John S. Colter

Synthesis of viral-specific polypeptides in Mengo virus-infected L cells: Evidence for asymmetric translation of the viral genome

Investigations of the synthesis of Mengo virus-specific polypeptides in L cells during the logarithmic phase of virus production have shown that, as is the case with other picornaviruses, the stable viral gene products are formed by posttranslational cleavage of larger primary proteins and that the pattern of cleavages by which they are produced is very similar to that proposed earlier for EMC virus (Butterworth et al., 1972b). However, analysis of the data suggests strongly that, on a molar basis, twice as much capsid as noncapsid viral protein is synthesized. This, in turn, suggests that translation of the viral genome is under some kind of control, the model most compatible with the data being one in which some viral protein(s) functions as termination factor(s).

Physico-chemical studies of three variants of Mengo virus and their constituent ribonućleates☆

Methods are described for the isolation in pure form of three variants of Mengo virus and their constituent ribonucleates. The resulting preparations have been subjected to a detailed physico-chemical investigation using the techniques of sedimentation velocity, diffusion at low centrifugal fields (method of Moller, 1964), optical rotatory dispersion and electron microscopy. It was established that all three variants are identical with respect to the measured hydro dynamic parameters (S20,w0 and D20,w0), and a similar conclusion was drawn for their RNA constituents. The particle weight of the virus, as estimated from the Svedberg equation, is 8.32 ± 0.7 × 106, while that of the RNA is 1.74 ± 0.2 × 106. In the electron microscope the three Mengo variants are indistinguishable from each other and appear as spherical particles with diameters of 26.2 to 27.2 mμ. The hydrated diameter calculated from the experimental diffusion coefficient is 29.3 mμ, which is consistent with a hydrated sphere having 0.23 g water per gram dry virus. The frictional ratio of the virus particle, estimated from S20,w0 and D20,w0, is 1.10, which also suggests that it is essentially spherical in shape. A comparison of the optical rotatory dispersion of the three variants of Mengo virus and their constituent ribonucleates, shows that the curves are essentially identical to one another, with similar cross-over points, positions of troughs and peaks, and amplitudes. This would suggest that the manner in which the proteins are arranged around the RNA chain in the virus is precise and similar in all three cases. By subtracting the contribution of the RNA from that of the virus, the optical rotatory dispersion curve of the protein in situ was obtained. The small amplitude of [m′]234 suggests that the protein in its native environment possesses negligible α-helical content.

Studies of the pH inactivation of three variants of Mengo encephalomyelitis virus

Abstract Three variants of Mengo virus (designated L, M, and S) are rapidly inactivated when incubated at slightly acid pHs. The inactivation reaction appears to have a rigorous requirement for chloride (or bromide) ions; no loss of infectivity occurs when the virions are incubated at slightly acid pHs in solutions of other salts, including sodium iodide and sodium fluoride. Inactivation is maximal at pH 6.2 and at a sodium chloride concentration of 0.1–0.2 M . The rate of inactivation is temperature dependent, being approximately doubled for each 10 ° rise in temperature over the range 7–37 °C. At 17 ° (but not at 37 °) the M variant may be shown to be inactivated more rapidly than are the other two. The inactivation of all three variants results in the dissociation of the viral capsids into protein subunits of uniform size having a sedimentation coefficient of the order of 15 S. If care is taken to block the activity of traces of nucleases that appear to be present in preparations of purified virus, it can be shown that this dissociation proceeds with the release of intact viral genomes from the virions.

Observations on the assay of infectious viral ribonucleic acid: Effects of DMSO and DEAE-dextran☆

Abstract DMSO and DEAE-D have been shown to stimulate the formation of infectious centers when added to media in which L cells and Mengo RNA are incubated. Maximum stimulation is obtained with DMSO when the compound is present at a concentration of 10–12.5% in the basic medium, 0.6 M sucrose/PBS, while DEAE-D is maximally effective at a concentration of 100 μg/ml in PBS. The latter is, by a wide margin, the most efficient medium for assaying the infectivity of Mengo RNA by the infectious center method. The stimulating effects of DMSO and DEAE-1) are not additive. The kinetics of infectious center formation in 0.6 M sucrose/PBS-10% DMSO and PBS containing 100 μg DEAE-D/ml have been studied, and the optimal time of incubation in the two media has been found to be 6 minutes and 2–3 minutes, respectively. Evidence is presented to support the premise that, in the suspended cell assay of viral RNA at least, only a very small percentage of the potentially infectious RNA molecules are detected.

Studies of Three Variants of Mengo Encephalomyelitis Virus. II. Inhibition of Interaction with L Cells by an Agar Inhibitor and by Protamine.

Abstract It is has been shown that the differences in the sizes of the plaques produced in L cell monolayers by three variants (S, M, and L) of Mengo encephalomyelitis virus, can be explained on the basis of differences in the sensitivities of the three variants to inhibition by an agar factor. In a suspended cell system, the interaction between L cells and S, M, and L Mengo is inhibited to the extent of 50% by agar inhibitor concentrations of 0.4, 3.3, and 35 μg/ml, respectively. Protamine, which, when added to agar overlay, reverses the inhibition of plaque formation imposed by the agar factor, is itself a potent inhibitor of cell-virus interaction in the suspended cell system. The S variant is most sensitive to inhibition by this polycation, while L and M Mengo exhibit sensitivities of the same order of magnitude. Data are presented which indicate that the agar factor blocks cell-virus interaction by directly immobilizing the virus particles, while protamine acts by blocking receptor sites on the cell surface.

