Wolfgang K. Joklik

Hybridization and sedimentation studies on “early” and “late” vaccinia messenger RNA

Abstract The vaccinia virus multiplication cycle can be divided into two portions: the early portion, which precedes viral DNA replication and lasts for about one-and-a-half hours; and the late portion which follows until maturation of progeny is complete. Vaccinia messenger RNA transcribed during these two periods, that is, early and late messenger RNA, has been studied by means of hybridization and density gradient sedimentation. The following results were obtained: 1. (1) Early vaccinia messenger RNA is distinctly smaller than late messenger RNA (10 to 12 s against 16 to 20 s). Late messenger RNA contains nucleotide sequences not present in early messenger RNA; however, all the sequences transcribed early are also transcribed late. If late messenger RNA is transcribed from the entire viral genome, one-half to two-thirds is transcribed early. 2. (2) Messenger RNA transcribed in the presence of cytosine arabinoside, which completely inhibits viral DNA replication, appears to be identical to early messenger RNA. The cytosine arabinoside RNA transcribed in HeLa and L cells contains the same base sequences. 3. (3) The pattern of transcription of early and late vaccinia messenger RNA in HeLa and L cells is quite different. In HeLa cells the total amount of late messenger RNA synthesized greatly exceeds the amount of early; in L cells the position is reversed. 4. (4) The effect of varying the multiplicity of infection on the pattern of early and late messenger RNA transcription has been studied. The rate of early mRNA synthesis is proportional to the multiplicity over a certain limited range. Once the maximum rate of mRNA transcription has been reached, which occurs the earlier the higher the multiplicity, the rate of transcription of messenger RNA falls to a steady low level equal to no more than 5% of the maximum value; this rate of transcription then persists until at least 19 hours after infection. 5. (5) The large messenger RNA molecules transcribed late contain nucleotide sequences also present in small early messenger RNA molecules. Some small messenger RNA molecules are also transcribed late: these contain at least some sequences characteristic of late messenger RNA. 6. (6) At five hours after infection the messenger RNA molecules present in polyribosomes contain all the sequences characteristic of early messenger RNA molecules. By eight hours after infection messenger RNA in polyribosomes is very significantly depleted with respect to sequences characteristic of early messenger RNA. 7. (7) The stability of early and late vaccinia messenger RNA in the presence of actinomycin D has been studied in HeLa and L cells. Early vaccinia messenger RNA is very stable in HeLa cells. Those late messenger RNA molecules which contain sequences characteristic of late messenger RNA are mostly unstable (half-life less than one hour); but those containing sequences characteristic of early messenger RNA are as stable as early messenger RNA itself. In L cells early and late vaccinia messenger RNA are equally stable (half-life two to three hours). mRNA is considerably more stable in extracts of HeLa cells than of L cells; and early mRNA is somewhat more stable in extracts of HeLa cells than is late mRNA. These results are discussed in relation to the pattern of macromolecular biosynthesis during the vaccinia virus multiplication cycle.

Isolation and preliminary genetic and biochemical characterization of temperature-sensitive mutants of reovirus

Abstract A set of temperature-sensitive mutants of reovirus type 3 has been isolated. The mutagens used were nitrous acid, nitrosoguanidine, and proflavine. Thirty-five mutants have been tested for their ability to recombine following pairwise infection. Five recombination groups have been detected. Twenty-nine mutants fall into one group, four into another, and three mutants recombine efficiently with every other mutant tested. Recombination frequencies are either zero or in excess of 2%. This result supports chemical evidence that the genome of reovirus consists of a number of segments. The mutants have been tested for their ability to induce the formation of virus-specific RNA at the nonpermissive temperature. The classification of mutants according to this criterion agrees exactly with that deduced from recombination tests.

