Determining the concentration and the absorptivity factor at 260 nm in sodium dodecyl sulfate of the adenovirus reference material using analytical ultracentrifugation

A concentration of 6.4  ±  0.2 (at 1 SD) × 10¹¹ virus particles (vp) per milliliter was determined for the adenovirus reference material (ARM) by averaging analytical ultracentrifugation concentration data obtained with refractometric detection with the completely independent concentration reported by the Adenoviral Reference Material Working Group (ARMWG). Using this concentration, the ARM absorptivity factor (in sodium dodecyl sulfate [SDS] at 260 nm) of 1.2 ± 0.1 (at 1 SD) × 10¹² vp/ml per OD_260nm per centimeter was obtained.     

Temperature-sensitive mutant of adenovirus type 2 blocked in virion assembly: accumulation of light intermediate particles.

A temperature-sensitive mutant of human adenovirus type 2, ts112, was isolated and characterized, ts112 was blocked in a late function required for virus maturation. At restrictive temperature, it accumulated light precursor particles that were able to mature into infectious virions upon temperature shift-down. Use of a mild extraction procedure and a reversible fixation by a cleavable diimido ester permitted the isolation and analysis of these labile intermediates in the adenovirus assembly. These accumulated particles had a sedimentation coefficient of about 600S and a buoyant density of 1.315 g/cm3 in CsCl. They contained a DNA fragment of 7--11S and two nonvirion proteins having molecular weights of 50,000 (50K) and 39.000 (39K), respectively. They resembled in composition and morphology the light intermediate particles found in wild-type adenovirus 2, which were identified as precursors of heavy intermediates, preceding the young virions. The ts112 lesion was apparently located at the exit of either the 50K and/or 39K proteins and at the entry of viral DNA.     

Characterization of heterologous protein-protein interactions using analytical ultracentrifugation.

Methods for quantitative characterization of heterologous protein-protein interactions by means of analytical ultracentrifugation (AUC) include sedimentation equilibrium, tracer sedimentation equilibrium, sedimentation velocity, and analytical band sedimentation. Fundamental principles governing the behavior of macromolecules in a centrifugal field are summarized, and the application of these principles to the interpretation of data obtained from each type of experiment is reviewed. Instrumentation and software for the acquisition and analysis of data obtained from different types of AUC experiments are described.     

Distinctive properties of the hepatitis B virus envelope proteins.

Using recombinant adenoviral vectors, we expressed and characterized the large, middle, and major envelope proteins of hepatitis B virus (HBV). Cells infected with the recombinant adenovirus which contained the large envelope gene (HS1.HP) expressed predominantly large envelope and small but detectable quantities of middle (4%) and major (6%) envelope proteins in the cell lysate. No HBV envelope proteins were detected in the culture medium from HS1.HP-infected cells. Cells infected with recombinant adenovirus which contained the middle envelope gene (HS2.HP) expressed and secreted the middle and major envelope proteins in a molar ratio of 3:1. Cells infected with the recombinant adenovirus which contained the major envelope gene (HS.HP) expressed and secreted major envelope proteins. The HBV envelope proteins secreted by cells infected with either HS2.HP or HS.HP were assembled in 22-nm particles, as shown by velocity sedimentation rate determination, buoyant densities, and electron microscopy. Cells coinfected with a recombinant adenovirus which contained the large envelope gene and with either HS2.HP or HS.HP expressed similar quantities of the large, middle, and major envelope proteins in the cell lysates. Secretion of the major and middle envelope proteins was inhibited more than 95% by the presence of the large envelope proteins. These results suggest that differential biosynthesis, transport, and processing of the envelope proteins occur during HBV infection, allowing efficient assembly and secretion of virions and hepatitis B surface antigen particles.     

Adenovirus early region 4 is required for efficient virus particle assembly.

H2dl807, a defective deletion mutant of human adenovirus type 2 lacking parts of early regions 3 and 4 and all of late region 5, was severely defective for virus particle assembly on HeLa cells, producing about 1% of the normal yield of particles. On Vero cells, H2dl807 produced only 5% as many particles as wild type, while on W162 cells, a Vero cell derivative which supports the growth of early region 4 mutants, H2dl807 produced nearly 40% of the wild-type level of particles. Two other defective deletion mutants, H2dl802 and H5dl1021, which lack parts of early region 3 and which are incapable of making fiber, the product of late region 5, were wild type for virus assembly. These data suggest that the cause of the assembly defect of H2dl807 is the lack of a diffusible early region 4 product. H2dl807-infected Vero cells accumulated nearly wild-type amounts of viral late proteins in the nucleus and cytoplasm. Thus, the defect of the mutant in assembly on Vero cells is not due to a general lack of late proteins. Finally, the fact that H2dl802 and H5dl1021 make wild-type amounts of virus particles suggests that fiber is not essential for adenovirus assembly.     

