryptophan fluorescence, and atomic force microscopy, all of which contributed evidence for a fully assembled capsid in the organic solvent. The solubilized MS2 was derivatized with stearic acid in chloroform, illustrating that bioconjugation reactions can be performed on organic-solubilized capsids using reagents that are completely insoluble in water. Furthermore, the organic-solubilized phage remained infectious after heating at 90 degrees C for 20 min, whereas phage in aqueous buffer or dried with nitrogen were nonviable following the heat treatment protocol. The extended range of available chemical modifications and the enhanced thermal stability of the organic-solubilized capsids bodes well for the formulation of storage-stable vaccines predicated on reactions in or exposure to organic media.

Biotechnology↗
PubMed1994

Protein glycosylation and myristylation in Chlorella virus PBCV-1 and its antigenic variants.

Chlorella virus PBCV-1 particles contain three glycoproteins, the major capsid protein Vp54 and two minor proteins Vp280 and Vp260. The major capsid protein is myristylated as well as glycosylated. Both modifications are in the carboxyl-terminal portion of the protein. A gene which is modified in a PBCV-1 antiserum-resistant mutant was cloned and sequenced. This gene has an open reading frame of 3099 bases and encodes one of the two large virion glycoproteins (Vp260). Vp260 contains 13 tandem repeats of 61 to 65 amino acids. The mutation deletes the equivalent of four of the amino acid repeat sequences and duplicates one of these sequences.

Amino Acid Sequence↗
PubMedDate not supplied

Hepatitis B virus preC mutants in human hepatocellular carcinoma tissues.

A comparative analysis of preC sequences of hepatitis B virus (HBV) in human hepatoma (hepatocellular carcinoma; HCC) tissues and non-tumoral liver samples from HCC patients was performed. Ten out of 17 HCC tissue samples exhibited an amino acid substitution at the level of the distal cysteine residue of the HBV preC region, while generation of a TAG translational stop codon was observed in 4 of these samples. Interestingly, substitution of the distal cysteine residue was not observed in non-tumoral liver (available from 8 of the 17 patients), thus suggesting either that a selection among different HBV variants occurs in HCC cells, or that modifications to the conformation and stability of the HBV capsid protein may play a role in the process of selection and escape of transformed liver cells.

Base Sequence↗
PubMed1978

Head maturation pathway of bacteriophages T4 and T2. IV. In vitro transformation of T4 head-related particles produced by mutants in gene 17 to capsid-like structures.

T4 mutants in gene 17 accumulate particles which contain the main head protein in the cleaved form (gp23*) arranged in an unexpanded lattice (empty small particles), together with other expanded capsids (empty large particles). The isolated empty small particles can be transformed in vitro, by lowering the ionic strength, to capsid-like structures. This structural transformaton is not coupled to chemical modification of the structural proteins of the empty small particles. In contrast to unexpanded particles that are easily dissociated, the transformed structures are as resistant to dissociation as other T-even head-related particles with expanded lattice. Furthermore, the transformed particles are able to bind in vitro hoc and soc proteins, rendering capsids indistinguishable from the normal T4 capsids both morphologically and by their stability against denaturing agents. Our results indicate that the in vitro transformation of the empty small particles might mimic important and characteristic aspects of the in vivo maturation of T4 heads, thus suggesting a possible role of the "cleaved but unexpanded" particle in the maturation pathway of the T4 shell.

Capsid↗
PubMed1998

Characterization of a recombinant human calicivirus capsid protein expressed in mammalian cells.

The capsid protein of the Hawaii strain of human calicivirus was expressed in the transient MVA/bacteriophage T7 polymerase hybrid expression system in order to examine its processing in mammalian cells. Selected amino acid modifications (an insertion, deletion, and substitution) at the predicted amino terminus of the capsid protein as well as the presence or absence of the ORF3 gene were examined for their effect on capsid expression. The protein was expressed efficiently in cell lines derived from three different species, with most of the expressed protein remaining localized within the cells. There was no evidence for N-linked glycosylation or myristylation of the 57 kDa capsid protein. Hawaii virus-like particles (HV VLPs), efficiently produced in the baculovirus expression system, were not observed in this expression system under the conditions in this study.

Animals↗
PubMed1998

Development of an antigen presentation system based on plum pox potyvirus.

The development of an antigen presentation system based on the plum pox potyvirus (PPV) is here described. The amino-terminal part of PPV capsid protein was chosen as the site for expression of foreign antigenic peptides. Modifications in this site were engineered to avoid the capability of natural transmission by aphids of this PPV vector. As a first practical attempt, different forms of an antigenic peptide (single and tandem repetition) from the VP2 capsid protein of canine parvovirus (CPV) were expressed. Both chimeras are able to infect Nicotiana clevelandii plants with similar characteristics to wild-type virus and remain genetically stable after several plant passages. The antigenicity of purified chimeric virions was demonstrated, proving the suitability of this system for diagnostic purposes. Moreover, mice and rabbits immunized with chimeric virions developed CPV-specific antibodies, which showed neutralizing activity.

