Viral Shell Research Articles

Maturation of the head of bacteriophage T4: I. DNA packaging events

Pulse-chase experiments in wild-type and mutant phage-infected cells provide evidence that the following particles called prohead I, II and III are successive precursors to the mature heads. The prohead I particles contain predominantly the precursor protein P23 and possibly P22 (mol. wt 31,000) and IP III (mol. wt 24,000) and have an s value of about 400 S. Concomitantly with the cleavage of most of P23 (mol. wt 55,000) to P23∗ (mol. wt 45,000), they are rapidly converted into prohead II particles which sediment with about 350 S. The prohead II particles contain, in addition to P23∗, the major constituents of the viral shella—a core consisting of proteins P22 and IP III. In cell lysates, prohead I and prohead II particles contain no DNA in a DNase-resistant form and are not bound to the replicative DNA. We cannot, however, positively rule out the possibility that these particles may have contained some DNA while in the cells. The prohead II particles are in turn converted into particles which sediment with about 550 S after DNase treatment (prohead III). During this conversion about 50% of normal DNA complement becomes packaged in a DNase-resistant form, and roughly 50% of the core proteins P22 and IP III are cleaved. In lysates the prohead III particles are attached to the replicative DNA. The prohead III particle appears to be the immediate precursor of the full mature head (1100 S). Cleavage of protein P22 to small polypeptides and conversion of IP III IP III∗ are completed at this time. No precursor proteins are found in the full heads. Studies with various mutant phage showed that the prohead II to III conversion is blocked by mutations in genes 16 and 17 and that the conversion of the prohead III particles to the mature heads is blocked by mutations in gene 49. Cleavage of the head proteins, however, occurs normally in these mutant-infected cells. We conclude that the cleavage of the major component of the viral shell, P23, into P23∗ precedes the DNA packaging event, whereas cleavage of the core proteins P22 and IP III appears to be intimately linked to the DNA packaging event. Models relating the cleavage processes to DNA encapsulation are discussed.

The structure of lcosahedral cytoplasmic deoxyriboviruses: II. An alternative model

A detailed comparative ultrastructural study has been made on several icosahedral cytoplasmic deoxyriboviruses (ICDV) of insects and one of vertebrates. The available evidence now seems to indicate that ICDV particles contain only a single structural unit membrane, associated with the viral nucleoid. ICDV shells are therefore redefined entirely in terms of icosahedral lattices, in contrast to an earlier model. A partial explanation is offered for the variability in appearance of ICDV shell surfaces. Previous evidence for the structural relatedness of ICDV's of different sizes and from different hosts is corroborated by the following additional observations: triangular arrays of capsomers (trisymmetrons) are present in mosquito iridescent virus (MIV) shells; mosquito and Corethrella iridescent virus (CoIV) cores are identical in morphology and are uncoated in an apparently intact form under similar conditions; sectioned MIV and frog virus (FV3) particles reveal definite structural homology; the morphogenesis of all ICDV's studied appears basically identical. Proposals concerning the nomenclature and classification of this group of viruses are briefly discussed.

Comparison of the structures of blue-green algal viruses LPP-1M and LPP-2 and bacteriophage T7

Quantitative electron microscopy and polyacrylamide gel electrophoresis have been applied to characterize and compare the structures of three viruses of prokaryotes. The molecular weights of blue-green algal viruses LPP-1M and LPP-2 and phage T7 are (in millions) 53.4 ± 0.2, 50.9 ± 0.4, and 44.7 ± 0.2, respectively. These values were derived from the median dry masses of populations of virus particles, measured by an electron microscopic technique. For all three viruses one protein, of around 40,000 daltons, comprises over 50% of the viral protein mass. Approximately 310 copies of this molecule are used in the construction of LPP-1M, 290 copies in LPP-2 and 460 copies in T7. The blue-green algal viruses differ from the bacteriophage in utilizing a second major structural protein in building the viral shell. Most other viral structural proteins are represented by no more than about a half dozen or a dozen copies. There appear to be no proteins represented only once in any of the viruses examined.