Studies on reovirus RNA: I. Characterization of reovirus genome RNA

Abstract Chemical treatment does not release the genome of reovirus in intact form. Irrespective of the procedure used, the same mixture of fragments is released with sedimentation coefficients of about 14, 12 and 10.5 s. On electrophoresis in polyacrylamide gels these size classes can be further resolved, the two former into two components each, the last into three. These fragments are double-stranded as judged by the following criteria: they exhibit a sharp melting profile with a Tm dependent on the ionic concentration; they are resistant to ribonuclease, provided that the concentrations of both monovalent and divalent ions, as well as that of the enzyme, are suitably adjusted; their sedimentation behavior is independent of the ionic concentration over a wide range; and their base composition displays equality of A and U, as well as of G and C. In addition to this mixture of double-stranded fragments there is released from virions an RNA of small size (2 to 3 s) which amounts to about 20% of the mass of the viral genome. The base composition of this RNA is 82% A, 13% U, 3% G and 3% C. This A-rich RNA is synthesized in step with the viral genome RNA. RNA molecules corresponding to intact viral genomes were not detected in infected cells at times when the viral genome was replicating. Irrespective of the method of breaking open the cells, or of the cell strain used, the same mixture of RNA fragments was obtained as was released from virions. These results suggest that the reovirus genome consists of a number of segments of double-stranded RNA; further, it would appear that the reovirus genome does not replicate as one intact molecule, but rather in the form of these same segments. The molecular weights of the L, M and S segments have been estimated by reference to three sets of data in the literature; the most likely values are 2.3 ± 0.2 × 106, 1.3 ± 0.2 × 106 and 8 ± 2 × 105. L, M and S segments are released from virions in the ratio of n : n : 1.5n. Owing to uncertainty concerning the size of the reovirus genome, the exact number of segments of which it is composed cannot be specified. The most likely value of n is 2, although a value of 3 or even 4 is not ruled out. The function of the A-rich material is not known. It is conceivable that it serves to link the double-stranded segments, giving rise to a structure which is so labile that it does not withstand any but the most gentle manipulation. However, the precise manner in which linking is accomplished, and the role, if any, of the Arich material in this process must await further work.

Studies on the genesis of polyribosomes: I. Origin and significance of the subribosomal particles

In addition to polyribosomes and 74 s ribosomal monomers, there exist in the cytoplasm of HeLa cells two classes of subribosomal particles, namely, 60 s particles which contain 28 s RNA, and 40 s particles which contain 16 s RNA. Evidence is presented that their presence in cytoplasmic cell fractions is not a consequence of dissociation of 74 s ribosomes due to a deficiency of magnesium ions after cells are disrupted. 60 s and 40 s particles are always present in strictly equivalent numbers and together account for about 10% of total ribosomal material in the cytoplasm. In contrast, the number of free 74 s ribosomal monomers may vary widely (although it is strictly constant when aliquots of the same cell suspension are examined); on occasions, cytoplasmic fractions containing more 60 s particles than 74 s ribosomes have been observed. However, the total number of 74 s ribosomes (“polymers” plus monomers) within the cell tends to be fairly constant. The smaller the number of free 74 s ribosomes, the larger is the number released from polyribosomes by treatment with puromycin. Such treatment, however, does not change the number of 60 s and 40 s particles, nor do 74 s ribosomes released from polyribosomes dissociate detectably to 60 s and 40 s particles within 60 minutes. Labeling with [ 14 C]uridine has shown that 60 s and 40 s particles are made in the nucleus and enter the cytoplasm as individual entities, the latter preceding the former. Combination of 60 s particles with 40 s particles to form 74 s ribosomes proceeds mainly, and most probably exclusively, in polyribosomes, that is, while the 40 s particle is attached to a messenger RNA molecule. The relative specific radioactivity of 74 s ribosomes in polyribosome form often exceeds that of free 74 s ribosomal monomers for at least 3 hr after synthesis of the constituent subribosomal particles. The population of free 74 s ribosomal monomers exchanges only slowly with the population of 74 s ribosomes in polyribosome form, the rate depending on the metabolic state of the cell. Those 74 s ribosomes which are liberated from the messenger RNA molecule by puromycin recombine with it preferentially on removal of puromycin. This is interpreted as a topographical effect; other explanations are possible. The implications of these findings are discussed.