Characterization of alfalfa-mosaic-virus protein polymerization in the presence of nucleic acid

The polymerization of alfalfa mosaic virus (AMV) protein in the presence of homologous nucleic acids and a number of other natural and synthetic nucleic acids was studied. The conditions for optimal assembly were found to be pH 6.0 and low ionic strength (I = 0.1 M) at room temperature, irrespective of the type of nucleic acid. The resulting nucleoprotein particles exhibited the same structural characteristics as the virus. This information emerged from optical diffraction and computer filtering of electron micrographs from the reconstituted particles. Irrespective of the type of nucleic acid present the polymerization of the protein resulting in a nucleoprotein particle is a cooperative process. Evidence for this was obtained by nitrocellulose filter binding assay, sodium dodecylsulphate/polyacrylamide gel electrophoresis, sedimentation velocity and electron microscopy of the reaction mixtures. The rates and efficiencies of reconstitution were of the same order of magnitude for a number of ribonucleic acids. Sedimentation data derived from AMV protein and AMV RNA mixtures suggested the existence of a specific nucleation product in the first stage of assembly. The results are discussed in terms of a tentative model of the assembly, in which at least two different steps (nucleation and elongation) can be distinguished, each characterized by an association constant.     

Genetic Analysis of Adenovirus Type 2 II. Preliminary Phenotypic Characterization of Temperature-Sensitive Mutants

The properties of temperature-sensitive mutants of adenovirus type 2 representing 12 complementation groups were studied. All mutants were normal with respect to adsorption as measured by viral inclusion formation and viral DNA synthesis as shown by velocity sedimentation in alkaline sucrose gradients. One mutant, however, formed viral inclusions of altered morphology at the nonpermissive temperature. The synthesis of the major capsid proteins was examined by immunodiffusion. On this basis, the complementation groups could be arranged as follows: (i) one group was negative for all three proteins; (ii) three groups failed to synthesize penton bases; (iii) eight groups were positive for hexons, pentons, and fibers. The assembly of virus particles at 39 C was examined by equilibrium sedimentation in CsCl; three groups were found defective, whereas two of the penton-negative groups were positive for virion production. Tests of the thermolability of virions at 50 C revealed eight groups labile whereas the remainder were insensitive to heat inactivation. None of five mutants inoculated in newborn rats induced tumors, although three of them were capable of in vitro transformation.     

Physical analysis of virus particles using electrospray differential mobility analysis

This review critically examines an emerging tool to measure viral clearance from biomanufacturing streams, monitor assembly of viruses and virus-like particles, rapidly identify viruses from biological milieu, assay virus neutralization, and prepare bionanoconjugates for bacterial detection. Electrospray differential mobility analysis (ES-DMA) is a tool of choice to simultaneously determine viral size and concentration because it provides full multimodal size distributions with subnanometer precision from individual capsid proteins to intact viral particles. The review contrasts ES-DMA to similar tools and highlights expected growth areas including at-line process sensing as a process analytical technology (PAT), bioseparating as a distinct unit operation, monitoring viral reactions, and interrogating virus-host protein interactions.     

Cytoskeletal proteins associated with intracytoplasmic human adenovirus at an early stage of infection

Taking advantage of the sedimentation properties of adenovirus particles, adenovirus-infected baby hamster kidney (BHK21) cells were reversibly fixed with cleavable diimidoester dimethyl 3,3'-dithiobispropionimidate (DTBP) at early times of infection (30 min). Cytoskeletal proteins associated with/or in close vicinity to virions were isolated as a complex cross-linked with carrier virus. Four major cellular proteins were thus found to co-purify with adenovirus particles. They were characterized by their coordinates on 2D maps and immunological reactivity. Two of them were identified as alpha-tubulin (58 kD), and vimentin subunits (56 kD). The two other species 68 and 66 kD might correspond to stress proteins. Affinity blotting on gels showed that both alpha-tubulin and vimentin were capable of binding with intact and penton-less adenovirions. Adenovirus components involved in the binding seemed to be mainly core proteins V and VII, and to a lesser extent, hexon. Analysis of neighbor relationships among proteins of the adenovirus-cytoskeletal protein cross-linked complex suggested that some capsid alterations occurred upon/or after entry of the virus into the cell, and that these structural modifications preferentially concerned the vertex components penton and IIIa, and the core protein V.     

In vitro assembly of human immunodeficiency virus type 1 Gag protein.