Stepwise degradation of cauliflower mosaic virus by pronase

Treatment of 32P-labeled cauliflower mosaic virus (CaMV) with 0.02% pronase for 2–5 min at 37° degraded the viral protein shell, producing particles of about 100 S and finally nucleic acid free from protein. Incubation of 35S-labeled CaMV under the above conditions resulted in the degradation of virus particles to about 100 S and finally protein remaining in the top of the gradient. The slower-sedimenting particles show the regular loss of protein moiety in a stepwise fashion with the time of incubation in pronase, suggesting that CaMV particles consist of some layers of protein shells. Tobacco mosaic virus (TMV), however, is not degraded by the same treatment with pronase as CaMV. Pronase digestion is rather specific for some viruses. The structure of CaMV is discussed in comparison with that of polyoma virus and simian virus 40.

Structures of turnip crinkle and tomato bushy stunt viruses: III. The chemical subunits: Molecular weights and number of molecules per particle

The chemical compositions of turnip crinkle virus and of tomato bushy stunt virus have beeen characterized by electrophoreais on acrylamide gels in the presence of sodium dodecyl sulphate. The structures of the two viruses are identical and consist of 180 protein subunits of molecular weight 38,000, and 12 protein subunits of molecular weight 28,000. The major protein subunit is identified with the structural subunit of the virus shell, and the minor component with the other surface material described in the earlier papers in this series.

The structure of viruses of the papilloma-polyoma type: V. Tubular variants built of pentamers

Tumour virus infection is generally accompanied by the production of hollow, tubular particles which appear to be polymorphic forms of the protein shell of the virus. The two classes of tube recognized previously—wide and narrow—have been studied by electron microscopy combined with optical diffraction and filtering. In the filtered image, which provides a picture of one side of a tube at a time, the wide tubes are seen to be composed of hexamers arranged on a hexagonal plane lattice rolled up into a cylindrical tube—a type of variant structure expected on the theory of virus assembly described by Caspar & Klug (1962). The narrow tubes have a quite novel kind of structure, which, although not anticipated, can also be understood in terms of the theory of virus assembly. The narrow tubes are seen in the filtered images to be built of pentamers arranged in a particular kind of pentagonal tessellation which arises as a result of the bonding of pentamers across 2-fold axes of the surface lattice; this is the same kind of bonding as is found in the hexamer tubes and in the normal virus shell. Two closely related categories of pentamer tube with the same local packing but differing in their helical parameters have been found. The arrangement of pentamers in the “zero-start” tubes can be thought of geometrically as consisting of puckered rings of six pentamers stacked helically above each other, whereas in the “one-start” tubes the rings become the successive turns of a continuous helix containing approximately 6.6 pentamers per turn.

Electrophoretic and buoyant density variants of southern bean mosaic virus

Five electrophoretic variants of southern bean mosaic virus have been found. Each was characterized by its mobility as a function of pH in two buffer systems. In monovalent buffers of 0.10 ionic strength, their isoelectric points ranged between pH 3.95 and 6.03. When sodium chloride was a constituent of the buffer system, the isoelectric points of each of the variants was lowered, suggesting that chloride ions were binding to the viral protein shell. Calculations using these data show that, depending on the variant, chloride ions bind between 7 and 38% of the total available amphoteric charges. A relationship exists among the variants since, for each, approximately the square of the fractional number of chloride ions bound is proportional to the slope of the mobility-pH curve. The “valency” of each variant is similar. Although each variant is homogeneous electrophoretically and in its buoyant density in CsCl, two of the variants display three components in other cesium salts. When the multiple components are recombined and recentrifuged in CsCl density gradient, only a single component is present. This indicates that multiple components do not represent degraded virus. Fourteen “species” have been identified among the five electrophoretic variants by their characteristic buoyant densities.