The replication and coating of vaccinia DNA

The replication and subsequent fate of vaccinia DNA in HeLa cells has been studied by examining the cytoplasmic fraction. Vaccinia DNA replication begins one to one and a half hours after infection, reaches a maximum rate at two to two and a half hours and then decreases sharply. More than 90% of viral DNA is synthesized by four and a half hours. The maximum rate of viral DNA replication is several times that of DNA replication in uninfected cells. The replication of viral DNA requires synthesis of protein, presumably a polymerase, even after viral DNA has been uncoated. Replicating viral, as well as parental, DNA is associated with very large structures or aggregates, which may be identical with the viral inclusions (factories) identified by microscopic, electronmicroscopic and autoradiographic techniques. Aggregates contain no detectable amounts of viral messenger RNA. They dissociate or break at concentrations of Mg 2+ of less than 1·5 m M . During the early stages of the infection cycle practically all viral DNA is associated with the aggregates; after three to four hours progressively increasing amounts of viral DNA become dissociated from them. Shortly afterwards, DNA becomes coated with protein in what appears to be a stepwise fashion. This process, which appears to proceed within the aggregates, has been analysed by the use of puromycin. The first stage(s) result in viral DNA becoming larger or heavier without losing its susceptibility to DNase. The next stage(s) result in the formation of particles resistant to DNase, but still smaller or lighter than virions. The proportion of viral DNA in these immature forms, which are very heterogeneous in size and density, remains constant throughout the maturation period. The final stage of the maturation process becomes detectable at five to six hours; very little of the protein(s) necessary for this step is synthesized before five hours. Only about one-third of the total DNA replicated during the early part of the infection cycle is actually incorporated into virions. Some implications of these findings are discussed.

The intracellular uncoating of poxvirus DNA: I. The fate of radioactively-labeled rabbitpox virus

1. The uncoating of rabbitpox virus DNA within the cell has been studied, using highly purified virus preparations labeled with [2- 14 C]thymidine, L -[ 14 C]leuoine and 32 P. The uncoating is recognized as the sensitization of poxvirus DNA to DNase. Once uncoated, viral DNA is stable in the sense that it is not broken down to acid-soluble material. 2. Two stages of the uncoating process may be recognized: the first stage, commencing immediately on infection and affecting almost all virus particles, results in the breakdown of practically all viral phospholipid and the disintegration of part of the protein coat of the virus. The second stage begins after a lag and results in the sensitization of viral DNA to DNase. Not all parficles which are degraded during the first stage are fully uncoated. The extent of uncoating differs for different cell lines. 3. The lag before uncoating proper begins is shortened if the multiplicity of infection is increased. However, the extent of uncoating is independent of multiplicity down to a multiplicity of less than 0·35 viral particles per cell. 4. Pre-infection abolishes the lag; in cells pre-exposed to unlabeled virus, subsequently infecting labeled virus is immediately uncoated. 5. Pre-infection with any of three poxvirus strains abolishes the lag for rabbitpox virus. 6. The results are interpreted as follows : on penetrating into a cell the poxvirus particle is rapidly partially degraded by enzymes pre-existing in the cell. For the DNA to be fully uncoated, however, a viral protein has to fulfill some function leading to the establishment of a special uncoating mechanism. This mechanism may explain the phenomenon of poxvirus reactivation.

THE INTRACELLULAR UNCOATING OF POXVIRUS DNA. II. THE MOLECULAR BASIS OF THE UNCOATING PROCESS.