Retroviral Gag protein is sufficient to produce Gag virus-like particles when expressed in higher eukaryotic cells. Here we describe the in vitro assembly reaction of human immunodeficiency virus Gag protein, which consists of two sequential steps showing the optimal conditions for each reaction. Following expression and purification, Gag protein lacking only the C-terminal p6 domain was present as a monomer (50 kDa) by velocity sedimentation analysis. Initial assembly of the Gag protein to 60 S intermediates occurred by dialysis at 4 degrees C in low salt at neutral to alkaline pH. However, higher order of assembly required incubation at 37 degrees C and was facilitated by the addition of Mg(2+). Prolonged incubation under these conditions produced complete assembly (600 S), equivalent to Gag virus-like particles obtained from Gag-expressing cells. Neither form disassembled by treatment with nonionic detergent, suggesting that correct assembly might occur in vitro. Electron microscopic observation confirmed that the 600 S assembly products were spherical particles similar to authentic immature human immunodeficiency virus particles. The latter assembly stage but not the former was accelerated by the addition of RNA although not inhibited by RNaseA treatment. These results suggest that Gag protein alone assembles in vitro, but that additional RNA facilitates the assembly reaction.     

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuumNot only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.     

Size distribution analysis of recombinant adenovirus using disc centrifugation

Recombinant adenovirus is one of the primary vectors for human gene therapy. However, the aggregation of unstable virus has been a recurring problem during the production of purified virus for human therapeutics. To facilitate the development of a robust manufacturing process for recombinant adenovirus vectors, a convenient and reliable size distribution analytical assay is necessary and we demonstrate here that disc centrifuge sedimentation is applicable to this purpose. Using the disc centrifuge system and the line start method, the assay can provide particle size distribution of adenovirus samples within 30 min. The assay can detect virus concentrations down to 0.01% (w/v) or 3 × 10¹¹ particles per ml. The apparent hydrodynamic diameter of recombinant adenovirus was determined to be about 0.063 μm. Furthermore, the disc centrifuge analysis was able to detect adenovirus dimers, trimers, and tetramers, consistent with a rigid sphere approximation for adenovirus, as well as a large aggregate of 0.35 μm. The appearance of viral aggregates is confirmed by increased light scattering based on A₃₂₀/A₂₆₀ ratios. The technique could be useful for monitoring the kinetics of aggregation for adenovirus and other DNA and RNA viruses in the submicron region. Therefore, this novel assay provides a critical tool for purification development of viral vectors for meeting therapeutic and research needs. Received 18 September 1997/ Accepted in revised form 15 May 1998     

A model for sedimentation in inhomogeneous media. II. Compressibility of aqueous and organic solvents

The effects of solvent compressibility on the sedimentation behavior of macromolecules as observed in analytical ultracentrifugation are examined. Expressions for the density and pressure distributions in the solution column are derived and combined with the finite element solution of the Lamm equation in inhomogeneous media to predict the macromolecular concentration distributions under different conditions. Independently, analytical expressions are derived for the sedimentation of non-diffusing particles in the limit of low compressibility. Both models are quantitatively consistent and predict solvent compressibility to result in a reduction of the sedimentation rate along the solution column and a continuous accumulation of solutes in the plateau region. For both organic and aqueous solvents, the calculated deviations from the sedimentation in incompressible media can be very large and substantially above the measurement error. Assuming conventional configurations used for sedimentation velocity experiments in analytical ultracentrifugation, neglect of the compressibility of water leads to systematic errors underestimating sedimentation coefficients by approximately 1% at a rotor speeds of 45000 rpm, but increasing to 2-5% with increasing rotor speeds and decreasing macromolecular size. The proposed finite element solution of the Lamm equation can be used to take solvent compressibility quantitatively into account in direct boundary models for discrete species, sedimentation coefficient distributions or molar mass distributions. Using the analytical expressions for the sedimentation of non-diffusing particles, the ls-g*(s) distribution of apparent sedimentation coefficients is extended to the analysis of sedimentation in compressible solvents. The consideration of solvent compressibility is highly relevant not only when using organic solvents, but also in aqueous solvents when precise sedimentation coefficients are needed, for example, for hydrodynamic modeling.     

Form-determining functions in Sindbis virus nucleocapsids: nucleosomelike organization of the nucleocapsid.

Purified intact Sindbis virus nucleocapsids were treated at different pH values or with various concentrations of divalent cations, cation chelators, salt, or formamide. The resulting structures were examined by velocity sedimentation, electron microscopy, and protein-protein cross-linking. Changes in each of the test conditions led to alterations in the sedimentation profile of treated nucleocapsids. Appropriate concentrations of formamide or divalent cations generated beaded strandlike structures similar in morphology to those generated from adenovirus cores and nucleosomes. The capsid protein and RNA remained associated with each other at NaCl concentrations less than or equal to 1 M or after treatment of the structures with alkaline pH up to and including pH 10.7. Protein and RNA were dissociated by salt concentrations of greater than 1 M, suggesting that the arginine-rich, amino-terminal portion of the capsid protein is responsible for binding the RNA. Protein-protein cross-linking also indicated that the capsid proteins remained associated in small aggregates under some of the conditions that caused dissociation of the nucleocapsid and suggested the presence of more than one type of protein-protein interaction in the nucleocapsids. Collectively, these data suggest that, like histones and adenovirus core proteins, the Sindbis virus capsid protein serves to package segments of the genome into nucleoprotein beads which are capable of interacting with each other to form the nucleocapsid structure.     