Effects of 2-thiouracil on the formation of double-stranded plant viral ribonucleic acid

2-Thiouracil treatment leads to a marked suppression of incorporation of 32P into duplex viral RNA in TMV-infected tobacco leaves and TYMV-infected chinese cabbage leaves. The same treatment has little effect on the incorporation of 32P into host RNA. 35S-labelled 2-thiouracil is incorporated into the normal RNA of both tobacco and chinese cabbage leaves and into TMV viral duplex RNA. Under similar conditions no incorporation could be detected into TYMV duplex RNA. In leaves infected with TYMV, 2-thiouracil treatment did not lead to the disappearance of duplex viral RNA present at the time treatments were begun. TYMV infection, or 2-thiouracil treatment of either healthy or TYMV-infected leaves had no marked effect on the proportion of leaf ribosomes in polyribosome form. Empty TYMV protein shells produced during 2-thiouracil treatment were found to have an altered ultraviolet absorption spectrum and changed stability to heating. We suggest that 2-thiouracil has two major effects on TYMV synthesis: (i) it prevents RNA synthesis on the duplex RNA, leading to a marked reduction in viral RNA production, (ii) it leads to the production of abnormal viral protein shells through interference with the coding mechanism either by being incorporated into a small amount of viral RNA used for message or into host transfer RNA or both.

A theory of the quaternary structure of dehydrogenases, dehydrogenating complexes, and other proteins

On the basis of a comparison of the molecular weights and other properties of dehydrogenases it has been suggested that they are all composed of subunits of molecular weights between 17,000 and 21,000. Only certain given numbers of subunits appear to be able to form enzymes. This fact is used to propose quaternary structures for the molecules. A physical model for the interaction between subunits is proposed according to which there is at most one specific point of interaction between neighbouring subunits. All other forces are unspecific and symmetrically distributed around the subunit. Structures are proposed for the α-unit of glutamic dehydrogenase and the pyruvate and α-ketoglutarate decarboxylation complexes consistent with the proposed model and with the assumption that they have icosahedral symmetry. The dissociations, molecular weights, and compositions of these entities can be explained on this basis. It is shown that if virus shells having cubic symmetry are built according to the principles suggested here they will always have icosahedral or pseudo-icosahedral symmetry, and be based on the P = 1 or P = 3 icosadeltahedra, in aggreement with experimental findings to date. Assuming that the active site of a dehydrogenase spans two subunits, and using a slightly modified version of the model already suggested, the number of active sites per enzyme molecule can be predicted. The predictions agree fairly well with experimental evidence to date. Some aspects of in vitro complementation of mutant forms of dehydrogenases are discussed.

INCOMPLETE SIMIAN PAPOVAVIRUS SV40. FORMATION OF NON-INFECTIOUS VIRAL ANTIGEN IN THE PRESENCE OF FLUOROURACIL.

A study was made of the effects of 5-fluorouracil (FU) and 5-fluorodeoxyuridine (FUDR) on the replication of the simian papovavirus SV40 in cercopithecus monkey kidney cells and on the production of virus antigen by these cells. Both drugs markedly suppressed the production of new infectious virus by SV40-infected cells. Synthesis of viral protein was also markedly suppressed by FUDR, but not by FU. In the presence of FU, infected cells produced large amounts of viral protein which were detected by the fluorescent antibody technique. The antigen was not distributed in a particulate fashion as in untreated cells. Diffuse virus antigen was observed in the nuclei of FU-treated cells, resembling the distribution of antigen near the end of the eclipse period in untreated, infected cultures. This stage of antigen production presumably preceded viral assembly. Virus particles with or without cores were rarely seen with the electron microscope in infected FU-treated cells, although large numbers of SV40 particles were readily visualized in untreated, infected cells. It appears that at least one antigenic protein of this papovavirus is synthesized abundantly in FU-treated cells, but is not assembled into virus shells in the presence of the inhibitor.