The uncoating of poxvirus DNA is a two-step process. The first stage begins immediately after penetration of virus particles and is effected by enzymes present in the uninfected cell; the products of this stage are virus cores. Viral DNA within cores is not accessible to DNase. The second stage of uncoating results in the breakdown of the cores to release naked poxvirus DNA. This stage is inhibited by fluorophenylalanine and puromycin, inhibitors of protein synthesis, as well as by actinomycin D and irradiation with u.v. light, inhibitors of messenger RNA synthesis. It is concluded that in order for virus cores to be broken down a protein has to be synthesized after infection. This protein is synthesized only in response to infection with virus particles containing un-denatured protein. Evidence is presented suggesting that the synthesis of this protein is under the control of the host-cell genome. A scheme is presented which is capable of explaining all known facts concerning poxvirus reactivation, initiation and uncoating. It postulates that a viral protein released during the first stage of uncoating causes derepression of a portion of host cell DNA, permitting the synthesis, through the mediation of messenger RNA formation, of a protein which is instrumental in degrading virus cores and thus releasing viral DNA in the free state.

Studies on the genesis of polyribosomes: II. The association of nascent messenger RNA with the 40 s subribosomal particle

The fate of nascent vaccinia messenger RNA has been determined by pulse-labeling with [3H]uridine for very short periods of time. The findings may be interpreted thus: vaccinia messenger RNA has a free half-life of about 30 sec, after which it combines with a 40 s subribosomal particle. Certain properties of the resultant complexes are described. They can be chased into polyribosomes; their free half-life in the cytoplasm is of the order of three minutes. It is proposed that they represent the first intermediates in the genesis of polyribosomes.

Studies on the structural proteins of vaccinia virus. I. Structural proteins of virions and cores.

Radioactively labeled vaccinia virions grown in L cells were dissociated to yield the constituent polypeptide chains, which were then subjected to electrophoresis in polyacrylamide gels. Mechanical fractionation of these gels yielded complex profiles in which at least 17 components, some major, some minor, could be reproducibly identified. The relative mass in each component has been calculated from the amount of radioactivity incorporated. The principal component (VSP-4) accounts for about 28% of the total viral protein mass. Cores derived from virions by chemical treatment contain the principal viral protein component, which is clearly multiple, as well as two minor ones (VSP-1 and VSP-2), which are probably single polypeptide species. These latter two components are the two slowest moving ones, and therefore most probably have the largest molecular weights. Treatment of virions with the nonionic detergent NP 40 liberates one major viral protein component (VSP-6). This component accounts for 18.7% of viral protein, the second highest amount, and is also multiple in nature.

Studies on “early” enzymes in HeLa cells infected with vaccinia virus☆

Abstract DNA polymerase activity in the cytoplasm begins to increase within 1 1 2 hours after infection of HeLa cells with vaccinia virus (strain WR). The properties of the enzyme in normal and infected cell cytoplasmic extracts have been studied. The enzymes differ with respect to the saturating concentration of DNA primer and the shape of the pH-activity curve: enzyme from infected cells is saturated by 25 μg primer per milliliter whereas the enzyme from normal cells is only saturated by 100 μg/ml; and although the optimum pH is between 7 and 8 for both enzymes, the enzyme from infected cells is relatively more active at pH 9 than that derived from normal cells. The activity of DNase acting on single-stranded DNA also increases on infection of HeLa cells with vaccinia WR, as does the activity of thymidine kinase. The synthesis of all three enzymes is induced at the same time (about 1 1 2 hours after infection), is arrested at any time during the rise period by the addition of puromycin, and is switched off at about 4 hours after infection. The switch-off mechanism for all three enzymes fails to operate when synthesis of progeny viral DNA is inhibited. However, there is one significant difference: whereas the synthesis of thymidine kinase is induced by virus inactivated with UV-irradiation, synthesis of DNA polymerase and DNase (single-stranded DNA) is not. The virus-induced synthesis of both DNA polymerase and thymidine kinase continues for at least 5 hours after the addition of actinomycin D at a not significantly reduced rate. This signifies that the messenger RNAs coding for these “early” enzymes are remarkably long lived.