A comparative study of BK and polyoma viruses

Abstract A comparative study of BK and polyma (Py) viruses has shown that the molecular anatomy of the two virions is strikingly similar. The capsids of both are composed of 72 capsomeres arranged in a T = 7d icosahedral lattice. Analysis of the DNAs of the two viruses suggest that the full-length DNA of BK virus may be slightly smaller than that of Py virus. The two virions contain the same number of structural polypeptides, but the molecular mass of BK-VP1 is about 4000 daltons less, and those of BK-VP2 and 3 about 4000 daltons more than those of the corresponding Py polypeptides. The observation that the major capsid polypeptide, BK-VP1, is smaller than Py-VP1, is compatible with the observed difference in the diameters of the two virions (BK = 405 ± 10A; Py = 430 ± 15A). The two viruses differ sharply in the relative efficiencies with which they hemagglutinate guinea pig and human erythrocytes. BK virus, unlike Py virus, replicates only in certain human or monkey cells; and of the cells examined in this study, human fetal kidney cells are the most satisfactory for the propagation of this agent.

Isolation and characterization of BK virus-transformed hamster cells.

Abstract BK virus (BKV) was used to transform baby hamster kidney (HK) and hamster embryo fibroblast (HE) cells in culture. Six clones of each of the BKV-transformed HK and HE cells were isolated and characterized with respect to a number of biological properties. None of the cloned lines was found to produce infectious BKV, and all 12 lines were shown to contain BKVT antigen, to have a lower serum dependency for growth and to grow to higher saturation densities than the corresponding untransformed cells, to have higher plating efficiencies than control cells, to have acquired the ability to produce colonies in soft agar, and to produce progressively growing tumors when injected subcutaneously into weanling hamsters. Differences were found to exist among the cell lines of each group with respect to these parameters, and, in general, transformed HK cells have a lower serum dependency, grow to higher saturation densities, and have higher plating efficiencies and a greater capacity to produce colonies in soft agar than do the transformed HE cells. Infection of secondary cultures of HE cells with various input multiplicities of BKV showed that transformation is a multiplicity-dependent phenomenon and can be achieved with an input multiplicity as low as 3 PFU/cell.

Studies of Three Variants of Mengo Encephalomyelitis Virus. III. Effect of Overlay and Polyanions on Plaque Size.

Abstract The effect of a number of different overlays on the sizes of plaques produced by three variants (S, M, and L) of Mengo encephalomyelitis virus has been examined. Agar has been shown to contain at least two fractions inhibiting S- and M-Mengo plaque development, only one of which can be extracted with saline. The respective merits of agarose (the neutral polysaccharide of agar) and methylcellulose as inhibitor-free overlays are compared. The addition of several anionic polymers (dextran sulfate, heparin, and chondroitin sulfate) to regular agar overlay has been shown to have little or no effect on the sizes of plaques produced in L cell monolayers by L- and S-Mengo. M-Mengo plaque size, however, is greatly affected: it may either be, decreased or increased, or both, by individual polyanions. Data are presented suggesting that molecular weight is a factor in determining the ability of a sulfated polysaccharide to enhance M-Mengo plaque size. Lower molecular weight preparations of sulfated hyaluronic acid enhance plaque size more efficiently than do higher molecular weight ones containing the same amount of sulfate. This, and other possible reasons for the varying effects of the polyanions on plaque size are discussed.

Structure of the Mengo virion: II. Physicochemical and electron microscopic analysis of degraded virus

Abstract Incubation of Mengo encephalomyelitis virus at slightly acidic pH in the presence of chloride or bromide ions results in the dissociation of the viral capsid into uniform protein subunits (13.4 S) with the release of the intact viral genome (Mak et al., 1970). Electron microscopic studies have shown that the 13.4 S subunit has a well defined, slightly ellipsoidal shape, with surface dimensions of 16.8 ± 0.3 × 14.2 ± 0.2 nm. It is concluded from these studies that the virus capsid is composed of 12 of these subunits, arranged in icosahedral symmetry. The 13.4 S subunits can be further dissociated into 4.7 S fragments by incubation in 2 M urea. The 4.7 S fragment has an approximately spherical shape, with a diameter of 6.8 ± 0.3 nm; and has the same polypeptide composition as does the 13.4 S subunit. These observations suggest that there are 5 such fragments in each 13.4 S subunit, for a total of 60 in the complete virus capsid. A model is proposed for the architecture of the Mengo virus particle, based on the physicochemical and electron microscopic data obtained for the intact virion and its dissociation products.

The structural proteins of Mengo virus variants

Abstract Sodium dodecyl sulfate (SDS)-acrylamide gel electrophoresis of the solubilized proteins from purified, radioactively labeled virions of three variants of Mengo virus (L-, M-, and S-Mengo) revealed that each contains three major structural polypeptide components. These components, designated I, II, and III, are present in the same relative molecular ratios in all three variants. M-Mengo also contains a minor structural component (IV) which was not detected in any preparations of S- or L-Mengo virions. Molecular weights of the viral polypeptides, estimated from their rates of migration in SDS-acrylamide gels relative to those of known marker proteins, were found to be 31,000, 28,000, and 20,000 for major components I, II, and III, respectively, and 10,000 for minor component IV of M-Mengo. Immunological studies employing Mengo antisera in double gel-diffusion plates revealed that the three major components (but not the minor component of M-Mengo) retained their immunological reactivity and formed precipitin bands characteristic of immunological identity. These data suggest that the major structural polypeptides are immunologically identical and that all three components contribute to the antigenic composition of the virion.