Amino Acid Sequence↗
PubMed1993

Cooperation of structural proteins during late events in the life cycle of polyomavirus.

The polyomavirus minor late capsid antigen, VP2, is myristylated on its N-terminal glycine, this modification being required for efficient infection of mouse cells. To study further the functions of this antigen, as well as those of the other minor late antigen, VP3, recombinant baculoviruses carrying genes for VP1, VP2, and VP3 have been constructed and the corresponding proteins have been synthesized in insect cells. A monoclonal antibody recognizing VP1, alpha-PyVP1-A, and two monoclonal antibodies against the common region of VP2 and VP3, alpha-PyVP2/3-A and alpha-PyVP2/3-B, have been generated. Reactions of antibodies with antigens were characterized by indirect immunofluorescence, immunoprecipitation, and immunoblot analysis. Immunofluorescent staining of mouse cells infected with polyomavirus showed all antigens to be localized in nuclei. When the late polyomavirus proteins were expressed separately in insect cells, however, only VP1 was efficiently transported into the nucleus; VP2 was localized discretely around the outside of the nucleus, and VP3 exhibited a diffused staining pattern in the cytoplasm. Coexpression of VP2, or VP3, with VP1 restored nuclear localization. Immunoprecipitation of infected mouse cells with either anti-VP1 or anti-VP2/3 antibodies precipitated complexes containing all three species, consistent with the notion that VP1 is necessary for efficient transport of VP2 and VP3 into the nucleus. Purified empty capsid-like particles, formed in nuclei of insect cells coinfected with all three baculoviruses, contained VP2 and VP3 proteins in amounts comparable to those found in empty capsids purified from mouse cells infected with wild-type polyomavirus. Two-dimensional gel analysis of VP1 species revealed that coexpression with VP2 affects posttranslational modification of VP1.

Amino Acid Sequence↗
PubMed1981

In vivo NH2-terminal acetylation of Sindbis virus proteins.

The in vivo incorporation of exogenous radioactive acetate into two proteins of Sindbis virus, the capsid protein and PE2, is described. Under appropriate labeling conditions, 40-50% of the label in the capsid protein is found in an N-acetyl group which constitutes the NH2-terminal modification of this blocked protein. The incorporated radiolabeled acetate was useful in the purification and analysis of peptides derived from the NH2-terminus of the capsid protein, and from these peptides the NH2-terminal sequence of the protein was determined to be N-acetyl-Met-Asx-, with the asx group most likely asparagine. The analysis of a peptide derived from the NH2-terminus of PE2 and containing 45% of the acetate-derived label in this protein leads us to conclude that at least a significant fraction of PE2 is also blocked by N-acetylation.

Acetates↗
PubMed1987

Surface structure and RNA-protein interactions of foot-and-mouth disease virus.

The surface structure of foot-and-mouth disease virus (FMDV) and the interaction of the individual capsid proteins with the virus RNA have been examined using modification reagents. By measuring the extent of modification of the lysine residues of intact and disrupted virus particles and the 12S protein subunit with Bolton & Hunter reagent it was found that 54% of the residues of VP1, 15% of the residues of VP2 and 37% of the residues of VP3, equivalent to five, two and four lysine residues respectively, are on the surface of the intact virus particle. Polypeptide VP4 was not modified in intact virus particles, indicating that it has no lysine residues on the surface of the virus. Modification with sodium metabisulphite, which causes a specific transamination reaction between cytidylic acid residues in ssRNA and closely associated basic amino acids, cross-linked all four structural proteins to the virus RNA. Both fragments of VP1, produced by treatment of the virus particle with trypsin, are also cross-linked to the RNA. These observations have been combined with the evidence that the immunogenic activity of VP1 may be contained in two discontinuous sites, at amino acids 141 to 160 and 200 to 213, in proposing a model for the arrangement of this polypeptide in the virus particle.

Antigens, Viral↗
PubMed1998

Amino acids in the capsid protein of tomato yellow leaf curl virus that are crucial for systemic infection, particle formation, and insect transmission.

A functional capsid protein (CP) is essential for host plant infection and insect transmission in monopartite geminiviruses. We studied two defective genomic DNAs of tomato yellow leaf curl virus (TYLCV), Sic and SicRcv. Sic, cloned from a field-infected tomato, was not infectious, whereas SicRcv, which spontaneously originated from Sic, was infectious but not whitefly transmissible. A single amino acid change in the CP was found to be responsible for restoring infectivity. When the amino acid sequences of the CPs of Sic and SicRcv were compared with that of a closely related wild-type virus (TYLCV-Sar), differences were found in the following positions: 129 (P in Sic and SicRcv, Q in Sar), 134 (Q in Sic and Sar, H in SicRcv) and 152 (E in Sic and SicRcv, D in Sar). We constructed TYLCV-Sar variants containing the eight possible amino acid combinations in those three positions and tested them for infectivity and transmissibility. QQD, QQE, QHD, and QHE had a wild-type phenotype, whereas PHD and PHE were infectious but nontransmissible. PQD and PQE mutants were not infectious; however, they replicated and accumulated CP, but not virions, in Nicotiana benthamiana leaf discs. The Q129P replacement is a nonconservative change, which may drastically alter the secondary structure of the CP and affect its ability to form the capsid. The additional Q134H change, however, appeared to compensate for the structural modification. Sequence comparisons among whitefly-transmitted geminiviruses in terms of the CP region studied showed that combinations other than QQD are present in several cases, but never with a P129.

