Hans J. Zweerink

Polypeptide components of virions, top component and cores of reovirus type 3

Abstract The capsid protein moiety of virions of the Dearing strain of reovirus type 3 consists of seven species of polypeptides as determined by polyacrylamide gel electrophoresis. The molecular weights of these polypeptides have been estimated by comparing their rate of migration in SDS-containing gels with that of marker proteins of known molecular weights. The number of molecules of each polypeptide in the virion has also been determined. There are 113 and 80 molecules, respectively, of polypeptides λ 1 (M.W. 155,000) and λ 2 (M.W. 140,000); 23 and 550 molecules, respectively, of polypeptides μ 1 (M.W. 80,000) and μ 2 (M.W. 72,000); and 31, 202, and 890 molecules, respectively, of polypeptides σ 1 (M.W. 42,000), σ 2 (M.W. 38,000), and σ 3 (M.W. 34,000). In addition virions also contain about 500 molecules of a very small polypeptide, the molecular weight of which is 5000–10,000. The polypeptides in reovirus top component (particles lacking RNA) are identical with those of virions. Treatment with chymotrypsin or trypsin converts virions to cores which lack the outer shell of capsomeres. Cores contain capsid polypeptides λ 1 , λ 2 , and σ 2 . Cores can similarly be prepared from top component. Top component cores contain the same polypeptides as virion cores, but the enzyme treatment necessary for the conversion cleaves polypeptide λ 1 . The molecular weights of the three size classes of capsid polypeptides are such that they are probably coded by monocistronic messenger RNA molecules transcribed from the L, M, and S segments of reovirus genome RNA. The capsid polypeptides thus use the information in 2 of the 3 L segments, 2 (or possibly only 1) of the 3 M segments, and 3 of the 4 S segments.

Studies on the intracellular synthesis of reovirus-specified proteins.

Abstract All reovirus capsid polypeptides are formed in measurable amounts in mouse L fibroblasts. Five of them (λ1, λ2, μ1, σ2 and σ3) are formed more or less abundantly; one (σ1) is formed in very small amounts. Capsid polypeptide μ2, the major constituent of the virion, is not a primary gene product, but is derived from polypeptide μ1 by removal of 10% of its mass. The synthesis of all capsid polypeptides appears to start synchronously. During much of the infection cycle (from 3 to 9 hours) only minor, though significant, changes in their relative rates of synthesis occur. No evidence was obtained for the synthesis of any virus-specified noncapsid polypeptides. The extent to which the ten individual species of reovirus mRNA are transcribed within the cell was also measured. The pattern of transcription differs markedly from the uncontrolled pattern exhibited by cores in vitro . Some mRNA species are clearly translated frequently, others very infrequently. Both transcription and translation of reovirus message is thus controlled. The incorporation of capsid polypeptides formed at various times during the infection cycle into virions was determined. Core polypeptide molecules which are formed early in the cycle tend to be incorporated to a greater extent than such molecules which are formed later on, while the reverse is true for polypeptide molecules comprising the outer shell. The kinetics of synthesis of capsid polypeptides was studied as a function of the multiplicity of infection and the temperature of incubation and the effect of reovirus infection on host protein synthesis was determined; host protein synthesis does not decrease until late during the infection cycle.

Fate of parental reovirus in infected cell.

Abstract Parental reovirus is not completely uncoated after infection to yield free genome RNA. Instead it is converted into a subviral particle which contains besides 10 segments of double-stranded RNA and A-rich RNA, all core polypeptides namely λ1, λ2, μ1, and σ2 and a polypeptide with a molecular weight of 65,000 which is derived from the outer capsomere protein μ2 by cleavage. Isolated partially uncoated particles catalyze the synthesis of virus specific messenger RNA. Late during the replication cycle partially uncoated particles are converted into particles with a slightly higher density in CsCl than the virus by the addition of newly synthesized polypeptide σ3.

Isolation and characterization of two types of bluetongue virus particles.

Abstract Two distinct types of particles were isolated during purification of bluetongue virus (BTV) from infected mouse L fibroblasts. Both were infectious and contained a double-stranded RNA genome composed of ten segments ranging in molecular weight from 2.7 × 106 to 0.28 × 106 daltons. BTV L (light), the particles with the greater specific infectivity, had a buoyant density of 1.36 g/cm3 in cesium chloride, a sedimentation coefficient of 550 S, and a diameter of 69.2 nm. Their surface is poorly defined. They are composed of four major capsid proteins (MW 110,000, 100,000, 58,000, and 32,000) and three minor capsid proteins (MW 155,000, 72,000, and 38,000). BTV D (dense), the particles with the lower specific infectivity, had a buoyant density of 1.38 g/cm3 in cesium chloride, a sedimentation coefficient of 470 S, and a diameter of 63.2 nm. Thirty-two capsomers were clearly visible on their surface. They contained two fewer capsid proteins (MW 110,000 and 58,000) than the other type of particles. In contrast to BTV L particles, they possessed RNA-dependent RNA polymerase activity.