Properties of subviral particles of hepatitis B virus

In the sera of patients infected with hepatitis B virus (HBV), in addition to infectious particles, there is an excess (typically 1,000- to 100,000-fold) of empty subviral particles (SVP) composed solely of HBV envelope proteins in the form of relatively smaller spheres and filaments of variable length. Hepatitis delta virus (HDV) assembly also uses the envelope proteins of HBV to produce an infectious particle. Rate-zonal sedimentation was used to study the particles released from liver cell lines that produced SVP only, HDV plus SVP, and HBV plus SVP. The SVP made in the absence of HBV or HDV were further examined by electron microscopy. They bound efficiently to heparin columns, consistent with an ability to bind cell surface glycosaminoglycans. However, unlike soluble forms of HBV envelope protein that were potent inhibitors, the SVP did not inhibit the ability of HBV and HDV to infect primary human hepatocytes.     

A New Procedure for Quantitative Measurements of Virus Particles in Crude Preparations

A new method is described for the quantitative measurement of virus concentration in crude preparations by density gradient centrifugation and electron microscopy. The centrifugation is carried out in a specially designed centrifuge tube which permits separation and sedimentation of virus particles at different levels according to their sedimentation velocity. The gradient of a mixture of heavy and normal water (D(2)O-H(2)O) is designed to sediment the virus particles with constant velocity so that the optimal time of centrifugation can easily be calculated. The virus particles are collected on carbon-coated nickel grids floating on mercury at the bottom of the centrifuge tube and are counted by means of electron microscopy. The efficiency of the method is demonstrated with a crude plant extract of tobacco mosaic virus.     

A Multilaboratory Comparison of Calibration Accuracy and the Performance of External References in Analytical Ultracentrifugation

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.     

Fluorescence detection for the XLI analytical ultracentrifuge

Analytical ultracentrifugation (AUC) provides first-principle hydrodynamic and thermodynamic information concerning the size, shape and interactions of macromolecules. The fundamental measurement needed in AUC is the macromolecular concentration as a function of radial position and time. Currently, the Beckman Coulter XLI analytical ultracentrifuge may be equipped with absorbance and refractive detectors, which provide complementary concentration determinations. For detecting trace quantities of materials, fluorescence detection offers unique advantages over either absorbance or interference detection. A prototype fluorescence detector for the XLI analytical ultracentrifuge has been developed and its characteristics determined. An Ar(+) laser provides a continuous 488-nm excitation beam. Radial resolution is achieved by scanning the focused beam along a radial axis. Detection of the fluorescence signal uses a co-axial, front-face optical configuration to reduce inaccuracies in the concentration caused by inner filter effects. A high-speed A/D data acquisition system allows the fluorescence intensity to be monitored continuously and at a sufficiently high angular resolution so that at any radial position the intensities from all of the samples may be acquired at each revolution. The fluorescence detector is capable of detecting concentrations as low as 300 pM for fluorescein-like labels. The radial resolution of the fluorescence detector is comparable to that of the absorbance system. Both sedimentation velocity and sedimentation equilibrium measurements may be made with the fluorescence detector. Results are presented comparing data acquired using the fluorescence with those acquired using the absorbance detector.     

Quantitation of adenovirus DNA and virus particles with the PicoGreen fluorescent Dye.

A microplate assay for the rapid quantitation of adenovirus DNA has been developed using the fluorescent dye PicoGreen, which selectively binds double-stranded DNA. The method was first applied to extracted adenoviral DNA and then extended to samples of intact, purified adenovirus after lysis of the viral capsid with the ionic detergent SDS. Utilizing the stoichiometric relationship between adenovirus DNA and intact particles, a physical particle count of intact virus is then derived for the sample. This PicoGreen-based assay has excellent reproducibility, linearity, and sensitivity. In its present form, this assay has a limit of quantitation of 10.3 ng/ml viral DNA, predicted to correspond to 2.6 x 10(8) virus particles/ml. This procedure was compared to a widely utilized spectroscopic method, in which samples are lysed with SDS and absorbance is read at 260 nm, and found to be 10- to 20-fold more sensitive. The dye binding assay also uses considerably less sample volume (<20%) than that needed for the spectroscopic method. Particle count values generated by the PicoGreen procedure are consistently lower (typically 1.5- to 2-fold) than this spectroscopic method. The applications and limitations of this method in the analysis of adenovirus samples are discussed.