Amino Acid Sequence↗
PubMed2004

Analysis of proteins stained by Alexa dyes.

Alexa dye staining of proteins is used for the fluorescence microscopy of single particles that are sometimes multimolecular protein complexes. To characterize the staining, post-staining determination must be made of which protein(s) in a complex have been Alexa-stained. The present communication describes the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for performing this determination. The Alexa-stained proteins are observed directly in gels by illumination with an ultraviolet transilluminator. The test multimolecular particle is bacteriophage T7. The protein capsid of T7 is a multimolecular complex that has both external and internal proteins. SDS-PAGE of Alexa-stained bacteriophage T7 produces fluorescent capsid proteins each of which usually comigrates with an unstained protein. However, one Alexa-induced modification of protein migration was observed by SDS-PAGE. Mass spectrometry shows that the protein with modified migration is the major protein of the outer shell of the T7 capsid. The procedures used are generally applicable. The distribution of Alexa staining among T7 capsid proteins depends on the size of the dye molecule used. The larger the dye molecule is, the greater the preference for external proteins.

Acetates↗
PubMed1986

Self-assembly of purified polyomavirus capsid protein VP1.

The polyomavirus major capsid protein VP1, purified after expression of the recombinant gene in E. coli, was isolated as oligomers resembling the dissociated capsomeres derived from viral capsids. Image analysis of low-dose electron micrographs demonstrates that these VP1 oligomers are exclusively pentamers. The purified VP1 pentamers associated to form capsid-like assemblies and polymorphic aggregates at high ionic strength. The capsid-like assemblies were stabilized at low ionic strength by the addition of calcium. Self-assembly of the unmodified, recombinant DNA-generated VP1 implies that the posttranslational charge modifications of VP1 and the minor virion protein components, VP2 and VP3, are not essential for capsid formation. The nonequivalently related subunits of the penta- and hexavalent capsomeres therefore must spontaneously switch their bonding specificity during assembly.

Capsid↗
PubMed2004

Interior surface modification of bacteriophage MS2.

An efficient strategy for the interior surface functionalization of MS2 viral capsids is reported, featuring a new hetero-Diels-Alder bioconjugation reaction. After virus isolation, the RNA genome was removed from the spherical particles by exposure to pH 11.8 conditions for a period of 4 h. Following this, 180 tyrosine residues on the interior surface of each "empty" capsid shell were modified by using a site-selective diazonium-coupling reaction. To attach additional functionality, the azo conjugate was reduced with Na2S2O4 to afford an ortho-amino tyrosine derivative. Oxidation of this moiety with NaIO4 produced an o-iminoquinone on the protein surface, which was found to undergo an efficient hetero-Diels-Alder reaction with N-(4-aminophenyl)acrylamide. This four-step procedure can be carried out in under 4 h, reaches high levels of conversion, and yields the desired conjugates in >60% overall yield.

Bioreactors↗
PubMed1983

Respiratory syncytial virus mRNA coding assignments.

The polypeptide coding assignments for six of the respiratory syncytial virus-specific mRNAs were determined by translation of the individual mRNAs in vitro. The coding assignments of the RNAs are as follows. RNA band 1 is complex and can be separated into at least two components on the basis of electrophoretic mobility (molecular weights [MWs] approximately equal to 0.21 X 10(6) and 0.31 X 10(6), respectively) that code for three polypeptides of 9.5, 11, and 14 kilodaltons (K). RNA 2 (MW, 0.39 X 10(6)) codes for a 34K polypeptide; RNA 3 (MW, 0.40 X 10(6)) codes for a 26K polypeptide; RNA 4 (MW, 0.47 X 10(6)) codes for a 42K polypeptide; and RNA 5 (MW, 0.74 X 10(6)) codes for a 59K polypeptide. By limited-digest peptide mapping, the 34, 26, and 42K polypeptides synthesized in vitro appeared to be unique. Additionally, peptide mapping showed that the 34, 26, and 42K polypeptides synthesized in vitro were indistinguishable from their counterparts synthesized in infected cells. Thus, the 34, 26, and 42K polypeptides coded for by mRNAs 2, 3, and 4, respectively, were identified as the respiratory syncytial virus phosphoprotein (34K), matrix protein (26K), and nucleocapsid protein (42K), respectively. RNA 5 was shown to code for a 59K polypeptide. The 59K polypeptide synthesized in vitro did not comigrate with any polypeptide specific to infected cells, suggesting that it is a candidate for co- or post-translational modification.

Capsid↗