Essential and nonessential noncapsid reovirus proteins

Abstract Nine species of reovirus-specified polypeptides have been detected in mouse L fibroblasts infected at 31°. Eight of these are primary gene products (designated λ1, λ2, μ0, μ1, σ1, σ2, σ2A, and σ3), while one (μ2) is derived from μ1 by cleavage which reduces the molecular weight by about 10%. Six of the primary gene products are capsid polypeptides, while two, namely μ0 and σ2A, are not; these are classed as essential noncapsid polypeptides. All these nine polypeptides are also formed at 37°, in addition to three others, polypeptides X, Y, and Z, which are cleavage products of either μ1 or μ2. Since they are not formed when virus multiplies at 31°, they are classed as nonessential noncapsid polypeptides. They are probably formed as a result of a cleavage mechanism triggered by cytopathic damage resulting from viral infection. The molecular weights of all reovirus-specified polypeptides are listed, and tentative assignments are made as to the identity of the polypeptide coded by each reovirus genome RNA segment.

Studies on the structure of reovirus cores: Selective removal of polypeptide λ2

Abstract Exposure of reovirus cores, or reovirus top component cores, to pH 11.8 at 4° removed nearly all of polypeptide λ2. This effect occurred only within a narrow pH range: exposure to pH 11.4 resulted in less than 50% removal of λ2, and exposure to pH 12.4 completely destroyed the cores. Exposure to pH 11.8 at 4° also removed the 12 spikes that are present on the surface of cores, and reduced transcriptase activity by more than 90%. Iodination studies demonstrated that λ1 was the more accessible of the two major polypeptides which remained in cores after alkali treatment.

An ultrastructural study of virions and cores of reovirus type 3

Abstract The results of an ultrastructural examination of virions and cores of reovirus type 3 are reported. The diameter of virions is 764 ± 43 A, that of cores 521 ± 31 A. The capsomers of both the outer an the inner shell (the core) are so arranged that 20 appear to be located peripherally. Those comprising the outer shell are 90 A in diameter and 10 A apart, those of the inner shell are 40 A in diameter. Both seem to be uniform in size and shape. Rare head-on views of both confirm these size estimates and also suggest that both are hollow truncated pyramids. Spatial interrelationships between neighboring capsomers on neither outer nor inner shells could be discerned clearly enough to permit assignation of any particular symmetry pattern, not even when virions were treated briefly with chymotrypsin, which enhances surface detail. The total number of capsomers in each shell follows directly from the fact that 20 are located on the periphery: the maximum number is 127. This is substantially in excess of the number (92) required for a model proposed by others which, in addition, requires 18 peripherally located capsomers. Another model, which postulates 180 capsomers located around 92 holes, is also not compatible with our results. Cores possess 12 projections or spikes located on 5-fold vertices, demonstrating that at least these reovirion components are arranged according to icosahedral symmetry principles. Their diameter is 100 A, and they project about half way to the outer surface of the virion. They appear to be hollow at their distal end. It is conceivable that they are the organelles through which transcripts of reovirus genome RNA are released. Dissolution of cores with sodium dodecyl sulfate releases viral RNA as a tightly associated mass of strands which appear to be coiled either randomly or concentrically. These results are discussed with reference to the structure of the reovirion capsid shells, and the possible structure and function of the core spikes.

Characterization of transcriptase and replicase particles isolated from reovirus-infected cells.

Abstract Double-stranded RNA-dependent single-stranded RNA polymerase (transcriptase) and single-stranded RNA-dependent double-stranded RNA polymerase (replicase) were isolated from reovirus-infected cells. Both transcriptase and replicase were associated with subviral particles that could be separated by velocity sedimentation in glycerol density gradients and by equilibrium sedimentation in CsCl density gradients. The transcriptase particles (ϱ = 1.42 g/cm 3 ) which sedimented at approximately 220 S contained double-stranded RNA and the virus-specific polypeptides λ1, λ2, μ0, and σ2. The majority of the replicase particles (ϱ = 1.35 g/cm 3 ) sedimented at approximately 180 S. These particles were composed of single-stranded, partially double-stranded and double-stranded RNA and the virus-specific polypeptides λ1, λ2, μ2, σ1, σ2, and σ3. Both particles were approximately 40 nm in diameter.

Cells infected with a cell-associated subacute sclerosing panencephalitis virus do not express M protein

Abstract Using immunoprecipitation techniques, we have analyzed measles-specific antibodies in the cerebrospinal fluids (CSF) as well as in sera from patients with subacute sclerosing panencephalitis (SSPE). We confirm previous reports that SSPE sera contain a relative lack of M protein-specific antibodies, and report that CSF also have antibodies specific for all measles polypeptides except the M protein. We have also found that Vero cells infected with one of the SSPE viruses used in this study, the cell-associated IP-3 strain, completely lack expression of detectable M protein. Thus, the lack of M protein-specific antibody in the CSF of SSPE patients may be the result of a lack of expression of this polypeptide in the cells of the central nervous system.

Reovirus morphogenesis: characterization of subviral particles in infected cells.

Abstract Subviral particles which sedimented at 400 and 600 S were identified in reovirus-infected cells. The 600 S particles (immature virions) contained 10 segments of double-stranded RNA, the major capsid polypeptides λ1, λ2, μ2, σ2, σ3, and a small amount of the nonstructural polypeptide μ0. They lacked A-rich oligonucleotides. Kinetic experiments demonstrated that the acquisition of these oligonucleotides by virions is a very late event in viral morphogenesis. The 400 S particles, which contained 10 segments of double-stranded RNA, consisted of two kinds of particles: (1) short-lived intermediates which contained the core polypeptides λ1, λ2 (in reduced amounts), σ2, and polypeptide μ0; and (2) particles that accumulated in infected cells and that contained the core polypeptides only. When observed in the electron microscope, the latter particles ranged in size from 25 to 54 nm. They lacked the spikes present on the surface of viral cores, but possessed surface detail similar to that on viral cores.